Identification and selection of plant-derived chondroitin sulfate and hyaluronic acid starting materials, and conversion of plant-derived starting materials to obtain components for use in foods, nutritional supplements, medical devices, or pharmaceuticals.

Abstract

The present invention provides a method for producing plant-derived hyaluronic acid or a salt thereof, and / or chondroitin or a salt thereof, which have low levels of impurities and / or by-products and high purity. [Solution] The present invention provides a method for producing a mixture (m) comprising, or consisting thereof, (a) hyaluronic acid or a salt thereof (HA) having a weight-average molecular weight of 10 kDa to 600 kDa; (b) chondroitin or chondroitin sulfate or a salt thereof (CS) having a weight-average molecular weight of 3 kDa to 50 kDa; and (c) at least one glycosaminoglycan selected from a combination of (a) and (b), extracted from a plant-derived starting material (e.g., fungi).

Description

Technical Field

[0001] The present invention relates to a mixture comprising at least one glycosaminoglycan obtained from a plant starting material, or consisting of, selected from the group consisting of hyaluronic acid or a salt thereof (hyaluronic acid anion salt; namely, salt or free HA) derived from plants with low impurities and / or by-products and having high purity, and / or chondroitin or a salt thereof (e.g., chondroitin sulfate or a salt thereof; namely, salt or free CS).

[0002] Furthermore, the present invention relates to the use of the above mixture as an additive, excipient, or ingredient in the manufacture of pharmaceutical products, medical devices, dietary supplements, therapeutic special foods (FSMP), foods or health supplements.

[0003] Furthermore, the present invention relates to a composition comprising (i) the above mixture comprising hyaluronic acid or a salt thereof (hyaluronic acid anion salt; namely, salt or free HA) derived from plants, and / or chondroitin or a salt thereof (e.g., chondroitin sulfate or a salt thereof; CS), or consisting of, and (ii) any technical additives and pharmaceutical or food-grade excipients.

[0004] Furthermore, the present invention relates to the composition comprising the above mixture for use as a medicine.

[0005] Furthermore, the present invention relates to the composition comprising the above mixture for use in a method for prophylactic or therapeutic treatment of humans and animals having a specific disorder or condition or disease selected from arthritis, osteoarthritis, arthrosis, joint pain, inflammation of hands, feet and joints, gastroesophageal reflux disease.

[0006] Furthermore, the present invention relates to a mixture comprising or containing plant-derived hyaluronic acid or a salt thereof (hyaluronic acid anionic salt; in short, salt or free HA), and / or chondroitin or a salt thereof (e.g., chondroitin sulfate or a salt thereof; i.e., CS), and a method for producing a composition comprising the mixture thereof.

[0007] Finally, the present invention relates to the use of fungi for producing plant-derived hyaluronic acid or a salt thereof, and / or chondroitin or a salt thereof (e.g., chondroitin sulfate or a salt thereof), which have low levels of impurities and / or by-products and high purity.

[0008] Hyaluronic acid is an anionic, non-sulfated glycosaminoglycan (GAG) that is abundantly distributed in the connective tissue, epithelial tissue, and nerve tissue of vertebrates. Hyaluronic acid has important structural, fluid, and physiological functions.

[0009] Rooster combs and human umbilical cords contain extremely high concentrations of hyaluronic acid, at 7,500 mg / L and 4,100 mg / L, respectively. For this reason, in the early 1980s, Endre A. Balazs and his collaborators developed a method for isolating and purifying hyaluronic acid from rooster combs and human umbilical cords. Since then, hyaluronic acid has been obtained industrially from rooster combs.

[0010] Chondroitin sulfate is a sulfated GAG consisting of alternating sugar units of N-acetylgalactosamine and glucuronic acid. A chondroitin sulfate chain can consist of hundreds of sugar units, each of which can be sulfated at different positions and in different amounts. Due to its high compressive strength, chondroitin sulfate is an important structural component of cartilage.

[0011] Chondroitin sulfate is given by the following general formula (I): [ka] [In the formula, at least one of R2, R4 and R6 is a sulfite group (SO3- It has a repeating unit (disaccharide) of ) ). Chondroitin monosulfate has only one of the R2, R4, or R6 groups being a sulfite group. The three possible chondroitin monosulfates are 6-chondroitin sulfate (R2=H; R4=H; R6=SO3 - ), 4-chondroitin sulfate (R2=H; R4=SO3 - ; R6=H) and 2-chondroitin sulfate (R2=SO3 - (R4=H; R6=H).

[0012] Many chondroitin sulfates are obtained from animal cartilage extracts, mainly from bovine and porcine tissues (e.g., trachea, ears, and nose), but other sources such as shark, fish, and bird cartilage may also be used.

[0013] Despite numerous undeniably effective medical applications of hyaluronic acid and chondroitin sulfate in mammals, particularly humans, today, production methods involving extraction from animal cells face increasing ethical, religious, and moral concerns and fears. The primary concerns and fears arise from the use of products secreted by or derived from animals in the production of hyaluronic acid or its salts, and / or chondroitin or its salts (e.g., chondroitin sulfate), especially when these compounds or salts are intended for nutritional, biomedical, or pharmaceutical purposes. Furthermore, while chondroitin sulfate and hyaluronic acid extracted from animal sources have high molecular weights, lower molecular weight chondroitin sulfate and / or hyaluronic acid have higher transdermal penetration and are more beneficial.

[0014] In prior art literature on chondroitin sulfate derived from non-animal sources, a method is known in which sulfate groups are inserted into non-sulfated chondroitin obtained by bacterial fermentation.

[0015] Therefore, in the fields of pharmaceutical products, medical devices, nutritional supplements, specialty therapeutic foods (FSMPs), health supplements, or foods, there is a strong need and demand from market participants for a method of obtaining hyaluronic acid or its salts and / or chondroitin or its salts (e.g., chondroitin sulfate) in a way that allows all consumers, including vegans, vegetarians, those with allergies, and those who avoid such products for religious or ideological reasons, to use products or pharmaceuticals containing hyaluronic acid or its salts and / or chondroitin or its salts, which are currently unavailable, as an alternative to existing manufacturing methods. Furthermore, there is a need to produce non-animal derived chondroitin sulfate and / or hyaluronic acid in a more economically advantageous and easily applicable manner than methods known in the field.

[0016] As a result of extensive and dedicated research and development activities, the applicant has developed manufacturing technologies and methods that can adequately address current limitations, obstacles, and problems.

[0017] Therefore, the present invention is formed from a mixture comprising, or selected from, a group comprising, at least one glycosaminoglycan obtained from a plant starting material, having the characteristics defined in the appendix claims, including, or selected from the group comprising, plant-derived hyaluronic acid or a salt thereof (hyaluronic acid anionic salt; in short, salt or free HA), and / or chondroitin or a salt thereof (e.g., chondroitin sulfate or a salt thereof, in short, salt or free CS), and combinations thereof.

[0018] Furthermore, the present invention is formed from the use of the above mixture as an additive, excipient, or component in the manufacture of pharmaceutical products, medical devices, nutritional supplements, therapeutic specialty foods (FSMPs), foods, or health supplements, the use thereof having the characteristics defined in the appended claims.

[0019] Furthermore, the present invention is formed from a composition comprising (i) plant-derived hyaluronic acid or a salt thereof (hyaluronic acid anionic salt; in short, salt or free HA), and / or chondroitin or a salt thereof (e.g., chondroitin sulfate (CS)), or a mixture thereof, and (ii) any technical additives and pharmaceutical or food-grade excipients.

[0020] Furthermore, the present invention is formed from a mixture and at least one technical additive or excipient, or composition, for use as a pharmaceutical (first medical use), having the features defined in the appended claims.

[0021] Furthermore, the present invention is formed from a mixture or composition comprising such mixtures (second medical use) for use in a method of preventive or therapeutic treatment of specific disorders, conditions or diseases selected from arthritis, osteoarthritis, arthropathy, arthralgia, inflammation of the hands and feet or joints, and gastroesophageal reflux disease, having the characteristics defined in the appendix claims, and the use thereof.

[0022] Furthermore, the present invention is comprised of methods for producing a composition comprising, or a mixture thereof, plant-derived hyaluronic acid or a salt thereof (hyaluronic acid anionic salt; in short, salt or free HA), and / or chondroitin or a salt thereof (e.g., chondroitin sulfate (CS)), having the characteristics defined in the appended claims.

[0023] Finally, the present invention is formed from the use of fungi as plant starting materials to produce hyaluronic acid or a salt thereof and / or a salt thereof (e.g., chondroitin sulfate or a salt thereof) having high purity, low levels of impurities and / or by-products, and having the characteristics defined in the appended claims. [Brief explanation of the drawing]

[0024] Preferred embodiments of the present invention are described below with reference to the accompanying drawings. Figures 1 to 4 show flowcharts of methods according to different embodiments (First Embodiment: P1), which are the subject of the present invention. Figures 5 and 6 show flowcharts of methods according to the Second Embodiment (P2), which are the subject of the present invention. Figures 7 and 8 show flowcharts of methods according to the Third Embodiment (P3), which are the subject of the present invention. Figures 9 and 10 show two HPLC spectra for determining unsaturated disaccharides in samples containing HA and CS, respectively.

