A functionalized tamarind seed polysaccharide and a preparation process thereof

Abstract

A functionalized tamarind seed polysaccharide, and a process for its preparation. In particular, said functionalized tamarind seed polysaccharide is a TSP having a selected functionalization degree and molecular weight, showing improved mucoadhesive properties, as well as stability and compatibility with pharmaceutical ingredients. Therefore, the invention also relates to a pharmaceutical composition, as well as a biomaterial, comprising said f-TSP.

Description

[0001] ADV-00748PTITWO

[0002] “A FUNCTIONALIZED TAMARIND SEED POLYSACCHARIDE AND A PREPARATION PROCESS THEREOF”

[0003] DESCRIPTION

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a functionalized tamarind seed polysaccharide (shortly referred to as “f-TSP”), and a process for its preparation. In particular, said f-TSP is a TSP having a selected functionalization degree and molecular weight, showing improved mucoadhesive properties, as well as stability and compatibility with pharmaceutical ingredients. Therefore, the invention also relates to a pharmaceutical composition comprising said f-TSP. Finally, the present invention also concerns a process of preparation of said f-TSP.

[0006] BACKGROUND ART

[0007] Mucus is a viscous and heterogeneous biological product that coats many epithelial surfaces. Mucus-secreting cells are widely spread in different locations in the body, including the nasal, ocular, buccal area and the gastrointestinal, reproductive and respiratory tracts.

[0008] Mainly, the mucus serves as a lubricant to minimize shear stresses and as a protection barrier against harmful substances. However, mucus can perform other important functions. Goblet cells located in the epithelium are unicellular mucus-secreting glands. Mucus is stored in large granules in the goblet cell and can be released by exocytosis or exfoliation of the whole cell. Mucus granules are mainly stored in the apical side of the goblet cell, which results in the characteristic balloon shape of these cells. Although the secretion of mucus can vary depending on age, sex, body location and health condition, the average mucus turnover is approximately 6 h. Goblet cells experience two types of granules exocytosis: basal secretion, which is characterized by a low level, continuous and unregulated secretion and stimulated secretion, which is a regulated exocytosis of granules in response to extracellular stimuli.

[0009] The stimulated pathway can dramatically increase the mucus secretion. In pathological conditions, secretion of mucus can considerably vary. For example, in ulceration or inflammation, the intestinal mucous layer is thinner. In physiological conditions, it has been observed that the mucous layer on the gastric and duodenal epithelial surfaces has a ADV-00748PTITWO thickness between 5 and 200 m in the rat, and twice this variation in the human.

[0010] Mucus consists mainly of water (up to 95% weight), inorganic salts (about 1% weight), carbohydrates and lipids (less than 1%) and glycoproteins (not more than 5% weight). Mucus glycoproteins are also called mucins and consist of a protein core with branched oligosaccharide chains attached over 63% of its length. Approximately 80% by weight of the glycoprotein consists of oligosaccharides, which make the mucin more hydrosoluble and also protects the protein core from proteolytic degradation.

[0011] Mucins are responsible for the gel-like properties of the mucus. Glycoprotein concentrations determine the cohesion of the mucus. When a critical mucin concentration is achieved, the hydrodynamic volumes of the molecules start overlapping and a gel is formed.

[0012] The main amino acids in the branched protein blocks are serine and threonine, which are linked to the oligosaccharide chains by O-glycosidic bonds. The sugar residues composing the oligosaccharide side chains are galactose, fucose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid. Generally the oligosaccharide chain is linked to the protein core through an ether bond between the a-1 position hydroxyl group from the N-acetylgalactosamine and the hydroxyl group from the serine or threonine amino acids. Oligosaccharide chains are normally 2-19 residues long and often fucose, sialic acid, sulfate esters of galactose and N-acetylglucosamine are the terminal groups. Opposite to the rich serine and threonine branched blocks, the unbranched blocks of the protein core have a normal amino acid composition.

[0013] Mucin glycoproteins exhibit molecular weights between 0.5 and 40 * 106Da, although the average MW is 1.8 x 106Da. They consist of four to six subunits linked together. Mucin subunits are joined together through disulfide bonds between the cysteine residues present in the non-glycosylated areas of the protein core. Intermolecular interactions have also been detected between mucin molecules. They are believed to be non-covalent, being the hydrogen-bonding, hydrophobic interactions and physical entanglement the main intermolecular interactions.

[0014] Mucoadhesion can be defined as the phenomenon of the attachment of natural or synthetic polymers to a mucosal surface. In order to develop and / or improve mucoadhesive materials, it is essential to understand the forces and mechanisms that lead to an effective bond between a polymer and a mucous layer. Yet, the mechanisms of mucoadhesion are ADV-00748PTITWO not completely clear. In general, it is agreed that the process involved in the mucoadhesion phenomenon can be described in three steps: first of all, the wetting and swelling of the polymer should allow an intimate contact with the tissue, secondly interpenetration of the polymer chains and entanglement between the polymer and the mucin chains should be attained and finally, the formation of weak chemical bonds should be possible. Certain polymeric hydrogels can exhibit mucoadhesive properties. Some of the characteristics that have shown to increase hydrogel mucoadhesive properties include: the presence of a high amount of hydrogen-bonding chemical groups, such as hydroxyls and carboxyls, anionic surface charges, high polymer molecular weight, high polymer chain flexibility and surface tensions that will induce spreading into the mucus layer.

[0015] There exist three main types of interactions between a polymer and the mucous layer: physical or mechanical bonds, secondary chemical bonds and covalent chemical bonds. Physical bonds imply the entanglement of mucin glycoproteins with the polymer chains, and the interpenetration of the mucin chains in the polymer matrix. This interpenetration of macromolecules will depend on the respective chain flexibility and diffusion coefficients.

[0016] Secondary chemical interactions include ionic bonds, van der Waals interactions and hydrogen bonding. Hydrogen bonding is probably the most important secondary chemical interaction in mucoadhesion. Some of the functional groups that form hydrogen bonds are hydroxyls, carboxyls, sulfate and amino groups. For this reason, polymers such as poly(vinyl alcohol), poly(acrylic acid) and poly(hydroxyalkyl methacrylate) have shown good mucoadhesive properties in the past. Even these types of forces are weak; numerous interaction sites lead to strong mucoadhesion.

[0017] Covalent bonds are attained by the chemical reaction of the polymer and the substrate. Even though this type of bond is permanent, the necessity of such type of system should be evaluated since both the mucus turnover and the epithelial desquamation would result in the detachment and loss of the polymer from the tissue anyway.

[0018] To date, no individual theory has been accepted to explain mucoadhesion as a phenomenon occurring via one singular mechanism. However, several theories have been developed and are used to describe the complex phenomenon of mucoadhesion.

[0019] Anyway, independently of the possible mechanisms, the potential use for mucoadhesive systems as drug carriers lies in its prolongation of the residence time at the absorption ADV-00748PTITWO site, allowing intensified contact with the epithelial barrier. On the other hand, adhesion of preparations onto mucous membrane can be impaired by the mucociliary clearance system. This clearance, a natural defence mechanism of the body against the deposition of impurities onto the mucous membrane, can also remove the preparation. Thus, by using bioadhesive molecules, it is possible to retain the preparation at the action site and to direct the drug to a specific site or tissue.

[0020] Mucoadhesive polymers have numerous hydrophilic groups, such as hydroxyl, carboxyl, amide, and sulfate. These groups attach to mucus or the cell membrane by various interactions such as, as said above, hydrogen bonding and hydrophobic or electrostatic interactions. These hydrophilic groups also cause polymers to swell in water and, thus, expose the maximum number of adhesive sites.

