PL244687B1 - Selenized bioactive polysaccharide fraction from Lentinula edodes, a pharmaceutical composition comprising selenized bioactive polysaccharide fraction, the selenized bioactive polysaccharide fraction for use as medicaments and a method of preparation thereof - Google Patents

Abstract

The present invention concerns a composition of fractions of selenized bioactive polysaccharides from Lentinula edodes, containing a mixture of fraction A and fraction B-C, wherein: fraction A consists of an α-1,4-D-glucan polymer; and fraction B-C consists of the following glucan polymers: 1,3-β-D-glucan, 1,6-β-D-glucan and β-1,3-branched 1,6-β-D-glucan, and where the content fraction A and fraction B-C in the composition is equal to about 85% by weight and about 4% by weight, respectively. Furthermore, the present invention relates to a pharmaceutical composition comprising a fraction of selenized bioactive polysaccharides, a fraction of selenized bioactive polysaccharides for use as therapeutic agents and a method of preparation.

Description

Description of the invention

Field of the invention

The present invention relates to a composition of a selenized bioactive polysaccharide fraction from Lentinula edodes, a pharmaceutical composition containing a selenized bioactive polysaccharide fraction, a selenized bioactive polysaccharide fraction for use as a medicine, and a method for preparing the same.

Background of the invention

Many Basidiomycota fungi have the ability to synthesize bioactive compounds, including anticancer agents, which can be isolated from fruiting bodies, mycelial cultures, or culture media. Lentinula edodes (Berk.) Pegl. is one of the most widely used medicinal mushrooms. This fungus is the source of two drugs approved in several countries. Both drugs are immunomodulators and are used in anticancer therapy. Additionally, L. edodes produces compounds with antibacterial, antiviral, cholesterol-lowering and anticoagulant activity. Water-soluble lignans obtained from the culture medium of L. edodes are being tested as potential drugs for the treatment of hepatitis B and AIDS. L. edodes protein consists of 18 amino acids, including all essential amino acids, in proportions suitable for humans. The fruiting bodies of the fungus contain significant amounts of vitamins C, B1, B2, PP, B12 and D. Immunomodulatory polysaccharides isolated from L. edodes are used in chemoprevention and as adjuvants in anticancer treatment due to their ability to alleviate the side effects of such therapies. This valuable mushroom is also used to produce dietary supplements (Turło, 2013).

Mushroom polysaccharides (MPS), in particular β-D-glucans, are of great interest because they are immunomodulatory substances recognized by the human immune system (Cheng et al., 2014; Liu et al. 2015; Rathore, Presad and Sharma, 2017). Most biologically active MPS are extracted from the fungal cell wall, which consists of inner layers of chitin and β-D-glucans (Latge, 2010; Hardison and Brown, 2012).

As mentioned in several articles, cell wall β-D-glucans are typically 1,3- and 1,6-linked chains with varying amounts of side chains at the O -6 or O -3 position (Synytsya and Novak, 2013; Dalonso, Goldman and Gern, 2015;Zhu, Du, Bian, & Xu, 2015).

Such a structure has been described for lentinan, a highly purified polysaccharide fraction extracted from the fruiting bodies of Lentinula edodes (Japanese sclerotium). This substance is a very powerful immune system enhancer (Wasser & Weis, 1999; Zheng, Jie, Hanchuan & Moucheng, 2005), approved for use in anticancer treatment as an adjunct to conventional therapy (Boon & Wong, 2004; Sullivan, Smith & Rowan, 2006; Yeung and Gubili, 2008). However, it seems that the β-1,3-β-1,6 MPS structure of the cell wall is not universal among Basidiomycota, as there are fungi such as A. ovinus that do not contain this type of β-D-glucan (Samuelsen et al. .2019). Moreover, strain variations, stage of the development cycle, culture method and conditions, medium composition, extraction method, and even drying method may result in variability in monosaccharide composition and combinations among polysaccharides (Ma, Chen, Zhu, and Wang, 2013; Diamantopoulou et al., 2014; Xu, Li, & Hu, 2014; Chien, Yen, Tseng, & Mau, 2015; Su, Lai, Lin, & Ng, 2016).

Additionally, in accordance with the Commission Decision of 2 February 2011 authorizing the placing on the market of mycelium extract from Lentinula edodes (Japanese sclera) as a new food ingredient in accordance with Regulation (EC) No 258/97 of the European Parliament and of the Council (notified as document C(2011) 442, Lentinula edodes mycelium extract was defined as follows: the new food ingredient is a sterile aqueous extract obtained from Lentinula edodes mycelium grown under submerged fermentation conditions. It is a light brown, slightly turbid liquid. Lentinan is β-(1-3 ) β-(1-6)-D-glucan, which has a molecular weight of approximately 5 x 10 ⁵ Daltons, a degree of branching of 2/5 and a triple helical tertiary structure. The composition of the mycelium extract from Lentinula edodes includes: humidity 98%, dry matter 2%, free glucose less than 20 mg/ml, total protein less than 0.1 mg/ml (Bradford method), ingredients containing N less than 10 mg/ml (Kjeldahl method) and lentinan 0.8-1.2 mg /ml.

The immunomodulatory effect of polysaccharides isolated from L. edodes has been widely described in the state of the art.

Patent publication TW201923087A discloses [beta]-1,3-1,6-glucan powder with high water solubility, and a glucan-containing composition using the powder or the like. β-1,3-1,6-glucan can be isolated and extracted by bacteria or from seaweeds, fungi, for example Lentinula edodes.

Moreover, the European patent application EP3277271A1 concerns compounds with unique antiviral properties found in mycelium and their analogues. The invention includes active ingredients present in a combination of products from the mycelium of many species of mushrooms in such a form that they have a cumulative effect of limiting the growth, spread and survival of viruses in animals, in particular in humans, birds and bees. The present invention also includes a combination of products from multiple species of fungi in a form useful for preventing, treating, alleviating, minimizing, ameliorating or reducing the occurrence of viruses, including oncogenic viruses, in animals, including humans. Such formulations may have additional benefits by acting as antibacterial, antiprotozoal, immunomodulatory, nutraceutical and/or prebiotic agents, as well as enhancing innate immune defense mechanisms and the host immune response, leading to healing.

The American patent application US5756318A discloses new polysaccharides derived from, isolated and purified from the culture broth of a liquid culture, including a mixture of culture medium containing plant tissues and mycelium of microorganisms belonging to the Basidiomycetes class. Polysaccharides have a molecular weight of 500 to 10,000 and consist of linearly (1 > 4) linked α-D-glucose units, the 2,3-hydroxyl groups of which are partially acetylated, in a ratio of approximately 30%, and have response modifier properties biological (BRM). They are useful as an immune system enhancer or immune system activator.

International publication WO2020000015A1 relates to immunomodulatory compositions containing one or more extracts of medicinal mushrooms, and the use of such compositions in the treatment or prevention of conditions or diseases associated with dysfunction of the immune system and/or for providing one or more health benefits to an animal subject. In some embodiments, the invention relates to compositions containing a plurality of extracts of a plurality of medicinal mushrooms, and the use of such compositions to provide an individual in need thereof with one or more health benefits.

International publication WO2019209706A2 provides a dried product containing biologically active compounds derived from mushrooms. In some embodiments, the dried product is characterized by the following properties - 60%-90% by weight of carbohydrates, including polysaccharides including a(1-4) glucans and b(1-3) glucans, as determined by 1H and 13C NMR spectra, polysaccharides with a median molecular weight of approximately 10,000 Da; containing 3%-5% protein by weight; 0.1%-3% lipids by weight; 2 or 3 retention time peaks determined by HPLC of the delipidated sample on an Agilent Hi-Plex Na column; and immunomodulatory biological activity.

Moreover, the American patent publication US20190249211A1 provides a method for obtaining β-glucan from mushrooms with high efficiency. The method includes: providing a liquid culture for growing mycelium by fermentation to increase the yield of mycelium and polysaccharide, wherein the liquid culture contains at least two ingredients selected from the group consisting of glucose, trehalose, dietary fiber and mannose or derivatives thereof; and disruption of the mycelium using an ultrasonic device with continuous repeated action; and removal of insoluble substances from the liquid culture. A method of obtaining β-glucan powder and solution from mushrooms with high purity of their products is also presented. By the method of the disclosure, the yield of β-glucan production from mushrooms is effectively increased, the loss of its activity is reduced and the stability of its product is improved. One of the myceliums is the mycelium of Lentinula edodes.

Additionally, patent application CN110437343A refers to a method of extracting lentinan.

The Polish patent document PL225547B1 discloses a biosynthetic method leading to the preparation of a pharmacologically active preparation from Lentinula edodes and the use of this preparation. The preparation obtained in this way combines the immunomodulatory effect of polysaccharides from L. edodes with a similar effect of selenium compounds, which act through a different mechanism. However, this study focuses on the properties of the unseparated fractions containing mixtures of different polysaccharides.

