Identification and selection of a plant starting material of a plant chondroitin sulfate and hyaluronic acid, and transformation of such plant starting material to obtain ingredients for use in foods, supplements, medical devices or drugs

Abstract

Process for extraction from a plant starting material, such as a fungus, for the preparation of a mixture (m) comprising or, alternatively, consisting of at least one glycosaminoglycan selected from: (a) hyaluronic acid or a salt thereof (HA) having a weight average molecular weight of comprised from 10 kDa to 600 kDa; (b) chondroitin or chondroitin sulfate or a salt thereof (CS) having a weight average molecular weight comprised from 3 kDa to 50 kDa; and (c) a combination of (a) and (b).

Description

(19) *EP004721763A2* (11) EP 4 721 763 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 08.04.2026 Bulletin 2026 / 15 (21) Application number: 26160058.9 (22) Date of filing: 08.06.2020 (51) International Patent Classification (IPC): A61K 47 / 36 (2006.01) (52) Cooperative Patent Classification (CPC): (C-Sets available) A61K 47 / 36; A23L 33 / 105; A61K 31 / 728; A61K 31 / 737; A61K 36 / 07; A61P 1 / 04; A61P 19 / 02; C08B 37 / 0069; C08B 37 / 0072; C12P 19 / 26; A61K 2236 / 00 (Cont.) (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR Designated Extension States: BA ME Designated Validation States: KH MA MD TN (30) Priority: 07.06.2019 IT 201900008409 (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 20736429.0 / 3 980 030 (71) Applicant: Vivatis Pharma GmbH 20097 Hamburg (DE) (72) Inventor: The designation of the inventor has not yet been filed (74) Representative: Marben S.r.l. Via Larga, 16 20122 Milano (IT) Remarks: This application was filed on 23‑02‑2026 as a divisional application to the application mentioned under INID code 62. (54) IDENTIFICATION AND SELECTION OF A PLANT STARTING MATERIAL OF A PLANT CHONDROITIN SULFATE AND HYALURONIC ACID, AND TRANSFORMATION OF SUCH PLANT STARTING MATERIAL TO OBTAIN INGREDIENTS FOR USE IN FOODS, SUPPLEMENTS, MEDICAL DEVICES OR DRUGS (57) Process for extraction from a plant starting ma- terial, such as a fungus, for the preparation of a mixture (m) comprising or, alternatively, consisting of at least one glycosaminoglycan selected from: (a) hyaluronic acid or a salt thereof (HA) having a weight average molecular weight of comprised from 10 kDa to 600 kDa; (b) chon- droitin or chondroitin sulfate or a salt thereof (CS) having aweight averagemolecularweight comprised from3kDa to 50 kDa; and (c) a combination of (a) and (b). EP 4 72 1 76 3 A 2 Processed by Luminess, 75001 PARIS (FR) (Cont. next page) (52) Cooperative Patent Classification (CPC): (Cont.) C-Sets A61K 31 / 728, A61K 2300 / 00; A61K 31 / 737, A61K 2300 / 00 2 EP 4 721 763 A2 Description

[0001] The present invention relates to a mixture comprising or, alternatively, consisting of at least one glycosami- noglycan, obtained from a plant starting material, selected from the group comprising or, alternatively, consisting of hyaluronic acid or a salt thereof (a hyaluronate anion salt) (in short, together or separate, HA), and / or chondroitin or a salt thereof such as a chondroitin sulfate or a salt thereof (in short, together or separate, CS), of plant origin having a high degree of purity and reduced content of contaminants and / or by-products.

[0002] Furthermore, the present invention relates to a use of the aforementioned mixture as an additive, excipient, or ingredient in thepreparationofpharmaceutical products,medical devices, nutraceutical products, food for specialmedical purposes (FSMPs), food products or dietary supplements.

[0003] Furthermore, the present invention relates to a composition comprising (i) said mixture comprising or, alter- natively, consistingof hyaluronic acid or a salt thereof (a hyaluronateanion salt) (in short, together or separate,HA), and / or chondroitin or a salt thereof, such as a chondroitin sulfate or a salt thereof, (in short, CS), of plant origin, and (ii) optionally technological additives and pharmaceutical or food grade excipients.

[0004] Furthermore, the present invention relates to said composition comprising said mixture for use as medicament.

[0005] Furthermore, thepresent invention relates tosaid composition comprisingsaidmixture for use inamethod for the preventive or curative treatment of humans and animals having specific disorders or pathologies or diseases, selected from arthritis, osteoarthritis, arthrosis, joint pain, inflammation of the limbs and joints, gastroesophageal reflux.

[0006] Furthermore, the present invention relates to a process for preparing said mixture, and said composition comprising said mixture, comprising or, alternatively, consisting of hyaluronic acid or a salt thereof (a hyaluronate anion salt) (in short, togetheror separate,HA), and / or achondroitinorasalt thereof, suchasachondroitin sulfateorasalt thereof, (in short CS) of plant origin.

[0007] Lastly, the present invention relates to the use of a fungus as a plant starting material for preparing a hyaluronic acidorasalt thereof and / or a chondroitinorasalt thereof, suchasachondroitin sulfateorasalt thereof, of plant originwitha high degree of purity and a reduced content of contaminants and / or by-products.

[0008] Hyaluronic acid is an anionic non-sulfate glycosaminoglycan (GAG) which is distributed abundantly in con- nective, epithelial and neural tissues of vertebrates. Hyaluronic acid has important structural, rheological and physio- logical functions.

[0009] The rooster crests and human umbilical cords have very high concentrations of hyaluronic acid, respectively 7,500 mg / l and 4,100 mg / l. For this reason, in the early years ’80, Endre A. Balazs and his collaborators developed a procedure for isolating and purifying hyaluronic acid from the rooster crests and from the human umbilical cords. Since then, hyaluronic acid has been produced from the rooster crests on an industrial scale.

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

[0011] Chondroitin sulfate has a repeating unit (disaccharide) of the following general formula (I): wherein at least one from among R2, R4 and R6 is a sulfite group (S03 -). In chondroitin mono-sulfate, only one of the R2, R4 or R6 groups is a sulfite group. The three possible mono-sulfated chondroitins are therefore 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] Most chondroitin sulfate is obtained from extracts of animal cartilage, mainly from bovine andporcine tissues (for example: trachea, ear and nose), but other sources such as shark, fish and bird cartilage can also be used.

[0013] Despite the numerous and undisputed - in terms of efficacy - medical applications of hyaluronic acid and chondroitin sulfate in mammals, in particular in humans, the processes of preparation through extraction from animal precursors are today faced with growing concerns and fears, also of an ethical, religious and moral nature. The main concerns and fears arise from the use of products derived from animals or of animal origin for the preparation of a hyaluronic acid or a salt thereof and / or a chondroitin or a salt thereof, such as a chondroitin sulfate, especially when these compounds or salts are intended for nutritional, biomedical or pharmaceutical applications. In addition, chondroitin sulfate 3 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 andhyaluronic acid extracted from animal sources havehighmolecularweights, while it would be advantageous to havea chondroitin sulfate and / or a hyaluronic acid with low molecular since they have better transcutaneous penetration properties.

[0014] In the prior art chondroitin sulfate, not of animal origin, obtained by inserting a sulfate group on a non-sulfated chondroitin obtained by means of bacterial fermentation processes, are known.

[0015] Therefore, in the field of pharmaceutical products, medical devices, nutraceutical products, food for special medical purposes (FSMPs),dietary supplementsor foodproducts, there isastrongneedanddemandbymarketoperators to have a hyaluronic acid or a salt thereof and / or a chondroitin or a salt thereof, such as a chondroitin sulfate, that are prepared in an alternative way with respect to the existing ones and that can be used across all categories of consumers including vegans, vegetarians, subjects suffering from allergies and anyone who - for religious or ideological reasons - access to products or medicaments containing hyaluronic acid or a salt thereof and / or a chondroitin or salt thereof is currentlyprecluded.Furthermore, theneed is felt toproducechondroitin sulfateand / orhyaluronicacidof non-animal origin with processes that are economically advantageous with respect to what is known in the art and easy to apply.

[0016] After a long and intense research and development activity, the Applicant developed a preparation technology and process capable of providing an adequate response to the existing limits, drawbacks and problems.

[0017] Thus, forming anobject of the present invention is amixture comprisingor, alternatively, consisting of at least one glycosaminoglycan, obtained from a plant starting material, selected from the group comprising or, alternatively, consisting of hyaluronic acid or a salt thereof (a hyaluronate anion salt) (in short, together or separate, HA), and / or chondroitin or a salt thereof such as a chondroitin sulfate or a salt thereof (in short, together or separate, CS), and the combinations thereof, of plant origin, having the characteristics as defined in the attached claims.

[0018] Furthermore, forming an object of the present invention is a use of the aforementioned mixture as an additive, excipient, or ingredient in the preparation of pharmaceutical products, medical devices, nutraceutical products, food for special medical purposes (FSMPs), food products or dietary supplements, said use having the characteristics as defined in the attached claims.

[0019] Forming another object of the present invention is a composition comprising (i) said mixture comprising or, alternatively consisting of hyaluronic acid or a salt thereof (a hyaluronate anion salt) (in short, together or separate, HA), and / or chondroitin or a salt thereof, suchas chondroitin sulfate, (in shortCS) of plant origin, and (ii) optionally technological additives and pharmaceutical or food grade excipients, having the characteristics as defined in the attached claims.

[0020] Forminganotherobject of thepresent invention isamixtureandat least one technological additiveorexcipient, or a composition for use as medicament (first medical use), having the characteristics as defined in the attached claims.

[0021] Forming another object of the present invention is a mixture or a composition comprising said mixture for use in a method for the preventive or curative treatment of humans and animals having specific disorders or pathologies or diseases, selected from arthritis, osteoarthritis, arthrosis, joint pain, inflammation of the limbs and joints, gastroesopha- geal reflux (second medical use), said use having the characteristics as defined in the attached claims.

[0022] Furthermore, forming another object of the present invention is a process for preparing said mixture, or said composition comprising said mixture, comprising or, alternatively, consisting of hyaluronic acid or a salt thereof (a hyaluronate anion salt) (in short, together or separate, HA), and / or a chondroitin or a salt thereof, such as a chondroitin sulfate, (in shortCS)of plant origin, having thecharacteristics asdefined in theattachedclaims. Lastly, forminganobject of thepresent invention isauseof a fungusasaplant startingmaterial for preparingahyaluronicacidor asalt thereof and / or a chondroitin or a salt thereof, suchas chondroitin sulfate or a salt thereof, with a high degree of purity anda reduced content of contaminants and / or by-products, having the characteristics as defined in the attached claims.

[0023] Preferred embodiments of the present invention will now be described hereinafter, with the reference to the attached drawings, wherein: - figures 1 to 4 show flow diagrams of the process, subject of the present invention, according to different embodiments (first embodiment, P1); - figures 5 and 6 show flow diagrams of the process, subject of the present invention, according to the second embodiment (P2); - figures 7and8 showflowdiagramsof the process, subject of the present invention, according to the third embodiment (P3); - figures9and10show twoHPLCspectra for thedeterminationofunsaturateddisaccharides inasamplecontainingHA and in a sample containing CS, respectively.