[0021] An ideal polymer for a bioadhesive drug delivery system should have the following characteristics:

[0022] - the polymer and its degradation products should be nontoxic and nonabsorbable,

[0023] - it should be non-irritant,

[0024] - it should preferably form a strong noncovalent bond with the mucus or epithelial cell surface,

[0025] - it should adhere quickly to moist tissue and possess some site specificity,

[0026] - it should allow easy incorporation of the drug and offer no hindrance to its release,

[0027] - the polymer must not decompose on storage or during the shelf life of the dosage form,

[0028] - the cost of the polymer should not be high so that the prepared dosage form remains competitive.

[0029] It would be therefore desirable to have a polymer showing those properties, or at least most of them, in order to advantageously increasing the efficacy of drug release through mucosae, by improving mucoadhesion.

[0030] SUMMARY OF THE INVENTION

[0031] Said object has been achieved by a functionalized tamarind seed polysaccharide (f-TSP), as stated in Claim 1.

[0032] In a further aspect, the present invention concerns a pharmaceutical composition comprising said functionalized TSP.

[0033] In a further aspect, the present invention concerns a biomaterial comprising said functionalized TSP. ADV-00748PTITWO

[0034] In another aspect, the present invention concerns the cosmetic use of said functionalized TSP.

[0035] In a further aspect, the present invention concerns a process of preparation of said functionalized TSP.

[0036] BRIEF DESCRIPTION OF THE FIGURES

[0037] The characteristics and advantages of the present invention will become clear in the following detailed description of the embodiments provided by way of non-limiting examples and illustrated in the drawings annexed hereto, wherein:

[0038] - Figure 1 shows the13C CP-MAS spectra of the native tamarind seed polysaccharide (shortly “TSP”), C-TSP 6, C-TSP 7 and C-TSP 8. The attribution of the peaks is reported;

[0039] - Figure 2 shows 'H spectra of TSP and C-TSP 3. The peaks of the anomeric protons of the residues and the position 2 of glucose are indicated. The superscript corresponds to the carbon number of the observed monosaccharide, which is in bold style, while the monosaccharide linked is in the parentheses.

[0040] - Figure 3 shows HSQC superimposition of TSP with partial assignments and C- TSP 3. The superscript corresponds to the carbon number of the observed monosaccharide, which is in bold style, while the monosaccharide linked is in the parentheses. Signals related to the presence of BDDE were signalled with arrows. Arabinose signals are circled in grey.

[0041] - Figure 4 shows 'H spectra of hydrolysed with xyloglucanase TSP and C-TSP 4.

[0042] - Figure 5 shows13C spectra of hydrolysed with xyloglucanase TSP and C-TSP 4. The peaks of the anomeric carbons of the residues are indicated. The superscript corresponds to the carbon number of the observed monosaccharide. Signals related to the presence of BDDE were signalled with arrows.

[0043] - Figure 6 shows the superimposition of FT-IR spectra of TSP, C-TSP 1 and C-TSP 8.

[0044] - Figure 7 shows viscosity curves of TSP, C-TSP 1, C-TSP 2 and C-TSP 3.

[0045] - Figure 8 shows viscosity curves of C-TSP_6 and C-TSP_7 at mg / mL at 20°C.

[0046] - Figure 9 shows viscosity curves of C-TSP 6 and C-TSP 7 at mg / mL at 37°C.

[0047] - Figure 10 shows amplitude sweep of C-TSP 6 and C-TSP 7 at 20 mg / mL at 37°C. Squared point are related to G’ values and dot points to G” values.

[0048] - Figure 11 shows frequency sweep of C-TSP 6 and C-TSP 7 at 20 mg / mL at 37°C. ADV-00748PTITWO

[0049] Squared point are related to G’ values and dot points to G” values.

[0050] Figure 12. SC (full coloured) and CT (striped colour) values of C-TSP 6 in red, C-TSP 7 in green and C-TSP 8 in blue in water

[0051] - Figure 12 shows the variations of viscosity as a function of the shear rate of a functionalized TSP according to the invention, as per Example 3.

[0052] - Figure 13. SC (full coloured) and CT (striped colour) values of C-TSP 6, C-TSP 7 and C-TSP 8, in NaCl 0.9%.

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054] The invention therefore relates to a tamarind seed polysaccharide functionalized with BDDE (shortly referred to as “f-TSP”), where BDDE is 1,4-butanediol diglycidyl ether. The term "tamarind seed polysaccharide” means the polysaccharide moiety obtainable from the seeds of Tamarindus indica. also referred to hereinafter for the sake of brevity as “TSP” (from the English term " Tamarindus indica Seed Polysaccharide ").

[0055] As it is known, the tamarind tree is common in India, in Africa, and throughout the Far East, where it is grown essentially for food purposes. The seed, which was originally a by-product, has since found various uses, sometimes ground up into a mealy product (currently known as "raw tamarind gum or “tamarind nut powder”), above all in the textile and paper industry, where it is used respectively as a sizing agent for yam and as a gluing agent, and in the food industry, where it is used as a thickener, gelling agent, stabiliser and binder in all kinds of products, much the same ways as further polysaccharide products, such as alginates, pectins, the guar gum or the locust bean meal. Raw tamarind gum (commercially available, for example, as Glyloid® produced by MP Gokyo Food & Chemical Co., Ltd. based in Osaka, Japan) typically contains, in addition to 65-73 wt% polysaccharide, also 15-23% protein material, 3-8% oils and fats and 2-4% ash, as well as traces of raw fibre, tannins, and further impurities.

[0056] One advantageous aspect is that the TSP solutions are suitable to be sterilized by a passage in autoclave (for example for 20 minutes at 120 °C) without undergoing thermal degradation, unlike as occurs, for example, with hyaluronic acid. The possibility of sterilization by simply a passage in autoclave renders the TSP-based preparations particularly convenient from a production viewpoint.

[0057] TSP (or “native TSP”) is a purified, neutral, water-soluble polysaccharide fraction comprising a polymeric molecule of galactoxyloglucan which is very hydrophilic and ADV-00748PTITWO features a ramified structure: attached to the main linear chain, formed of glucose repeating units, are small monosaccharide units of xylose and disaccharide units of xylose-galactose, in the latter case, the galactose is at the end of the side chain. The three monomers are present in a molar ratio of 3: 1 :2 and constitute approximately 65% of the components of the seed, as reported below:

[0058] As can be observed, the “mucin-like” molecular structure determines significant mucoadhesive properties of the polysaccharide, derived from the formation of bonds, of various kinds, with said mucins.

[0059] TSP can be isolated by means of chemical methods and enzymatic methods, by using protease or a combination of protease and high intensity ultrasounds. In the chemical method, Tamarind seed powder are added to cold distilled water to prepare a suspension which is then poured into boiling distilled water. The solution thus formed is left to boil and stirred continually; after resting for one night, said solution, undergoes centrifugation. The supernatant is separated and poured into a volume of pure alcohol amounting to double the amount of said supernatant. Thus, a precipitate is obtained which is then washed with pure ethanol and air-dried. Finally, the dried polymer is ground up, sieved, and stored in a dryer until use. In the enzymatic method, the powder obtained from the seeds is mixed with ethanol and then treated with protease; subsequently, said powder is centrifuged and ethanol is added to the supernatant for precipitation. Finally, the polymer ADV-00748PTITWO is separated and dried.

[0060] Natural polymers, such as TSP, have advantages over synthetic and semi -synthetic polymers like low cost, natural origin, less side effects, locally availability and higher patient tolerance.

[0061] However, these natural substances also suffer with the drawbacks like purity, source and microbial contamination. At the same time, TSP has the additional problem of poor workability, due to its complex structure and requires long pre-treatment in order to be suitable for subsequent preparations.