Additional structural analysis of selenized polysaccharides - lentinan analogue was presented in the work of Malinowska et al. (Malinowska, E., Klimaszewska, M., Strączek, T., Schneider, K., Kapusta, C., Podsadni, P., Łapienis G., Kleps, J., Dawidowski, M., Górska, S., Pisklak, D.M., Turło J. (2018). Selenized polysaccharides-biosynthesis and structural analysis. Carbohydrate Polymers 198, 407-417). The authors of this publication expected that the inclusion of selenium in the polysaccharide would enhance the immunostimulatory activity of the isolated fraction compared to lentinan. It has also been suggested that the isolated Se-polysaccharide is a non-toxic immunosuppressant with strong antioxidant activity that even improves cell viability (Kaleta, B., Gorski, A., Zagozdzon, R., Cieslak, M., Kazmierczak-Baranska, J., Nawrot, B., Turlo, J. (2019). Selenium-containing polysaccharides from Lentinula edodes - Biological activity. Carbohydrate Polymers, 223). The biological effect was completely different than the biological effect related to lentinan. It was also suggested that it depends partly on the inclusion of selenium in the polysaccharide molecule, but probably also on the different structure of the fractions isolated from mycelial cultures compared to lentinan. Preliminary structural studies showed that the isolated immunologically active fraction is a protein-containing mixture of polysaccharides with a high molar mass (Mw 3.9x106 g/mol and 2.6x105 g/mol), α- and β-glucans, composed of glucose or mannose. Spectral analysis of the X-ray absorption structure (XAFS) in the near-edge region (XANES) confirmed that selenium in the Se-polysaccharides structure is present in the -II oxidation state and that Se is organically bound. Simulation analysis in the EXAFS region suggested that selenium is most likely bound via a glycosidic bond in a β-1,3 or α-1,4-glycosidic bond or substituted for oxygen in the pyranoside ring (Malinowska et al., 2018). However, as in the case of the above publications, the presented research concerned structural studies related to unseparated fractions, which are a mixture of various polysaccharides, and it was not clarified which of the components of the complex fraction was responsible for its immunosuppressive activity, unusual for polysaccharides of fungal origin. Moreover, the complex composition of the preparation of selenized polysaccharides synthesized by Lentinula edodes would make it difficult to use this preparation in pharmaceutical applications, as it is required that each medical device approved for use is subject to thorough inspection of purity and components.

Therefore, the aim of the present invention is to provide a composition of fractions of selenized bioactive polysaccharides from Lentinula edodes, containing a mixture of fraction A and fraction B-C, in which: fraction A consists of an α-1,4-D-glucan polymer; and fraction B-C consists of the following glucan polymers: 1,3^-D-glucan, 1,6^-D-glucan and β-1,3-branched 1,6^-D-glucan, and in which the content of fraction A and fraction B-C in the composition is approximately 85% by weight and approximately 4% by weight, respectively.

As used herein, the term "about" is to be understood to include close values such as equal to ± 6% of such value, i.e. the term about 85% by weight is to be understood to include a range of 85% ± 6% of the value of 85%, i.e. a range of 79 .90% to 90.10%, and where about 4% is to be understood as covering the range of 4% ± 6% of the value of 4%, i.e. the range of 3.76% to 4.24%. Preferably, the content of fraction A in the composition is in the range of 84%-86%, while the content of fraction B-C in the composition is in the range from 3.76% to 4.24%. Even more preferably, the content of fraction A is 85.35% and the content of fraction B-C is 4.23%.

The present invention also provides a method for preparing the composition of selenized bioactive polysaccharide fractions from Lentinula edodes according to the present invention, which includes the following steps:

(a) cultivating Lentinula edodes in a culture medium enriched with at least 30 μg/ml selenium;

b) isolating mycelium;

c) extraction with boiling methanol 1:4 w/v;

d) extracting the methanol-insoluble fraction from step c) with hot water;

e) precipitating the fraction extracted in step d) with 1 volume of ethanol;

f) solubilizing the precipitate formed in step e) in water and precipitating it with 0.2 M cetyltrimethylammonium hydroxide;

g) extracting the precipitate from step f) with 20% acetic acid;

h) extracting the insoluble fraction from step g) with 50% acetic acid;

i) solubilizing the insoluble fraction from step h) with 6% NaOH;

j) precipitation with 3 volumes of ethanol;

k) deproteinizing the precipitated step from step j); and

l) purification of the polysaccharide fraction from the deproteinized sediment.

In a preferred embodiment of the present invention, step 1) comprises the use of ion exchange chromatography and gel permeation chromatography. More preferably, the first polysaccharide fraction (fraction A) eluted from the gel permeation chromatography column is retained, which fraction contains at least 80% by weight of the α-1,4-D-glucan polymer forming the α-helix structure; the second polysaccharide fraction (fraction A/B-C) eluted from the gel permeation chromatography column is retained, which fraction contains a mixture of the following glucan polymers: α-1,4-D-glucan, 1,3-3-D-glucan, 1,6 -3-D-glucan and 3-1,3-branched 1,6-3-D-glucan; or a third polysaccharide fraction (fraction B-C) eluted from the gel permeation chromatography column is retained, which fraction contains a mixture of three glucan polymers: 1,3-3-D-glucan, 1,6-3-D-glucan and 3-1, 3-branched 1,6-3-D-glucan.

The present invention further provides a composition of selenized bioactive polysaccharide fractions from Lentinula edodes obtained by the method of the present invention, said composition comprising a mixture of α-1,4-D-glucan polymer fraction A; and fractions B-C of 1,3-3-D-glucan, 1,6-3-D-glucan and 3-1,3-branched 1,6-3-D-glucan, and where the content of fraction A and fraction B-C in the composition is equal to about 85% by weight and about 4% by weight, respectively. Preferably, said composition is obtained by a method in which a second polysaccharide fraction eluted from the gel permeation chromatography column is retained, which fraction contains a mixture of the following glucan polymers: α-1,4-D-glucan, 1,3-3-D-glucan , 1,6-3-D-glucan and 3-1,3-branched 1,6-3-D-glucan.

Furthermore, the present invention provides a composition of the selenized bioactive polysaccharide fractions of the invention for use as a therapeutic agent.

In a preferred embodiment of the present invention, the selenized bioactive polysaccharide fraction composition of the invention is used as a therapeutic agent for selective inhibition of T cell proliferation. More preferably, selective inhibition of T cell proliferation is required for conditions selected from the group consisting of allogeneic organ transplantation, autoimmune diseases and allergies T cell dependent. Most preferably, the autoimmune diseases are selected from rheumatoid arthritis, multiple sclerosis, celiac disease, glomerulonephritis, inflammatory bowel disease and psoriasis. In another most preferred embodiment, the T cell mediated allergy is contact dermatitis.

In a preferred embodiment of the present invention, said composition according to the invention is for oral administration, preferably in the form of tablets, capsules, granules for reconstitution or in the form of a solution; intravenous; intraperitoneal or external, preferably in the form of ointments, creams, suppositories, vaginal rings, rectal enemas.

The present invention also provides a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable excipient which, as active ingredient, contains a composition of selenized polysaccharide fractions.

By the method of the present invention it is possible to obtain a composition of the fraction of selenized bioactive polysaccharides from Lentinula edodes which is, due to the unexpected synergy effect provided by fraction A and fraction B-C, more active and effective. Moreover, the present invention overcomes the disadvantages of methods known in the art and of the compositions obtained by such methods, since it provides compositions that are much purer and could be used directly as an active ingredient in a pharmaceutical composition without any additional treatment, in particular additional cleansing. This is particularly important because such a composition could be used to treat rheumatoid arthritis, multiple sclerosis, celiac disease, glomerulonephritis, inflammatory bowel disease and psoriasis.

Brief description of the figures

Preferred, non-limiting examples which embody certain aspects of the invention are herein described with reference to the following figures.

Fig. 1. A. Chihara method for lentinan isolation; B Modified method for the isolation of Se-enriched lentinan analogue (Se-Le-30 fraction).

Fig. 2. Selected fragments of 1H-13C HSQC-DEPT NMR spectra of fraction II (A, B) and fraction III (C, D).

Fig. 3 Structure of polysaccharides presented in fraction II separated from Se-L-30.

Figure 4. RI records from SEC of Se-Le-30 fractions, A (I), A/B-C (II), and B-C (III). The lines for fractions A, A/B-C and B-C were smoothed to remove noise.

Fig. 5. Deconvolution of the RI signal of product B-C.

Fig. 6. Structural models of the tested polysaccharides; lateral view (left) and longitudinal view (right). A. Polysaccharide A (1,4-a-D-glucan). B. 1-3-3-D-glucan - component of the B-C fraction. C. 1-6-3-D-glucan - component of the B-C fraction. D. 1 -3-3-branched 1 -6-3-D-glucan - component of the B-C fraction. E. Distances (A) between oxygen atoms, suggesting the possibility of stabilizing the helical structure of 1-3-3-branched 1-6-3-D-glucan with hydrogen bonds. Created using the Chem3D Pro module from ChemOffice Professional 2017 (PerkinElmer). Energy minimization was performed using the MM2 method.