[0025] The expression "HS" found in the text of this specification is used to denote hyaluronic acid or its salts, or hyaluronate, or combinations thereof. On the other hand, the expression "CS" is used to denote chondroitin, chondroitin salts, preferably chondroitin sulfate or its salts, or mixtures thereof.

[0026] The terms "plant starting material" and "starting material of plant origin" found in this specification are synonyms and are therefore used interchangeably.

Mode for Carrying Out the Invention

[0027] The present invention is formed from a mixture (m) containing or consisting of at least one glycosaminoglycan obtained from a plant-derived starting material. The plant-derived material is selected from the group consisting of or containing one or more natural fungi.

[0028] The glycosaminoglycan is (a) hyaluronic acid or its salts, hyaluronic acid anion salts (i.e., HA); (b) chondroitin or its salts (e.g., chondroitin sulfate or its salts, i.e., CS); (c) a combination of (a) and (b) and is selected from the group consisting of or containing them.

[0029] The plant-based starting material is fungi, which are fungi that grow and exist in nature, for example, in forests where they can be collected, but they can also be cultivated in greenhouses.

[0030] These fungi belong to the subkingdom Dicaria, preferably to the phylum Basidiomycota.

[0031] Dicaria is a subkingdom of fungi that includes the Ascomycota and Basidiomycota phyla. Basidiomycota (RT Moore, 1980) is one of the broadest phyla that make up the Fungi kingdom.

[0032] In one embodiment, the plant starting material is a fungus of the Tremella fuciformis species, or the plant starting material contains or consists of a fungus of the Tremella fuciformis species.

[0033] White fungus (Berk. 1856), also known as white wood ear or silver ear, is a fungus native to tropical and subtropical regions, growing on the trunks of dead hardwoods. It is in high demand, particularly in Japanese and Chinese cuisine and traditional medicine, and is therefore also cultivated. White fungus has white, gelatinous fruiting bodies (basidiocarps) that resemble thalli.

[0034] The use of Tremella fuciformis in the present invention is particularly beneficial because it allows for the acquisition of both low molecular weight HA and CS from plant starting materials using the extraction techniques described herein (First Embodiment (P1), Second Embodiment (P2), and Third Embodiment (P3)). The HA and / or CS glycosaminoglycans contained in the mixture (m) and obtained from the method of the present invention have a particularly effective and unique characteristic of having a lower molecular weight than the HA and / or CS obtained from animal cartilage as described in the prior art literature. Low molecular weight HA and / or CS glycosaminoglycans have high transdermal penetration.

[0035] More precisely, the hyaluronic acid or its salt (hyaluronic acid salt) obtained by the method of the present invention (P1 and / or P2) has a weight-average molecular weight of 10 kDa to 600 kDa, preferably 100 kDa to 500 kDa, more preferably 200 kDa to 400 kDa or 100 kDa to 300 kDa, for example about 50 kDa, 150 kDa, or 250 kDa, or 300 kDa, or 350 kDa, or 450 kDa, or 550 kDa. Preferably, the HA contains 0.01% to 5%, preferably 0.1% to 3%, more preferably 0.5% to 2%, for example 1% or 2% by weight of chondroitin (preferably unsulfated chondroitin) based on the total weight of HA extracted from the fungi.

[0036] According to a beneficial aspect of the present invention, HA having a weight-average molecular weight within the above range has high transdermal penetration power because its molecular size is extremely small.

[0037] Chondroitin or its salts (e.g., chondroitin sulfate or its salts (CS)) obtained by the methods of the present invention (P1 and / or P3) have a weight-average molecular weight of 1 kDa to 50 kDa or 5 kDa to less than 50 kDa, preferably 3 kDa to 40 kDa, more preferably 5 kDa to 25 kDa or 5 kDa to 10 kDa, for example, about 4 kDa, or 6 kDa, or 8 kDa, or 10 kDa, or 12 kDa, or 14 kDa, or 16 kDa, or 18 kDa, or 22 kDa, or 24 kDa.

[0038] In a further beneficial aspect of the present invention, CS having a weight-average molecular weight within the range described above has also been proven effective in reducing bone damage caused by osteoarthritis of the knee and hip.

[0039] The CS contained in the mixture (m) of the present invention is 1 kDa (1,000.00 Da = 1 x 10⁻¹⁰). 3The chondroitin sulfate comprises a weight-average molecular weight of )~50kDa, preferably 3kDa~40kDa, more preferably 5kDa~25kDa, for example, about 4kDa, or 6kDa, or 8kDa, or 10kDa, or 12kDa, or 14kDa, or 16kDa, or 18kDa, or 22kDa, or 24kDa.

[0040] Preferably, the CS has a charge density of 0.70 to 0.99 or 0.70 to 1.50, preferably 0.75 to 0.98 or 0.75 to 1.20, more preferably 0.80 to 0.97, for example 0.85, 0.87, 0.90, 0.92, 0.94, or 0.96.

[0041] More preferably, the CS obtained by the P1 and / or P3 method contains 50% to 99.5%, preferably 50% to 95%, more preferably 75% to 88%, more preferably 78% to 86%, for example, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% by weight of 6-chondroitin sulfate, based on the total weight percent of the CS (or disaccharide contained in chondroitin sulfate) as determined, for example by HPLC.

[0042] In addition to 6-chondroitin sulfate, the CS preferably includes unsulfated chondroitin.

[0043] Preferably, the product contains 0.1% to 25%, preferably 0.5% to 20% or 5% to 20%, more preferably 7% to 15%, and even more preferably 8% to 13%, by weight percent of the total CS (or disaccharide contained in chondroitin sulfate) as determined, for example by HPLC, such as about 0.2%, 0.3%, 0.4%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5.5%, 6%, 8%, 9%, 10%, 11%, or 12% by weight of unsulfated chondroitin.

[0044] In addition to 6-chondroitin sulfate and unsulfated chondroitin, the CS preferably comprises 2,6-chondroitin disulfate.

[0045] Preferably, the mixture contains 2,6-chondroitin disulfate in an amount of 0.1% to 10%, preferably 0.2% to 8%, more preferably 0.3% to 5%, for example, about 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 4.5% by weight, relative to the total weight percent of the CS (or disaccharide contained in chondroitin sulfate) as determined, for example, by HPLC.

[0046] In addition to 6-chondroitin sulfate, unsulfated chondroitin, and 2,6-chondroitin disulfate, the CS preferably comprises 4-chondroitin sulfate.

[0047] Preferably, the product contains 4-chondroitin sulfate in an amount of 0.01% to 5%, preferably 0.05% to 3%, more preferably 0.1% to 1.5%, for example, about 0.02%, 0.03%, 0.04%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% by weight, relative to the total weight percent of the CS (or disaccharide contained in chondroitin sulfate) as determined, for example, by HPLC.

[0048] In addition to 6-chondroitin sulfate, unsulfated chondroitin, 2,6-chondroitin disulfate, and 4-chondroitin sulfate, the CS preferably comprises 4,6-chondroitin disulfate.

[0049] Preferably, the product contains 4,6-chondroitin disulfate in an amount of 0.01% to 5%, preferably 0.05% to 3%, more preferably 0.1% to 1.5%, for example, about 0.02%, 0.03%, 0.04%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% by weight, relative to the total weight percent of the CS (or disaccharide contained in chondroitin sulfate) as determined, for example, by HPLC.

[0050] In addition to 6-chondroitin sulfate, unsulfated chondroitin, and 2,6-chondroitin disulfate, 4-chondroitin sulfate, and 4,6-chondroitin disulfate, the CS preferably includes 2,4-chondroitin disulfate.

[0051] Preferably, the product contains 2,4-chondroitin disulfate in an amount of 0.01% to 5%, preferably 0.05% to 3%, more preferably 0.1% to 1.5%, for example, about 0.02%, 0.03%, 0.04%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0% by weight, relative to the total weight percent of the CS (or disaccharide contained in chondroitin sulfate) as determined, for example, by HPLC.

[0052] In addition to 6-chondroitin sulfate, unsulfated chondroitin, 2,6-chondroitin disulfate, 4-chondroitin sulfate, 4,6-chondroitin disulfate, and 2,4-chondroitin disulfate, the CS preferably includes hyaluronic acid or hyaluronic acid salt (preferably unsulfated). Preferably, the HA is present in an amount of 0.01% to 5%, preferably 0.05% to 3%, more preferably 0.1% to 1.5%, for example, 0.8% or 1.0% by weight relative to the total weight of the CS.