[0062] Surprisingly, it was found that, when TSP is functionalized with BDDE, the resulting polymer not only overcomes the drawbacks above-mentioned, but also improved the properties of the initial TSP, such as mucomimetic, mucoadhesive, and bioadhesive properties.

[0063] 1,4-Butanediol diglycidyl ether (BDDE) is an organic chemical in the glycidyl ether family. It is aliphatic and a colorless liquid. It has two epoxide (oxirane) groups per molecule, as shown below:

[0064] Preferably, the functionalized TSP (f-TSP) of the invention is obtained by reacting TSP and BDDE in a molar ratio of 1 :0.25 to 1 :5.

[0065] Preferably, TSP has a weight average molecular weight of 600-800 kDa, more preferably, 600-650 kDa.

[0066] Alternatively, the present invention also concerns TSP functionalized with bi- or polyfunctional epoxy selected from 1,2-ethylenediol diglycidyl ether, l-(2,3- epoxypropyl)-2,3-epoxycyclohexane, N,N-diglycidylaniline, epoxy-substituted pentaerythritol, and mixtures thereof. In other alternative embodiments, the present invention also concerns TSP functionalized with divinyl sulfone. In further alternative embodiments, the present invention also concerns TSP functionalized with a biscarbodiimide of formula YI-N=C=N-Y2-N=C=N-Y3, where Yi and Y3 are, independently of each other, hydrogen, linear or branched aliphatic group Cl -CIO, alkoxy group Cl -CIO, cycloaliphatic group Cl -CIO, aryl Cl -CIO, heteroaryl Cl -CIO, aralkyl ADV-00748PTITWO

[0067] C1-C10, heteroaralkyl C1-C10, and Y2 is a bifunctional moiety deriving from linear or branched aliphatic group Cl -CIO, alkoxy group Cl -CIO, cycloaliphatic group Cl -CIO, aryl Cl -CIO, heteroaryl Cl -CIO, aralkyl Cl -CIO, heteroaralkyl Cl -CIO. Preferably, said biscarbodiimide is selected from 1,6-hexam ethylene bis(ethylcarbodiimide), 1,8- octamethylene bis(ethylcarbodiimide), 1,10 decamethylene bis(ethylcarbodiimide), 1,12 dodecamethylene bis(ethylcarbodiimide), PEG-bis(propyl (ethylcarbodiimide)), 2,2'- dithioethyl bis(ethylcarbodiimide), l,l'-dithio-p-phenylene bis(ethylcarbodiimide), para- phenylene-bis(ethylcarbodiimide), l,l'-dithio-m-phenylene bis(ethylcarbodiimide) and mixtures thereof. In additional alternative embodiments, the present invention also concerns TSP functionalized with hydrazine.

[0068] In first preferred embodiments, functionalized TSP of the invention is obtained by reacting TSP and BDDE in a molar ratio of lower than 1 : 1.

[0069] More preferably, functionalized TSP of the invention is obtained by reacting TSP and BDDE in a molar ratio of 1 :0.25 to 1 :0.9.

[0070] These first preferred embodiments have been found to be water-soluble, so as to be suitable for applications where a low viscosity and solubility are appreciable and desirable, such as in products designed for topical administration to conjunctiva mucosa. Preferably, functionalized TSP of these first preferred embodiments has a weight average molecular weight (Mw) at least 20% higher than the weight average molecular weight of TSP before functionalization.

[0071] More preferably, functionalized TSP of these first preferred embodiments has a weight average molecular weight 22-28% higher than the weight average molecular weight of TSP before functionalization.

[0072] Most preferred are functionalized TSP having a weight average molecular weight of 800- 900 kDa.

[0073] Preferably, functionalized TSP of these first preferred embodiments has a number average molecular weight (Mn) at least 10% higher than the number average molecular weight of TSP before functionalization.

[0074] More preferably, functionalized TSP of these first preferred embodiments has a number average molecular weight 12-22% higher than the number average molecular weight of TSP before functionalization.

[0075] Most preferred are functionalized TSP having a number average molecular weight of 450- ADV-00748PTITWO

[0076] 550 kDa.

[0077] Preferably, functionalized TSP of these first preferred embodiments has an intrinsic viscosity (p) at least 15% higher than the an intrinsic viscosity of TSP before functionalization.

[0078] More preferably, functionalized TSP of these first preferred embodiments an intrinsic viscosity (p) 18-25% higher than the an intrinsic viscosity of TSP before functionalization.

[0079] Most preferred are functionalized TSP having an intrinsic viscosity (p) of 6.0-8.0 dl / g.

[0080] The molecular weight distribution and intrinsic viscosity above, are measured by size exclusion chromatography with triple detector array (HP-SEC-TDA).

[0081] In second preferred embodiments, functionalized TSP of the invention is obtained by reacting TSP and BDDE in a molar ratio of equal to or higher than 1 : 1.

[0082] More preferably, functionalized TSP of the invention is obtained by reacting TSP and BDDE in a molar ratio of 1 : 1 to 1 :3.

[0083] These second preferred embodiments have been found to be hydrogels, so as to be suitable for applications where a higher viscosity and swelling / releasing properties are appreciable and desirable, such as in products designed for topical administration to oral, nasal, vaginal, or middle ear mucosa.

[0084] Preferably, functionalized TSP of these second preferred embodiments has a viscosity at a shear rate (0.1 s'1, at 20°C and at 37°C) from 50 to 5,000 kPa.s.

[0085] Preferably, the functionalized TSP features a storage (or elastic) modulus G’(co) higher than a loss (or plastic) modulus G” (co) at 37°C, thus indicating a gel structure, due to a stable network of forces, which thus fulfils the assumption of physical stability of the dispersion.

[0086] The sample viscoelastic behaviour was investigated in the oscillation mode, to determine the storage modulus G’(co) and the loss modulus G” (co) at 37°C, i.e. physiological temperature. First, preliminary tests were conducted to determine the upper amplitude limit of the linear viscoelastic region (LVE), testing the samples over an extended strain field (0.1-100%), at constant angular frequency (10 rad / s). Second, after the determination of the LVE (1%) the samples were tested by performing a frequency sweep test over the 100 rad / s-0.1 rad / s frequencies, at a constant strain.

[0087] Preferably, functionalized TSP of these second preferred embodiments has a swelling ADV-00748PTITWO capacity (SC) of 35-60.

[0088] More preferably, functionalized TSP of these second preferred embodiments has a swelling capacity of 45-55.

[0089] Preferably, functionalized TSP of these second preferred embodiments has a releasing capacity (SC) of 25-45.

[0090] More preferably, functionalized TSP of these second preferred embodiments has a releasing capacity of 30-40.

[0091] The swelling capacity (SC) and releasing capacity (CT) are calculated by the teabag method.

[0092] In another aspect, the present invention concerns a pharmaceutical composition comprising the functionalized tamarind seed polysaccharide above described and pharmaceutically acceptable excipients.