[0024] It should be observed that, in the context of the present description, the expression "HS" is used to indicate hyaluronicacidora salt thereof, or ahyaluronate, or combinations thereof. Theexpression "CS", on theotherhand, is used to indicate a chondroitin, a chondroitin salt, preferably a chondroitin sulfate or a salt thereof, or mixtures thereof.

[0025] It should be observed that, in this description, the terms "plant starting material" and "starting material of plant origin" are synonyms, and are therefore used interchangeably. 4 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 DETAILED DESCRIPTION OF THE INVENTION

[0026] Forming an object of the present invention is a mixture (m) comprising or, alternatively, consisting of at least one glycosaminoglycan obtained from a starting material of plant origin. Said material of plant origin is selected from the group comprising or, alternatively, consisting of one or more natural fungi.

[0027] Said glycosaminoglycan is selected from the group comprising or, alternatively, consisting of: (a) a hyaluronic acid or a salt thereof, a hyaluronate anion (in short, HA); (b) a chondroitin or a salt thereof, such as a chondroitin sulfate (in short, CS) or a salt thereof; (c) a combination of (a) and (b).

[0028] Theplant startingmaterial is a fungus. The fungus is a fungus that growsand is found in nature, for example it can be found and collected in the woods, but it can also be cultivated in the greenhouse.

[0029] The fungus belongs to the Dikarya subkingdom, preferably it is of the Basidiomycota division.

[0030] Dikarya is a fungi subkingdom that includes theAscomycota and Basidiomycota divisions. The Basidiomycetes (Basidiomycota R.T. Moore, 1980) is one of the largest phyla that form the kingdom of fungi.

[0031] According toanembodiment, theplant startingmaterial isa fungusof thespeciesTremella fuciformisorsaidplant starting material comprises or, alternatively, consists of a fungus of the species Tremella fuciformis.

[0032] TheTremella fuciformisBerk. (1856) (also knownasasnow fungus, or silver ear fungus) is a fungusoriginating in tropical and subtropical areas, where it thrives on dead hardwood logs, and it is also cultivated to cope with the extremely high demand - especially in Japan and China - in cooking and traditional medicine. Tremella fuciformis produces white gelatinous fruit-bearing bodies (basidiocarps), similar to fronds.

[0033] The use of Tremella fuciformis in the present invention is particularly advantageous given that, starting from this plant starting material, the extraction technology developed herein (first embodiment (P1), second embodiment (P2) and third embodiment (P3)), allow to produce both HA and CS completely of plant origin (and with low molecular weight). The HA and / or CS glycosaminoglycans contained in the mixture (m) and obtained from the process of the present invention have distinctive characteristics which make them particularly effective, above all by virtue of their reduced molecular weight, with respect to HA and / or CS obtainable from animal cartilage according to the prior art. A low molecular weight allows to have HA and / or CS glycosaminoglycans with improved transcutaneous penetration properties.

[0034] More precisely, hyaluronic acid or the salt thereof (hyaluronate) obtained by the process of the present invention (P1 and / or P2) has a weight average molecular weight comprised from 10 kDa to 600 kDa, preferably comprised from 100 kDa to 500 kDa, even more preferably comprised from 200 kDa to 400 kDa or from 100 kDa to 300 kDa, for example a weight average molecular weight of about 50 kDa, 150 kDa, or 250 kDa, or 300 kDa, or 350 kDa, or 450 kDa, or 550 kDa. Preferably, said HA contains a percentage by weight of chondroitin (preferably of non-sulfated chondroitin) comprised from 0.01% to 5%, preferably comprised from 0.1% to 3%, even more preferably comprised from 0.5% to 2%, for example 1% or 2%, with respect to the total weight of said HA extracted from fungus.

[0035] According to anadvantageousaspect of the present invention, theHAhavingaweight averagemolecularweight falling within such ranges has a high transcutaneous penetration power due to the limited size of the molecule.

[0036] Chondroitin or the salt thereof, such as chondroitin or the salt thereof (CS) obtained by the process of the present invention (P1and / orP3)hasaweightaveragemolecularweight comprised from1kDa to50kDaor fromgreater than5kDa to less than 50 kDa, preferably comprised from 3 kDa to 40 kDa, even more preferably comprised from 5 kDa to greater than 5 kDa to 25 kDa or from greater than 5 kDa to 10 kDa, for example a weight average molecular weight of 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.

[0037] According to a further advantageous aspect of the present invention, the CS with a weight average molecular comprised in the ranges mentioned herein also proved to be effective in reducing bone damage from osteoarthritis of the knee and hip.

[0038] The CS contained in the mixture (m) of the present invention comprises a chondroitin sulfate having a weight average molecular weight comprised from 1 kDa (1,000.00 Da=1x103 ) to 50 kDa, preferably comprised from 3 kDa to 40 kDa, even more preferably comprised from 5 kDa to 25 kDa, for example a weight average molecular weight of 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.Preferably, saidCShas a chargedensity of from 0.70 to 0.99or from 0.70 to 1.50, preferably comprised from0.75 to 0.98 or from0.75 to 1.20, even more preferably comprised from 0.80 to 0.97, for example 0.85, 0.87, 0.90, 0.92, 0.94, or 0.96.

[0039] More preferably, said CS (obtained from the P1 and / or P3 process) has a percentage by weight of 6-chondroitin sulfate comprised from 50% to 99,5%, preferably comprised from 50% to 95%, more preferably comprised from 75% to 88%, even more preferably comprised from 78% to 86%, for example about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or98%,with respect to the totalweight of saidCS (orwith respect to the total of disaccharidescontained in chondroitin sulfate; % determined, for example, by means of HPLC).

[0040] Besides 6-chondroitin sulfate, said CS preferably comprises non-sulfate chondroitin (non-sulfated chondroitin). 5 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55

[0041] Preferably, the non-sulfated chondroitin has a weight percentage comprised from 0.1% to 25%, preferably from 0.5% to 20% or from 5% to 20%, more preferably comprised from 7% to 15%, even more preferably comprised from 8% to 13%, for exampleabout 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%or12%,with respect to the totalweight of saidCS (orwith respect to the total of disaccharidescontained in chondroitin sulfate; % determined, for example, by HPLC).

[0042] Besides 6-chondroitin sulfate and non-sulfated chondroitin, said CS preferably comprises 2.6-chondroitin disulfate. Preferably, the 2,6-chondroitin disulfate has a percentage by weight comprised from 0.1% to 10%, preferably comprised from 0.2% to 8%, even more preferably comprised from 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%, with respect to the total weight of said CS (or with respect to the total of disaccharides contained in chondroitin sulfate; % determined, for example, by means of HPLC).

[0043] Besides 6-chondroitin sulfate, non-sulfated chondroitin and 2,6-chondroitin disulfate, said CS preferably comprise4-chondroitin sulfate (orwith respect to the total of disaccharides contained in chondroitin sulfate;%determined, for example, by means of HPLC).

[0044] Preferably, 4-chondroitin sulfate has a weight percentage comprised from 0.01% to 5%, preferably comprised from 0.05% to 3%, even more preferably comprised from 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%, with respect to the total weight of saidCS(orwith respect to the total of disaccharidescontained in chondroitin sulfate;%determined, for example, bymeans of HPLC).

[0045] Besides 6-chondroitin sulfate, non-sulfated chondroitin, 2,6-chondroitin disulfate and 4-chondroitin sulfate, said CS preferably comprises 4,6-chondroitin disulfate.

[0046] Preferably, 4,6-chondroitin disulfate has a weight percentage comprised from 0.01% to 5%, preferably com- prised from 0.05% to 3%, even more preferably comprised from 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%, with respect to the total weight of saidCS (orwith respect to the total of disaccharides contained in chondroitin sulfate;%determined, for example, by means of HPLC).

[0047] Besides 6-chondroitin sulfate, non-sulfated chondroitin and 2,6-chondroitin disulfate, 4-chondroitin sulfate and 4,6chondroitindisulfate, saidCSpreferably comprise2,4-chondroitindisulfate (orwith respect to the total of disaccharides contained in chondroitin sulfate; % determined, for example, by means of HPLC).

[0048] Preferably, 2,4-chondroitin disulfate has a weight percentage comprised from 0.01% to 5%, preferably com- prised from 0.05% to 3%, even more preferably comprised from 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%, with respect to the total weight of saidCS (orwith respect to the total of disaccharides contained in chondroitin sulfate;%determined, for example, by means of HPLC).

[0049] Besides 6-chondroitin sulfate, non-sulfated chondroitin, 2,6-chondroitin disulfate, 4-chondroitin sulfate, 4,6- chondroitin disulfate and 2,4-chondroitin disulfate, said CS preferably comprises hyaluronic acid or hyaluronate (pre- ferably non-sulfated). Preferably, said HA is present at a weight percentage comprised from 0.01% to 5%, preferably comprised from0.05% to3%, evenmore preferably comprised from0.1% to1.5%, for example 0.8%or 1.0%,with respect to the total weight of CS.

[0050] According to an embodiment, the CS contained in the mixture (m) and obtained by the process of the present invention (P1 and / or P3) comprises: - a 6-chondroitin sulfate at a weight percentage comprised from 50% to 99.5%, preferably from 50% to 95±0.5%,more preferably comprised from 75% to 88%, even more preferably comprised from 78% to 86%; - a non-sulfated chondroitin at a weight percentage comprised from 0.1% to 25%, preferably from 0.5% to 20%, more preferably comprised from 7% to 15%, even more preferably comprised from 8% and 13%; - a 2,6-chondroitin disulfate at a weight percentage comprised from 0.1% to 10%, preferably comprised from 0.2% to 8%, even more preferably comprised from 0.3% to 5%; and, furthermore, - a 4-chondroitin sulfate at a weight percentage comprised from 0.01% to 5%, preferably comprised from 0.05% to 3%, even more preferably comprised from 0.1% to 1.5%, - a 4,6-chondroitin disulfate at a weight percentage comprised from 0.01% to 5%, preferably comprised from 0.05% to 3%, even more preferably comprised from 0.1% to 1.5%, and - a 2,4-chondroitin disulfate at a weight percentage comprised from 0.01% to 5%, preferably comprised from 0.05% to 3%, even more preferably comprised from 0.1% to 1.5%.

[0051] By way of example, the CS contained in the mixture (m) and obtained by the process of the present invention has a compositionCS.1, CS.2,CS.3,CS.4, CS.5 or a composition CS.6, according Table 1 below (the values being expressed as a weight percentage of each component with respect to the total weight of said CS.n with n=1‑6). 6 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 Table 1 CS contained in the mixture (m) CS.1 CS.2 CS.3 CS.4 CS.5 CS.6 Non-sulfated chondroitin 10.6 11.5 10.8 8.9 2.9 1.7 6-chondroitin sulfate 85.3 79.7 84.7 87.9 92.6 95.7 4-chondroitin sulfate 0.0 0.2 0.4 0.6 0.1 0.4 2,6-chondroitin disulfate 4.1 0.5 2.4 1.9 3.4 0.5 4,6-chondroitin disulfate 0.0 0.9 0.8 0.3 0.4 0.9 2,4-chondroitin disulfate 0.0 1.0 0.9 0.4 0.6 0.8 Charge density 0.94 0.85 0.95 0.92 0.94 0.96

[0052] Forming an object of the present invention is a composition comprising: (i) the aforementioned mixture (m) comprising or, alternatively, consisting of (a) HA having a weight average molecular weight comprised from 10 kDa to 600 kDa (preferably from 100 kDa to 500 kDa, even more preferably from 200 kDa to 400 kDa, Or from 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) CS having a weight average molecular weight comprised from 1 kDa to 50 kDa or from greater than 5 kDa to less than 50 kDa (preferably comprised from 3 kDa to 40 kDa, even more preferably comprised from 5 or greater than 5 kDa to 25 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), and (ii) optionally technological additives and pharmaceutical or food grade excipients. Such a composition may be a pharma- ceutical composition,amedical devicecomposition (EU)2017 / 745,anutraceutical functioncomposition,aspecial food for medical purpose (SFMPs) composition, a dietary supplement composition, or a food product composition, or a novel food composition (EU) 2015 / 2283.