Fig. 7. Effect of polysaccharides (Se-L, Se-Le-30, A, B-C and A/B-C) on the proliferation of peripheral blood mononuclear cells (PBMC). A. Without stimulation (self-stimulation). B. Stimulated with anti-CD3 monoclonal antibody (OKT3). C. Stimulated with phytohemagglutinin (PHA). D. Stimulated with a suspension of Staphylococcus aureus Cowan (SAC). Results are presented as percentage of control proliferation (without polysaccharides). *p<0.05 vs. control (K). Error bars represent standard deviation. The results for the Se-L fraction were described by Kaleta et al. (2019).

Fig. 8. RALS records for Se-Le-30 and fractions A, A/B-C and B-C.

Fig. 9. SEC records. a) Se-Le-30, b) fraction A/B-C, c) fraction A, d) fraction B-C.

Fig. 10. Deconvolution of the Se-Le-30 RI signal. The curves corresponding to fractions A and B-C are indicated by arrows.

Fig. 11. Deconvolution of the RI signal of product A/B-C. The curves corresponding to fractions A and B-C are indicated by arrows.

Fig. 12. Deconvolution of the RI signal of product A. The curve corresponding to fraction B-C is indicated by an arrow.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure relates. For example, in Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, ed. 3., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press, provide definitions generally accepted in the art for many of the terms used in this disclosure.

As used herein and in the appended claims, the singular forms include plural referents unless the context clearly indicates otherwise. The terms without numerical indication, as well as the terms "one or more" and "at least one" may be used interchangeably herein.

As used herein, the terms "treat" or "treatment" or "treatment" refer to (i) reducing the potential for a disease or disorder (e.g., a disease associated with increased T cell activity or proliferation), (ii) reducing the incidence of disease or disorder, (iii) reducing the severity of the disease or disorder, preferably to the extent that the individual suffers less or no longer suffers from discomfort and/or altered functioning, (iv) reducing an indicator or marker of the disease or disorder, such as decreased levels of T-cell markers in blood or serum, or (v) a combination thereof.

As used herein, the terms "individual", "individual" or "patient" refer to any individual, particularly a mammalian individual, for whom diagnosis, prognosis or therapy of a disease or disorder is desired. As used herein, the terms "subject", "individual" or "patient" include any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g. mammals and non-mammalian animals such as mice, non-human primates, sheep, dogs, cats, horses, cows, bears, chickens, amphibians, reptiles etc. In preferred In embodiments, the subject is a human.

As used herein, the term "administering" or "administering" a drug or therapeutic agent includes providing, using, or administering a therapy or drug to an individual, including self-administration by the individual.

As used herein, the term "therapeutically effective amount" refers to an amount of a compound or mixture (e.g., a polysaccharide fraction) that will effectively "treat" a disease or disorder in an individual and/or prevent or reduce the risk, potential, possibility, or incidence of the disease or disorders.

The therapeutically effective amount for treatment may in practice depend on factors including levels of pro-inflammatory factors in the blood or serum, the severity of the disease or disorder in the subject prior to treatment, the presence or absence of various conditions (e.g., the presence or absence or severity of cardiovascular disorders and/or diseases or allergic reactions), the age and sociocultural gender identity of the individual, and can be corrected by a person of ordinary skill in the field (e.g. a doctor).

These treatments may include the treatment of diseases requiring modulation of T-cell activity, including, but not limited to, allogeneic organ transplantation, as well as a number of autoimmune diseases, especially those with known abnormal T-cell activity, such as rheumatoid arthritis, multiple sclerosis, celiac disease, glomerulonephritis nephritis, inflammatory bowel disease, psoriasis, etc., as well as in T cell-mediated allergy such as contact dermatitis, etc.

"Deproteinization" is intended to mean any method known in the art for reducing protein content or removing proteins from a mixture, i.e., the Savage method (Ruthes, A.C.; Smiderle, F.R.; lacomini, M. D - Glucans from Edible Mushrooms: A Review on the Extraction , Purification and Chemical Characterization Approaches. Carbohydr. Polym. 2015, 117, 753-761, doi:10.1016/j.carbpol.2014.10.051).

Glucan is a polysaccharide made of D-glucose, connected by glycosidic bonds. A glycosidic bond or glycosidic linkage is a type of covalent bond that connects a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate. α- and β-glycosidic bonds differ in the relative stereochemical configuration of the anomeric position and the center of stereoisomerism furthest from C1 in the saccharide. An α-glycosidic bond is formed when both carbon atoms have the same stereochemical configuration, while a β-glycosidic bond occurs when two carbon atoms have different stereochemical configurations.

The term "analog", as used in this description, should be understood as a chemical compound with a similar structure, which may, however, exhibit different biological activity.

As used herein, the term "Se-L" refers to the lentinan analog isolated by the Yap and Ng method.

As used herein, the term "Se-Le-30 fraction" refers to the lentinan analogue isolated by the modified Chihara method.

As used herein, the term "fraction A" or "fraction I" refers to α-1,4-D-glucan characterized by an elution time expressed in milliliters at a flow rate of 0.5 ml/min: ion exchange chromatography : 6-24 ml and gel permeation chromatography: 62-74 ml.

As used herein, the term "fraction A/B-C" or "fraction II" refers to α-1,4-D-glucan, 1,3^-D-glucan, 1,6^-D-glucan and β-1,3-branched 1,6^-D-glucan, characterized by an elution time expressed in milliliters at a flow rate of 0.5 ml/min: ion exchange chromatography: 79.5-97.5 ml and gel permeation chromatography: 62 -76 ml.

As used herein, the term "fraction B-C" or "fraction III" refers to 1,3^-D-glucan, 1,6^-D-glucan and β-1,3-branched 1,6^- D-glucan, characterized by an elution time expressed in milliliters at a flow rate of 0.5 ml/min: ion exchange chromatography: 87-109.5 ml and gel permeation chromatography: 72-90 ml. (Calculated from fractions: LC-1KP after DEAE A4B1 on HW C7D1, LC-1KP after DEAE D8E5 on HW C7D2, LC-1EM after DEAE D13E13 on HW C11D9).

Hot water means water with a temperature in the range of 90-100°C.

The polysaccharide fractions obtained by the method of the present invention can be administered by various routes, e.g. orally, intravenously, topically or by other routes. The polysaccharide fractions may be administered alone or in a pharmaceutical composition containing a pharmaceutically acceptable excipient.

The polysaccharide fractions of the present invention or pharmaceutical compositions thereof may be present in various dosage forms. They may be administered in liquid form (e.g., solution, dispersion, suspension, sterile solution for injection or infusion, etc.) or in solid form (e.g., powder, such as lyophilized). If the polysaccharide fractions of the present invention or pharmaceutical compositions thereof are to be administered orally, they may be in the form of, for example, tablets, capsules, lozenges or other forms.

Where a pharmaceutically effective amount of the polysaccharide fractions of the invention is administered orally, e.g., contained in a pharmaceutically acceptable oral dosage form, such oral dosage forms may also contain an appropriate amount of one or more pharmaceutically acceptable excipients, including a diluent, suspending agent, solubilizer, a binding agent, a disintegrating agent, a preservative, a coloring agent, a lubricant and the like. The pharmaceutical excipient may be a liquid such as water or oil, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipient may be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. Additionally, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In some embodiments, the pharmaceutically acceptable excipient is sterile when administered to a human subject. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol and the like. The pharmaceutical compositions, if desired, may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Examples of pharmaceutically acceptable carriers and excipients that can be used to formulate various dosage forms are known in the art, such as those described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986).

Detailed description of the invention

The aim of the present invention is to provide a Se-polysaccharide fraction that exhibits improved immunosuppressive activity compared to the isolated Se-polysaccharide-Se-L protein fraction.

Lentinan, a Japanese immunostimulant drug for which the inventors intended to obtain a selenized analogue, was first isolated by G. Chihara from the fruiting bodies of L. edodes in a multi-step fractionation procedure using strong bases and organic acids (Chihara et al., 1970). It was revealed that when isolating Se-polysaccharides from the mycelium of L. edodes in this way, a significant part of selenium may be lost. Selenium-containing polysaccharides (selenoglycosides) may be unstable in strongly alkaline and strongly acidic environments. Therefore, the lentinan isolation method of Yap and Ng (2001) was chosen. The advantage of this method is the mild conditions of the isolation process, which lead to higher polysaccharide yield and lower selenium loss. However, the disadvantage is lower homogeneity and reduced purity of the obtained fraction compared to the Chihara method (87% vs. 96%).

Due to the fact that the inventors wanted to exclude the immunosuppressive effect of the previously tested Se-L fraction caused by components other than polysaccharides (proteins or other unspecified impurities), it was decided to obtain a pure analogue of lentinan using the Chihara isolation method. To compensate for the loss of selenium during isolation, the mycelial culture was grown in a medium containing a higher concentration of selenium (30 ppm). By increasing the selenium concentration in the culture medium to 30 ppm, a very significant increase in the Se concentration in the mycelium of L. edodes was obtained: 1753 μg/g compared to 1040 μg/g for the culture enriched with 20 ppm. A significant increase in selenium concentration was also observed in the Se-L fraction isolated by the Ng and Yap method (305 μg/g vs. 190 μg/g). However, the lentinan Se analog Le-30 isolated by the Chihara method contained a much lower concentration of selenium (48 μg/g). Therefore, the change in the isolation method resulted in a significant loss of selenium, but also had the expected positive effect, a significant increase in the purity of the fraction in terms of protein and sugars other than glucose content.