[0093] The term "excipient" means a compound or a mixture of compounds suitable for pharmaceutical use, respectively. For example, an excipient for use in a pharmaceutical grade formulation generally must not cause an adverse response in a subject, nor must it significantly inhibit the efficacy of the functionalized TSP contained therein. Suitable excipients are acidifiers, acidity regulators, anti-caking agents, antioxidants, bulking agents, resistance agents, gelling agents, glazing agents, modified starches, sequestrants, thickeners, sweeteners, thinners, disaggregants, glidants, dyes, binders, lubricants, stabilisers, adsorbents, humectants, flavours, film-forming substances, emulsifiers, wetting agents, release retardants and mixtures thereof. Preferably, said excipients are olive oil, mineral oil, liquid paraffin, white petrolatum, polyoxyethylene, emulsifying wax, stearyl alcohol, isostearyl alcohol, cetylstearyl alcohol, stearic acid, glyceryl stearate, sodium lauryl sarcosinate, glycerine, diethylene glycolmonoethyl ether, polyethylene glycol, polyethylene glycol, polyethylene glycol stearates, Carbopol, carbomers, Poloxamer 407, Macrogol 400, purified bentonite, myristyl propionate, dimethicone, titanium dioxide, anionic, cationic and non-ionic surfactants, water, potassium sorbate, sodium benzoate, s-polylysine, sucralose, maltodextrin, citric acid, sodium carbonate, calcium carbonate, magnesium carbonate, magnesium stearate, natural starch, partially hydrolysed starch, modified starch, lactose, calcium phosphate, calcium carbonate, calcium sulfate, polyvinylpyrrolidone, silica, colloidal silica, precipitated silica, magnesium silicates, aluminium silicates, sodium lauryl sulfate, magnesium lauryl ADV-00748PTITWO sulfate, methacrylate copolymers, sodium dehydroacetate, xanthan gum, guar gum, tara gum, carob gum, fenugreek gum, Arabic gum, alginic acid, sodium alginate, propylene glycol alginate, sodium croscarmellose, polyvinylpolypyrrolidone, glyceryl behenate, indigo carmine, cellulose, modified cellulose, calcium carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, ethyl cellulose, gelatine, hydroxyethyl cellulose, hydroxypropyl cellulose, polydextrose, carrageenan, methylcellulose, sucrose, sucrose esters, sorbitol, xylitol, dextrose, maltitol, tragacanth gum, pectin, agar-agar, carboxypolymethylene, hydroxypropyl methylcellulose, tragacanth gum, mannitol, or mixtures thereof.

[0094] In another aspect, the present invention relates to a biomaterial comprising the functionalized tamarind seed polysaccharide, preferably the second embodiments as above described, and a natural, a semisynthetic or a synthetic polymer, wherein the natural polymer is selected from the group consisting of a collagen, a coprecipitate of collagen and glycosaminoglycan, a cellulose, a polysaccharides in the form of a gel selected from the group consisting of chitin, chitosan, pectin, pectic acid, agar, agarose, xanthan, gellan, alginic acid, an alginate, polymannan, a polyglycan, starch, and a natural gum, wherein the semisynthetic polymer is a collagen crosslinked with a crosslinking agent selected from the group consisting of an aldehyde, a precursor of an aldehyde, a dicarboxylic acid, a dicarboxylic acid halogenide, a diamine, a cellulose derivative, hyaluronic acid, chitin, chitosan, gellan, xanthan, pectin, pectic acid, a polyglycan, polymannan, agar, agarose, natural gum and glycosaminoglycans, and wherein the synthetic polymer is selected from the group consisting of polylactic acid, polyglycolic acid, a copolymer of polylactic acid, a derivative of polylactic acid, a copolymer of polyglycolic acid, a derivative of polyglycolic acid, polydioxan, polyphosphazene, a polysulphonic resin, polyurethane and PTFE.

[0095] In a further aspect, the present invention concerns the cosmetic use of the functionalized tamarind seed polysaccharide as above described, as thickener, gelling agent, stabiliser, moisturizing agent, solubilizer, and / or smoothing agent.

[0096] When the functionalized TSP of the invention is used in a cosmetic product, the latter is preferably in the form of a solution, lotion, emulsion, suspension, gel, ointment, cream, paste, solution spray, transdermal patch, spray-on patch, foam, or wet wipe, wherein the composition is preferably a suspension or is dissolved in one or more suitable excipients. ADV-00748PTITWO

[0097] Examples of suitable cosmetically acceptable excipients are mineral oil, liquid paraffin, white vaseline, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, stearyl alcohol, isostearyl alcohol, cetylstearyl alcohol, stearic acid, glyceryl stearate, sodium lauryl sarcosinate, glycerine, diethylene glycol monoethyl ether, polyethylene glycols, polyethylene glycol stearates, starch, carbopol, carbomers, methyl paraben, Poloxamer 407, Macrogol 400, purified bentonite, propyl paraben, myristyl propionate, dimethicone, titanium dioxide, anionic, cationic and non-ionic surfactants, water, and mixtures thereof.

[0098] The functionalized TSP of the invention can be prepared according to the process comprising the steps of: i) providing a solution of TSP in NaOH, ii) heating the solution at 45-65°C, then adding BDDE and stirring for at least one hour, iii) dialyzing the resulting functionalized TSP in water, and drying.

[0099] Preferably, the weight average molecular weight of the (native) TSP of step i) is 600-800 kDa, more preferably, 600-650 kDa. Actually, TSP having lower molecular weights tend to depolymerize, so that a functionalization reaction could further promote degradation, while TSP having higher molecular weights are in principle less suitable for pharmaceutical applications.

[0100] In the most preferred embodiments, the preparation process is performed as follows.

[0101] Tamarind seed polysaccharide is suspended in NaOH IM and stirred until complete solubilization at room temperature. Then, the solution is heated to 50°C and BDDE at different mole / residue ratios of polysaccharide / BDDE (5-120 pL) are added. After 2 hours, the resulting products are dialyzed in water (c.o.=6-8 kDa) to remove NaOH and residual BDDE. The functionalized TSP is then dried using acetone and lyophilized.

[0102] It should be also understood that all the combinations of preferred aspects of the functionalized TSP, as well as of the products containing the same, their preparation and uses, as above reported, are to be deemed as hereby disclosed, and similarly preferred.

[0103] It should also be also understood that all combinations of the preferred aspects of the functionalized TSP of the invention, preparation processes, and uses disclosed above are to be understood as herein described.

[0104] Below are working examples of the present invention provided for illustrative purposes. ADV-00748PTITWO

[0105] EXAMPLES

[0106] 1. Materials and Methods

[0107] 1.1. Materials

[0108] TSP was provided by FARMIGEA (Pisa, Italy). Sodium azide, Sodium nitrate, sodium hydroxide, acetone, 1,4-Butanediol diglycidyl ether (BDDE) and Cellulase from Aspergillus niger (80 KU / g) from Sigma Aldrich (Milan, Italy); Xyloglucanase (GH5) (Paenibacillus sp.) was purchased from Megazyme International (Ireland); deuterium oxide (99.9%) from Euriso-top (Saint-Aubin, France). Deionized water (conductivity less than 0.1 pS) was prepared with an osmosis inverse system (Culligan, Milan, Italy). PolyCAL-Pullulan Std-102K and PolyCAL-DextranStd-T67K (Malvern Panalytical, UK). When not specified, the reagents are >98%.

[0109] 1.2. Methods

[0110] 1.2,1. Preparation of functionalized TSP

[0111] Tamarind seed polysaccharide (TSP- 400 mg) was suspended in 8 mL of NaOH IM and stirred until complete solubilization at room temperature. Then, the solution was heated to 50°C and BDDE at different mole / residue ratios of polysaccharide / BDDE (5-120 pL) were added. After 2 hours, the resulting products were dialyzed in water (c.o.=6-8 kDa) to remove NaOH and residual BDDE. The samples were then dried using acetone and lyophilization. In Table 1, the product obtained are reported. The molar ratio was calculated using for the TSP the molecular weight of the repetitive unit, 1207 g / mol, and for the BDDE the molecular weight of 202.25 g / mol and density of 1.1 g / mL.