[0053] Such composition can be used as medicament, or as a composition for use in the preventive and / or curative treatment of arthritis, osteoarthritis, arthrosis, joint pain, inflammation of the limbs and joints, gastroesophageal reflux.

[0054] Forming an object of the present invention is a technology and a process for preparing hyaluronic acid or hyaluronate (HA) (processP1and / orP2) and / or chondroitin sulfateor chondroitin or a salt thereof (CS) (processP1and / or P3), said process comprising at least one step of extracting hyaluronic acid or hyaluronate and / or chondroitin sulfate or chondroitin fromastartingmaterial of plant origin, for exampleastartingmaterial of plant origin comprisingor, alternatively, consisting of at least one natural fungus belonging to the Dikarya subkingdom, preferably Basidiomycota division , more preferably Tremella fuciformis species.

[0055] The processes of the present invention (first embodiment (P1), second embodiment (P2) and third embodiment (P3)) do not include the steps of fermentation and / or digestion with bacteria, as for example the bacterial fermentations reported in patent documents WO 2012 / 152872 A1 and EP 2852437 B1 for the preparation of chondroitin or chondroitin sulfate.

[0056] Various embodiments of this process are exemplified in the flow diagrams of figures 1 to 8.

[0057] According to a first embodiment (P1), the process subject of the present invention comprises the following steps: (i) identifying one or more natural fungi as a plant starting material of a glycosaminoglycan; for example, one or more natural fungi in dry or dried form, preferably comprising or, alternatively, consisting of at least one fungus belonging to the Dikarya subkingdom, preferably Basidiomycota division, more preferably Tremella fuciformis species; (ii) optionally, carrying out the crushing or pulverisation of the plant starting material; (iii) extraction of said glycosaminoglycan (HA or CS) from the plant starting material obtained from step (i) or from step (ii) using an extraction solvent, preferably an aqueous solvent, even more preferably water (for example distilled or double distilled water) to obtain an aqueous extract of said glycosaminoglycan; (iv) addition of a solvent, preferably ethanol, to the aqueous extract obtained from step (iii) to obtain a liquid product; (v) carrying out a centrifugation and / or a filtration of the liquid product obtained from step (iv) to obtain a liquid phase and a solid residue; (vi) carrying out a processing of the liquid phase obtained from the centrifugation and / or filtration of step (v) by means of the following step (vi.a), and / or processing the solid residue obtained from the centrifugation and / or filtration of step (v) by means of the following steps (vi.b), (vi.c), (vi.d) and (vi.e): (vi.a) drying, preferably concentration anddrying, the liquid phaseobtained fromstep (v) to obtain hyaluronic acid or the salt thereof having a weight average molecular weight comprised from 10 kDa to 600 kDa; and / or 7 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 (vi.b) recovery and purification of the solid residue obtained from step (v) to obtain chondroitin or the salt thereof (CS) having a weight average molecular weight comprised from 1 kDa to 50 kDa; (vi.c) treating chondroitin or the salt thereof (CS) obtained from step (vi.b)with a source of sulfuric acid, preferably selected from the group comprising or, alternatively, consisting of sulfuric acid, a sulfur-trioxide pyridine complex, a sulfur-trioxide dimethyl-formamide complex and the mixtures thereof, to obtain an acidified product; (vi.d) neutralisation of the acidified product obtained from step (vi.c) using a basic agent to obtain a neutralised product; (vi.e) concentration and drying of the neutralised product obtained from step (vi.d) to obtain chondroitin sulfate or a salt thereof having a weight average molecular weight comprised from 1 kDa to 50 kDa.

[0058] The plant starting material subjected to extraction in step (iii) could be intact (i.e. a single piece, for example a whole fungus), or it could be crushed (into pieces or flakes) or pulverised (into granules, powder or pellets) in step (ii). The flow diagrams of figure 1 and figure 2 show embodiments of the process subject of the present invention according to the first embodiment (P1) in which the natural fungus, or the plurality thereof, identified in step (i) it is extracted in step (iii) or steps (iii.a) and (iii.b) to obtain the aqueous extract.

[0059] The flow diagrams of figure 3 and figure 4 show embodiments of the process subject of the present invention according to the first embodiment (P1) (process carried out according to techniques and apparatuses known to the man skilled in theart) inwhich thenatural fungus, or the plurality thereof, identified in step (i), is crushed or pulverised in step (ii). Subsequently, the natural fungus, or the plurality thereof, crushed or pulverised in step (ii) is extracted in step (iii) or steps (iii.a) and (iii.b) to obtain the aqueous extract.

[0060] In the case where the plant starting material is crushed or pulverised (according to a technique and apparatus known to the man skilled in the art) in step (ii), an average distribution of the particle size of said plant starting material is preferably comprised from 500 µm and 2,500 µm, more preferably comprised from 800 µm to 1,800 µm, even more preferably comprised from 900 µm to 1,200 µm.

[0061] According to anembodiment, the plant starting material supplied in step (iii) (fromstep (i) or (ii)) is a plantmaterial crushed into pieces or flakes, or pulverised either into granules or into pellets.

[0062] The plant starting material supplied in step (iii) (from step (i) or (ii)) is preferably dry or dried, that is to say it is a plant starting material containing an amount by weight of water comprised from about 2% to 20%, preferably comprised from 5% to 15%, even more preferably comprised from 8% to 10%, with respect to the total weight of the plant starting material.

[0063] In step (iii), the extraction solvent is selected from an aqueous solvent and water.

[0064] The aqueous solvent (or aqueous solution) is preferably a hydroalcoholic mixture in which the alcohol (for example ethanol) is present at aweight percentage comprised from0.1% to50%,morepreferably comprised from0.5% to 25%, even more preferably comprised from 1% to 15%, with respect to the total weight of the extraction solvent, for example at a percentage by weight of about 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% 7.5%, 10%, 20%.

[0065] The water is preferably distilled or double distilled water.

[0066] In step (iii), the plant startingmaterial is loaded continuously or in batches into a container or extractionapparatus for example providedwithmechanical stirringmeans, heatingmeans, filteringmeansaswell as temperature andpressure control means.

[0067] In step (iii), the plant starting material is then extracted in said extraction container or apparatus by means of the extraction solvent, so that hyaluronic acid or the salt thereof passes into solution in the liquid phase, inside the aqueous extract, and so that chondroitin remains in the solid residue.

[0068] The extraction of step (iii) is carried out using a [weight of the plant starting material] : [volume of the extraction solvent] ratio comprised from 1:1: to 1:90, preferably comprised from 1:10 to 1:90, more preferably comprised from 1:20 to 1:75, even more preferably comprised from 1:40 to 1:60, for example of 1:3, 1:5, 1:15, 1:25, 1:45, 1:50 or 1:55. The extraction of step (iii) is carried out within a period of time comprised from 1 minute to 12 hours, preferably from 10 minutes to 9 hours, even more preferably from 15 minutes to 4 hours, for example in about 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, or 180 minutes.

[0069] Step (iii) is preferably carried out at atmospheric pressure (P=1 Atm. at 20‑25°C), and at a temperature of the extraction solvent comprised from10°C to90°C, preferably comprised from20°C to60°C,evenmorepreferably from35°C to 55°C, for example of about 25°C, 30°C, 40°C, 44°C, 48°C, Or 50°C.

[0070] Preferably, a pHvalueof theextraction solvent in step (iii) is comprised from3 to10, preferably from3.5 to 9,more preferably from 4 to 8, even more preferably comprised from 5 to 7, for example at a pH value of about 4.5; 5.5; 6; 6.5; 7.5; 8.5; or 9.5.

[0071] In the extraction of step (iii) a proteolytic enzyme is preferably used to degrade the surface pectins of the material of plant origin, and thus increase the yield of the process. Preferably, the proteolytic enzyme comprises or, alternatively, consists of bromelain or bromelain extract.

[0072] Bromelain is an enzyme extract of the fruit and / or stem of pineapple containing proteolytic enzymes and other 8 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 substances in smaller quantities.

[0073] In order to increase the yield of the extraction, step (iii) is preferably carried out in two steps, as described below: (iii.a) a first extraction from the plant starting material with a first volume of extraction solvent at a temperature comprised from10°C to 90°C, preferably comprised from20°C to60°C, evenmorepreferably comprised from35°C to 55°C, for a period of time comprised from 1 minute or 30 minutes to 12 hours, preferably from 10 minutes to 9 hours, even more preferably from 15 minutes to 4 hours, to obtain a first aqueous extract; and (iii.b) asecondextraction fromtheplant startingmaterial (or fromasolid residueof saidfirst extractionstep (iii.a))witha secondvolumeof extraction solvent at a temperature comprised from80°C to120°C, preferably comprised from90°C to 110°C, preferably comprised from 95°C to 105°C, even more preferably comprised from 98°C to 102°C, preferably under pressureor at reducedpressure, for a periodof timecomprised from10minutes to 6hours or from30minutes to 8 hours, preferably from 20 minutes to 4 hours, even more preferably from 40 minutes to 2 hours, for example for a period of time of about 30 minutes, 60 minutes, or 90 minutes, to obtain a second aqueous extract. Preferably, in the first extraction (iii.a) a first volume of extraction solvent is used comprised from 25 to 75 times the weight of the plant starting material, preferably comprised from 35 and65 times, even morepreferably comprised from 45 to 55 times, for example about 30 times, or 50 times.

[0074] Preferably, in the second extraction (iii.b) a second volume of extraction solvent is used comprised from 10 and 150 times, preferably comprised from 75 to 125 times the weight of the plant starting material, more preferably comprised from85 to115 times,evenmorepreferablycomprised from95 to105 times, for exampleabout15,20,25, 30,35,40,45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 times.

[0075] According to said first embodiment of the HA and / or CS (P1) preparation process, the first aqueous extract obtained from step (iii.a) and the second aqueous extract obtained from step (iii.b) are then combined, and the solvent according to step (iv) is added thereto.

[0076] In step (iv), the solvent, preferably ethanol, is added to the aqueous extract obtained from step (iii), or to the first aqueous extract obtained from step (iii.a) and to the second aqueous extract obtained from step (iii.b) to obtain the liquid product.

[0077] In step (v), the liquid product obtained from step (iv) and the plant starting material present (for example as supernatant or as precipitate) in such product, are centrifuged and / or they are made to pass through a filtering means (first filtration) which retains the solid part (a solid residue), and which lets the liquid phase to pass through.

[0078] In step (vi), subsequent to step (v), the liquid phase obtained by centrifugation and / or filtration is processed through step (vi.a), and / or the solid residue is processed through steps (vi.b), (vi.c), (vi.d) and (vi.e).