The previously determined Se - L fraction (Kaleta et al., 2019) showed an immunosuppressive effect that is opposite to the effect of the Japanese drug lentinan, which is isolated using the same method from the fruiting bodies of the same mushroom species. Structural studies of the Se - L fraction indicated that it contains a mixture of 1,4-α-, 1,6-β- and 1,3-3-glucans and mannans (Malinowska et al., 2018), while lentinan is (1 .3; 1.6)-β-glucan (Zhang et al., 2011). The proportion of 1,6 and 1,3^-glycosidic bonds in the mycelium fraction was also different than in the case of lentinan (Malinowska et al., 2018).

Se-Le-30 faction

Similarly to the Se - L fraction, data on the type of glycosidic bonds were recorded for the Se-Le-30 fraction. NMR and TRISEC studies showed that the Se-Le-30 fraction was not homogeneous. Product A was the main component of the Se-Le-30 fraction, and product B-C, with a much lower molar mass, was present only in small amounts. The approximate weight ratio of polysaccharides A and B-C in the Se-Le-30 fraction was 26:1. Additionally, the Se-Le-30 fraction contained approximately 6% mannose and 4% protein (possibly in the form typical of fungal cell wall mannoproteins) by weight. The selenium content in the Se-Le-30 fraction was almost 4 times lower than in the previously isolated Se-L fraction (Malinowska et al., 2018).

Fraction A (I)

The analysis of sugars and the determination of the absolute configuration showed that both isolated polysaccharides (A and B-C) consist of D-glucose.

Methylation analysis showed that polysaccharide A is linear. Sequence analysis of monosaccharide residues within the repeating unit of this polysaccharide obtained by assigning residue interactions observed in 2D NOESY and HMBC spectra revealed associations between C-1 and C-4. The complete assignment of the ^1H and ^13C resonances and the sequence and bonding information obtained from a series of 2D NMR experiments suggested that residue A is α-linked. This indicated that A represents a >4)-aD-Glcp-(1> moiety. Obtained data on the structure of fraction A indicated that this natural polysaccharide of fungal origin, most likely a component of the cell wall of L. edodes, has a structure similar to amylose, a polysaccharide of plant origin. Its molar mass determined using TRISEC was significantly higher than the average masses typical for amylose. According to Joye (2019), the range of molar masses of amylose is quite wide and varies between 8 x 104 and 106 g/mol depending on the plant species, varieties and maturity of the starch tested.

The >80% content of A, an amylose-like α-1,4-D-glucan, in the Se-Le-30 polysaccharide fraction isolated from L. edodes mycelium was surprising. The amylose-like polysaccharide has not yet been described as the major hot-water-soluble cell wall component of L. edodes. However, McCleary and Draga (2016) determined the content of α- and β-glucans in commercial preparations of mushroom origin, including L. edodes extracts; they contain up to 80% a-glucans, instead of the β-glucans declared by the manufacturer. The authors explained the presence of α-glucans as an artifact caused by the culture and isolation method. They found that commercial mycelium is grown on a sterilized grain base. In the latter process, grain infected with mycelium is collected and polysaccharide fractions are isolated. Based on analytical data, they suggested that grain starch is the main α-glucan present in the final product (McClery and Draga, 2016).

This was possible in the case of mycelium grown on solid medium (cereal). However, the present invention discloses mycelium that has been grown in a liquid medium that does not contain starch or solids. After collection, it was thoroughly filtered and washed, which means that contamination with the culture medium was not possible. Polysaccharide A, a water-soluble 1,4-a-D-glucan, was a component of L. edodes mycelium.

The secondary structure of polysaccharide A predicted by molecular modeling is an α-helix (Figure 6A), analogous to amylose, which also typically adopts a natural helical structure (Joye, 2019). Natural polysaccharides have a polydisperse molar mass, but using fractionation techniques such as size exclusion chromatography can achieve a low polydisperse index. This is the case of fraction A with a low polydispersion index (B = 1.2), which indicates its homogeneity. Deconvolution of the RI signal of product A allowed to determine the residual content of the B-C fraction equal to 2.73%. Generally speaking, purification by GPC did not result in the isolation of completely pure polysaccharides. In particular, it was very difficult to separate fraction B-C from fraction A, despite the very significant difference in molar mass, probably, without being bound by any theory, due to intermolecular interactions. A similar problem occurs when separating amylose from amylopectin using gel permeation chromatography. Perhaps amylose is cross-linked to amylopectin and thus is eluted with amylopectin (Ai and Jane, 2018), or is interspersed with amylopectin molecules. Similar interactions may occur between polysaccharides A and B-C.

Fraction B-C (III)

The same analytical methods used to examine the structure of fraction A (sugar analysis and determination of absolute configuration, methylation analysis, 2D 1H, ^13C NMR) showed that the BC fraction is a mixture of three glucans: 1,3-3-D-glucan, 1, 6-3-D-glucan and 3-1,3-branched 1,6-3-D-glucan. The molar mass determined by TRISEC was significantly lower than that of fraction A. The homogeneity of the BC fraction was also lower, indicating a higher polydispersion index (B = 2.18).

1,6-3-glucan has been shown to be an important component of the cell walls of S. cerevisiae and C. albicans (Aimanianda et al., 2009; Klis et al., 2006; Lesage and Bussey, 2006). Nevertheless, a number of publications report the presence of 1-6-3-glucans in the cell wall of higher fungi (Malinowska et al., 2018; Li et al., 2019; Samuelsen et al., 2019). Structural analysis of aqueous and alkaline extracts from A. ovinus fruiting bodies suggested the presence of two different β-D-glucan skeleton structures: β-D-glucan with 1-6 bonds with single β-D-Glcp residues; at the O-3 and β positions of D-glucan with (1>3) linkages with branches (Samuelsen et al., 2019). The B-C polysaccharide with a similar structure was present in low proportions in the tested Se-Le-30 fraction isolated from L. edodes, which is probably due to its low solubility in hot water. A similar compound was much more effectively isolated by Samuelsen et al. (2019) and by Li et al. (2019) with a hot alkali solution.

Unlike lentinan, a polysaccharide isolated from the fruiting bodies of L. edodes by G. Chihara - a β-D-glucan with a skeleton with 1-3-β bonds and side chains connected by 1-6-β bonds, the polysaccharide fraction isolated using the same procedure from submersible culture of L. edodes mycelium turned out to be a mixture of linear 1-4-a-D-glucans, 1-3^-D-glucans, 1-6-β-D-glucans and branched 1-6^-D-glucan with skeletons with 1-3-β bonds.

EXAMPLES

MATERIALS AND METHODS

Biosynthesis and isolation of Se-enriched polysaccharide fractions

Mushroom strain and cultivation conditions

The Lentinula edodes (Berk.) Pegler strain used in this study was ATCC 48085. The seed culture was performed under conditions described in previous reports (Turło et al., 2010; 2011). The mycelium used as a raw material for polysaccharide extraction was grown under submerged conditions in a 10-liter fermenter (BioTec FL 110, Stockholm, Sweden). Culture media were enriched with 30 μg/ml selenium by adding sodium selenite (Na2SeO3, Sigma, tested on cell cultures). The initial pH of the medium was 6.5. The medium was inoculated with 5% (v/v) seed culture and cultured at 26°C. Fermentation was carried out for 10 days under the following conditions: aeration rate, 0.5 volume per volume per minute (vvm); mixing speed, 200 rpm; and working volume, 8 l. The mycelium was collected by filtration, washed three times with distilled water and freeze-dried.

Extraction of the polysaccharide fraction enriched in Se Se-Le-30 using the Chihara method

The Se-enriched polysaccharide fraction, an analogue of the Japanese anticancer drug lentinan, was isolated from the Se-enriched mycelium of L. edodes using a modified Chihara method (Chihara et al., 1970). The original method was complemented by pre-extraction of lipids, small carbohydrate molecules and other non-polysaccharide compounds in boiling methanol (1:4 w/v) for 4 hours (Figure 1).

It is also possible to use a simplified method allowing for lower losses of selenium, in which, after the precipitation step with alcohol, the obtained precipitate is lyophilized, then re-dissolved in hot water, precipitated again with alcohol, lyophilized and subjected to chromatographic separation.

Isolation/purification of the polysaccharide fraction enriched in Se-Le-30

Polysaccharides were purified according to the publications of Górska et al. (2016). Briefly, the lyophilized crude Se-Le-30 extract was purified by ion exchange chromatography on a DEAESephadex A-25 column (1.6x20 cm; Pharmacia Biotech, USA) and then by gel permeation chromatography on a Toyopearl HW-55S column (1.6x10 cm; Tosoh Bioscience LLC, Germany) adapted to the FPLC system (Amersham Pharmacia Biotech, Sweden). The fractions were eluted with a NaCl gradient (0-2 M in 20 mM Tris buffer, pH 8.2) and 0.1 M ammonium acetate buffer, respectively. Column eluates were monitored at 220, 260, and 280 nm with a UV-VIS absorbance detector and a Knauer differential refractometer and for carbohydrate content (Dubois, Giller, Rebers, & Smith, 1956). The carbohydrate containing fractions were collected.