[0112] Table 1 Concentration of the BDDE used for the preparations ADV-00748PTITWO

[0113] 1.2,2. NMR

[0114] TSP and C-TSPs’13C cross polarization with magic angle spinning (CP-MAS) spectra and the measurement of the spin lattice relaxation time, Tip, were performed on AVANCE IIIHD spectrometer operating at a proton frequency of 500 MHz (Bruker), equipped with 4 mm for solid state probe, at room temperature. About 50 mg of the powered samples were placed directly in the rotor.

[0115] 13C CP-MAS spectra were acquired using the Bruker cp pulse program, with the following parameters: contact time (P15) 1.4 ms, Irradiation frequency (01) 78.258 ppm, spectral width of 301.2410 ppm, time domain (TD) 3026, relaxation delay (DI) 8s, 2048 number of scans (NS).

[0116] 'H Tip were acquired using Bruker cphtlrho pulse program, with the following parameters: contact time (P15) 1.4 ms, Irradiation frequency (01) 78.258 ppm (F2) and 6.2 (Fl), spectral width of 301.2410 ppm (F2) and 10 ppm (Fl), time domain (TD) 3026 (F2) and 12 (Fl), relaxation delay (DI) 8s, 512 number of scans (NS).

[0117] TSP’s 'H and HSQC spectra were obtained with a Bruker AVANCE 600 III (Bruker, Karlsruhe, Germany) at 313 K. About 8 mg of sample were dissolved in 0.6 mL of deuterium oxide (D2O). TSP sample was stirred overnight to ensure a complete solubilization before transfer in a 5 mm NMR tube for the analysis.

[0118] 'H-NMR were acquired with pre-saturation of residual HOD, using the Bruker zgcppr pulse program, with the following parameters: number of scans 16, relaxation delay 12 s DI, time domain 32 k points and a spectral width of 18 ppm with transmitter offset 4.7 ppm.

[0119] Heteronuclear single quantum correlation (HSQC) experiments were acquired using the Bruker hsqcetgpsisp2.2 pulse program, with GARP4 decoupling. The following acquisition parameters were set: 24 number of scans, 16 dummy scans, relaxation delay 2 s, time domain 2048 (F2) and 256 (Fl), spectral width 8 ppm (F2) and 160 ppm (Fl), transmitter offset 4.7 ppm (F2) and 80 ppm (Fl), and number of tl increments equal to 320. The 1 JC-H tune value was set to 150 Hz.

[0120] C-TSP 3 HSQC spectra were obtained with a Bruker AVANCE NEO 500MHz spectrometer (Bruker, Karlsruhe, Germany) equipped with 5 mm TCI cry oprobe, at 303 K. About 8 mg of sample were dissolved in 0.6 mL of deuterium oxide (D2O). C-TSP ADV-00748PTITWO samples was stirred overnight to ensure a complete solubilization before transfer in a 5 mm NMR tube for the analysis.

[0121] 'H NMR were acquired with pre-saturation of residual HOD, using the Bruker zgcppr pulse program, with the following parameters: number of scans 16, relaxation delay 12 s DI, time domain 32 k points and a spectral width of 18 ppm with transmitter offset 4.7 ppm.

[0122] HSQC were acquired using Bruker hsqcedetgpsisp2.2 pulse program, with the following parameters: number of scans 32, dummy scan 16, relaxation delay 2 s, time domain 2048 (F2) and 256 (Fl), spectral width 9.9 ppm (F2) and 160 ppm (Fl), transmitter offset 4.7 ppm (F2) and 80 ppm (Fl).

[0123] Hydrolyzed with xyloglucanase TSP sample’s 'H and13C spectra were obtained with a Bruker AVANCE IIIHD spectrometer operating at a proton frequency of 500 MHz (Bruker), equipped with 5 mm BBO probe, at 303K. About 20 mg of sample were dissolved in 0.6 mL of deuterium oxide (D2O) with 0.002 % trimethylsilyl-3-propionic acid and transferred in a 5 mm NMR tube for the analysis.

[0124] 1H NMR was acquired with presaturation of residual HOD, using the Bruker zgcppr pulse program, with the following parameters: number of scans 128, relaxation delay 12 s DI, time domain 32 k points and a spectral width of 18 ppm with transmitter offset 4.7 ppm.13C was acquired using the Bruker zgig pulse program, with the following parameters: number of scans 24 k, relaxation delay 1.5 s DI, time domain 32 k points and a spectral width of 301 ppm with transmitter offset 90 ppm.

[0125] Hydrolyzed with xyloglucanase C-TSP 4 sample’ s1H and13C spectra were obtained with a Bruker AVANCE IIIHD spectrometer operating at a proton frequency of 500 MHz (Bruker), equipped with 5 mm BBO probe, at 303K. About 20 mg of sample were dissolved in 0.6 mL of deuterium oxide (D2O) and transferred in a 5 mm NMR tube for the analysis.

[0126] 1H NMR was acquired with presaturation of residual HOD, using the Bruker zgcppr pulse program, with the following parameters: number of scans 64, relaxation delay 12 s DI, time domain 32 k points and a spectral width of 18 ppm with transmitter offset 4.7 ppm.13C was acquired using the Bruker zgig pulse program, with the following parameters: number of scans 16 k, relaxation delay 1.5 s DI, time domain 32 k points and a spectral width of 301 ppm with transmitter offset 90 ppm. ADV-00748PTITWO

[0127] All the spectra were processed with BrukerTopspin software version 4.1.1.

[0128] 1.2.3. Fourier-transform infrared spectroscopy (FT-IR)

[0129] The infrared spectra of TSP and C-TSP were recorded using an Alpha spectrometer (Bruker, Bremen, Germany), in the range of 4,000-400 cm-1at room temperature. An ATR (attenuated total reflection) (Bruker, Bremen, Germany), platinum diamond was used for the measurement. A resolution of 4 cm'1and a phase resolution of 32 were employed. The sample scan time was 100 and the background scan time was 50. The data were analysed using OPUS software version 7.0 (Bruker, Bremen, Germany)

[0130] 1.2.4. Molecular weight distribution by size exclusion chromatography with triple detector array (HP-SEC-TDA)

[0131] Chromatographic acquisitions were performed with Viscotek system model TDA305 (Malvern Panalytical, UK) and on Omni SEC Multi-detector System (Malvern Panalytical, UK) equipped both systems with a triple detector array exploiting simultaneous action of refraction index detector (RI), Right and Low Angle Light Scattering (RALS and LALS) and Viscometer (DP).

[0132] Measurements were performed at 40°C using 2 x TSKGMPWXL colums,13 pm, 7.8 mm ID x 30 cm L, in series (Tosoh Bioscience, Tokio, Japan). NaNOs O. I M+NaNs 0.05%, prefiltered (0.22 pm Mixed cellulose Ester filter), was used as mobile phase at a flow rate of 0.6 mL / min. Chromatographic profiles were elaborated using the OmniSEC software version 4.6.2 for Viscotek system TDA 305 and OMNISEC software version 11.40 for OmniSEC Multi -detector System.

[0133] RI increments, referred to as dn / dc, equal to 0.164 was used.

[0134] The detectors were calibrated with Pullulan standard, with certified molecular weight, poly dispersion index and intrinsic viscosity (PolyCAL-PullulanStd-102K Malvern Panalytical, UK).

[0135] Samples were solubilized at about 1 mg / mL in 0.1M NaNOs +NaNs 0.05%.

[0136] 1.2.5. Rheometer

[0137] The rheological properties of soluble resulting products were studied using a Modular Compact Rheometer MCR 92 (Anton Paar GmbH, Graz, Austria), with measure system DG26.7 (double gap geometry) at of 37°C.