[0053] In one embodiment, CS contained in the mixture (m) and obtained by the method of the present invention (P1 and / or P3) is as follows: 6-chondroitin sulfate in weight % of 50% to 99.5%, preferably 50% to 95±0.5%, more preferably 75% to 88%, and even more preferably 78% to 86%; 0.1% to 25%, preferably 0.5% to 20%, more preferably 7% to 15%, and even more preferably 8% and 13% by weight of unsulfated chondroitin; 0.1% to 10%, preferably 0.2% to 8%, more preferably 0.3% to 5% by weight of 2,6-chondroitin disulfate; and further 4-chondroitin sulfate in weight percent of 0.01% to 5%, preferably 0.05% to 3%, more preferably 0.1% to 1.5%; 4,6-chondroitin disulfate in weight percent of 0.01% to 5%, preferably 0.05% to 3%, more preferably 0.1% to 1.5%; and 2,4-chondroitin disulfate in weight percent of 0.01% to 5%, preferably 0.05% to 3%, and more preferably 0.1% to 1.5%. Includes.

[0054] For example, CS contained in mixture (m) and obtained by the method of the present invention include compositions CS.1, CS.2, CS.3, CS.4, CS.5, or composition CS.6 as listed in Table 1 below (the values ​​represent the weight % of each component relative to the total weight of CS.n; n=1~6). [Table 1]

[0055] The present invention relates to (i) (a) HA having a weight-average molecular weight of 10 kDa to 600 kDa (preferably 100 kDa to 500 kDa, more preferably 200 kDa to 400 kDa, or 100 kDa to 300 kDa, for example about 150 kDa, or 250 kDa, or 300 kDa, or 350 kDa, or 450 kDa, or 550 kDa), and / or (b) 1 kDa to 50 kDa or 5 kDa to less than 50 kDa (preferably 3 kDa). (i) a composition comprising (m) a mixture of the above, which includes CS having a weight-average molecular weight of Da~40kDa, more preferably 5kDa~25kDa, for example about 4kDa, or 6kDa, or 8kDa, or 10kDa or 12kDa, or 14kDa, or 16kDa, or 18kDa, or 22kDa, or 24kDa), and (ii) any technical additives and pharmaceutical or food-grade excipients.

[0056] The above composition may be a pharmaceutical composition, a medical device composition (EU) 2017 / 745, a nutritional supplement composition, a specialty therapeutic food (FSMP) composition, a health supplement composition, or a food composition, or a novel food composition (EU) 2015 / 2283.

[0057] The above composition may be used as a pharmaceutical or composition for the prevention and / or treatment of arthritis, osteoarthritis, arthropathy, joint pain, inflammation of the hands and feet and joints, and gastroesophageal reflux disease.

[0058] The present invention comprises techniques and methods for producing hyaluronic acid or hyaluronic acid salts (HA) (Methods P1 and / or P2) and / or chondroitin sulfate or chondroitin or salts thereof (CS) (Methods P1 and / or P3), characterized by at least one step of extracting hyaluronic acid or hyaluronic acid salts and / or chondroitin sulfate or chondroitin from a plant-derived starting material (a plant-derived starting material comprising or consisting of at least one natural fungus belonging to the subkingdom Dicaryia, preferably Basidiomycota, more preferably Tremella species).

[0059] The methods of the present invention (first embodiment (P1), second embodiment (P2), and third embodiment (P3)) do not involve bacterial fermentation and / or digestion steps in the production of chondroitin or chondroitin sulfate as shown, for example, in Patent Document WO 2012 / 152872 A1 and EP 2852437 B1.

[0060] Various embodiments of this method are illustrated in the flowcharts in Figures 1 to 8.

[0061] In the first embodiment (P1), the method that is the subject of the present invention includes the following steps. (i) Identify one or more natural fungi in a dry state as plant starting materials for glycosaminoglycans (for example, one or more natural fungi including or consisting of at least one fungus belonging to the subkingdom Dicaryia, preferably Basidiomycota, more preferably Tremella species); (ii) Press or grind the plant starting material as appropriate; (iii) Extract the glycosaminoglycan (HA or CS) of the plant starting material obtained from step (i) or step (ii) using an extraction solvent, preferably an aqueous solvent, more preferably water (e.g., distilled water or redistilled water) to obtain an aqueous extract of the glycosaminoglycan; (iv) Add the solvent (preferably ethanol) to the aqueous extract obtained from step (iii) to obtain the liquid product; (v) The liquid product obtained from step (iv) is centrifuged and / or filtered to obtain the liquid phase and solid residue; (vi) The liquid phase obtained from the centrifugation and / or filtration in step (v) is treated in the following step (vi.a), and / or the solid residue obtained from the centrifugation and / or filtration in step (v) is treated in the following steps (vi.b), (vi.c), (vi.d) and (vi.e): (vi.a) The liquid phase obtained from step (v) is dried (preferably concentrated and dried) to obtain hyaluronic acid or a salt thereof having a weight-average molecular weight of 10 kDa to 600 kDa; and / or (vi.b) The solid residue obtained from step (v) is collected and purified to obtain chondroitin or its salt (CS) having a weight-average molecular weight of 1 kDa to 50 kDa; (vi.c) The chondroitin or salt (CS) obtained from step (vi.b) is treated with a sulfuric acid source (preferably sulfuric acid, sulfur trioxide pyridine complex, sulfur trioxide dimethylformamide complex, and mixtures thereof, or selected from the group consisting thereof) to obtain an acidification product; (vi.d) The acidification product obtained from step (vi.c) is neutralized with a basic reagent to obtain a neutralization product; (vi.e) The neutralization product obtained from step (vi.d) is concentrated and dried to obtain chondroitin sulfate or a salt thereof having a weight-average molecular weight of 1 kDa to 50 kDa.

[0062] The plant starting material extracted in step (iii) may be in a complete state (i.e., a single state, e.g., a whole fungus) or may be pressed (into a mass or fragment) or ground (into granules, powder or pellets) in step (ii).

[0063] The flowcharts in Figures 1 and 2 illustrate a first embodiment (P1) of the method which is the subject of the present invention, in which a natural fungus, or a plurality thereof, identified in step (i) is extracted in step (iii) or steps (iii.a) and (iii.b) to obtain an aqueous extract.

[0064] The flowcharts in Figures 3 and 4 illustrate an embodiment of the method (a method known to those skilled in the art) that is the subject of the present invention, in which a first embodiment (P1) is obtained in which a natural fungus or more identified in step (i) is pressed or crushed in step (ii). The natural fungus or more pressed or crushed in step (ii) is then extracted in step (iii) or in steps (iii.a) and (iii.b) to obtain an aqueous extract.

[0065] If the plant-derived starting material is compressed or crushed in step (ii) (by a method known to those skilled in the art), the average particle size distribution of the plant-derived starting material is preferably 500 μm to 2,500 μm, more preferably 800 μm to 1,800 μm, and even more preferably 900 μm to 1,200 μm.

[0066] In one embodiment, the plant starting material obtained from step (iii) (step (i) or (ii)) is compressed into lumps or fragments, or ground into granules or pellets.

[0067] The plant-derived starting material obtained from step (iii) (step (i) or (ii)) is preferably in a dry state. That is, the plant-derived starting material contains about 2% to 20%, preferably 5% to 15%, and more preferably 8% to 10%, of the total weight of the plant-derived starting material.

[0068] In step (iii), the extraction solvent is selected from aqueous solvents and water.

[0069] The aqueous solvent (or aqueous solution) is preferably a hydroalcohol mixture containing 0.1% to 50%, more preferably 0.5% to 25%, and even more preferably 1% to 15%, of alcohol (e.g., ethanol) relative to the total weight of the extraction solvent, for example, about 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 7.5%, 10%, and 20%.

[0070] The water is preferably distilled water or redistilled water.

[0071] In step (iii), the plant starting material is loaded into a container or extraction apparatus (e.g., an apparatus that performs stirring, heating, filtering, and temperature and pressure control) in a continuous or multi-step manner.

[0072] In step (iii), the plant starting material is extracted with an extraction solvent in a container or extraction apparatus, hyaluronic acid or its salt passes through the extraction aqueous solution in the liquid phase, and chondroitin remains in the solid residue.

[0073] In step (iii), an extraction is performed in which the ratio of [weight of plant starting material]:[volume of extraction solvent] is 1:1 to 1:90, preferably 1:10 to 1:90, more preferably 1:20 to 1:75, and even more preferably 1:40 to 1:60, for example 1:3, 1:5, 1:15, 1:25, 1:45, 1:50, or 1:55. The extraction in step (iii) is performed for 1 minute to 12 hours, preferably 10 minutes to 9 hours, more preferably 15 minutes to 4 hours, for example about 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, or 180 minutes.

[0074] Step (iii) is preferably carried out at atmospheric pressure (P=1 Atm, 20-25°C), and the temperature of the extraction solvent is 10-90°C, preferably 20-60°C, more preferably 35-55°C, for example, about 25°C, 30°C, 40°C, 44°C, 48°C, or 50°C.

[0075] Preferably, the pH value of the extraction solvent in step (iii) is 3 to 10, preferably 3.5 to 9, more preferably 4 to 8, and even more preferably 5 to 7, for example, about 4.5, 5.5, 6, 6.5, 7.5, 8.5, or 9.5.

[0076] In step (iii) extraction, a proteolytic enzyme is preferably used to decompose the surface pectin of the plant-derived material, thereby improving the yield of the method. Preferably, the proteolytic enzyme includes or consists of bromelain or bromelain extract.