Structural analysis

The molar mass of the analyzed fractions was estimated using triple detection size exclusion chromatography (SEC) (TRISEC), as described in the work by Malinowska et al. Namely, three TSK-GEL columns were used (G5000 PWxl + 3000 PWxl + 2500 PWxl; 7.8x300 mm; Tosoh) with a Knauer K-501 pump, a refractometric (RI) detector (LDC) and a TDA 270 dual detector (laser light scattering [RALS + LALS] detector and viscosity detector; Viscotek, USA). The analysis was performed at 26°C with a mobile phase of 0.1% NaN3 and a flow rate of 1.0 ml/min. The injection volume was 100 μ!. Before injection, the samples were filtered through 0.2 μm pore size membrane filters. OmniSEC software (Viscotek, USA) was used to calculate the numerical average molar mass (Mn) for dn/dc = 0.145 (^-glucans in NaN3 solution). The procedure for calculating molar mass using OmniSEC software has been described previously (Huang et al., 2004).

Determination of monosaccharide composition using RP-HPLC

The monosaccharide composition of polysaccharides was determined using the reversed-phase high-performance liquid chromatography (RP HPLC) method described in the work by Malinowska et al. Before analysis, the polysaccharides were hydrolyzed for 5 hours in 3M TFA at 120°C.

Determination of Se content using RP HPLC

The RP HPLC procedure used to determine the Se content was a modified fluorimetric method for determining Se after derivatization with 2,3-diaminonaphthalene (formation of 3,5-benzopiazselenol) with fluorescence detection, as described in the publication by Turło et al. and Malinowska et al.

Determination of protein content in lentinan analogue (Se-Le-30)

Protein content was determined using two independent methods described in the publication by Malinowska et al. The results of the well-known Bradford spectrophotometric method (Bradford, 1976) were verified by measuring the nitrogen content in the analyzed fractions using a CHNS elemental composition analyzer (Vario EL III, Elementar, Germany). Protein content was calculated using a conversion factor of 6.25 total nitrogen to protein.

Analysis of sugars and methylation and determination of absolute configuration

Polysaccharide samples (0.5 mg) were hydrolyzed with 10 M HCl at 80°C for 25 minutes and evaporated in a stream of N2. The obtained monosaccharides were converted into alditol acetates according to Sawardeker, Sloneker and Jeanes (1956). For methylation analysis, polysaccharide samples were permethylated according to the method described by Ciukanu and Kerek (1984). The product was purified by water-chloroform extraction. Methylated polysaccharides were hydrolyzed in 2 M TFA at 120°C for 2 h and evaporated under N2. Finally, the methylated monosaccharides were reduced with NaBD4 and acetylated for analysis - partition gas chromatography-mass spectrometry (GLC-MS) - using the same conditions as for the analysis of sugars and butyl glycosides, as described below. The absolute configurations of the sugars were determined by GLC-MS of their acetylated glycosides using (S)-/+2-butanol, essentially as described by Gerwig, Kamerling, and Vliegenthart (1979). Alditol acetates and acetylated 2-butylglycosides were analyzed by GLC-MS using an ITQ 700 Thermo Scientific system equipped with a ZB-5HT capillary column (Phenomenex) with a temperature gradient from 150 to 270°C at 8°C/min.

Determination of protein content in the lentinan analogue, Se-Le-30

Protein content was determined using two independent methods described in the publication by Malinowska et al. The results of the well-known Bradford spectrophotometric method (Bradford, 1976) were verified by measuring the nitrogen content in the analyzed fractions using a CHNS elemental composition analyzer (Vario EL III, Elementar, Germany). Protein content was calculated using a conversion factor of 6.25 total nitrogen to protein.

Analysis of sugars and methylation and determination of absolute configuration

Polysaccharide samples (0.5 mg) were hydrolyzed with 10 M HCl at 80°C for 25 minutes and evaporated in a stream of N2. The obtained monosaccharides were converted into alditol acetates according to Sawardeker, Sloneker and Jeanes (1956). For methylation analysis, polysaccharide samples were permethylated according to the method described by Ciukanu and Kerek (1984). The product was purified by water-chloroform extraction. The methylated polysaccharides were hydrolyzed in 2 M TFA at 120°C for 2 h and evaporated under an N 2 atmosphere. Finally, the methylated monosaccharides were reduced with NaBD4 and acetylated for analysis - partition gas chromatography-mass spectrometry (GLC-MS) - using the same conditions as for the analysis of sugars and butylglycosides as described below. The absolute configurations of the sugars were determined by GLC MS of their acetylated glycosides using (S)-/+2-butanol, essentially as described by Gerwig, Kamerling, and Vliegenthart (1979). Alditol acetates and acetylated 2-butylglycosides were analyzed by GLC-MS using an ITQ 700 Thermo Scientific system equipped with a ZB-5HT capillary column (Phenomenex) with a temperature gradient from 150 to 270°C at 8°C/min.

NMR spectral analysis

NMR spectra were obtained on a Bruker 600 MHz Avance III spectrometer using a 5-mm QCI ¹ H/ ¹³ C/ ¹⁵ N/ ³¹ P probe equipped with a z gradient. NMR spectra were obtained for a ² H2O solution of polysaccharide at 25°C using acetone ( 5h 2.225, 5c 31.05 ppm) as internal standard. The polysaccharide (10 mg of fractions I and II and 2 mg of fraction III) was subjected to repeated exchange with ² H2O, with intermediate freeze-drying. Data acquisition and processing were performed using Bruker Topspin (version 3.1) and SPARKY software (Goddard and Kneller, 2001). Signals were assigned using one-dimensional (1H, ¹³ C, ³¹ P) and two-dimensional (2D) experiments: correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser spectroscopy (NOESY), single-quantum heteronuclear coherence spectroscopy with detection 1H (HSQC) with or without carbon decoupling, HSQC-TOCSY and heteronuclear long-range 1H- ¹³ C correlation spectroscopy (HMBC). TOCSY experiments were performed with mixing times of 30, 60, and 100 ms, NOESY with mixing times of 100 ms and 300 ms, and HMBC with a mixing time of 60 ms.

Molecular modeling

Structural models of the tested polysaccharides were built using the Chem3D Pro module from ChemOffice Professional 2017 (PerkinElmer). Energy minimization was carried out using the MM2 method implemented in the software.

Effect of polysaccharides on the proliferation of human peripheral blood mononuclear cells (PBMC)

To investigate the effect of polysaccharide fractions and isolated polysaccharides on the proliferation of human lymphocytes, the method described in Kaleta et al. was used. Briefly, peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood of healthy donors by density gradient centrifugation on Histopaque-1077 (Sigma). PBMCs were cultured in Parker's medium (Biomed) supplemented with 2 mM L-glutamine (Sigma), 0.1 mg/ml gentamicin (KRKA), β-mercaptoethanol (Sigma), 0.23% Hepes (Sigma), and 10% heat-inactivated fetal bovine serum (FBS, Gibco).

Polysaccharide fractions from L. edodes (Se-L, Se-Le-30, I (A), II (A/BD) and III (BD)) were diluted in 0.9% NaCl (Fresenius Kabi) to obtain concentrations of 0, 1-0.001 mg/ml. PBMCs were seeded into 96-well flat-bottom microplates at a density of 1 x ¹⁰⁶ cells/well and stimulated with specific mitogens for T and B lymphocytes: anti-CD3 mAb (OKT3, 1 μg/ml, BD Pharmingen, mitogen for T lymphocytes), phytohemagglutinin (PHA, 20 μg/ml, Sigma, a mitogen for T lymphocytes) and a suspension of Staphylococcus aureus strain Cowan (SAC, 0.004% w/v, Calbiochem, a mitogen for B lymphocytes). Polysaccharides were added to the cultures and cells in portions of 100 μl of the prepared dilution per well. Control cultures contained an equivalent amount of 0.9% NaCl. As an internal control, an analogous test was performed without any mitogens (self-stimulation). PBMCs were cultured for 72 hours at 37°C in a humidified atmosphere with 5% CO2, treated with 1 µCi/well of [ ^3H ]-thymidine (113 Ci/nmol, NEN) for 18 h and harvested using a Skatron Cell Harvester. Thymidine incorporation into the DNA of proliferating cells was analyzed in a liquid scintillation counter (Wallac Microbeta). Results were expressed as counts per minute (cpm). All experiments were performed in triplicate.

All participants provided written informed consent to participate in the study. The study was approved by the local ethics committee. The procedures were performed in accordance with the 1975 Declaration of Helsinki, as revised in 2000.

Statistical analysis

The Mann-Whitney U test and Spearman's correlation using Statistica 9.0 (StatSoft Inc) were used. Differences from control cultures were considered significant at p < 0.05.

RESULTS

Composition of the Se-Le-30 polysaccharide fraction

Selenium content

The selenium concentration in Se-enriched mycelium grown in medium supplemented with 30 μg Se/ml was 1753 μg/g. The selenium concentration in the Se-Le-30 fraction isolated by the Chihara method was 48 pg/g.