[0138] Viscosity measurements were performed in rotation mode, they were investigated in the range of 1-1000 s'1, with a logarithmic ramp, and ten points per decade were acquired. ADV-00748PTITWO

[0139] Samples were solubilized in deionized water to a concentration of 2 mg / mL, about 20 mg were solubilized in 10 mL of deionized water.

[0140] For the hydrogels, the rheological properties were studied using a Modular Compact Rheometer MCR 92 (Anton Paar GmbH, Graz, Austria), with measure system PP50 (plate / plate geometry) at the temperature of 20°C and 37°C. Viscosity measurements were performed in rotation mode, they were investigated in the range of 1-1000 s'1, with a logarithmic ramp, and ten points per decade were acquired. Samples were analysed at 20 mg / mL.

[0141] The sample viscoelastic behaviour was investigated in the oscillation mode, to determine the storage modulus G’(co) and the loss modulus G” (co) at 37°C. First, preliminary tests were conducted to determine the upper amplitude limit of the linear viscoelastic region (LVE), testing the samples over an extended strain field (0.1-100%), at constant angular frequency (10 rad / s).

[0142] Second, after the determination of the LVE (1%) the samples were tested by performing a frequency sweep test over the 100 rad / s-0.1 rad / s frequencies, at a constant strain.

[0143] 1.2,6. Swelling (teabag method)

[0144] The powdered samples (50-100 mg) are placed into a tea bag, in nylon gauze with fine meshes, with a known weight, and the bag is dipped in about 100 mL of water or NaCl 0.9% aqueous solution for a prescribed period of 4 hours. Then the teabag is dripped for few minutes to remove excessive fluid and weighted. The swelling capacity (SC) at time t is calculated using the following equation:

[0145] Eq. (1) where A(g) is the weight of the filter and swollen sample, X(g) is the dry sample, F(g)the filter.

[0146] Then a centrifugal test (CT) was also performed, centrifugating the tea-bag with the swollen sample for 2’ at 3000 rpm at room temperature, to verify the ability of the samples of releasing liquid. The CT was calculated with the following equation:

[0147] Eq. (2) where B(g) is the weight of the filter and the sample after centrifugation, X(g) is the dry ADV-00748PTITWO sample, F(g)the filter.

[0148] 1.2.7. Enzymatic depolymerization

[0149] Enzymatic depolymerizations were carried out using Cellulase from Aspergillus niger and Xyloglucanase (GH5) form Paenibacillus sp. 20 mg of TSP and C-TSP were solubilized in 1.8 mL of deionized water at 37°C overnight and 20 mg of cellulase were dissolved in 2 mL of water at low agitation overnight. After the complete solubilization 0.2 mL of the cellulase solution were added to the TSP’ s solution. After 24h from addition of the enzyme, the solution was heated at 100°C for 10 minutes to denature the enzyme, filtrated to remove the precipitated enzyme (LLG-Syringe filter, CA pore size 0.20 pm, 0 13 mm) and then the solution lyophilized.

[0150] The depolymerization with xyloglucanase of TSP and C-TSP was performed adapting the method used by Zhang and Ai (Zhang, et al., 2020). TSP and C-TSP were solubilized at the concentration of 10 mg / mL in deionized water at 40 °C, dissolving 20 mg of C-TSP in 2 mL of deionized water overnight. Then, 20 pL of xyloglucanase (Paenibacillus sp., 20 U) were added to the solution. After 24 h, the reaction mixture was heated at 100 °C for 10 min to inactivate the enzyme, filtrated to remove the precipitated enzyme (LLG- Syringe filter, CA pore size 0.20 pm, 0 13 mm) and then lyophilized.

[0151] 1.2.8. Interaction with mucin

[0152] The adhesion ability of polymers to mucous membranes can be determined in vitro through a rheological method by measuring the differences in viscosity of solutions containing only the sample of interest, only mucin, and both. Once these viscosity values are obtained, the mucoadhesion index in percent (A%) is derived through the following expression (Rayahin et al., 2015): where r|muc is the mucin viscosity, r|poiis the viscosity of the polysaccharide under consideration, and r|muc+poi is the viscosity of the solution containing the polysaccharide and mucin. The viscosities used for the calculation of the mucoadhesion were the values at 10 1 / s of shear rate.

[0153] The interaction with mucin was evaluated comparing the viscosity of TSP and C-TSP 3 without and with mucin. A solution of mucin 5% (w / w) was prepared solubilized 250 mg of mucin in 5 mL of deionized water. ADV-00748PTITWO

[0154] TSP and C-TSP 3 solutions were prepared at 10 mg / mL in deionized water and measured the viscosity.

[0155] For experiments with mucin, 70 mg of TSP or C-TSP 2 were solubilized in 3.5 mL of deionized water, after complete solubilization, 3.5 mL of mucin solution were added to obtain a final concentration of 10 mg / mL for the polysaccharide and of 2.5% of mucin (w / w). Viscosity measurements were performed in rotation mode, they were investigated in the range of 1-1000 s'1, at 20°C with a logarithmic ramp, and ten points per decade were acquired.

[0156] 2. Results

[0157] 2.1. The functionalized TSP

[0158] The f-TSP preparation was carried out by utilizing BDDE as a reagent, being already known for crosslinking hyaluronic acid (HA) to give gels, as reported in literature (Guarise, Pavan, Pirrone, & Renier, 2012). For the activation of the BDDE, to open the epoxide groups, a basic environment is necessary. Indeed, a sodium hydroxide solution was used as solvent.

[0159] The reaction of the invention is different to other crosslinking synthesis reported in literature for HA, e.g. for the molarity of NaOH, the time of the reaction, the temperature, and the concentration of the polysaccharide. In table 2, the main differences are illustrated.

[0160] Table 2. Different parameters of the crosslinking reaction ADV-00748PTITWO

[0161] 2.2. NMR

[0162] NMR solid state was performed for gels (C-TSP 6-8) due to the high viscosity and highly crosslinked structure, which did not allow the solubilization of the products.13C cross polarization with magic angle spinning, CP -MAS, is technique used to study the structure of samples.

[0163] In Figure 1, the resulting13C CP -MAS spectra are reported. As shown, the C-TSP samples presented a similar profile to the TSP; so, after the reaction the products maintained the TSP’s main structure. Moreover, in C-TSPs the BDDE’s peaks appeared, demonstrating the presence of BDDE in the products. The presence of broad peaks indicated that the samples have an amorphous structure.

[0164] The spin-lattice relaxation time3H Tip values were measured for TSP, C-TSP_6,_7 and _8. This parameter gives information about populations of nuclei in phase, related to the structure of the samples: crystalline samples show high values of Tip (100 ms), instead amorphous samples lower values of Tip (10 ms):

[0165] Table 3. 'H T1 p values of TSP, C-TSP 6, C-TSP 7 and C-TSP 8

[0166] Tip values minor than 10 ms indicated an amorphous structure of the samples. For all the samples, the values are very similar so there is on homogeneity of phase. BDDE derivatized samples have slightly inferior values indicating smaller phase sizes.

[0167] For soluble functionalized products, 'H and HSQC spectra were performed. As example, the superimposition of 'H and HSQC spectra of C-TSP 3 of TSP are reported in Figure 2 and 3. ADV-00748PTITWO

[0168] In proton spectra of Figure 2, no significant chemical shift changes were observed following the functionalization reaction, expect for the signal at 1.65 ppm attributed to the CH2 protons in the internal position of BDDE. In both samples, the high viscosity and molecular weight reduce the spectral resolution, especially in C-TSP 3 where from 4 to 3.5 ppm peaks are not well defined. For this reason, only the well -separated peaks were attributed: in the anomeric region xylose linked to the galactose at 5.16 ppm and linked only with the glucose at 4.95 ppm, glucose and galactose at 4.55 ppm and the hydrogen in position 2 of glucose at 3.40 ppm.