[0079] In the preferred step (vi.a), the liquid phase obtained from step (v) is dried, optionally concentrated and dried, to obtain a hyaluronic acid or the salt thereof having a weight average molecular weight comprised from 10 kDa to 600 kDa.

[0080] The liquid phase to be dried, preferably to be concentrated and dried, in step (vi.a) must preferably be free of sediments, when allowed to cool to 20‑25°C. Therefore, in the presence of sediments, the liquid phase obtained from step (v) is further centrifuged and / or filtered before step (vi.a).

[0081] The concentration of the centrifuged and / or filtered liquid phase is preferably carried out at a temperature comprised from 60°C to 90°C, more preferably comprised from 65°C to 85°C, even more preferably comprised from 70°C to 80°C, for example at 70°C, at 75°C or at 80°C. Besides the temperature, the duration of the concentration step also depends on the desired amount of substances dissolved in the liquid phase.

[0082] The concentration of the centrifugedand / or filtered liquid phase preferably provides for an increase in an amount of dissolved substances (including HA) in the liquid phase up to a range comprised from 1 g to 35 g per 100 ml of liquid phase, preferably from 5 g to 25 g, even more preferably comprised from 8 g to 18 g.

[0083] Preferably, the liquid phase obtained from the concentration step (vi.a) has a relative density (defined as the [density of the centrifuged and / or filtered liquid phase]: [density of the liquid phase at the end of the concentration]) ratio comprised from 1.01 to 1.20, preferably comprised from 1.02 to 1.15, even more preferably comprised from 1.05 to 1.08.

[0084] Preferably, the concentration of the liquid phase is carried out at reduced pressure (lower than 1 atm. att 25°C), morepreferablyat apressurecomprised from ‑1.5mPa to ‑0.1mPa,evenmorepreferably comprised from ‑1.0mPa to ‑0.5 mPa, for example to ‑0.8 mPa.

[0085] Preferably, the HA obtained from step (vi.a) has a purity (% by weight with respect to the total weight of HA) comprised from 90% to 100%, preferably comprised from 95% to 99.5%, even more preferably comprised from 97% to 99% (% determined, for example, by means of HPLC).

[0086] In the preferred steps (vi.b), (vi.c), (vi.d) and (vi.e), the solid residue obtained from centrifugation and / or filtration of step (v) is processed to obtain chondroitin sulfate or a salt having a weight average molecular weight comprised from 1 kDa to 50 kDa.

[0087] In the preferred step (vi.b), the solid residue obtained from centrifugation and / or by filtration of step (v) is recoveredandpurified toobtain chondroitin or salt thereof (CS) havingaweight averagemolecularweight comprised from 9 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 1 kDa to 50 kDa.

[0088] In the preferred step (vi.c) (sulfonation (or sulfation) step, intended as a reaction step capable of inserting a sulfate groupon the dimer of chondroitin) subsequent to step (vi.b), the solid residue is treated with the sulfuric acid source (preferably selected from the group comprising or, alternatively, consisting of sulfuric acid, a sulfur-trioxide pyridine complex, a sulfur-trioxidedimethyl-formamidecomplexandmixtures thereof), to obtain theacidifiedproduct (inwhich said sulfonation step is carried out according to a technique and apparatus known to the man skilled in the art).

[0089] The amount of the sulfuric acid source used in step (vi.c) is such to obtain a weight percentage of 6-chondroitin sulfate comprised from 51% to 99%, or about 95±0.5% (preferably comprised from 78% to 85%, or 86%) with respect to the total content of disaccharides of CS in the solid residue of step (vi.b).

[0090] Preferably, in step (vi.c) there are used from1ml to 50 ml of the sulfur-trioxide dimethyl-formamide complex (SO3 DMF), preferably from 2 ml to 40 ml, even more preferably from 4 ml to 30 ml, for every 100 g of chondroitin or salt thereof (CS)obtained fromstep (vi.b), for example5ml, 10ml, 15ml, 18ml, 22mlor25mlof thesulfur-trioxidedimethyl-formamide complex every 100 g of the chondroitin or of the salt thereof (CS) obtained from step (vi.b). More preferably, the sulfur- trioxide dimethylformamide complex is added to the chondroitin or to the salt thereof (CS) obtained from step (vi.b) in several steps, for example by adding from 2 ml to 8 ml, then adding from 8 ml to 12 ml, and lastly adding further from 8 ml to 12 ml of the sulfur-trioxide dimethyl-formamide complex.

[0091] The treatment of step (vi.c) is carried out for a period of time comprised from 1 minute to 4 hours, preferably comprised from 10 minutes to 2 hours, even more preferably comprised from 20 minutes to 1 hour, at a temperature comprised from 20°C to 80°C, preferably comprised from 30°C to 70°C, even more preferably comprised from 40°C to 60°C. In step (vi.d), the product obtained from step (vi.c) is neutralised with a basic agent.

[0092] Therefore, in step (vi.d) the source of sulfuric acid still free in the acidified product of step (vi.c) (i.e. the source of sulfuric acid not bound to chondroitin as chondroitin sulfate in the acidified product) is eliminated by neutralising with the basic agent to obtain the neutralised product.

[0093] In the present description, the expression "neutralised" or "neutralisation" is used to indicate a pH value of 6 to 8, preferably comprised from 6.4 to 7.6, even more preferably comprised from 6.6 to 7.4, for example at a pH value of 7.0 ±0.2.

[0094] The basic agent used in step (vi.d) is preferably an inorganic basic agent.

[0095] The basic agent is preferably selected from the group comprising or, alternatively, consisting of: ammonia, sodium hydroxide, potassium hydroxide and mixtures thereof.

[0096] Preferably, the sodium hydroxide usable in step (vi.d) is at a concentration of 1 M, 2 M or 4 M.

[0097] In the preferred strep (vi.e), subsequent to step (vi.d), the neutralised product obtained from step (vi.d) is concentratedanddried to obtain chondroitin sulfate havingaweight averagemolecularweight comprised from1kDa to50 kDa

[0098] The concentration of step (vi.e) provides for a relative density (defined as the [density of the neutralized product obtained from step (vi.d)]: [density of the concentrated product obtained from step (vi.e)]) ratio comprised from 1.0 to 1.30, preferably comprised from 1.01 to 1.20, even more preferably comprised from 1.05 to 1.15.

[0099] Preferably, the concentration of step (vi.e) is carried out by means of dialysis and / or through vacuum concen- tration. More preferably, dialysis is carried out by means of a dialysis bag, so as to remove small impurities which may be present.

[0100] The concentration step (vi.e) is preferably terminated when the solid content in the concentrated product obtained fromstep (vi.e) is comprised from10 g to 60gper 100ml, preferably comprised from 20g to 50 gper 100ml, even more preferably comprised from 35 g to 45 g per 100 ml, for example 40 g / 100 ml.

[0101] The drying of step (vi.e) is carried out subsequently to the concentration of step (vi.e), preferably by means of a vacuum oven.

[0102] Forming an object of the present invention is a use of a plant starting material, preferably a fungus, more preferably of the Dikarya subkingdom, even more preferably of the Basidiomycota division, further preferably of the Tremella fuciformis species, for preparing a hyaluronic acid or a salt thereof (HA), a hyaluronate anion salt, and / or a chondroitin or a salt thereof, such as a chondroitin sulfate or a salt thereof (CS).

[0103] The embodiments of the mixture, of the use of said mixture, of the composition comprising said mixture as additive, or excipient, or ingredient (or non-active ingredient), of the use of the composition as medicament, of the use of the composition in the treatment of specific disorders or diseases or pathologies, of the process for preparing said mixture or of said composition comprising said mixture, of the use of said composition for use as additive, or excipient, or ingredient, and of the aforementioned plant starting material could be subjected - by a man skilled in the art - to substitutions or modifications regarding the described characteristics according to the contingencies. These embodi- ments are also to be considered included in the scope of protection formalised in the following claims. Furthermore, it should be observed that any embodiment may be implemented independently from the other embodiments described.

[0104] Embodiments present invention (FRn) are outlined below: 10 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 FR1. A mixture (M) comprising or, alternatively consisting of a glycosaminoglycan obtained from a plant starting material; said glycosaminoglycan being selected from the group comprising or, alternatively consisting of: (a) hyaluronic acid or a salt thereof, a hyaluronate anion, (HA) having a weight average molecular weight comprised from 10 kDa to 600 kDa; (b) chondroitin or a salt thereof (CS) such as chondroitin sulfate, having a weight average molecular weight comprised from 1 kDa to 50 kDa; (c) a combination of (a) and (b). FR2. The mixture (M) according to the preceding FR, wherein: (a) the hyaluronic acid or the salt thereof (HA) has a weight average molecular weight comprised from 100 kDa to 500 kDa, preferably comprised from 200 kDa to 400 kDa; and / or (b) thechondroitinor thesalt thereof (CS)hasaweightaveragemolecularweight comprised from1kDa to50kDa, preferably comprised from 3 kDa to 40 kDa, even more preferably comprised from 5 kDa to 25 kDa. FR3. The mixture (M) according to any one of the preceding FRs, wherein said material of plant starting material is a fungus, preferably of the Dikarya subkingdom, even more preferably of the Basidiomycota division. FR4. The mixture (M) according to the preceding FR, wherein the fungus is of the Tremella fuciformis species. FR5. Use of the mixture (M) according to anyone of the preceding FRs, as additive, excipient, or ingredient in the preparation of pharmaceutical products, medical devices, nutraceutical products, food for special medical purposes (SFMPs), dietary supplements or food products. FR6. A composition comprising: (i) the mixture according to any one of FR1‑4, and (ii) technological additives or pharmaceutical or food grade excipients. FR7. The composition according to the preceding FR, for use as medicament. FR8. The composition according to FR6, for use in the preventive or curative treatment of human beings or animals, having specific disorders or diseases, selected from arthritis, osteoarthritis, arthrosis, joint pain, inflammations of the limbsand joints, or gastroesophageal reflux, and / or for useasanadditive, or excipient, or ingredient in thepreparation of pharmaceutical products, products for medical devices, nutraceutical products, food for special medical purpose (FSMPs), food products or food supplements. FR9. A process for the preparation of hyaluronic acid or hyaluronate (HA), and / or chondroitin sulfate or chondroitin (CS), said process comprising at least one step for extracting hyaluronic acid or hyaluronate, and / or chondroitin sulfate or chondroitin, from a starting material of plant origin. FR10. The process according to the preceding FR, comprising the following steps: (i) identifying one or more natural fungi as the plant starting material of a glycosaminoglycan; (ii) optionally crushing or pulverising the plant starting material; (iii) extractionof said glycosaminoglycan from theplant startingmaterial obtained fromstep (i) or fromstep (ii) with an extraction solvent, preferably an aqueous solvent, even more preferably water to obtain an aqueous extract of said glycosaminoglycan; (iv) addition of a solvent, preferably ethanol, to the aqueous extract obtained from step (iii) to obtain a liquid product; (v) centrifugation and / or filtration of the liquid product obtained from step (iv) to obtain a liquid phase and a solid residue; (vi) processing the liquid phase obtained from the centrifugation and / or filtration of step (v) by means of the following step (vi.a), and / or processing the solid residue obtained from the centrifugation and / or filtration of step (v) by means of the following steps (vi.b), (vi.c), (vi.d) and (vi.e): (vi.a) drying, preferably concentration anddrying, the liquid phase obtained from step (v) to obtain hyaluronic acid or the salt thereof having a weight average molecular weight comprised from 10 kDa to 600 kDa; and / or (vi.b) recovery and purification of the solid residue obtained from step (v) to obtain chondroitin or the salt thereof (CS) having a weight average molecular weight comprised from 1 kDa to 50 kDa; (vi.c) treating chondroitin or the salt thereof (CS) obtained from step (vi.b) with a source of sulfuric acid, preferably selected from the group comprising or, alternatively, consisting of sulfuric acid, a sulfur-trioxide pyridine complex, a sulfur-trioxide dimethyl-formamide complex and the mixtures thereof, to obtain an acidified product; (vi.d) neutralisation of the acidified product obtained from step (vi.c) using a basic agent to obtain a neutralised product; 11 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 (vi.e) concentration and drying of the neutralised product obtained from step (vi.d) to obtain chondroitin sulfate having a weight average molecular weight comprised from 1 kDa to 50 kDa. FR11. Use of a plant starting material, preferably a fungus, more preferably of the Dikarya subkingdom, even more preferably of the Basidiomycota division, further preferably of the Tremella fuciformis species, for preparing a hyaluronic acid or a salt thereof, a hyaluronate anion salt, and / or a chondroitin or a salt thereof, such as a chondroitin sulfate.