Monosaccharide composition

The monosaccharide composition of the Se-enriched fraction, Se-Le-30, was determined after complete hydrolysis using trifluoroacetic acid. Similarly to the previously described Se-L fraction (Malinowska et al., 2018), mannose and glucose constituted 99.5% of the total monosaccharides by weight. However, the mannose content in the Se-Le-30 fraction was lower than for the previously described Se-L fraction (6% vs. 14%).

Protein content

The total protein content in the Se-Le-30 fraction was 3.3%, determined using the Bradford method, which was almost 2 times lower than in the Se-L fraction isolated using the Ng and Yap method (8.4%) (Malinowska et al. 2018). The protein content in the Se-Le-30 fraction, calculated on the basis of elemental analysis and conversion of total nitrogen into true protein, was similar to the Bradford method (4.8% for Se-Le-30 and 10.2% for Se-L) . The use of conversion factors for total nitrogen in true protein is controversial and considerable variability in conversion factors has been found for different foods (Mariotti, Tome, & Mirand, 2008). It was assumed that the isolated polysaccharide fraction was free from nitrogen-containing impurities and a conversion factor of 6.25 was used.

Structural analysis of separated polysaccharides from the crude Se-L-30 extract.

The crude polysaccharide extract (Se-L-30) containing Se was purified by ion exchange chromatography on DEAE-Sephadex A-25 and finally by gel filtration according to Górska et al. (2016). Three fractions (I, II, III) were obtained and analyzed by chemical analysis (sugar analysis, methylation and configuration determination) and NMR spectroscopy. The analysis of sugars and the determination of the absolute configuration showed that all fractions consist of D-glucose residues with a pyranose ring (D-Glcp). Methylation analysis of fraction I showed the presence of 4-substituted glucopyranose (1,4,5-triO-acetyl-2,3,6-tri-O-methyl-D-glucitol-1-d). In fraction III, 6-substituted glucopyranose (1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-D-glucitol-1-d), 3,6-disubstituted glucopyranose (1 ,3,5,6-tetra-O-acetyl-2,4-di-O-methyl-D-glucitol-1-d), 3-substituted glucopyranose (1,3,5-tri-O-acetyl-2 ,4,6-tri-O-methyl-D-glucitol-1-d) and terminal glucopyranose (1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol -1-d) in molar ratios 2:0.4:1.8:1.8. In fraction II, which is a mixture of both fractions I and III, all of the above-mentioned monosaccharide derivatives were identified.

Complete structural analysis by 2D NMR spectroscopy was performed on all fractions (I-III). The 1H-1H COSY spectra allowed the identification of H-2 residue signals, followed by the 1H-1H TOCSY spectra with different mixing times and the 1H-13C HSQC-DEPT spectrum allowed the assignment of H-3 signals to H-6, 6' for each residue from three factions. The structural analysis of fraction II is described in detail later in the description, as it is a mixture of polysaccharides presented in both fractions I and III.

The HSQC-DEPT spectrum of fraction II contained signals for four anomeric protons and carbon atoms, respectively (Figures 2A,B and Table 1).

Residue A (5h/5c 5.33/99.7 ppm, ¹ Jc-i,hi ~172 Hz) was recognized as 4-substituted α-D-Glcp based on the characteristic proton spin system. The downshift value of the C-4 field (5c 76.7 ppm) indicated that it is a substituted residue at position 4 (Górska et al. 2016; Mandai et al. 2012).

Residue B (5h/5c 4.48/102.6 ppm, ¹ Jc-i,hi ~165 Hz) was recognized as 3-substituted β-D-Glcp based on large vicinal couplings between all protons in the sugar ring and characteristic high shifts chemical C-3 signal at 5c 84.5 ppm.

PL 244687 Β1

Residue C (5h/5c 4.46/103.0 ppm, ~163 Hz) and residue C' (5h/5c 4.63/103.0 ppm, ~163 Hz) were recognized as -^6-substituted β-D -Glcp based on large vicinal couplings between all protons in the sugar ring and substitution-specific chemical shifts of C-6 signals at 5h/5c 3.80, 4.15/68.8, and at 5h/5c 3.77, 4, 13/68.9 ppm for C and C', respectively. A significant difference in the chemical shift values was observed only for the anomeric protons of the C and C' residues, at 5h 4.46 and 4.63 ppm, respectively. The presence of two residues -Β-β-D-Glcp in fraction II can be explained by the heterogeneity of the fraction and the presence of a mixture of three polysaccharide structures.

The monosaccharide sequences in fraction II were determined using the ^{1H- 1H} NOESY and ^{1H- 13C} HMBC experiments. NOESY spectra showed strong inter-residue correlation peaks between the following transglycoside protons: H-1 from A/H-4 from A, H1 from B/H3 from B, H-1 from C/H-6 from C, and H-1 from C7H -3 with B. The HMBC fraction II experiment confirmed the substitution positions of all monosaccharide residues except the glycosidic bond between B residues in β(1 ->3) glucan.

Table 1. ^1H and ^13C NMR chemical shifts of fraction II separated from the crude Se-L-30 extract.

Sugar rest Chemical shifts (ppm) HI/ Cl H2/ C2 H3/ C3 H4/ C4 H5/ C5 H6, H6' C6 AND 5.33 3.54 3.90 3.57 3.77 3.77, 3.80 —>4)-a-D-Glcp-(l—> 99.7 71.6 73.4 76.7 71.2 60.5 B 4.48 3.45 3.69 3.48 3.46 3.67, 3.85 -»3)-P-D-Glcp-(l-> 102.6 72.8 84.5 69.2 75.5 60.7 C 4.46 3.26 3.39 3.40 3.56 3.80, 4.15 —>6)-p-D-Glcp-(l—> 103.0 73.1 75.8 69.4 74.8 68.8 C' 4.63 3.31 3.44 3.40 3.56 3.77, 4.13 ->6)-p-D-Glcp-(l-> 103.0 73.3 75.4 69.5 74.8 68.9

Spectra were obtained for ² H2O solutions at 25°C, and acetone (5h 2.225, 5c 31.05 ppm) was used as an internal standard.

Each of the -^δ-β-D-Glcp residues (C and C') belongs to different polysaccharide structures presented in fraction II. While the C residue forms the main β(1^6)-glucan, the C' residue replaces the B residue at position 3 in another polysaccharide - β(1^3)-glucan, presented in fraction II. The amount of C' residue compared to B is significantly lower.

The fractions obtained after separating the crude Se-Le-30 extract were: (I) a(1->4) glucan homopolysacharide fraction; (II) fraction with a mixture of shorter chains of a(1->4) glucan, β(1->6) glucan, β(1->3) glucan, and also (1(1^6)-(X1^3) -glucan; (III) fraction containing a mixture of shorter chains (1(1^6)-glucan, β(1 ->3) glucan and (X1^6)-(1(1^3)-glucan. At the same time, signals were identified 3,6-disubstituted β-D-Glcp (residue E) and β-D-Glcp (residue D) (Figure 2C, D), however, the sequence of this branched molecule has not been determined. It should be emphasized that in fraction III, β(1) predominated —>6) and β(1 —>3) glucan. Residues E and D were observed in fractions II, III in the methylation analysis.

Determination of homogeneity and molar mass of lentinan analogue (Se-Le-30) and isolated polysaccharides

The Rl records from SEC of the Se-Le-30 fractions, A (I), A/b-C (II), and B-C (III) are shown in Figure 4. The corresponding RAL records of these products are shown in Figure 8.

Additional SEC recordings from the Rl, light scattering (LS), and viscosity detectors are shown in Figure 9. Bimodal signals from the LS detectors were observed for all samples analyzed. Sy

PL 244687 Β1 bimodal momentum for Rl was observed only for the Se-Le-30 fraction (Fig. 9A). For all analyzed samples, both LS signals were shifted to lower retention volume values (higher molar masses) in relation to the signals from Rl and the viscometer. The signals measured from the four detectors for each product do not completely overlap, resulting in a broader molar mass distribution (Table 2).

Table 2

SEC analysis (Rl, RALS, LALS and viscosity) of the analyzed products

A sample Retention volume [ml] A/n A7 _W D Comments Se-Le-30 18.58 1.69 x 10 ^ć 3.62 x 10 ^ć 2.15 Bimodal signal for all detectors ABC 18.48 1.72 χ 10 ^ΰ 2.18 x 10 ^c 1.27 Bimodal signal for LS and viscosity detectors AND 18.49 1.82 x 10 ^ć 2.25 x 10 ^ć 1.24 Bimodal signal for LS detectors B-C 20.05 5.06 x 10 ⁴ 1.10 X 10 ⁵ 2.18 Bimodal signal for LS detectors

The OmniSEC calculation was based on the glucan standard (glu-245). D (dispersity) represents Mw/Mn

Molar masses for the analyzed products were determined using OmniSEC for the polysaccharide fraction as follows: Se-Le-30: Mn = 1.69 x 10 ⁶ g/mol, Mw = 3.62 χ 10 ⁶ g/mol, £> = 2, 15 (dispersity); A/BC: Mn = 1.72 x 10 ⁶ g/mol, Mw = 2.18 χ 10 ⁶ g/mol, D = 1.27; A: Mn = 1.82 x 10 ⁶ g/mol, Mw = 2.25 x 10 ⁶ g/mol, D = 1.24; and BC: Mn = 5.06 x 10 ⁴ g/mol, M _w = 1.10 x 10 ⁵ g/mol, 0 = 2.18. The BC fraction had a much lower molar mass. Broad recordings for Rl and viscosity were observed, as well as bimodal recordings for the LS detectors. The nature of the SEC curves indicates that the BC fraction does not have a uniform structure. It was assumed that it is a mixture of linear and branched polysaccharides. The behavior of the BC fraction is consistent with its molecular modeling.