[0169] In HSQC spectrum of C-TSP 3 of Figure 3, new signals are observed due to the presence of the BDDE. As in proton spectrum (Figure 2) the cross peak at 1.6 / 28 ppm attributed to the CEE protons in the internal position of BDDE is present, but also signals at 3.61 / 65.6 ppm, 3.99 / 71.6 ppm and 3.88 / 73.3 are observed probably related to the functionalization reaction. As in proton spectra, the resolution is low due to the high viscosity of the samples and also, as previously demonstrated with high molecular weight hyaluronic acid (Sofia, et al., 2024), two-dimensional experiments are not quantitative due to the different mobility of the residues.

[0170] For this reason, an enzymatic degradation was performed to reduce the molecular weight of the polysaccharide, while maintaining the functionalization, to allow a more effective NMR study. Xyloglucanase and cellulase, enzymes with different specificity towards xyloglucans, were used: xyloglucanase hydrolyses the P-1,4 bond, forming mainly the repetitive unit of TSP (Zhang, et al., 2020), cellulase involves the cleavage the P-1,4 linkage between glucose (Ejaz, Sohail, & Ghanemi, 2021).

[0171] As example, the superimposition of and13C spectra of C-TSP 4 of TSP hydrolysed with xyloglucanase are reported in Figure 4 and 5.

[0172] The reduction of the molecular weight after the hydrolysis allowed a higher resolution of the spectra and also a quantitative analysis of the total amount of BDDE in the samples, calculating the degree of modification (MoD) by the integration of the anomeric region and the BDDE’s signals in and13C spectra. The values of MoD are reported in Table 4.

[0173] Table 4. MoD values (%) of C-TSP samples calculated from the integration of and 13C of hydrolysed samples. ADV-00748PTITWO

[0174] Similar values were obtained from the integration of 'H and13C spectra. Furthermore, integration values are proportional to the amount of BDDE used.

[0175] 2.3. FT-IR

[0176] FT-IR analysis was performed to verify the structure of the samples after the reaction with BDDE. In Figure 6, the superimposition of TSP, C-TSP 1 and C-TSP 8 is reported.

[0177] The similar profile in Figure 6 of the C-TSP samples showed the maintenance of the TSP’s structure after the reaction.

[0178] 2.4. HP-SEC-TDA

[0179] HP-SEC-TDA with multi-detector systems Right Angle and Low Angle Light Scattering (Refractive Index and Viscosimeter), was used to determine the molecular weight distribution, intrinsic viscosity and hydrodynamic radius of TSP and C-TSP_1,_2,_3, which were the only samples with a suitable viscosity for the analysis with this technique. In Table 5, HP-SEC-TDA results are reported.

[0180] Table 5. HP-SEC-TDA results Functionalized C-TSP 2 and C-TSP 3 showed a higher molecular weight and p than the TSP, due to the effect of BDDE. Instead, C-TSP 1 presented similar values to TSP probably because the concentration of the BDDE was very low and consequently the functionalization was lower compared to the other samples. ADV-00748PTITWO

[0181] 2.5. Rheological properties

[0182] Viscosity analysis of soluble functionalized products were performed at 37°C, the physiological temperature, using a double gap geometry, at the concentration of 2 mg / mL. In Figure 7, the viscosity curves of TSP and C-TSP 1, _2 and _3 are reported, where it can be observed that soluble functionalized products maintained the same low viscosity of TSP.

[0183] Viscosities of gels were studied using a parallel plate geometry at two temperatures: 25°C and 37°C. As expected, the viscosities of the samples were very high, between IxlO6and IxlO5mPa s at the shear rate of 1 1 / s, for both the temperature.

[0184] In Figures 8 and 9, the viscosity curves of C-TSP 6 and C-TSP 7 at 20 mg / mL are reported at respectively 20°C and 37°C.

[0185] No great differences were observed in the curves at the two temperatures: at 37°C the viscosities slightly decreased for both temperatures. So, this parameter did not affect much the functionalized structure of the samples, which was quite stable. As expected, at the same concentration C-TSP 7 showed a higher viscosity than C-TSP 6 due to the higher concentration of BDDE used.

[0186] For gels, the elastic moduli G’, storage modulus, and G”, loss modulus, both expressed in Pascal, were also studied because they are important parameters for gels formulation. The values of these moduli are measured by amplitude sweep test, during which the sample is subjected to an increasing shear stress while maintaining a constant temperature and stress frequency. Samples with G‘ lower than G’‘ show a fluid structure (viscoelastic liquid material) while when G’ is greater than G” the structure is solid (viscoelastic solid material). Through this analysis it is possible to identify the shear rate range in which the sample exhibits linear viscoelastic behaviour, so where no material damage is observed. In this way, the linear viscoelastic region (LVE) of the sample is determined, which is crucial for defining the shear rate levels to be applied for a further test called frequency sweep. In the graphs of the amplitude sweep curves, it is sometimes possible to observe the presence of a flow point where the modulus curves meet and the values of G‘ and G” are equal. Beyond this point, an inversion of the moduli occurs where the one that was smaller becomes larger than the other, indicating that the sample has been damaged to such an extent that the change in its physical nature occurs. Once the linear viscoelastic region has been determined, it is possible to perform the frequency sweep test, during ADV-00748PTITWO which the samples are subjected to an oscillatory stress with varying angular frequency. In this way, the viscoelasticity of the material can be measured and information on the internal structure of the polymers and the long-term stability of the dispersions can be gathered, evaluating the behaviour of the sample over time. With high frequencies, rapid movement on a short-term scale is simulated, while with low frequencies, slow movement on a long-term or resting scale is simulated. The results of these analyses are plotted in graphs on a logarithmic scale representing the angular frequency (co) expressed in radians per second (rad / s) or frequency (f) in Hertz (Hz), as a function of the G‘ and G” moduli. To verify the behaviour of the material being analysed, the values of the moduli G‘ and G’‘ are observed at high and low frequencies: if at lower frequencies, G”>G’, the sample will have predominantly viscous behaviour, if at higher frequencies, G’>G”, it will have predominantly elastic properties. To assess the long-term storage behaviour of the dispersion, one looks at the moduli G‘ and G’‘ in the low-frequency region; a value of G’>G” indicates a gel structure, due to a stable network of forces, which thus fulfils the assumption of physical stability of the dispersion; conversely, if G’‘>G’ means that the material is liquid and susceptible to segregation. In frequency sweep graphs, the crossover point, the point at which the elastic modulus curves meet, can be related to the strength of a formulation over time. In fact, the lower the crossover point is at low frequencies, the more resistant the tested sample will be over time, as frequency is inversely proportional to time.

[0187] In Figure 10 and 11 the curves obtained from amplitude and frequency sweep tests of C- TSP_6 and C-TSP 7 at 37°C are reported.

[0188] As expected, in amplitude sweep curves of both samples the G’ modulus was higher than G”, so samples showed a gel structure. The values of the elastic moduli of C-TSP 7 were higher than the ones of C-TSP 6, due to the higher concentration of BDDE used, as happened for the viscosity. A flow point, which determine a change on the structure of the samples, was detected for both samples but at the maximum value of the shear strain (100%).

[0189] As for the amplitude sweep, the values of G’ obtained by the frequency sweep test were higher than G” in both of samples, conforming the solid-like behaviour of the functionalized products both at high and low frequencies. In C-TSP 6 the frequency sweep was reported from 1 to 100 rad / s because the measurement at lower angular ADV-00748PTITWO frequency were not acceptable. In these curves, the crossover point was non detected, indicating resistant structures over time of the samples.