[0105] A second embodiment of the process of the present invention (in short, P2) relates to a process for preparing hyaluronic acid or a salt thereof (HA), the process comprises or, alternatively, consists of the following steps: (i) identifying oneor more natural fungi as a plant starting material of hyaluronic acid; for example, one or more natural fungi in dry or dried form, preferably comprising or, alternatively, consisting of at least one fungus belonging to the Dikarya subkingdom, preferably Basidiomycota division, more preferably Tremella fuciformis species; (ii) optionally, carrying out the crushing or pulverisation of the plant starting material (average particle size distribution preferably comprised from 500 µm to 1,800 µm, preferably from 700 µm to 1,000 µm, for example about 20 mesh = 841 µm); (pre-iii) carrying out an enzymatic hydrolysis of the plant starting material obtained from step (i) or step (ii) in an aqueous hydrolysis solvent, preferably water, at a temperature comprised from 10°C to 90°C wherein an enzyme, preferably a proteolytic enzyme, is added to the plant starting material dispersed in a volume of hydrolysis solvent to obtain a mixture of step (pre-iii); (iii) extracting said mixture of step (pre-iii) with an aqueous extraction solvent, preferably water, at a temperature comprised from 91°C to 110°C, preferably comprised from 95°C to 110°C, even more preferably from 98°C to 105°C (for example about 100°C or boiling temperature of the extraction solvent), to obtain an aqueous extract; (iv) adding a precipitation solvent (precipitation by adding a solvent), preferably an alcoholic solvent, more preferably ethanol, to the aqueous extract obtained from step (iii) to obtain a liquid product of step (iv); preferably, precipitating with ethanol by slowly adding 95% ethanol to said aqueous extract obtained from step (iii) at a volume / volume ratio comprised from2 to 4, preferably 3, andmaintaining under stirringwithin aperiod of time comprised from8hours to 16 hours, for example about 12 hours; (vii) processing said liquid product of step (iv) by applying step (vii.a) and, optionally, step (vii. b): (vii. a) removing said precipitation solvent (for example by distillation or heating to a pressure value lower than the room temperature), preferably ethanol, to obtain a liquid product of step (vii. a); (vii.b) adding water to said liquid product of step (vii.a) (for example to dissolve solid products) to obtain a liquid product of step (vii.b); (viii) drying said liquid product of step (vii.b), preferably concentrating and / or drying (for example removing water and the possible residual precipitation solvent (e.g. ethanol) by concentration and / or drying and / or freeze-drying, preferably freeze-drying) to obtain a product PR1 comprising or, alternatively, consisting of hyaluronic acid or a salt thereof havingaweight averagemolecularweight comprised from10 kDa to 600 kDa, preferably from50kDa to 350 kDa, more preferably from 100 kDa to 300 kDa, and a purity comprised from 85% to about 100% with respect to the total weight of said product PR1, preferably comprised from 95% to 99.5%, even more preferably comprised from 97% to 99% (% determined, for example, by means of HPLC).

[0106] A third embodiment of the process of the present invention (in short, P3) relates to a process for preparing chondroitin sulfate, preferably 6-chondroitin sulfate, the process comprising or, alternatively consisting of the following steps: (i) identifying oneor more natural fungi as a plant starting material of hyaluronic acid; for example, one or more natural fungi in dry or dried form, preferably comprising or, alternatively, consisting of at least one fungus belonging to the Dikarya subkingdom, preferably Basidiomycota division, more preferably Tremella fuciformis species; (ii) optionally, carrying out the crushing or pulverisation of the plant starting material (average particle size distribution preferably comprised from 500 µm to 1,800 µm, preferably from 700 µm to 1,000 µm, for example about 20 mesh = 841 µm); (pre-iii) carrying out an enzymatic hydrolysis of the plant starting material obtained from step (i) or step (ii) in an aqueous hydrolysis solvent, preferably water, at a temperature comprised from 10°C to 90°C wherein an enzyme, preferably a proteolytic enzyme, is added to the plant starting material dispersed in a volume of hydrolysis solvent to obtain a mixture of step (pre-iii); (iii) extracting said mixture of step (pre-iii) with an aqueous extraction solvent, preferably water, at a temperature 12 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 comprised from 91°C to 110°C, preferably comprised from 95°C to 110°C, even more preferably from 98°C to 105°C (for example about 100°C or boiling temperature of the extraction solvent), to obtain an aqueous extract; (vi.c.) treatingwithasourceof sulfuric acid (sulfonationstep) saidaqueousextract toobtaina liquidproductof step (vi.c), wherein said source of sulfuric acid is preferably selected from the group comprising or, alternatively, consisting of sulfuric acid, a sulfur-trioxide pyridine complex, a sulfur-trioxide dimethyl-formamide complex and mixtures thereof; more preferably the sulfur trioxide dimethylformamide complex (SO3‑DMS); (vi.d) neutralising said liquid product of step (vi.c) by adding a base to obtain a neutralised product, wherein said base is preferably an inorganic base, more preferably NaOH o KOH o Ca(OH)2 or Mg(OH)2, up to neutral pH; (vi.e) concentrating and drying said neutralised product obtained from step (vi.d) to obtain a product PR2 comprising or, alternatively, consisting of chondroitin sulfate or a salt thereof (in short, CS), having a weight average molecular weight comprised from 1 kDa to 45 kDa or 50 kDa (or from greater than 5 kDa to less than 50 kDa), preferably comprised from 3 kDa to 40 kDa, even more preferably comprised from 5 kDa or greater than 5 kDa to 25kDaor from5kDaor greater than5 kDa to10 kDa, for example about 5 kDa, or 6 kDa, or 7 kDa, or 8 kDa, or 9 kDa, or 10 kDa.

[0107] The term "chondroitin sulfate or a salt thereof (in short, CS)" obtained from said third embodiment of the process of the present invention (P3) is used to indicate the combination of non-sulfated chondroitin and mono‑, di‑ and / or tri- sulfated chondroitin in the various possible positions, preferably predominantly 6-chondroitin sulfate.

[0108] Alternatively, the term "chondroitin sulfate or a salt thereof (in short, CS)" obtained from said third embodiment of the process of the present invention (P3) is used to indicate the group of mono‑, di‑ and / or tri-sulfated chondroitin in the various possible positions, preferably predominantly 6-chondroitin sulfate.

[0109] Preferably, said chondroitin sulfate (CS) obtained from said third embodiment of the process of the present invention (P3) hasaweight averagemolecularweight in the rangesdescribed (preferably from5kDaor greater than5 kDa to 10 kDa, for example about 6 kDa, or 7 kDa, or 8 kDa, or 9 kDa), comprises 6-chondroitin sulfate at a weight percentage comprised from51% toabout 95±0.5%,preferably from75% to90%,evenmorepreferably from78% to86%,with respect to the total weight of said chondroitin sulfate (CS) (or with respect to the total of disaccharides contained in chondroitin sulfate), and it has a purity comprised from 80% to 99.99% (for example, 94.5%, 94.6%, 94.7%, 94.8% or 94.9%) with respect to the total weight of the product PR2 obtained from step (vi.e), preferably comprised from 85% to 98% (for example, 86%, 87%, 88%, 89%or 89,5%), evenmore preferably comprised from 90%and94.9%, for example 91%, 92%, 93%, 94%, or 94.5% (% determined, for example, by means of HPLC).

[0110] Even more preferably, said chondroitin sulfate (CS) obtained from said third embodiment of the process of the present invention (P3) has a weight average molecular weight in the ranges described (preferably from 5 kDa or greater than 5 kDa to 10 kDa, for example about 6 kDa, or 7 kDa, Or 8 kDa, or 9 kDa), comprises 6-chondroitin sulfate at a weight percentage comprised from51% toabout 95±0.5%, (preferably from75% to90%,morepreferably from78% to86%), and 4-chondroitin sulfate at a weight percentage comprised from 0.01% to about 5%, (preferably from 0.05% to 3%, more preferably from 0.1% to 1.5%) with respect to the total weight of said chondroitin sulfate (CS) (or with respect to the total of disaccharides contained in chondroitin sulfate), and it has a purity comprised from 80% to 99.99% with respect to the total weight of the product PR2 obtained from step (vi.e), preferably comprised from 85% to 98% (for example, 86%, 87%, 88% or 89%), more preferably comprised from 90% to 95%, for example 91%, 92%, 93%, 94% or 94.5% (% determined, for example, by means of HPLC).

[0111] The weight average molecular weight of HA and / or CS can be calculated according to methods and instruments common and known to the man skilled in the art, for example, by means of high performance size exclusion chromato- graphy (HPSEC); preferably, the weight average molecular weight of HA and / or CS can be determined by means of HPSEC provided with integrated specialised gel permeation chromatography (GPC) software.

[0112] In the step (pre-iii) of enzymatic hydrolysis, present in the second and in the third embodiments of the process of the present invention (P2 and P3), the enzyme (for example pectinase and / or cellulase and / or proteinase) is added to the plant starting material as described in the present invention, in a volume of hydrolysis solvent (for example an aqueous solventorwater comprised from25 to100 times theweight of theplant startingmaterial, preferablycomprised from35 to75 times,more preferably comprised from45 to55 times (for example 50 times), andheated to a temperature comprised from 10°C to 90°C, preferably from 20°C to 65°C, more preferably from 45°C to 55°C (e.g. about 50°C), for a period of time comprised from 0.5 hours to 12 hours, preferably from 1 hour to 8 hours, even more preferably from 2 hours to 6 hours (for example about 4 hours), to obtain said mixture of step (pre-iii) to be subjected to the extraction step (iii) or to the first extraction step (iii.a) (as described hereinafter).

[0113] Furthermore,advantageously, saidenzymatic hydrolysis step (pre-iii) is carriedoutat apHvalueof theenzymatic hydrolysis solution comprised from2 to 9, preferably from3 to 5or from 5 to 8,more preferably from3 to 4 (for example 3.5) or from 6 to 7; and / or in said enzymatic hydrolysis step (pre-iii) an amount of enzyme (aqueous solution of the enzyme at 1%‑20%, or 2%‑10%, or 3%‑6% weight / weight or weight / volume) is usedat a volumepercentage comprised from 0.001% 13 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 to1%,preferably from0.005%to0.1%,morepreferably from0.008%to0.05%(forexample0.01),with respect to themass of theplant startingmaterial to beextracted (volume / mass) or, alternatively,with respect to the volumeof the solution of the plant starting material to be extracted in the hydrolysis solvent (volume / volume).