Fractions A, A/B-C and B-C were isolated and purified on chromatographic columns. However, this procedure did not ensure the isolation of completely pure products without any contaminants. A more detailed analysis of the Rl records for the Se-Le-30, A and A/B-C fractions (after the deconvolution procedure; Figs. 10-12) allowed to determine the content of the remaining fraction, B-C. Product A is the main component of the Se-Le-30 fraction, and product B-C (with a much lower molar mass) is present only in small amounts. Therefore, in the Se-Le-30 fraction, the content of A and B-C was 77.86% and 2.86%, respectively (Fig. 10). In the A/B-C fraction, a higher amount of A (85.35%) and B-C (4.23%) was observed (Fig. 11). Finally, deconvolution of the Rl signal of product A allowed the determination of the residual content of the B-C fraction at 2.73% (Fig. 12). The overall purity of fraction A was estimated at 86.48%. These calculations should be treated with caution due to the large difference in the content of both products.

Deconvolution of the RI signal of the B-C product made it possible to determine the overall purity of the B-C fraction at the level of 85.03%. We were also able to estimate the content of three other products (peaks 1-3) with slightly higher molar masses: 2.03% (18.5 ml), 3.32% (18.8 ml) and 9.63% (19 .2 ml) (Fig. 5).

Additionally, based on the linear calibration to β-glucans, the following molar masses were calculated for the appropriate peaks: 96,500 g/mol (peak 1), 74,000 g/mol (peak 2), 56,700 g/mol (peak 3), 31 300 g/mol (fraction B-C).

Molecular modeling

MM2 calculations showed that all components of the polysaccharide fractions, A and B-C, tend to adopt broad helical conformations (Figure 6). Polysaccharide A is a linear 1,4-α-D-glucan and is expected to adopt an amylose-like structure (Figure 6A). The deformation of the helical structure by replacing the oxygen atom in the glycosidic bond with Se was previously described (Malinowska et al., 2018). However, the incorporation of Se may not affect the overall structure of the polysaccharide A chain due to the very low Se content in the samples tested; the effect of Se inclusion is only local. A, being an amylose-like polysaccharide, is expected to form left-handed helices with six glucose units per turn and stabilized by hydrogen bonds.

According to MM2 calculations, α-helix was also the most likely conformation in 1-3-3-D-glucan, 1-6^-D-glucan, and 1-3-3-branched 1-6-3-D-glucan, components of the polysaccharide fraction B-C (Fig. 6B-D). However, unlike 1-3-3-D-glucan, the helical structure of 1-6-3-D-glucan and 1-3-3-branched 1-6-3-D-glucan can be flexible.

One reason is the additional degree of freedom resulting from the ω bond (C6-C5 in 1-6-glucans) compared to 1-3-, 1-4-, and 1-2-glucans in which only φ bonds (Ci -O) and ψ (O-C3, O-C4, O-C2) (Fig. 6D). The second reason is the limited ability to form intramolecular hydrogen bonds (Fig. 6D).

To investigate whether the presence of 1-3-β branches affects the adopted conformation of the B-C polysaccharide, experiments were performed with MM2 for its unbranched 1-3-β counterpart (Fig. 6C-D). The results show similar conformations for both branched and unbranched polysaccharides.

Characterization of the polysaccharide fraction

The individual fractions were characterized as follows. Fraction A refers to α-1,4-D-glucan characterized by an elution time expressed in milliliters at a flow rate of 0.5 ml/min: ion exchange chromatography: 6-24 ml and gel permeation chromatography: 62-74 ml. Fraction B-C refers to 1,3^-D-glucan, 1,6^-D-glucan and β-1,3-branched 1,6^-D-glucan characterized by an elution time expressed in milliliters at a flow rate of 0 .5 ml/min: ion exchange chromatography: 87-109.5 ml and gel permeation chromatography: 72-90 ml. Fraction A/B-C refers to α-1,4-D-glucan, 1,3^-D-glucan, 1,6^-D-glucan and β-1,3-branched 1,6-β-D- glucan characterized by an elution time expressed in milliliters at a flow rate of 0.5 ml/min: ion exchange chromatography: 79.5-97.5 ml and gel permeation chromatography: 62-76 ml. (Calculated from fractions: LC-1KP after DEAE A4B1 on HW C7D1, LC-1KP after DEAE D8E5 on HW C7D2, LC-1 EM after DEAE D13E13 on HW C11D9).

Effect of polysaccharides on the proliferation of human peripheral blood mononuclear cells

The effect of the Se-Le-30 fraction (lentinan analogue) and fractions A and B-C on the proliferation of unstimulated, stimulated OKT3, PHA and SAC PB MCs is shown in Fig. 7. The results are compared with those previously obtained for the Se-L fraction (Malinowska et al. ., 2018).

As shown in the work of Kaleta et al., for the Se-L fraction, autostimulation showed a certain tendency to reduce PMBC proliferation. The opposite trend was observed for the Se-Le-30 fraction, but these effects were not significant (Fig. 7A). The Se-L fraction showed some tendency to reduce the proliferation of SAC-stimulated PMBCs ( Kaleta et al., 2019 ), but this effect was not observed for the Se-Le-30 fraction isolated by the Chihara method and purified polysaccharides A and B-C ( Figure 7D ).

OKT3-stimulated T cell proliferation was significantly inhibited by all tested fractions (Se-Le-30, A, B-C and A/BC) at a concentration of 100 μg/ml, but at a concentration of 10 μg/ml this effect was only visible in the Se fractions -Le-30, B-C and, unexpectedly, A/B-C. In the case of the Se-Le-30 fraction, the inhibition rate at 100 and 10 μg/ml was approximately 82% and 48%, respectively. For polysaccharides A and B-C, the inhibition rate at 100 μg/ml was approximately 82% and 83%, respectively.

However, the difference between fractions was significant at 10 μο/ml; fraction A was practically inactive, while the degree of inhibition in fraction B-C was 47%. The relatively high standard deviations in the Se-L and Se-Le-30 fractions (Fig. 7) were due to the differential response to mitogen of lymphocyte cultures from different donors. No significant inhibitory effect of polysaccharides A and B-C and the A/B-C mixture was observed on PHA-stimulated PBMCs (Fig. 7C).

Surprisingly, for the A/B-C fraction the effect on OKT3-stimulated PBMC proliferation was significantly stronger than for polysaccharides A and B-C separately (Fig. 7). Without being bound by any theory, the stronger inhibition of proliferation by the A/B-C fraction as opposed to the A or B-C fraction alone may be due to synergy and/or the formation of active intermolecular structures. Therefore, the expert would not feel motivated to combine the non-inhibitory A fraction with the B-C fraction to obtain the selective and most potent A/B-C fraction.

Assessment of cell viability using trypan blue staining confirmed that the inhibitory effect on OKT3- and PHA-stimulated PBMC proliferation was not caused by the toxicity of the tested polysaccharides.

Biological activity

The effect of the isolated Se-polysaccharide-protein (Se-L) fraction on the proliferation of human PBMCs was examined in a publication by Kaleta et al. OKT3- and PHA-induced lymphocyte proliferation was studied as a means of assessing T-cell responses. These two mitogens use different mechanisms to promote lymphocyte effector functions T; PHA stimulates T cell proliferation through interactions with the glycoprotein N-acetylgalactosamine present on these cells (Lindahl-Kiessling and Peterson, 1969), while OKT3 stimulates T cells through signaling through CD3 (Van Wauwe, De Mey and Goossens, 1980). SAC-induced lymphocyte proliferation was also used to assess B cell response capacity.

Here, the same experiments were performed for the Se-Le-30, A (I), B-C (III) and A/B-C (II) fractions. The results clearly demonstrate that polysaccharides A and B-C were mainly responsible for the immunosuppressive activity of the previously described Se-L fraction (Kaleta et al., 2019). The selenium content also has a significant impact on activity.

The inhibition of T cell proliferation by the currently isolated fractions was weaker but more selective than that of the Se-L fraction (Fig. 7). At higher concentrations (100 μg/ml), no significant difference was found between the activities of the Se-Le-30, A, B-C and A/B-C fractions. However, at lower concentrations (10 μg/ml) the differences become significant. Polysaccharide B-C is almost twice as active as polysaccharide A. Surprisingly, the mixture of polysaccharides A and B-C is more active than the individual fractions, suggesting possible synergy or the formation of active intermolecular structures.

The effect of selenium on the activity of polysaccharides is clearly visible, especially at lower concentrations (10 μg/ml). The 4-fold reduction in selenium concentration in the Se-Le-30 fraction compared to Se-L correlated approximately with a 4-fold reduction in the inhibition of T cell proliferation.