[0190] 2.6. Swelling

[0191] The sweeling capacity (SC) is the ability of a polysaccharide to absorb an amount of liquid. This characteristic is important for some uses of the cross-linked samples, for examples as superabsorbent. Another important parameter is the capacity of releasing the solution absorbed (CT), which is essential for drug delivery. These two values were calculated by relating the absorbed / released weight of the solution to the weight of the cross-linked product.

[0192] Swelling and releasing capacities were determined for C-TSP 6, _7 and _8 in water and in physiological solution of NaCl 0.9%. SC and CT values are reported in Figures 12 and 13.

[0193] All the samples tested showed a very similar behaviour in water and in NaCl 0.9% solution, so they maintained the same capacity independently of the presence of salts. C- TSP_6 and C-TSP 7 had the same SC values with a good capacity of absorption. Instead, C-TSP 8 showed a lower capacity of swelling probably due to a higher degree of crosslinking which reduced the ability of absorption. The same trend occurred for CT values: C-TSP 6 and C-TSP 7 had a higher capacity of releasing the solution than C- TSP 8 both in water and NaCl. In all the samples, CT values were lower than SC values.

[0194] 2. 7. Interaction with mucin

[0195] Mucoadhesion index values for TSP and C-TSP 3 are reported in table 6.

[0196] Table 6. Mucoadhesion index values

[0197] After the functionalization reaction, even in a sample with a low concentration of BDDE, a significant increasing of the mucoadhesion index was observed, i.e. almost 2.5 times higher. Without wishing to be bound by any theory, it is believed that the functionalization positively affect to affinity with the mucin, thus promoting and boosting mucoadhesive properties. ADV-00748PTITWO

[0198] Bibliography

[0199] Ejaz, U., et al. (2021). Cellulases: From Bioactivity to a Variety of Industrial Applications. Biomimetics, 44.

[0200] Guarise, C., et al. (2012). SEC determination of cross-link efficiency in hyaluronan fillers. Carbohydrate Polymers, 428-434.

[0201] Sofia, N., et al. (2024). A novel biomimetic probe for galectin-3 recognition: Chemical synthesis and structural characterization of a P-galactose branched sodium hyaluronate. Proteoglycan research.

[0202] Zhang, H., et al. (2020). An amendment to the fine structure of galactoxyloglucan from Tamarind (Tamarindus indica L.) seed. International Journal of Biological Macromolecules, 1189-1197.

Claims

ADV-00748PTITWOCLAIMS1. A tamarind seed polysaccharide functionalized with BDDE (f-TSP), where BDDE is 1,4-butanediol diglycidyl ether.

2. The functionalized tamarind seed polysaccharide of claim 1, wherein f-TSP is obtained by reacting TSP and BDDE in a molar ratio of 1 :0.25 to 1 :5.

3. The functionalized tamarind seed polysaccharide of claim 1 or 2, wherein f-TSP is obtained by reacting TSP and BDDE in a molar ratio of lower than 1 :1, preferably of 1 :0.25 to 1 :0.9.

4. The functionalized tamarind seed polysaccharide of claim 3, having a weight average molecular weight (Mw) at least 20% higher than the weight average molecular weight of TSP before functionalization, preferably 22-28% higher, as measured by size exclusion chromatography with triple detector array (HP-SEC-TDA).

5. The functionalized tamarind seed polysaccharide of claim 3 or 4, having a number average molecular weight (Mn) at least 10% higher than the number average molecular weight of TSP before functionalization, preferably 12-22% higher, as measured by size exclusion chromatography with triple detector array (HP-SEC-TDA).

6. The functionalized tamarind seed polysaccharide of any one of claims 3-5, having an intrinsic viscosity (p) at least 15% higher than the an intrinsic viscosity of TSP before functionalization, preferably 18-25% higher, as measured by size exclusion chromatography with triple detector array (HP-SEC-TDA).

7. The functionalized tamarind seed polysaccharide of claim 1 or 2, wherein f-TSP is obtained by reacting TSP and BDDE in a molar ratio of equal to or higher than 1 : 1, preferably of 1 : 1 to 1 :3.

8. The functionalized tamarind seed polysaccharide of claim 7, having a viscosity at a shear rate (0.1 s'1, at 20°C and at 37°C) from 50 to 5,000 kPa.s, and featuring a storage (or elastic) modulus G’(co) higher than a loss (or plastic) modulus G” (co) at 37°C.ADV-00748PTITWO9. The functionalized tamarind seed polysaccharide of claim 7 or 8, having a swelling capacity (SC) of 35-60, preferably 45-55, and a releasing capacity (SC) of 25-45, preferably 30-40, as calculated by the teabag method.

10. A tamarind seed polysaccharide functionalized with:- bi- or polyfunctional epoxy selected from 1,2-ethylenediol diglycidyl ether, l-(2,3- epoxypropyl)-2,3-epoxycyclohexane, N,N-diglycidylaniline, epoxy-substituted pentaerythritol, and mixtures thereof, or- divinyl sulfone, or- a biscarbodiimide selected from 1,6-hexamethylene bis(ethylcarbodiimide), 1,8- octamethylene bis(ethylcarbodiimide), 1,10 decamethylene bis(ethylcarbodiimide), 1,12 dodecamethylene bis(ethylcarbodiimide), PEG-bis(propyl (ethylcarbodiimide)), 2,2'- dithioethyl bis(ethylcarbodiimide), l,l'-dithio-p-phenylene bis(ethylcarbodiimide), para- phenylene-bis(ethylcarbodiimide), l,l'-dithio-m-phenylene bis(ethylcarbodiimide) and mixtures thereof.

11. A pharmaceutical composition comprising the functionalized tamarind seed polysaccharide of any one of claims 1-10 and pharmaceutically acceptable excipients.

12. A biomaterial comprising the functionalized tamarind seed polysaccharide of any one of claims 7-10 and a natural, a semisynthetic or a synthetic polymer, wherein the natural polymer is selected from the group consisting of a collagen, a coprecipitate of collagen and glycosaminoglycan, a cellulose, a polysaccharides in the form of a gel selected from the group consisting of chitin, chitosan, pectin, pectic acid, agar, agarose, xanthan, gellan, alginic acid, an alginate, polymannan, a polyglycan, starch, and a natural gum, wherein the semi synthetic polymer is a collagen crosslinked with a crosslinking agent selected from the group consisting of an aldehyde, a precursor of an aldehyde, a dicarboxylic acid, a dicarboxylic acid halogenide, a diamine, a cellulose derivative, hyaluronic acid, chitin, chitosan, gellan, xanthan, pectin, pectic acid, a polyglycan, polymannan, agar, agarose, natural gum and glycosaminoglycans, and wherein the synthetic polymer is selected from the group consisting of polylactic acid, polyglycolic acid, a copolymer of polylactic acid, a derivative of polylactic acid, a copolymer of polyglycolic acid, a derivative of polyglycolic acid, polydioxan, polyphosphazene, a polysulphonic resin, polyurethane andADV-00748PTITWOPTFE.

13. Cosmetic use of the functionalized tamarind seed polysaccharide of any one of claims 1-10, as thickener, gelling agent, stabiliser, moisturizing agent, solubilizer, and / or smoothing agent, in cosmetic products for external topical use.

14. A process for preparing the functionalized tamarind seed polysaccharide of any one of claims 1-9, the process comprising the steps of: i) providing a solution of TSP in NaOH, ii) heating the solution at 45-65°C, then adding BDDE and stirring for at least one hour, iii) dialyzing the resulting functionalized TSP in water, and drying.

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