[0114] According to a preferred example, the enzymatic hydrolysis step (pre-iii) is carried out under the following conditions (approximately): volume 0.01%; temperature 50°C; actual pH 3.5; duration 4 hours.

[0115] Said 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 proteinase.

[0116] Examples of enzymes usable in the process of the present invention are: - commercial product Pectinex ® Ultra Tropical, composition: enzyme: pectin lyase or pectinase, preservatives: potassium sorbate, stabilisers: sucrose, glycerol, sorbitol, sodium chloride, potassium chloride; compound activity: pectin lyase or pectinase (PECTU) = 5,000 PECTU / g; approximate density 1.18 (g / ml); pectin lyase is an enzyme which catalyses the elimination cleavage of (1,4)‑alpha-D-galacturonate methyl ester which gives oligosaccharides with 4-deoxy‑6‑ O-methyl-alpha-D-galact‑4-enurosyl groups at the non-reducing ends thereof; other activities: cellulase, polygalacturonase, beta-glucanase (endo‑1,3(4)‑). - commercial product Pectinex® Ultra SP-L, composition % w / w: 45% glycerol (CAS N° 56‑81‑5), 45% water (CAS N° 7732‑18‑5), 5% polygalacturonase (CAS N° 9032‑75‑1; defined as enzyme concentration (on dry weight basis)), 5% potassium chloride (CAS N° 7447‑40‑7); compound activity: polygalacturonase (PGNU) = 3300 PGNU / g; approx- imate density 1,17 (g / ml); polygalacturonase is an enzyme that hydrolyses (1,4)‑alpha-D-galactosiduronic bonds in pectate and other galacturonans. - commercial product Viscozyme ®, composition % w / w: 56.8% water (CAS N° 7732‑18‑5), 9% beta-glucanase (endo‑1,3(4)‑) (CAS N° 62213‑14‑3; defined as enzyme concentration (on dry weight basis)), 24% sucrose (CAS N° 57‑50‑1), 10% sodium chloride (CAS N° 7647‑14‑5), 0.20% potassium sorbate (CAS N° 24634‑61‑5); compound activity: beta-glucanase (endo‑1,3(4)‑) (FGB) = 100 FBG / g; approximate density 1.21 (g / ml); endo-beta-glucanase is an enzyme which hydrolyses (1,3)‑ or (1,4)‑ bonds in beta-D-glucans, other activities: xylanase, cellulase, hemi- cellulase.

[0117] In the second and third embodiments of the process of the present invention (P2 and / or P3), the extraction step (iii) preferably comprises or, alternatively, consists of the first extraction steps (iii.a) comprising or, alternatively, consisting of extracting with a first volume of extraction solvent at a temperature comprised from 91°C to 110°C (for example about 100°C) or a boiling temperature of the extraction solvent for a period of time comprised from 0.5 hours to 12 hours, preferably from 1hour to 9 hours,more preferably from1 hour to 4 hours (for example about 2 hours or 3 hours), to obtain a first aqueous extract; followed by the secondextraction step (iii.b) comprising or, alternatively, consisting of extracting with asecondvolumeof extractionsolvent at a temperaturecomprised from90°C to110°C (for exampleabout100°C)orboiling temperatureof theextractionsolvent for aperiodof timecomprised from0.5hours to8hours, preferably from0.5hours to4 hours,more preferably from 1hour to 3 hours (for example 1.5 hours, or 2 hours, or 2.5 hours), to obtain a secondaqueous extract; said first extract and said second extract are combined to obtain a final aqueous extract; and, optionally, a step (iii.c) of concentrating said final aqueous extract to obtain a concentrated aqueous extract follows.

[0118] In the second and third embodiment of the process of the present invention (P2 and P3), preferably, in the first extraction (iii.a) a first volume of extraction solvent is used comprised from 25 to 100 times the weight of the plant starting material, preferably comprised from 35 and 65 times, even more preferably comprised from 45 to 55 times, for example 50 times. For example, said first extraction volume comprised from 25 to 100 times the weight of the plant starting material is added to the volumeof enzymatic hydrolysis solvent, reachinga total volumeof solvent comprised from50 to200 times the weight of the plant material (for example about 100 times).

[0119] In the secondand third embodiment of the process of the present invention (P2andP3), preferably, in the second extraction (iii.b) a secondvolumeof extraction solvent is usedcomprised from25 to75 times theweight of theplant starting material, preferably comprised from 35 and 65 times, more preferably comprised from 45 to 55 times, (for example about 50 times).

[0120] In the second and third embodiments of the process of the present invention (P2 and P3), after the first extraction step (iii.a) a filtration of step (iii.a) is carriedout: the filtrate corresponds to the first aqueousextract obtained fromstep (iii.a) and the residue is subjected to thesecondextraction step (iii.b). After thesecondextraction step (iii.b), a filtrationstep (iii.b) is carried out and the filtrate corresponds to the second aqueous extract obtained from step (iii.b). For example, said filtrations of step (iii.a) and (iii.b) (or filtrations of the extraction step (iii)) are carried out with filters from 140 to 270 mesh, preferably 200 mesh. Optionally, in said second and third embodiments (P2 and P3), the first aqueous extract obtained from step (iii.a) and the second aqueous extract obtained from step (iii.b) are combined and subjected to the vacuum concentration step (iii.c), for example at a temperature comprised from 60°C to 90°C, preferably from 70°C to 80°C, more preferably at about 75°C, up to a relative density comprised fromabout 0.8 to 1.5, preferably from1.00 to 1.20 (for example about 1.05‑1.08). 14 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55

[0121] In the third embodiment of the process of the present invention (P3), preferably, in step (vi.c) (sulfonation step) there are used from 1 ml to 50 ml of the sulfur-trioxide dimethyl-formamide complex (SO3 DMF), preferably from 2 ml to 40 ml, evenmorepreferably from4ml to30ml, for every100gof chondroitin or a salt thereof (CS)obtained fromstep (vi.b), for exampleabout 5ml, 10ml, 15ml, 18ml, 22ml or 25ml of the sulfur-trioxidedimethyl-formamidecomplexevery100gof the chondroitin or of the salt thereof (CS) obtained from step (vi.b). More preferably, the sulfur-trioxide dimethylformamide complex is added to the chondroitin or to the salt thereof (CS) obtained from step (vi.b) in several steps, for example by adding from 2 ml to 8 ml, then adding from 8 ml to 12 ml, and lastly adding further from 8 ml to 12 ml of the sulfur-trioxide dimethyl-formamide complex. The treatment of step (vi.c) is carried out for a period of time comprised from 1 minute to 4 hours, preferably comprised from 10 minutes to 2 hours, even more preferably comprised from 20 minutes to 60 minutes (for example about 30 minutes), at a temperature comprised from 20°C to 80°C, preferably comprised from 30°C to 70°C, even more preferably comprised from 40°C to 60°C (for example about 50°C). According to a preferred example, said sulfonation step (vi.c) is carried out by adding to about 100 ml of aqueous extract deriving from step (iii) or (iii.b) or (iii.c) (solid content 10‑15g / 100 ml and relative density 1.05‑1.08) SO3‑DMF (5 ml, 15 ml, 25 ml) at a temperature comprised from 40°C to 60°C (for example at 50°C) and for a period of time comprised from 20 minutes to 60 minutes (for example 30 minutes).

[0122] In the third embodiment of theprocessof thepresent invention (P3), preferably, thebasic agent used in step (vi.d) is preferably an inorganic basic agent selected from thegroupcomprisingor, alternatively, consistingof: ammonia, sodium hydroxide, potassium hydroxide and mixtures thereof, preferably sodium hydroxide (for example, at a concentration of 1 M, 2 M or 4 M).

[0123] In the third embodiment of the process of the present invention (P3), preferably, said step (vi.e) comprises a membrane filtration by means of dialysis, for example by means of a 1,000 Da dialysis bag for a period of time comprised from 18 hours to 36 hours, preferably 24 hours, until a relative density comprised from 1.3 to 1.5, preferably about 1.1, is reached, followed by drying, for example in a vacuum oven.

[0124] According to an aspect of the invention, in said first and third embodiment of the process of the present invention (P1 and P3), the chondroitin sulfate or a salt thereof (CS) of the present invention has a weight average molecular weight comprised from greater than 5 kDa to less than 50 kDa, preferably from greater than 5 kDa to 25 kDa, and it comprises: - a 6-chondroitin sulfate at a weight percentage comprised from 50% to 95±0.5%, preferably from 75% to 90%; - a non-sulfated chondroitin at a weight percentage comprised from 5% to 20%, preferably from 7% to 15%; - a 2,6-chondroitin disulfate at a weight percentage comprised from 0.1% to 10%, preferably from 0.2% to 8%; and - a 4-chondroitin sulfate at a weight percentage comprised from 0.01% to 5%, preferably from 0.05% to 3%, all the percentagesbeing expressedwith respect to the total of disaccharides contained in chondroitin sulfate orwith respect to the total weight of chondroitin sulfate.

[0125] According to a preferred aspect of the invention, chondroitin sulfate or a salt thereof (CS) of the present invention (process P1 and / or P3) has a weight average molecular weight comprised from greater than 5 kDa to 10 kDa, and wherein said chondroitin sulfate or the salt thereof comprises: - a 6-chondroitin sulfate at a weight percentage comprised from 78% to 86%; - a non-sulfated chondroitin at a weight percentage comprised from 8% to 13%; - a 2,6-chondroitin disulfate at a weight percentage comprised from 0.3% to 5%; and - a4-chondroitin sulfateat aweight percentagecomprised from0.1% to1.5%,all thepercentagesbeingexpressedwith respect to the total of disaccharides contained in chondroitin sulfate or with respect to the total weight of chondroitin sulfate.

[0126] According to a preferred aspect of the invention, chondroitin sulfate or a salt thereof (CS) of the present invention (process P1 and / or P3) has a weight average molecular weight comprised from greater than 5 kDa to less than 50 kDa, preferably from greater than 5 kDa to less than 25 kDa, and wherein said chondroitin sulfate or the salt thereof comprises: - a 6-chondroitin sulfate at a weight percentage comprised from 50% to 95±0.5%, preferably from 75% to 90%; and - a 4-chondroitin sulfate at a weight percentage comprised from 0.01% to 5%, preferably from 0.05% to 3%, all the percentagesbeing expressedwith respect to the total of disaccharides contained in chondroitin sulfate orwith respect to the total weight of chondroitin sulfate.

[0127] According to a further preferred aspect of the invention, chondroitin sulfate or a salt thereof (CS) of the present invention (processP1and / orP3)hasaweight averagemolecularweight comprised fromgreater than5kDa to10kDa,and wherein said chondroitin sulfate or the salt thereof comprises: 15 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 - a 6-chondroitin sulfate at a weight percentage comprised from 78% to 86%; and - a4-chondroitin sulfateat aweight percentagecomprised from0.1% to1.5%,all thepercentagesbeingexpressedwith respect to the total of disaccharides contained in chondroitin sulfate or with respect to the total weight of chondroitin sulfate.