[0077] Bromelain is an enzymatic extract from the fruit and / or pineapple stem, containing proteolytic enzymes and small amounts of other substances.

[0078] To increase the extraction volume, step (iii) is preferably carried out in the following two steps. (iii.a) Extract the plant starting material with an extraction solvent at a temperature of 10°C to 90°C, preferably 20°C to 60°C, more preferably 35°C to 55°C, for 1 minute or 30 minutes to 12 hours, preferably 10 minutes to 9 hours, more preferably 15 minutes to 4 hours, to obtain a first extract aqueous solution; and (iii.b) The plant starting material (solid residue obtained from the first extraction in step (iii.a)) is extracted with the extraction solvent at a temperature of 80°C to 120°C, preferably 90°C to 110°C, more preferably 95°C to 105°C, and even more preferably 98°C to 102°C, preferably under atmospheric pressure or reduced pressure, for 10 minutes to 6 hours or 30 minutes to 8 hours, preferably 20 minutes to 4 hours, more preferably 40 minutes to 2 hours, for example about 30 minutes, 60 minutes, or 90 minutes, to obtain a second extract aqueous solution. Preferably, in the first extraction in (iii.a), the volume of the extraction solvent used is 25 to 75 times, preferably 35 to 65 times, more preferably 45 to 55 times, for example about 30 times or 50 times, relative to the weight of the plant starting material.

[0079] Preferably, in the second extraction of (iii.b), the extraction solvent is used in a volume that is 10 to 150 times, preferably 75 to 125 times, more preferably 85 to 115 times, and even more preferably 95 to 105 times, relative to the weight of the plant starting material, for example, about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 times the weight.

[0080] In the first embodiment (P1) of the method for producing HA and / or CS, the first extract aqueous solution obtained from step (iii.a) and the second extract aqueous solution obtained from step (iii.b) were then combined and the solvent described in step (iv) was added.

[0081] In step (iv), a solvent (preferably ethanol) is added to the aqueous extract solution obtained from step (iii), or to the first aqueous extract solution obtained from step (iii.a) and the second aqueous extract solution obtained from step (iii.b) to obtain a liquid product.

[0082] In step (v), the liquid product obtained from step (iv) and the plant-derived starting material present in the product (e.g., supernatant or precipitate) are filtered by centrifugation and / or filtration (first filtration) to allow the liquid phase to pass through, leaving the solid portion (solid residue).

[0083] In step (vi), following step (v), the liquid phase obtained by centrifugation and / or filtration is treated in step (vi.a), and / or the solid residue is treated in steps (vi.b), (vi.c), (vi.d), and (vi.e).

[0084] In the preferred step (vi.a), the liquid phase obtained from step (v) is dried, and if appropriate, concentrated and dried to obtain hyaluronic acid or a salt thereof having a weight-average molecular weight of 10 kDa to 600 kDa.

[0085] The liquid phase dried (preferably concentrated and dried) in step (vi.a) should preferably have the precipitate removed when cooled to 20-25°C. Therefore, if a precipitate is present, the liquid phase obtained from step (v) should be further centrifuged and / or filtered before step (vi.a).

[0086] The concentration of the centrifugation and / or filtration of the liquid phase is preferably carried out at a temperature of 60°C to 90°C, more preferably 65°C to 85°C, and even more preferably 70°C to 80°C, for example, 70°C, 75°C, or 80°C. In addition to the temperature, the time of the concentration step also varies depending on the amount of the desired substance dissolved in the liquid phase.

[0087] The concentration of the centrifugation and / or filtration of the liquid phase preferably increases the amount of the substance (such as HA) dissolved in the liquid phase to a range of 1g to 35g, preferably 5g to 25g, and more preferably 8g to 18g per 100mL of liquid phase.

[0088] Preferably, the liquid phase obtained from the concentration step (vi.a) has a relative density of 1.01 to 1.20, preferably 1.02 to 1.15, and more preferably 1.05 to 1.08 (defined as [density of the liquid phase after centrifugation and / or filtration]:[density of the liquid phase after final concentration]).

[0089] Preferably, the concentration of the liquid phase is carried out under reduced pressure (less than 1 atm, 25°C), more preferably -1.5 mPa to -0.1 mPa, more preferably -1.0 mPa to -0.5 mPa, for example down to -0.8 mPa.

[0090] Preferably, the HA obtained from step (vi.a) has a purity of 90% to 100%, preferably 95% to 99.5%, and more preferably 97% to 99% (percentages determined by HPLC) (relative to the total weight of HA).

[0091] In preferred steps (vi.b), (vi.c), (vi.d), and (vi.e), the solid residue obtained from centrifugation and / or filtration in step (v) is processed to obtain chondroitin sulfate or salt having a weight-average molecular weight of 1 kDa to 50 kDa.

[0092] In the preferred step (vi.b), the solid residue obtained from the centrifugation and / or filtration in step (v) is recovered and purified to obtain chondroitin or a salt thereof (CS) having a weight-average molecular weight of 1 kDa to 50 kDa.

[0093] A preferred step (vi.c) is followed by a reaction step intended to insert a sulfate group into the chondroitin dimer (sulfonation (or sulfation), in which the solid residue is treated with a sulfuric acid source (preferably selected from the group including or comprising sulfuric acid, sulfur trioxide pyridine complex, sulfur trioxide dimethylformamide complex and mixtures thereof) to obtain an acidification product (the sulfonation step is carried out by techniques and apparatus known to those skilled in the art).

[0094] The amount of sulfuric acid source used in step (vi.c) is such that it contains 6-chondroitin sulfate in a weight percentage of 51% to 99%, or about 95 ± 0.5% (preferably 78% to 85%, or 86%), relative to the total weight of the disaccharides of CS in the solid residue of step (vi.b).

[0095] Preferably, in step (vi.c), 1 mL to 50 mL, preferably 2 mL to 40 mL, more preferably 4 mL to 30 mL, for example 5 mL, 10 mL, 15 mL, 18 mL, 22 mL, or 25 mL of sulfur trioxide dimethylformamide complex (SO3DMF) is used for every 100 g of chondroitin or its salt (CS) obtained from step (vi.b). More preferably, in some steps, for example, 2 mL to 8 mL of sulfur trioxide dimethylformamide complex is added to the chondroitin or its salt (CS) obtained from step (vi.b) first, then 8 mL to 12 mL, and finally 8 mL to 12 mL.

[0096] The process in step (vi.c) is carried out at a temperature of 20°C to 80°C, preferably 30°C to 70°C, more preferably 40°C to 60°C for 1 minute to 4 hours, preferably 10 minutes to 2 hours, more preferably 20 minutes to 1 hour. In step (vi.d), the product obtained from step (vi.c) is neutralized with a basic reagent.

[0097] Therefore, in step (vi.d), the sulfuric acid source liberated from the acidification product of step (vi.c) (i.e., the sulfuric acid source that is not bound to chondroitin as chondroitin sulfate in the acidification product) is removed by neutralization with a basic reagent to obtain a neutralization product.

[0098] In this specification, the expressions “neutralized” or “neutralized” are used to indicate a pH value of 6 to 8, preferably 6.4 to 7.6, more preferably 6.6 to 7.4, for example, a pH value of 7.0 ± 0.2.

[0099] The basic reagent used in step (vi.d) is preferably an inorganic basic reagent.

[0100] The basic reagent is preferably selected from the group comprising ammonia, sodium hydroxide, potassium hydroxide, and mixtures thereof.

[0101] Preferably, the sodium hydroxide used in step (vi.d) is at a concentration of 1 M, 2 M, or 4 M.

[0102] In a preferred step (vi.e) following step (vi.d), the neutralization product obtained from step (vi.d) is concentrated and dried to obtain chondroitin sulfate having a weight-average molecular weight of 1 kDa to 50 kDa.

[0103] The concentration in step (vi.e) results in a relative density (defined as [density of neutralization product obtained from step (vi.d)]:[density of concentrated product obtained from step (vi.e)]) of 1.0 to 1.30, preferably 1.01 to 1.20, and more preferably 1.05 to 1.15.

[0104] Preferably, the concentration in step (vi.e) is carried out by dialysis and / or vacuum concentration.

[0105] More preferably, dialysis is performed using a dialysis bag to remove any small amounts of impurities that may be present.

[0106] The concentration step (vi.e) is preferably terminated when the concentrated product obtained from step (vi.e) contains 10g to 60g, preferably 20g to 50g, more preferably 35g to 45g, for example 40g of solid per 100mL.

[0107] The drying step (vi.e) is carried out, preferably in a vacuum oven, following the concentration in step (vi.e).

[0108] The present invention is formed by using plant starting materials (preferably fungi, more preferably the Dicaryia subkingdom, even more preferably the Basidiomycota phylum, and even more preferably the Tremella fuciformis species) for producing hyaluronic acid or a salt thereof (HA), hyaluronic acid anionic salt, and / or chondroitin or a salt thereof (e.g., chondroitin sulfate or a salt thereof (CS)).