Selective immunosuppressive activity is not typical for polysaccharides of fungal origin. Probable mechanisms are unknown and have not been described. In the case of the previously isolated Se-L fraction (Malinowska et al., 2018), significant inhibition of T cell proliferation was observed, most likely via the CD3 receptor, but suppression was also observed in PHA-stimulated PBMCs, although this effect was significantly weaker (Kaleta et al., 2019). In the autostimulation and SAC stimulation settings, the S-Le fraction showed some tendency to reduce PMBC proliferation, although this was not significant.

The inhibition of T cell proliferation for the currently isolated fractions (Se-Le-30, A, B-C) in the OKT3 assay, without any effect in the PHA assay, suggests a mechanism mediated by CD3 receptors without interactions with the glycoprotein N-acetylgalactosamine. Therefore, it was concluded that impurities in the Se-L fraction, which are not present in the currently tested polysaccharides, are responsible for the interaction with B lymphocytes and the glycoprotein N-acetylgalactosamine.

It was hypothesized, without committing to any theory, that the completely different biological effect of lentinan depends, at least in part, on the inclusion of selenium in the polysaccharide molecule, but also on the different structure of the fractions isolated from mycelium cultures compared to lentinan. Lentinan is a 1-6-3-branched 1-3-3-D-glucan with a Mw of 500 kDa. Its helical structure is stabilized by the formation of strong internal hydrogen bonds, reducing the hydration of the polysaccharide. This structure is inflexible and rigid (Burkus and Tamelli, 2005; Zhang et al., 2011). In contrast, the main component of the Se-enriched lentinan analogue amylose-like polysaccharide A is linear 1-4-a-D-glucan. It also has a helical structure, but much more flexible and hydrophilic than lentinan (1,3-3-D-glucan) (Almond, 2005). This explains the good solubility and extraction possibility of compound A (a polysaccharide with a mass of Mw > 2 million Da) in hot water.

The B-C polysaccharide fraction, the more active component of the Se-Le-30 fraction (lentinan analogue), is a mixture of unbranched 1-6^-D-glucan, unbranched 1-3-3-D-glucan and 1-3-3-branched 1- 6-3-D-glucan - all probably in helical conformation (Fig. 6). It was found that numerous branches created by single β-D-glucose molecules in 1-3-3-branched 1-6-β-D-glucan have no effect on the adopted conformation (Fig. 6C). However, the helical structure of the two components of the B-C polysaccharide fraction (unbranched 1-6^-D-glucan and 1-3^-branched 1-6^-D-glucan) should be flexible and labile due to the additional degree of freedom in the ω bond (C6- C5 in 1-6-glucans; Fig. 6D) compared to 1-3-, 1-4-, 1-2-glucans. The distances between some oxygen atoms (2.8-2.9 A) suggest the possibility of stabilizing the helical structure using hydrogen bonds (Fig. 6D). The low content of B-C polysaccharide in the Se-Le-30 fraction suggests a low content in the L. edodes cell wall and/or a low possibility of extraction with hot water. The latter is very possible, as two papers have described the isolation of the analogous 1-3^-branched 1-6^-D-glucan from fungal cell walls using a hot alkaline solution (Samuelsen et al., 2019; Li et al., 2019 ).

The structural elements of a polysaccharide determine their biological activity, and the relationship between activity and structure is well known for 1,3^-D-glucans. For other groups of fungal polysaccharides, knowledge is poor (Zhang, Cui, Cheung, & Wang, 2007).

Notably, the results do not indicate the absence of 1-3^-D-glucans in the cell wall of Se-enriched mycelium from L. edodes. These compounds are probably characterized by lower solubility in hot water than the 1-3^-D glucans isolated by Chihara (1970).

The fungal cell wall is a dynamic structure that constantly evolves in response to environmental conditions (Latge, 2010). The method, culture conditions and composition of the medium may influence the structure of cell wall polysaccharides. The results of the current research show that even a well-known and widely described species of fungus can biosynthesize a polysaccharide with a completely different structure and biological activity than that described so far in submerged culture in a specific liquid medium.

Modulation of T cell activity may be useful after allogeneic organ transplantation, as well as in a number of autoimmune diseases, especially those with known abnormal T cell activity, such as rheumatoid arthritis, multiple sclerosis, celiac disease, glomerulonephritis, inflammatory bowel disease, psoriasis, etc. , as well as in T-cell mediated allergy such as contact dermatitis etc.

Furthermore, the results of the present study show that there is no uniformity or predictability in the structural features or functional characteristics of biologically active polysaccharides. The biosynthetic pathways, the structure of polysaccharides obtained using the same mushroom species, and the mechanisms of biological activity are highly variable and confusing to researchers. Additionally, biotechnological methods of cultivating higher fungi offer advantages in many aspects over methods of growing plants. Mycelium cultures in bioreactors are carried out under repeatable conditions, which leads to a stable composition of the cultivated biomass. This facilitates the standardization of preparations derived from mushrooms, such as for pharmaceutical use.

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Claims (15)

1. Composition of fractions of selenized bioactive polysaccharides from Lentinula edodes, containing a mixture of fraction A and fraction B-C, in which: fraction A consists of α-1,4-D-glucan polymer; and fraction B-C consists of the following glucan polymers: 1,3-3-D-glucan, 1,6-3-D-glucan and 3-1,3-branched 1,6-3-D-glucan, and in which the content of fraction A and fraction B-C in the composition is approximately 85% by weight and approximately 4% by weight, respectively. 2. A method for producing a composition of selenized bioactive polysaccharide fractions from Lentinula edodes as defined in claim. The process of claim 1, characterized in that it comprises the following steps: a) cultivating Lentinula edodes in a culture medium enriched with at least 30 μg/ml selenium; b) isolating mycelium; c) extraction with boiling methanol 1:4 w/v; d) extracting the methanol-insoluble fraction from step c) with hot water; e) precipitating the fraction extracted in step d) with 1 volume of ethanol; f) solubilizing the precipitate formed in step e) in water and precipitating it with 0.2 M cetyltrimethylammonium hydroxide; g) extracting the precipitate from step f) with 20% acetic acid; h) extracting the insoluble fraction from step g) with 50% acetic acid; i) solubilizing the insoluble fraction from step h) with 6% NaOH; j) precipitation with 3 volumes of ethanol; k) deproteinizing the precipitate from step j); and l) purification of polysaccharide fractions from deproteinized sediment. 3. The method according to claim 2, characterized in that step 1) comprises the use of ion exchange chromatography and gel permeation chromatography. 4. The method according to claim The method of claim 3, characterized in that the first polysaccharide fraction eluted from the gel permeation chromatography column is retained, which fraction contains at least 80% by weight of the α-1,4-D-glucan polymer forming the α-helix structure. 5. The method according to claim 1. 3, characterized in that the second polysaccharide fraction eluted from the gel permeation chromatography column is retained, which fraction contains a mixture of the following glucan polymers: α-1,4-D-glucan, 1,3-3-D-glucan, 1,6 -3-D-glucan and β-1,3-branched 1,6^-D-glucan. 6. The method according to claim 1. 3, characterized in that a third polysaccharide fraction eluted from the gel permeation chromatography column is retained, which fraction contains a mixture of three glucan polymers: 1,3^-D-glucan, 1,6^-D-glucan and β-1,3 -branched 1,6-β-Ο-glucan. 7. The composition of the selenized polysaccharide fraction from Lentinula edodes obtained by the method according to any of the claims. 2-6, wherein the composition comprises a mixture of fraction A of the α-1,4-D-glucan polymer; and fractions B-C of 1,3^-D-glucan, 1,6^-D-glucan and β-1,3-branched 1,6^-D-glucan, where the content of fraction A and fraction B-C in the composition is equal, respectively about 85% by weight and about 4% by weight. 8. The composition according to claim 1. 7, which is obtained by the method as defined in claim. 5. 9. The composition of the selenized polysaccharide fraction as defined in claim. 1 or as defined in claim 8 for use as a therapeutic agent. PL 244687 Β1 10. The composition of the selenized polysaccharide fraction as defined in claim. 1 or as defined in claim 8 for use as a therapeutic agent in the selective inhibition of T cell proliferation. 11. Composition of selenized polysaccharide fractions for use according to claim 1. The method of claim 10, wherein selective inhibition of T cell proliferation is required for conditions selected from the group consisting of allogeneic organ transplantation, autoimmune diseases and T cell mediated allergies. 12. Composition of selenized polysaccharide fractions for use according to claim. The method of claim 11, wherein the autoimmune diseases are selected from rheumatoid arthritis, multiple sclerosis, celiac disease, glomerulonephritis, inflammatory bowel disease and psoriasis. 13. Composition of selenized polysaccharide fractions for use according to claim. The method of claim 11, wherein the T-cell mediated allergy is contact dermatitis. 14. A selenized polysaccharide fraction composition for use according to any one of claims. 9-13, characterized in that it is for oral, intravenous, intraperitoneal or external administration. 15. A pharmaceutical composition containing an active ingredient and a pharmaceutically acceptable excipient, characterized in that the active ingredient is a composition of selenized polysaccharide fractions as defined in any one of claims. 1 or 7 or 8.