[0128] According to a further preferred aspect of the invention, chondroitin sulfate or a salt thereof (CS) of the present invention (processP1and / orP3)hasaweight averagemolecularweight comprised fromgreater than5kDa to less than50 kDa, preferably from greater than 5 kDa to less than 25 kDa, and it comprises: - a 6-chondroitin sulfate at a weight percentage comprised from 50% to 95±0.5%, preferably from 75% to 90%; - a non-sulfated chondroitin at a weight percentage comprised from 5% to 20%, preferably from 7% to 15%; - a 2,6-chondroitin disulfate at a weight percentage comprised from 0.1% to 10%, preferably from 0.2% to 8%; - a 4-chondroitin sulfate at a weight percentage comprised from 0.01% to 5%, preferably from 0.05% to 3%; - a 4,6-chondroitin disulfate at a weight percentage comprised from 0.01% to 5%, preferably from 0.05% to 3%; and - a 2,4-chondroitin disulfate at a weight percentage comprised from 0.01% to 5%, preferably comprised from 0.05% to 3%, all the percentages being expressed with respect to the total of disaccharides contained in chondroitin sulfate or with respect to the total weight of chondroitin sulfate.

[0129] According to an embodiment, chondroitin sulfate or a salt thereof (CS) of the present invention (process P1 and / or P3) has a weight average molecular weight comprised from greater than 5 kDa to 10 kDa, and wherein said chondroitin sulfate or the salt thereof comprises: - a 6-chondroitin sulfate at a weight percentage comprised from 78% to 86%; - a non-sulfated chondroitin at a weight percentage comprised from 8% to 13%; - a 2,6-chondroitin disulfate at a weight percentage comprised from 0.3% to 5%; and, furthermore, - a4-chondroitin sulfate, 4,6-chondroitindisulfateand2.4-chondroitindisulfate, eachataweightpercentagecomprised from 0.1% to 1.5%. EXPERIMENTAL PART

[0130] I. Process for preparing hyaluronic acid according to the second embodiment (P2). (I) prepare dried fungi belonging to the species Tremella fuciformis, (ii) crush or pulverise said dried fungi (about 20 mesh) by grinding to obtain crushed / pulverised dry fungi; (pre-iii) carry out enzymatic hydrolysis, using a pectinase as an enzyme, by adding to the crushed / pulverised dry fungi and to an enzyme pectinase (Pectinex® Ultra Tropical, volume 0.01%) of distilled water (50 volume / weight) and heating at about 50°C for about 3 hours to obtain a hydrolysis mixture; (iii. a) carryout afirst extractionbyaddingdistilledwater (50volume / weight) to saidhydrolysismixtureandheating at about 100°C(boiling) for about 2.5 hours, and filtering with a 200-mesh sieve and collecting a filtrate and a solid residue; (iii.b) carry out a second extraction carried out on said solid residue obtained from said first extraction (iii.a) by adding distilled water (50 volume / weight) and heating to about 100°C (boiling) for about 2 hours, followed by filtering with 200 mesh sieve and collecting a filtrate; and combine the filtrate obtained from said first extraction (iii.a) with the filtrate obtained from said second extraction (iii.b) to obtain an aqueous extract; (iii. c) concentrate said aqueous extract at about 75°C to obtain a concentrated aqueous extract having a relative density of about 1.05; followed by (iv) slowly adding, under stirring, to said concentrated aqueous extract, 95% ethanol (volume / volume = 3) and leaving for 12 hours; remove the ethanol and keep the extract; (vii) add distilled water to said extract and freeze-dry to obtain a product PR1 comprising or, alternatively, consistingof hyaluronic acidor a salt thereof (HA)havinganaveragemolecularweight of from100 to300kDaand a purity by weight percentage comprised from 95% to 99% with respect to the total weight of said product PR1. II. Process for preparing chondroitin sulfate according to the third embodiment (P3). (i) prepare dried fungi belonging to the species Tremella fuciformis; (ii) crush or pulverise the dried fungi (20 mesh) by grinding to obtain crushed / pulverised dry fungi; 16 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 (pre-iii) carry out enzymatic hydrolysis, using a pectinase as an enzyme, by adding to the crushed / pulverised dry fungi and to an enzyme pectinase (Pectinex® Ultra Tropical, volume 0.01%) of distilled water (50 volume / weight) and heating at about 50°C for about 2 hours to obtain a hydrolysis mixture (pH from 5‑7); (iii.a) carry out a first extraction by adding distilled water (50 volume / weight) to the hydrolysis mixture and heating at100°C (boiling) for about2.5hours, followedbyfiltrationwitha200-meshsieve (if necessary centrifugingbefore filtering) and collecting a filtrate and a solid residue; (iii.b) carry out a second extraction carried out of said solid residue obtained from said first extraction by adding distilled water (50 volume / weight) and heating at about 100°C (boiling) for about 1.5 hours, followed by filtering with 200 mesh sieve and collecting a filtrate; and combine the filtrate obtained from said first extraction (iii.a) with said filtrate obtained from said second extraction (iii.b) to obtain an aqueous extract; (iii. c) concentrate said aqueous extract at about 75°C to obtain a concentrated aqueous extract having a relative density of about 1.05‑1.08; followed by (vi.c) carry out a sulfonation (or sulfation) reaction by adding SO3 DMF 5 ml, 15 ml, 55 ml, for every 100 ml of concentrated aqueous extract, at about 75°C for about 30 minutes; (vi.d) neutralise with NaOH up to about a pH value of 7; (vi.e) place the obtained solution in a dialysis bag (1,000 Da) for at least 24 hours, until a relative density of the solution of about 1.1 is reached. Dry in a vacuum oven to obtain a product PR2 comprising or, alternatively, consisting of chondroitin sulfate or a salt thereof (CS) having an average molecular weight comprised from greater than 5 kDa to 10 kDa (for example, about 8 kDa), wherein said CS has a composition similar to the compound CS.1 reported in Table 1 and a purity at a weight percentage comprised from 89% to 94.5%, with respect to the total weight of said product PR2. Claims 1. A process for preparing a mixture comprising or, alternatively, consisting of at least one glycosaminoglycan selected fromachondroitin sulfate or a salt thereof (CS), said process comprising at least one step for extracting from astarting material of plant origin with an aqueous solvent or water, wherein said starting material of plant origin comprises or, alternatively, consists of at least one natural fungus belonging to the Dikarya subkingdom, Basidiomycota division, and to the species Tremella fuciformis (Berk. 1856). 2. The process according to claim 1, comprising the following steps: (i) identifying said at least one natural fungus as a plant starting material of said at least one glycosaminoglycan; (ii) optionally, carrying out a crushing or pulverisation of the plant starting material to obtain said at least one pulverised and crushed fungus; (iii) carrying out an extraction of said glycosaminoglycan from said at least one fungus obtained from step (i) or from step (ii) using an extraction solvent, preferably an aqueous solvent, even more preferably water to obtain an aqueous extract of said glycosaminoglycan; (iv) adding a solvent, preferably analcohol,more preferably ethanol to the aqueousextract obtained fromstep (iii) to obtain a liquid product; (v) carrying out a centrifugation and / or a filtration of said liquid product obtained from step (iv) to obtain a liquid phase and a solid residue; (vi) carryingoutaprocessingof said solid residueobtainedbycentrifugationand / or filtrationof step (v) through the following steps (vi.b), (vi.c), (vi.d) and (vi.e): (vi.b) recovering and purifying said solid residue obtained from step (v) to obtain a product of step (vi.b); (vi.c) treating said product of step (vi.b) with a source of sulfuric acid, preferably wherein said source of sulfuric acid is selected from the group comprising or, alternatively, consisting of: sulfuric acid, a sulfur- trioxide pyridine complex, a sulfur trioxide dimethyl-formamide complex and mixtures thereof, more pre- ferably SO3‑DMF, to obtain an acidified product; (vi.d) neutralising said acidified product obtained from step (vi.c) using a basic agent, preferably sodium hydroxide, to obtain a neutralised product; (vi.e) concentrating and drying said neutralised product obtained from step (vi.d) to obtain a product PR2 comprising or, alternatively, consisting of chondroitin sulfate or a salt thereof having a weight average molecular weight comprised from 1 kDa to 50 kDa. 3. The process according to claim 2, wherein the step (iii) for the extraction of said at least one glycosaminoglycan from 17 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 the plant starting material comprises or, alternatively, consists of the following steps: (iii.a) carrying out a first extraction from the plant starting material with a first volume of extraction solvent at a temperature comprised from 10°C to 90°C for a period of time comprised from 0.5 hours to 12 hours to obtain first aqueous extract and a solid residue; and (iii.b) carrying out a second extraction of said solid residue obtained from step (iii.a) with a second volume of extractionsolventata temperaturecomprised from90°C to110°C, foraperiodof timecomprised from0.5hours to 8 hours, to obtain a second aqueous extract; and combining said first extract and said second extract to obtain an aqueous extract. 4. The process according to claim 3, wherein in the step (iii) of carrying out an extraction comprising or, alternatively, consisting of said step (iii.a) of carrying out a first extraction and said step (iii.b) of carrying out a second extraction, an enzyme, preferably a proteolytic enzyme, is used. 5. The processaccording to any oneof claims 1‑4,wherein said chondroitin sulfate, or the salt thereof, obtained from the processhasaweight averagemolecularweight comprised from5kDaorgreater than5kDa to25kDa;preferably from 5 kDa or greater than 5 kDa to 10 kDa; and / or wherein said chondroitin sulfate, or the salt thereof, obtained from the process comprises or, alternatively, consists of 6-chondroitin sulfate at a % by weight comprised from 50% to 95±0.5%, with respect to the total of disaccharides contained in chondroitin sulfate or salt thereof. 6. Useof a fungusbelonging to theDikarya subkingdom,preferably to theBasidiomycotadivision,morepreferably to the speciesTremella fuciformis (Berk. 1856), asaplant startingmaterial for preparingachondroitin sulfateorasalt thereof having a weight average molecular weight comprised from 1 kDa to 50 kDa; preferably from 5 kDa or greater than 5kDa to 25 kDa; more preferably from 5 kDa or greater than 5kDa to 10 kDa. 7. Useof a fungusbelonging to theDikarya subkingdom,preferably to theBasidiomycotadivision,morepreferably to the speciesTremella fuciformis (Berk. 1856), asaplant startingmaterial for preparingachondroitin sulfateorasalt thereof according to claim 6, wherein said chondroitin sulfate or said salt thereof comprises or, alternatively, consists of 6- chondroitin sulfate at a % by weight comprised from 50% to 95±0.5%, with respect to the total of disaccharides contained in chondroitin sulfate or salt thereof. 8. Achondroitin sulfate or a salt thereof, having a weight average molecular weight comprised from 1 kDa to less than 50 kDa, preferably comprised from 5 kDa to greater than 5 kDa to 25 kDa or from greater than 5 kDa to 10 kDa. 9. The chondroitin sulfate or a salt thereof according to claim 8, wherein said chondroitin sulfate or a salt thereof comprises 6-chondroitin sulfate at a % by weight comprised from 50% to 95%, the % being expressed with respect to the total of disaccharides contained in chondroitin sulfate. 10. The chondroitin sulfate or salt thereof (CS) according to claims 8 and 9 having a weight average molecular weight comprised from greater than 5 kDa to 25 kDa, preferably greater than 5 kDa to 10 kDa, and wherein said chondroitin sulfate or the salt thereof comprises: 6-chondroitin sulfate at a % by weight comprised from 75% to 90%, preferably from 78% to 86%, and 4-chondroitin sulfate at a % by weight comprised from 0.05% to 3%, preferably from 0.1% to 1.5%, the % being expressed with respect to the total of disaccharides contained in chondroitin sulfate. 11. Useof thechondroitin sulfateor a salt thereof accordinganyoneof claims8‑10asadditiveor excipient, or ingredient in the preparation of pharmaceutical products, medical devices, nutraceutical products, food for special medical purposes (SFMPs), dietary supplements or food products. 12. A composition comprising: (i) the chondroitin sulfate or a salt thereof according to any one of claims 8‑10, and (ii) technological additives or pharmaceutical or food grade excipients. 13. The composition according to the preceding claim for use as medicament. 14. The composition according to claim 13 for use in a method for preventive and / or curative treatment of arthritis, osteoarthritis, arthrosis, joint pain, inflammation of the limbs and of the joints, or gastroesophageal reflux, in human or animal subjects. 18 EP 4 721 763 A2 5 10 15 20 25 30 35 40 45 50 55 19 EP 4 721 763 A2 20 EP 4 721 763 A2 21 EP 4 721 763 A2 22 EP 4 721 763 A2 23 EP 4 721 763 A2 24 EP 4 721 763 A2 25 EP 4 721 763 A2 26 EP 4 721 763 A2 REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard. Patent documents cited in the description • WO 2012152872 A1