[0109] In embodiments of the use of the mixture, in a composition containing the mixture as an additive, excipient, or component (or inactive component), in the use of the composition as a pharmaceutical, in the use of the composition in the treatment of a particular disorder, disease, or condition, in a method of producing the mixture, or in the use of a composition containing the mixture, as an additive, excipient, or component, the above-mentioned plant starting material may be substituted or modified by those skilled in the art with respect to the features described herein. These embodiments are also considered to be included within the scope of protection of the appended claims.

[0110] Furthermore, any embodiment may be carried out independently of other described embodiments.

[0111] An outline of an embodiment (FRn) of the present invention is described below. FR1. A mixture (M) containing or consisting of glycosaminoglycans obtained from plant starting materials, wherein the glycosaminoglycans are (a) Hyaluronic acid or its salts, or hyaluronic acid anions (HA) having a weight-average molecular weight of 10 kDa to 600 kDa; (b) Chondroitin or its salt (CS) having a weight-average molecular weight of 1 kDa to 50 kDa (e.g., chondroitin sulfate); (c) Combinations of (a) and (b) Selected from a group that includes or consists of the following. FR2. The mixture (M) described in FR above, where (a) Hyaluronic acid or its salt (HA) has a weight-average molecular weight of 100 kDa to 500 kDa, preferably 200 kDa to 400 kDa; and / or (b) Chondroitin or its salt (CS) has a weight-average molecular weight of 1 kDa to 50 kDa, preferably 3 kDa to 40 kDa, and more preferably 5 kDa to 25 kDa. FR3. A mixture (M) according to any one of the above FRs, wherein the plant starting material is a fungus, preferably a Dicaryia subkingdom, more preferably a Basidiomycota. FR4. The mixture (M) described in FR above, where the fungus is Tremella fuciformis. FR5. Use of any of the above FRs (M) as an additive, excipient, or component in the manufacture of a pharmaceutical product, medical device, dietary supplement, specialty therapeutic product (SFMP), health supplement, or food. A composition comprising (i) a mixture described in any one of FR6. FR1 to FR4, and (ii) a technical additive or a pharmaceutical or food-grade excipient. FR7. The composition described in FR above, for use as a pharmaceutical. FR8. The composition according to FR6 for use in the preventive or therapeutic treatment of a person or animal having a specific disorder or disease selected from arthritis, osteoarthritis, arthropathy, arthralgia, inflammation of the hands and feet or joints, or gastroesophageal reflux disease, and / or for use as an additive, excipient, or component in the manufacture of a pharmaceutical product, a product for a medical device, a dietary supplement, a specialty therapeutic food (FSMP), or a health supplement. FR9. A method for producing hyaluronic acid or hyaluronic acid salt (HA), and / or chondroitin sulfate or chondroitin (CS), comprising at least one step of extracting hyaluronic acid or hyaluronic acid salt, and / or chondroitin sulfate or chondroitin from a plant-derived starting material. FR10. The method described in the above FR, including the next step. (i) Identify one or more natural fungi as plant starting materials for glycosaminoglycans; (ii) Press or grind the plant starting material as appropriate; (iii) Extract the glycosaminoglycan derived from the plant starting material obtained in step (i) or step (ii) with an extraction solvent (preferably an aqueous solvent, more preferably water) to obtain an aqueous extract of the glycosaminoglycan; (iv) Add the solvent (preferably ethanol) to the aqueous extract obtained from step (iii) to obtain the liquid product; (v) The liquid product obtained from step (iv) is centrifuged and / or filtered to obtain the liquid phase and solid residue; (vi) The liquid phase obtained from the centrifugation and / or filtration of step (v) is treated by the following step (vi.a), and / or the solid residue obtained from the centrifugation and / or filtration of step (v) is treated by the following steps (vi.b), (vi.c), (vi.d) and (vi.e): (vi.a) The liquid phase obtained from step (v) is dried, preferably concentrated and dried, to obtain hyaluronic acid or a salt thereof having a weight-average molecular weight of 10 kDa to 600 kDa; and / or (vi.b) The solid residue obtained from step (v) is collected and purified to obtain chondroitin or its salt (CS) having a weight-average molecular weight of 1 kDa to 50 kDa; (vi.c) The chondroitin or salt (CS) obtained from step (vi.b) is treated with a sulfuric acid source (preferably selected from the group including or comprising sulfuric acid, sulfur trioxide pyridine complex, sulfur trioxide dimethylformamide complex and mixtures thereof) to obtain an acidification product; (vi.d) The acidification product obtained from step (vi.c) is neutralized with a basic reagent to obtain a neutralization product; (vi.e) The neutralization product obtained from step (vi.d) is concentrated and dried to obtain chondroitin sulfate having a weight-average molecular weight of 1 kDa to 50 kDa. FR11. Use of plant starting materials (preferably fungi, more preferably the Dicaryia subkingdom, even more preferably the Basidiomycota phylum, and even more preferably the Tremella fuciformis species) for the production of hyaluronic acid or its salts, hyaluronic acid anionic salts, and / or chondroitin or its salts (e.g., chondroitin sulfate).

[0112] A second embodiment (P2) of the method of the present invention includes, or comprises, the following steps relating to a method for producing hyaluronic acid or a salt thereof (HA). (i) Identify one or more natural fungi as plant-derived starting materials for hyaluronic acid. For example, one or more natural fungi in a dried state preferably include or consist of at least one fungus belonging to the subkingdom Dicaria, preferably the phylum Basidiomycota, and more preferably the species Tremella fuciformis. (ii) The plant-based starting material is compressed or pulverized as appropriate (the average particle size distribution is preferably 500 μm to 1,800 μm, preferably 700 μm to 1,000 μm, for example, about 20 mesh = 841 μm); (Pre-iii) Enzymatic hydrolysis of the plant starting material obtained from step (i) or step (ii) is carried out in an aqueous hydrolysis solvent (preferably water) at a temperature of 10°C to 90°C. Here, the enzyme is preferably a protease, and in addition to the plant starting material dispersed in the hydrolysis solvent, the mixture from step (Pre-iii) is obtained; (iii) Extract the mixture from step (pre-iii) in an aqueous extraction solvent (preferably water) at 91°C to 110°C, preferably 95°C to 110°C, more preferably 98°C to 105°C (e.g., about 100°C or the boiling point of the extraction solvent) to obtain an aqueous extraction solution; (iv) Add a precipitation solvent, preferably an alcoholic solvent, more preferably ethanol, to the aqueous extraction solution obtained from step (iii) to obtain the liquid product of step (iv). Preferably, slowly add 95% ethanol to the aqueous extraction solution obtained from step (iii) in a volume ratio of 2 to 4, preferably 3, and continue stirring for 8 to 16 hours, for example about 12 hours, to obtain a precipitate from the ethanol; (vii) The liquid product of step (iv) is treated in step (vii.a) and, if appropriate, in step (vii.b): (vii.a) Remove the precipitated solvent, preferably ethanol, (for example, by distillation or heating under pressure at a temperature lower than room temperature) to obtain the liquid product of step (vii.a); (vii.b) Add water to the liquid product of step (vii.a) (for example, to dissolve a solid product) to obtain the liquid product of step (vii.b); (viii) Dry, preferably concentrate and / or dry, the liquid product of step (vii.b) to remove (e.g., water and any remaining precipitate solvent (e.g., ethanol) by concentration and / or drying and / or freeze-drying, preferably freeze-drying) to obtain product PR1 containing or consisting of hyaluronic acid or a salt thereof, having a weight-average molecular weight of 10 kDa to 600 kDa, preferably 50 kDa to 350 kDa, more preferably 100 kDa to 300 kDa, and a purity of 85% to about 100%, preferably 95% to 99.5%, more preferably 97% to 99% (percentages determined by HPLC) relative to the total weight of product PR1.

[0113] A third embodiment (P3) of the method of the present invention includes, or comprises, the following steps relating to a method for producing chondroitin sulfate, preferably 6-chondroitin sulfate. (i) Identify one or more natural fungi as plant-derived starting materials for hyaluronic acid. For example, one or more natural fungi in a dried state preferably include or consist of at least one fungus belonging to the subkingdom Dicaria, preferably the phylum Basidiomycota, and more preferably the species Tremella fuciformis; (ii) The plant-based starting material is compressed or pulverized as appropriate (the average particle size distribution is preferably 500 μm to 1,800 μm, more preferably 700 μm to 1,000 μm, for example, about 20 mesh = 841 μm); (Pre-iii) Enzymatic hydrolysis of the plant starting material obtained from step (i) or step (ii) is carried out in an aqueous hydrolysis solvent, preferably water, at a temperature of 10°C to 90°C. Here, the enzyme is preferably a protease, and in addition to the plant starting material dispersed in the hydrolysis solvent, the mixture of step (Pre-iii) is obtained; (iii) Extract the mixture from step (pre-iii) with an aqueous extraction solvent, preferably water, at 91°C to 110°C, preferably 95°C to 110°C, more preferably 98°C to 105°C (e.g., about 100°C or the boiling temperature of the extraction solvent) to obtain an aqueous extraction solution; (vi.c) The extracted aqueous solution is treated with a sulfuric acid source (sulfonation step) to obtain the liquid product of step (vi.c). Here, the sulfuric acid source is preferably selected from the group including or comprising sulfuric acid, sulfur trioxide pyridine complex, sulfur trioxide dimethylformamide complex and mixtures thereof. More preferably selected from sulfur trioxide dimethylformamide complex (SO3-DMS); (vi.d) Add a base to the liquid product of step (vi.c) to neutralize it and obtain a neutralized product. The base used to neutralize the pH is preferably an inorganic base, more preferably NaOH, KOH, Ca(OH)2, or Mg(OH)2; (vi.e) The neutralization product obtained from step (vi.d) is concentrated and dried to obtain product PR2, which contains or consists of chondroitin sulfate or a salt (CS) thereof, having a weight-average molecular weight of 1 kDa to 45 kDa or 50 kDa (or 5 kDa to less than 50 kDa), preferably 3 kDa to 40 kDa, more preferably 5 kDa to 25 kDa or 5 kDa to 10 kDa, for example, about 5 kDa, or 6 kDa, or 7 kDa, or 8 kDa, or 9 kDa, or 10 kDa.