[0055] • EP 2852437 B1

[0055] (19) *EP004721763A3* (11) EP 4 721 763 A3 (12) EUROPEAN PATENT APPLICATION (88) Date of publication A3: 03.06.2026 Bulletin 2026 / 23 (43) Date of publication A2: 08.04.2026 Bulletin 2026 / 15 (21) Application number: 26160058.9 (22) Date of filing: 08.06.2020 (51) International Patent Classification (IPC): A61K 31 / 728 (2006.01) A61K 31 / 737 (2006.01) A61P 1 / 04 (2006.01) A61P 19 / 02 (2006.01) (52) Cooperative Patent Classification (CPC): (C-Sets available) A61K 47 / 36; A23L 33 / 105; A61K 31 / 728; A61K 31 / 737; A61K 36 / 07; A61P 1 / 04; A61P 19 / 02; C08B 37 / 0069; C08B 37 / 0072; C12P 19 / 26; A61K 2236 / 00 (Cont.) (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR Designated Extension States: BA ME Designated Validation States: KH MA MD TN (30) Priority: 07.06.2019 IT 201900008409 (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 20736429.0 / 3 980 030 (71) Applicant: Vivatis Pharma GmbH 20097 Hamburg (DE) (72) Inventors: • CERANA, Giorgio Stefano 20097 Hamburg (DE) • BOS, Peter 20097 Hamburg (DE) (74) Representative: Marben S.r.l. Via Larga, 16 20122 Milano (IT) (54) IDENTIFICATION AND SELECTION OF A PLANT STARTING MATERIAL OF A PLANT CHONDROITIN SULFATE AND HYALURONIC ACID, AND TRANSFORMATION OF SUCH PLANT STARTING MATERIAL TO OBTAIN INGREDIENTS FOR USE IN FOODS, SUPPLEMENTS, MEDICAL DEVICES OR DRUGS (57) Process for extraction from a plant starting ma- terial, such as a fungus, for the preparation of a mixture (m) comprising or, alternatively, consisting of at least one glycosaminoglycan selected from: (a) hyaluronic acid or a salt thereof (HA) having a weight average molecular weight of comprised from 10 kDa to 600 kDa; (b) chon- droitin or chondroitin sulfate or a salt thereof (CS) having aweight averagemolecularweight comprised from3kDa to 50 kDa; and (c) a combination of (a) and (b). EP 4 72 1 76 3 A 3 Processed by Luminess, 75001 PARIS (FR) (Cont. next page) (52) Cooperative Patent Classification (CPC): (Cont.) C-Sets A61K 31 / 728, A61K 2300 / 00; A61K 31 / 737, A61K 2300 / 00 2 EP 4 721 763 A3 3 EP 4 721 763 A3 5 10 15 20 25 30 35 40 45 50 55 4 EP 4 721 763 A3 5 10 15 20 25 30 35 40 45 50 55 5 EP 4 721 763 A3 5 10 15 20 25 30 35 40 45 50 55 摘要 本发明公开了从植物起始材料如真菌进行提取以制备混合物(m)的 方法，该混合物包括糖胺聚糖或由其组成，该糖胺聚糖是选自：(a) 透明质酸或其盐(HA)，其重量平均分子量为 10kDa 至 600kDa；(b) 软骨素或硫酸软骨素或其盐 (CS)，其重量平均分子量为 3kDa 至 50kDa；以及(c)(a)和(b)的组合中的至少一种糖胺聚糖。

Claims

1. A process for preparing a mixture comprising or, alternatively, consisting of at least one glycosaminoglycan selected from a chondroitin sulfate or a salt thereof (CS), said process comprising at least one step for extracting from a starting material of plant origin with an aqueous solvent or water, wherein said starting material of plant origin comprises or, alternatively, consists of at least one natural fungus belonging to the Dikarya subkingdom, Basidiomycota division, and to the species Tremella fuciformis (Berk. 1856).

2. The process according to claim 1, comprising the following steps: (i) identifying said at least one natural fungus as a plant starting material of said at least one glycosaminoglycan; (ii) optionally, carrying out a crushing or pulverisation of the plant starting material to obtain said at least one pulverised and crushed fungus; (iii) carrying out an extraction of said glycosaminoglycan from said at least one fungus obtained from step (i) or from step (ii) using an extraction solvent, preferably an aqueous solvent, even more preferably water to obtain an aqueous extract of said glycosaminoglycan; (iv) adding a solvent, preferably an alcohol, more preferably ethanol to the aqueous extract obtained from step (iii) to obtain a liquid product; (v) carrying out a centrifugation and / or a filtration of said liquid product obtained from step (iv) to obtain a liquid phase and a solid residue; (vi) carrying out a processing of said solid residue obtained by centrifugation and / or filtration of step (v) through the following steps (vi.b), (vi.c), (vi.d) and (vi.e): (vi.b) recovering and purifying said solid residue obtained from step (v) to obtain a product of step (vi.b); (vi.c) treating said product of step (vi.b) with a source of sulfuric acid, preferably wherein said source of sulfuric acid is selected from the group comprising or, alternatively, consisting of: sulfuric acid, a sulfur-trioxide pyridine complex, a sulfur trioxide dimethyl-formamide complex and mixtures thereof, more preferably SO3-DMF, to obtain an acidified product; (vi.d) neutralising said acidified product obtained from step (vi.c) using a basic agent, preferably sodium hydroxide, to obtain a neutralised product; (vi.e) concentrating and drying said neutralised product obtained from step (vi.d) to obtain a product PR2 comprising or, alternatively, consisting of chondroitin sulfate or a salt thereof having a weight average molecular weight comprised from 1 kDa to 50 kDa.

3. The process according to claim 2, wherein the step (iii) for the extraction of said at least one glycosaminoglycan from the plant starting material comprises or, alternatively, consists of the following steps: (iii.a) carrying out a first extraction from the plant starting material with a first volume of extraction solvent at a temperature comprised from 10°C to 90°C for a period of time comprised from 0.5 hours to 12 hours to obtain first aqueous extract and a solid residue; and (iii.b) carrying out a second extraction of said solid residue obtained from step (iii.a) with a second volume of extraction solvent at a temperature comprised from 90°C to 110°C, for a period of time comprised from 0.5 hours to 8 hours, to obtain a second aqueous extract; and combining said first extract and said second extract to obtain an aqueous extract.

4. The process according to claim 3, wherein in the step (iii) of carrying out an extraction comprising or, alternatively, consisting of said step (iii.a) of carrying out a first extraction and said step (iii.b) of carrying out a second extraction, an enzyme, preferably a proteolytic enzyme, is used.

5. The process according to any one of claims 1-4, wherein said chondroitin sulfate, or the salt thereof, obtained from the process has a weight average molecular weight comprised from 5 kDa or greater than 5 kDa to 25 kDa; preferably from 5 kDa or greater than 5 kDa to 10 kDa; and / or wherein said chondroitin sulfate, or the salt thereof, obtained from the process comprises or, alternatively, consists of 6-chondroitin sulfate at a % by weight comprised from 50% to 95±0.5%, with respect to the total of disaccharides contained in chondroitin sulfate or salt thereof.

6. Use of a fungus belonging to the Dikarya subkingdom, preferably to the Basidiomycota division, more preferably to the species Tremella fuciformis (Berk. 1856), as a plant starting material for preparing a chondroitin sulfate or a salt thereof having a weight average molecular weight comprised from 1 kDa to 50 kDa; preferably from 5 kDa or greater than 5kDa to 25 kDa; more preferably from 5 kDa or greater than 5kDa to 10 kDa.

7. Use of a fungus belonging to the Dikarya subkingdom, preferably to the Basidiomycota division, more preferably to the species Tremella fuciformis (Berk. 1856), as a plant starting material for preparing a chondroitin sulfate or a salt thereof according to claim 6, wherein said chondroitin sulfate or said salt thereof comprises or, alternatively, consists of 6-chondroitin sulfate at a % by weight comprised from 50% to 95±0.5%, with respect to the total of disaccharides contained in chondroitin sulfate or salt thereof.

8. A chondroitin sulfate or a salt thereof, having a weight average molecular weight comprised from 1 kDa to less than 50 kDa, preferably comprised from 5 kDa to greater than 5 kDa to 25 kDa or from greater than 5 kDa to 10 kDa.

9. The chondroitin sulfate or a salt thereof according to claim 8, wherein said chondroitin sulfate or a salt thereof comprises 6-chondroitin sulfate at a % by weight comprised from 50% to 95%, the % being expressed with respect to the total of disaccharides contained in chondroitin sulfate.

10. The chondroitin sulfate or salt thereof (CS) according to claims 8 and 9 having a weight average molecular weight comprised from greater than 5 kDa to 25 kDa, preferably greater than 5 kDa to 10 kDa, and wherein said chondroitin sulfate or the salt thereof comprises: 6-chondroitin sulfate at a % by weight comprised from 75% to 90%, preferably from 78% to 86%, and 4-chondroitin sulfate at a % by weight comprised from 0.05% to 3%, preferably from 0.1% to 1.5%, the % being expressed with respect to the total of disaccharides contained in chondroitin sulfate.

11. Use of the chondroitin sulfate or a salt thereof according any one of claims 8-10 as additive or excipient, or ingredient in the preparation of pharmaceutical products, medical devices, nutraceutical products, food for special medical purposes (SFMPs), dietary supplements or food products.

12. A composition comprising: (i) the chondroitin sulfate or a salt thereof according to any one of claims 8-10, and (ii) technological additives or pharmaceutical or food grade excipients.

13. The composition according to the preceding claim for use as medicament.

14. The composition according to claim 13 for use in a method for preventive and / or curative treatment of arthritis, osteoarthritis, arthrosis, joint pain, inflammation of the limbs and of the joints, or gastroesophageal reflux, in human or animal subjects.

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