[0114] In the third embodiment (P3) of the method of the present invention, the term "chondroitin sulfate or its salt (CS)" refers to a combination of unsulfated chondroitin and mono, di, and / or trisulfated chondroitin at various possible positions, preferably mainly 6-chondroitin sulfate.

[0115] Alternatively, the term "chondroitin sulfate or its salt (CS)" as used in the third embodiment (P3) of the method of the present invention refers to a mono, di, and / or trisulfated chondroitin group, preferably mainly 6-chondroitin sulfate.

[0116] Preferably, the chondroitin sulfate (CS) obtained from the third embodiment (P3) of the method of the present invention has a weight-average molecular weight in the above range (preferably 5kDa to 10kDa, for example about 6kDa, or 7kDa, or 8kDa, or 9kDa), and contains 51% to about 95±0.5%, preferably 75% to 90%, more preferably 78% to 86% of 6-chondroitin sulfate (CS) (or disaccharides contained in chondroitin sulfate) based on the total weight. The product contains leutin sulfate and has a purity of 80% to 99.99% (e.g., 94.5%, 94.6%, 94.7%, 94.8%, or 94.9%), preferably 85% to 98% (e.g., 86%, 87%, 88%, 89%, or 89.5%), more preferably 90% and 94.9% (e.g., 91%, 92%, 93%, 94%, or 94.5%) relative to the total weight of the product PR2 obtained from step (vi.e).

[0117] More preferably, the chondroitin sulfate (CS) obtained from the third embodiment (P3) of the method of the present invention has a weight-average molecular weight in the above range (preferably 5 kDa to 10 kDa, for example about 6 kDa, or 7 kDa, or 8 kDa, or 9 kDa), and contains 51% to about 95 ± 0.5% (preferably 75% to 90%, more preferably 78% to 86%) of 6-chondroitin sulfate (CS) (or disaccharides contained in chondroitin sulfate) based on the total weight. The product contains 0.01% to about 5% (preferably 0.05% to 3%, more preferably 0.1% to 1.5%) of 4-chondroitin sulfate and has a purity of 80% to 99.99%, preferably 85% to 98% (e.g., 86%, 87%, 88%, or 89%), more preferably 90% to 95% (e.g., 91%, 92%, 93%, 94%, or 94.5%) relative to the total weight of product PR2 obtained from step (vi.e) (percentages are determined by HPLC).

[0118] The weight-average molecular weight of HA and / or CS can be calculated by methods and apparatus common to those skilled in the art (e.g., high-speed size exclusion chromatography (HPSEC)). Preferably, the weight-average molecular weight of HA and / or CS can be determined by HPSEC with built-in software specifically for gel permeation chromatography (GPC).

[0119] In the (pre-iii) step of enzymatic hydrolysis as described in the second and third embodiments (P2 and P3) of the method of the present invention, an enzyme (e.g., pectinase and / or cellulase and / or protease) is added to the plant starting material described in the present invention, and heated in a hydrolysis solvent (e.g., aqueous solvent or water) in a volume of 25 to 100 times, preferably 35 to 75 times, more preferably 45 to 55 times (e.g., 50 times) the weight of the plant starting material for 0.5 to 12 hours, preferably 1 to 8 hours, more preferably 2 to 6 hours (e.g., about 4 hours) at a temperature of 10°C to 90°C, preferably 20°C to 65°C, more preferably 45°C to 55°C (e.g., about 50°C) to obtain the mixture of step (pre-iii), and then the extraction step (iii) or the first extraction step (iii.a) is carried out as described herein.

[0120] In a more beneficial manner, the enzymatic hydrolysis of step (pre-iii) is carried out at a pH of 2-9, preferably 3-5 or 5-8, more preferably 3-4 (e.g., 3.5) or 6-7, and / or in the enzymatic hydrolysis step (pre-iii), the amount of enzyme (1%-20%, or 2%-10%, or 3%-6% by weight / weight or by weight / volume enzyme aqueous solution) is used in a volume (%) of 0.001%-1%, preferably 0.005%-0.1%, more preferably 0.008%-0.05% (e.g., 0.01), relative to the mass of the extracted plant starting material (volume / mass) or the volume of the extracted plant starting material solution in the hydrolysis solvent (volume / volume).

[0121] In preferred embodiments, the enzymatic hydrolysis step (pre-iii) is carried out under approximately the following conditions: volume 0.01%; temperature 50°C; actual pH 3.5; duration 4 hours.

[0122] The enzyme used in step (pre-iii) of the second and / or third embodiment (P2 and / or P3) may be a pectinase and / or cellulase and / or protease.

[0123] Examples of enzymes that can be used in the method of the present invention are listed below. • Commercially available Pectinex® Ultra Tropical, composition: Enzyme: Pectin lyase or pectinase, Preservative: Potassium sorbate, Stabilizers: Sucrose, Glycerol, Sorbitol, Sodium chloride, Potassium chloride; Active compound: Pectin lyase or pectinase (PECTU) = 5,000 PECTU / g; Approximate density 1.18 (g / mL); Pectin lyase is an enzyme that catalyzes the cleavage and elimination of (1,4)-alpha-D-galacturonate methyl ester, yielding an oligosaccharide having a 4-deoxy-6-O-methyl-alpha-D-galact-4-enurosyl group at its non-reducing end; Other active compounds: Cellulase, Polygalacturonase, β-Glucanase (endo-1,3(4)-) • Commercially available Pectinex® Ultra SP-L, composition %(w / w): 45% glycerol (CAS No. 56-81-5), 45% water (CAS No. 7732-18-5), 5% polygalacturonase (CAS No. 9032-75-1; enzyme concentration defined by dry weight), 5% potassium chloride (CAS No. 7447-40-7); Active compound: polygalacturonase (PGNU) = 3300 PGNU / g; Approximate density 1.17 (g / mL); Polygalacturonase is an enzyme that hydrolyzes (1,4)-alpha-D-galacturonane bonds in pectic acid and other galacturonans. • Commercially available Viscozyme®, composition %(w / w): 56.8% water (CAS No. 7732-18-5), 9% β-glucanase (endo-1,3(4)-) (CAS No. 62213-14-3; enzyme concentration defined by dry weight), 24% sucrose (CAS No. 57-50-1), 10% sodium chloride (CAS No. 7647-14-5), 0.20% potassium sorbate (CAS No. 24634-61-5); Active compound: β-glucanase (endo-1,3(4)-) (FGB) = 100 FBG / g; Approximate density 1.21 (g / mL); Endo-β-glucanase is an enzyme that hydrolyzes the (1,3)- or (1,4)- bonds of β-D-glucan; Other active compounds: Xylanase, cellulase, hemicellulase

[0124] In the second and third embodiments (P2 and / or P3) of the method of the present invention, the extraction step (iii) comprises, or consists of, a first extraction step (iii.a) which includes extracting a first extraction aqueous solution at 91°C to 110°C (e.g., about 100°C) or the boiling temperature of the extraction solvent for preferably 0.5 to 12 hours, preferably 1 to 9 hours, more preferably 1 to 4 hours (e.g., about 2 or 3 hours), followed by a second extraction step (iii.b) which includes extracting a second extraction aqueous solution at 90°C to 110°C (e.g., about 100°C) or the boiling temperature of the extraction solvent for preferably 0.5 to 8 hours, preferably 0.5 to 4 hours, more preferably 1 to 3 hours (e.g., 1.5 hours, or 2 or 2.5 hours), or consists of, a second extraction step (iii.b) which includes extracting a second extraction aqueous solution at 90°C to 110°C (e.g., about 100°C) or the boiling temperature of the extraction solvent for preferably 0.5 to 8 hours, preferably 0.5 to 4 hours, more preferably 1 to 3 hours (e.g., 1.5 hours, or 2 or 2.5 hours), followed by a step (iii.c) in which the first and second extraction solutions are combined to form a final extraction aqueous solution, and the final extraction aqueous solution is appropriately concentrated to obtain a concentrated extraction aqueous solution.

[0125] In the second and third embodiments (P2 and P3) of the method of the present invention, preferably in the first extraction (iii.a), a first extraction solvent is used in an amount of 25 to 100 times, preferably 35 to 65 times, more preferably 45 to 55 times, for example 50 times, the weight of the plant starting material. For example, a first extraction solvent in an amount of 25 to 100 times the weight of the plant starting material is added to the enzymatic hydrolysis solvent, and the total volume of the solvent reaches 50 to 200 times (for example about 100 times) the weight of the plant material.

[0126] In the second and third embodiments (P2 and P3) of the method of the present invention, preferably in the second extraction (iii.b), a second extraction solvent is used in an amount of 25 to 75 times, preferably 35 to 65 times, and more preferably 45 to 55 times (for example, about 50 times) the weight of the plant starting material.

[0127] In the second and third embodiments (P2 and P3) of the method of the present invention, after the first extraction step (iii.a), a filtration step (iii.a) is performed, and this filtrate corresponds to the first extracted aqueous solution obtained in step (iii.a). The obtained residue is then subjected to a second extraction step (iii.b). After the second extraction step (iii.b), a filtration step (iii.b) is performed, and this filtrate corresponds to the second extracted aqueous solution obtained in step (iii.b). For example, the filtration in steps (iii.a) and (iii.b) (filtration of extraction step (iii)) is performed with a filter of 140 to 270 mesh, preferably 200 mesh. Depending on the second and third embodiments (P2 and P3), the first extract aqueous solution obtained from step (iii.a) and the second extract aqueous solution obtained from step (iii.b) are combined and subjected to step (iii.c), which is a vacuum concentration (for example, at 60°C to 90°C, preferably 70°C to 80°C, more preferably about 75°C, until the relative density is about 0.8 to 1.5, preferably 1.00 to 1.20 (for example, about 1.05 to 1.08)).

[0128] In a third embodiment (P3) of the method of the present invention, preferably in step (vi.c) (sulfonation step), sulfur trioxide dimethylformamide complex (SO3DMF) is used in an amount of 1 mL to 50 mL, preferably 2 mL to 40 mL, more preferably 4 mL to 30 mL (for example, about 5 mL, 10 mL, 15 mL, 18 mL, 22 mL, or 25 mL) per 100 g of chondroitin or its salt (CS) obtained from step (vi.b). More preferably, the sulfur trioxide dimethylformamide complex is added to the chondroitin or its salt (CS) obtained from step (vi.b) in several steps (for example, 2 mL to 8 mL, then 8 mL to 12 mL, and finally another 8 mL to 12 mL). The treatment in step (vi.c) is carried out at 20°C to 80°C, preferably 30°C to 70°C, more preferably 40°C to 60°C (e.g., about 50°C) for 1 minute to 4 hours, preferably 10 minutes to 2 hours, more preferably 20 minutes to 60 minutes (e.g., about 30 minutes). In a preferred example, the sulfonation step (vi.c) is carried out by adding SO3-DMF (5 mL, 15 mL, 25 mL) at 40°C to 60°C (e.g., 50°C) to about 100 mL of the extracted aqueous solution (10 to 15 g of solid / 100 mL; relative density 1.05 to 1.08) obtained from step (iii), (iii.b), or (iii.c), for 20 minutes to 60 minutes (e.g., 30 minutes).

[0129] In the third embodiment (P3) of the method of the present invention, the basic reagent used in step (vi.d) is preferably an inorganic basic reagent, and is selected from the group comprising or consisting of ammonia, sodium hydroxide, potassium hydroxide and mixtures thereof, and is preferably sodium hydroxide (for example, at a concentration of 1M, 2M, or 4M).

[0130] In a third embodiment (P3) of the method of the present invention, step (vi.e) is preferably membrane filtration by dialysis (for example, in a dialysis bag of 1,000 Da for 18 to 36 hours, preferably 24 hours, until the relative density is 1.3 to 1.5, preferably about 1.1), followed by drying (for example, in a vacuum oven).

[0131] In the first and third embodiments (P1 and P3) of the method of the present invention, the weight-average molecular weight of the chondroitin sulfate or its salt (CS) of the present invention is 5 kDa to less than 50 kDa, preferably 5 kDa to 25 kDa. 50% to 95 ± 0.5%, preferably 75% to 90% by weight of 6-chondroitin sulfate; 5% to 20%, preferably 7% to 15% by weight, of unsulfated chondroitin; 0.1% to 10%, preferably 0.2% to 8% by weight of 2,6-chondroitin disulfate; and 0.01% to 5%, preferably 0.05% to 3% by weight of 4-chondroitin sulfate This includes, and all of the above percentages are expressed relative to the total weight of disaccharides or chondroitin sulfate contained in chondroitin sulfate.

[0132] In a preferred embodiment of the present invention, the weight-average molecular weight of chondroitin sulfate or its salt (CS) of the present invention (Methods P1 and / or P3) is 5 kDa to 10 kDa, and the above chondroitin sulfate or its salt is 78% to 86% by weight of 6-chondroitin sulfate; 8% to 13% by weight of unsulfated chondroitin; 0.3% to 5% by weight of 2,6-chondroitin disulfate; and 0.1% to 1.5% by weight of 4-chondroitin sulfate This includes, and all of the above percentages are expressed relative to the total weight of disaccharides or chondroitin sulfate contained in chondroitin sulfate.

[0133] In a preferred embodiment of the present invention, the weight-average molecular weight of chondroitin sulfate or its salt (CS) of the present invention (Methods P1 and / or P3) is 5 kDa to less than 50 kDa, preferably 5 kDa to less than 25 kDa, and the above chondroitin sulfate or its salt is 50% to 95 ± 0.5%, preferably 75% to 90% by weight of 6-chondroitin sulfate; and 0.01% to 5%, preferably 0.05% to 3% by weight of 4-chondroitin sulfate This includes, and all of the above percentages are expressed relative to the total weight of disaccharides or chondroitin sulfate contained in chondroitin sulfate.

[0134] In a more preferred embodiment of the present invention, the weight-average molecular weight of the chondroitin sulfate or its salt (CS) of the present invention (Methods P1 and / or P3) is 5 kDa to 10 kDa, and the above chondroitin sulfate or its salt is 78% to 86% by weight of 6-chondroitin sulfate; and 0.1% to 1.5% by weight of 4-chondroitin sulfate This includes, and all of the above percentages are expressed relative to the total weight of disaccharides or chondroitin sulfate contained in chondroitin sulfate.

[0135] In a more preferred embodiment of the present invention, the weight-average molecular weight of chondroitin sulfate or its salt (CS) of the present invention (Methods P1 and / or P3) is 5 kDa to less than 50 kDa, preferably 5 kDa to less than 25 kDa. 50% to 95 ± 0.5%, preferably 75% to 90% by weight of 6-chondroitin sulfate; 5% to 20%, preferably 7% to 15% by weight, of unsulfated chondroitin; 0.1% to 10%, preferably 0.2% to 8% by weight of 2,6-chondroitin disulfate; 0.01% to 5%, preferably 0.05% to 3% by weight of 4-chondroitin sulfate; 0.01% to 5%, preferably 0.05% to 3% by weight of 4,6-chondroitin disulfate; and 0.01% to 5%, preferably 0.05% to 3% by weight of 2,4-chondroitin disulfate This includes, and all of the above percentages are expressed relative to the total weight of disaccharides or chondroitin sulfate contained in chondroitin sulfate.

[0136] In one embodiment, the weight-average molecular weight of chondroitin sulfate or its salt (CS) in the present invention (Methods P1 and / or P3) is 5 kDa to 10 kDa, and chondroitin sulfate or its salt is 78% to 86% by weight of 6-chondroitin sulfate; 8% to 13% by weight of unsulfated chondroitin; 0.3% to 5% by weight of 2,6-chondroitin disulfate; and further It contains 0.1% to 1.5% by weight of 4-chondroitin sulfate, 4,6-chondroitin disulfate, and 2,4-chondroitin disulfate.

[0137] Experimental method I. Method for producing hyaluronic acid according to the second embodiment (P2) (I) Prepare dried fungi belonging to the Tremella species; (ii) The dried fungi are ground (to about 20 mesh) to obtain compressed / pulverized dried fungi; (Pre-iii) Enzymatic hydrolysis using pectinase as the enzyme: Add distilled water (50 vol / weight) containing the enzyme pectinase (Pectinex® Ultra Tropical, 0.01% by volume) to the compressed / crushed dried fungi, heat at approximately 50°C for approximately 3 hours to obtain a hydrolysis mixture; (iii.a) First extraction: Add distilled water (50 vol / mW) to the hydrolysis mixture, heat (boil) at approximately 100°C for approximately 2.5 hours, filter through a 200-mesh sieve, and collect the filtrate and solid residue; (iii.b) Second extraction: Add distilled water (50 vol / weight) to the solid residue obtained from the first extraction (iii.a), heat (boil) at approximately 100°C for approximately 2 hours, then filter through a 200-mesh sieve, collect the filtrate, and combine the filtrates obtained from the first extraction (iii.a) and the second extraction (iii.b) to obtain an aqueous extraction solution; (iii.c) The extracted aqueous solution is concentrated at approximately 75°C to obtain a concentrated extracted aqueous solution with a relative density of approximately 1.05; then (iv) Slowly add 95% ethanol (vol / vol = 3) to the concentrated extract aqueous solution while stirring, and let stand for 12 hours to remove the ethanol and retain the extract; (vii) Distilled water is added to the extract and freeze-dried to obtain product PR1 containing or consisting of hyaluronic acid or a salt thereof (HA) having an average molecular weight of 100-300 kDa and a purity of 95-99% relative to the total weight of product PR1.

[0138] II. Method for producing chondroitin sulfate according to the third embodiment (P3) (i) Prepare dried fungi belonging to the Tremella species; (ii) The dried fungi are ground (to about 20 mesh) to obtain compressed / pulverized dried fungi; (Pre-iii) Enzymatic hydrolysis using pectinase as the enzyme: Add distilled water (50 vol / weight) containing the enzyme pectinase (Pectinex® Ultra Tropical, 0.01% by volume) to the pressed / crushed dried fungi, heat at approximately 50°C for approximately 2 hours to obtain a hydrolysis mixture (pH 5-7); (iii.a) First extraction: Add distilled water (50 vol / mW) to the hydrolysis mixture, heat (boil) at 100°C for about 2.5 hours, then filter through a 200-mesh sieve (centrifugation before filtration if necessary), and collect the filtrate and solid residue; (iii.b) Second extraction: Add distilled water (50 vol / weight) to the solid residue obtained from the first extraction, heat (boil) at approximately 100°C for approximately 1.5 hours, then filter through a 200-mesh sieve, collect the filtrate, and combine the filtrates obtained from the first extraction (iii.a) and the second extraction (iii.b) to obtain an aqueous extraction solution; (iii.c) The extracted aqueous solution is concentrated at approximately 75°C to obtain a concentrated extracted aqueous solution with a relative density of approximately 1.05 to 1.08; then (vi.c) Sulfonation (or sulfation) reaction: Add 5 mL, 15 mL, and 55 mL of SO3DMF to each 100 mL of concentrated extract aqueous solution and stir at approximately 75°C for approximately 30 minutes; (vi.d) Neutralize to approximately pH 7 with NaOH; (vi.e) The obtained solution is placed in a dialysis bag (1,000 Da) and left for at least 24 hours until the relative density of the solution reaches approximately 1.1. It is dried in a vacuum oven to obtain product PR2, which contains or consists of chondroitin sulfate or a salt thereof (CS) having an average molecular weight of 5 kDa to 10 kDa (e.g., approximately 8 kDa). Here, the CS has a composition similar to compound CS.1 shown in Table 1 and is 89% to 94.5% pure relative to the total weight of product PR2.

Claims

1. A method for producing at least one glycosaminoglycan selected from hyaluronic acid or a salt thereof (HA) and / or chondroitin sulfate or a salt thereof (CS) having a weight-average molecular weight of 10 kDa to 600 kDa, the method comprising at least one step of extracting a plant-derived starting material containing or consisting of at least one natural fungus belonging to the Dicaryia subkingdom, preferably the Basidiomycota, with an aqueous solvent or water.

2. The method according to claim 1, wherein at least one species of the fungus belongs to Tremella fuciformis (Berk. 1856).

3. below, (i) Identify at least one of the natural fungi as a plant starting material for at least one of the glycosaminoglycans; (ii) The plant starting material is pressed or crushed as appropriate to obtain at least one of the crushed and pressed fungi; (iii) Extract glycosaminoglycans from at least one fungus obtained from step (i) or step (ii) using an extraction solvent, preferably an aqueous solvent, more preferably water, to obtain an aqueous solution of glycosaminoglycans; (iv) Add a solvent, preferably an alcohol, more preferably ethanol, to the aqueous extract obtained from step (iii) to obtain a liquid product; (v) The liquid product obtained from step (iv) is centrifuged and / or filtered to obtain the liquid phase and solid residue; (vi) The liquid phase obtained from the centrifugation and / or filtration of step (v) is treated by the following step (vi.a), and / or the solid residue obtained from the centrifugation and / or filtration of step (v) is treated by the following steps (vi.b), (vi.c), (vi.d) and (vi.e); (vi.a) The liquid phase obtained from step (v) is dried, preferably concentrated and dried, to obtain product PR1 containing or consisting of hyaluronic acid or a salt thereof having a weight-average molecular weight of 10 kDa to 600 kDa; (vi.b) The solid residue obtained from step (v) is recovered and purified to obtain the product of step (vi.b); (vi.c) The product of step (vi.b) is a sulfuric acid source, preferably a sulfuric acid source selected from the group comprising or consisting of sulfuric acid, sulfur trioxide pyridine complex, sulfur trioxide dimethylformamide complex and mixtures thereof, more preferably SO 3 - Treat with DMF to obtain the acidification product; (vi.d) The acidification product obtained in step (vi.c) is neutralized with a basic reagent, preferably sodium hydroxide, to obtain a neutralization product; (vi.e) The neutralization product obtained from step (vi.d) is concentrated and dried to obtain product PR2, which contains or consists of chondroitin sulfate or a salt thereof having a weight-average molecular weight of 1 kDa to 50 kDa; The method according to claim 1 or 2, comprising the step.

4. In step (iii) of extracting at least one glycosaminoglycan from a plant starting material, the following steps are taken: (iii.a) Perform an initial extraction from the plant starting material and extraction solvent at a temperature of 10°C to 90°C for 0.5 to 12 hours to obtain a first extract aqueous solution and a solid residue; and (iii.b) A second extraction is performed on the solid residue and extraction solvent obtained from step (iii.a) at a temperature of 90°C to 110°C for 0.5 to 8 hours to obtain a second extraction aqueous solution. The first and second extraction aqueous solutions are then combined to obtain an extraction aqueous solution. A method according to any one of the above claims, comprising or consisting of the steps.

5. The method according to claim 4, wherein an enzyme, preferably a proteolytic enzyme, is used in step (iii) of extraction.

6. The method according to any one of claims 1 to 5, wherein the hyaluronic acid or salt obtained by the above method has a weight-average molecular weight of 100 kDa to 300 kDa.

7. The method according to claim 3, wherein the purity of the hyaluronic acid or salt obtained by the above method is 85% to 100%, preferably 95% to 99.5%, and more preferably 97% to 99%, relative to the total weight of product PR1.

8. The method according to any one of claims 1 to 5, wherein the chondroitin sulfate or salt obtained by the above method has a weight-average molecular weight of 5 kDa to 25 kDa, preferably 5 kDa to 10 kDa, and / or the chondroitin sulfate or salt obtained by the above method contains or comprises 50% to 95 ± 0.5% of 6-chondroitin sulfate relative to the total weight of disaccharides contained in the chondroitin sulfate or salt.

9. Use of fungi belonging to the subkingdom Dicaryia, preferably Basidiomycota, and more preferably Tremella fuciformis (Berk. 1856), as plant starting materials for producing hyaluronic acid or a salt thereof having a weight-average molecular weight of 10 kDa to 600 kDa, preferably 100 kDa to 500 kDa, and more preferably 100 kDa to 300 kDa.

10. Use of fungi belonging to the subkingdom Dicaryia, preferably the phylum Basidiomycota, and more preferably the species Tremella fuciformis (Berk. 1856), as plant starting materials for producing chondroitin sulfate or a salt thereof having a weight-average molecular weight of 1 kDa to 50 kDa, preferably 5 kDa to 25 kDa, and more preferably 5 kDa to 10 kDa.

11. Chondroitin sulfate or a salt thereof having a weight-average molecular weight of 5 kDa to less than 50 kDa, comprising 50% to 95 ± 0.5% of 6-chondroitin sulfate and 0.01% to 5% of 4-chondroitin sulfate relative to the total weight of disaccharides contained in the chondroitin sulfate.

12. Chondroitin sulfate or a salt thereof (CS) according to claim 11, having a weight-average molecular weight of 5 kDa to 25 kDa, preferably 5 kDa to 10 kDa, comprising 75% to 90%, preferably 78% to 86%, of 6-chondroitin sulfate and 0.05% to 3%, preferably 0.1% to 1.5%, of 4-chondroitin sulfate, based on the total weight of disaccharides contained in the chondroitin sulfate.

13. Use of chondroitin sulfate or a salt thereof according to claim 11 or 12 as an additive or excipient or component in pharmaceutical products, medical devices, nutritional supplements, therapeutic specialty foods (FSMPs), health supplements or foods.

14. (i) a chondroitin sulfate or a salt thereof as described in claim 11 or 12, and (ii) a composition comprising a technical additive or a pharmaceutical or food-grade excipient.

15. The composition according to the above claim for use as a pharmaceutical.

16. The composition according to claim 15, for use in a method of preventing and / or treating arthritis, osteoarthritis, arthropathy, joint pain, inflammation of the hands and feet or joints, or gastroesophageal reflux disease in human or animal subjects.

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