Derivatives of tamarind seed polysaccharides and their preparation process

Abstract

Derivatives of tamarind seed polysaccharide (TSP) and processes for their preparation are disclosed. Specifically, the derivatives are sulfated TSP having selected degrees of sulfate and molecular weight, exhibiting improved processability, stability, and compatibility with pharmaceutical ingredients. Accordingly, pharmaceutical compositions and biomaterials containing the sulfated TSP are also disclosed. In addition, cosmetic applications of sulfated TSP are reported. Finally, the process for preparing the sulfated TSP is described.

Description

[Technical Field]

[0001] This invention relates to derivatives of tamarind seed polysaccharide (TSP) and processes for preparing them. Specifically, the derivatives are sulfated TSP having selected degrees of sulfate and molecular weight, exhibiting improved processability, stability, and compatibility with pharmaceutical ingredients.

[0002] Therefore, the present invention also relates to pharmaceutical compositions and biomaterials containing the aforementioned sulfated TSP.

[0003] Finally, the present invention also relates to a process for preparing the aforementioned sulfated TSP. [Background technology]

[0004] The term "tamarind seed polysaccharide" refers to the polysaccharide portion that can be obtained from the seeds of Tamarindus indica, and for brevity, it will also be referred to as "TSP" below (from the English term "Tamarindus indica Seed Polysaccharide").

[0005] As is well known, the tamarind tree is commonly found throughout India, Africa, and the Far East, where it is cultivated primarily for food. The seeds, originally a by-product, have since been sometimes ground into a meal-like product (now known as "raw tamarind gum" or "tamarind nut powder"), and have found various uses, particularly in the textile and paper industries where they are used as sizing agents and adhesives for spun yarns, respectively, and in the food industry where they are used as thickeners, gelling agents, stabilizers, and binders in all kinds of products in much the same way as other polysaccharide products such as alginates, pectin, guar gum, or locust bean meal. Raw tamarind gum (for example, marketed as Grilloid®, manufactured by Dainippon Pharmaceutical Co., Ltd. in Osaka, Japan) typically contains 65-73 wt% polysaccharides, 15-23% protein material, 3-8% oils and fats, and 2-4% ash, as well as trace amounts of unrefined fiber, tannins, and further impurities.

[0006] One advantageous aspect is that TSP solutions are preferably sterilized by autoclaving (e.g., at 120°C for 20 minutes) without undergoing thermal decomposition, unlike, for example, hyaluronic acid. The possibility of sterilization by simple autoclaving makes TSP-based preparations particularly convenient from a manufacturing standpoint.

[0007] Furthermore, TSP exhibits considerable mucosal mimicry, mucosal adhesion, and bioadhesion properties.

[0008] TSP (or "raw TSP") is a purified, neutral, water-soluble polysaccharide fraction containing polymer molecules of galactoxyloglucan. It is highly hydrophilic and characterized by a branched structure. A linear backbone formed from glucose repeating units is attached to small xylose monosaccharide units and xylose-galactose disaccharide units. In the latter case, galactose is at the end of the side chain. As illustrated in Figure 1, the three monomers are present in a molar ratio of 3:1:2 and account for approximately 65% ​​of the seed's composition. As can be observed, the "mucin-like" molecular structure determines the polysaccharide's excellent mucosal adhesion properties, which stem from the formation of various types of bonds with the aforementioned mucin.

[0009] TSP can be isolated by chemical and enzymatic methods, using proteases or a combination of proteases and high-intensity ultrasound. In the chemical method, tamarind seed powder is added to cold distilled water to prepare a suspension. This is then added to boiling distilled water. The resulting solution is continuously boiled and stirred. After standing overnight, the solution is centrifuged. The supernatant is separated and added to pure alcohol in a volume equal to twice the volume of the supernatant. Thus, a precipitate is obtained, which is then washed with pure ethanol and air-dried. Finally, the dried polymer is crushed, 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 a protease. The powder is then centrifuged, and ethanol is added to the supernatant for precipitation. Finally, the polymer is separated and dried.

[0010] Natural polymers such as TSP offer advantages over synthetic and semi-synthetic polymers, including lower cost, natural origin, fewer side effects, local availability, and better patient tolerance.

[0011] However, these natural substances also suffer from drawbacks such as purity, source, and microbial contamination. At the same time, TSPs have the additional problem of poor processability due to their complex structure and, as noted above, require lengthy pretreatment to be suitable for subsequent preparation. This was also confirmed by the very recent publication by Effendi AD et al. dated March 2022 ("Polysaccharides from Tamarindus indica L. as natural kinetic hydrate inhibitor at high subcooling environment," Journal of Petroleum Exploration and Production Technology (2022), Vol. 12: pp. 2711-2722). Here, on page 2714, a very complex and time-consuming procedure for extracting TSP from tamarind seed powder is disclosed. Specifically, the authors report the following procedure:

[0012] "First, 20 g of TSP powder was immersed in 800 mL of distilled water for 24 hours. To ensure the release of mutile material into the distilled water, the mixture was heated at 100°C for 1 hour and then allowed to stand for 2 hours. To remove all impurities, the solution was centrifuged at 6000 rpm for 20 minutes. The supernatant was separated from the remainder of the mixture. An equal volume of acetone was added to the supernatant for mutile material precipitation. The precipitate was collected using a stainless steel filter and dried in an oven at 50°C for 4 hours. The dried polymer was stored in a desiccator until it was needed again. The dried polymer was mixed with distilled water to create polysaccharide inhibitors of various concentrations." The authors explain that tamarind xyloglucan has properties similar to other polysaccharides in that its individual chains are still water-soluble even when not fully hydrated, but it was able to form a colloidal dispersion in aqueous solution due to its balanced hydrophobic and hydrophilic properties. Consequently, this is likely the reason behind the very long and complex procedure.

[0013] Later, on page 2715, the authors add: "Although polysaccharides are economical and easy to obtain, they are typically insoluble in water or have high viscosity after dissolving in water, making them difficult to inject into pipelines during use. Either their water solubility must be increased or new injection procedures must be developed (Wang et al., 2019). Solubleness is related to the hydrophilic and hydrophobic chains present on the polymer. Polymers with a predominance of hydrophilicity will allow for better solubilization. This is indirectly related to the inhibition mechanism by adsorption. Therefore, the solubility of TSPs is important as it allows TSPs to be mixed with multiphase fluids consisting mainly of water and hydrocarbons. Accordingly, dry polysaccharides were subjected to solubility tests using distilled water as the solvent under two separate conditions: room temperature water and boiling water." For this purpose, the following procedure is described on page 2717. "Since polysaccharides typically have low solubility, dry TSP was subjected to solubility tests using distilled water as the solvent under two separate conditions: room temperature water and boiling water. The study found that the dry polymer was poorly soluble in room temperature water but appeared to solubilize rapidly in boiling water. The results indicate that TSP requires solubilization with boiling water for effective injection into sites where hydrate formation is likely. Since efficient KHI depends on the adsorption affinity of KHI, the solubilization of TSP is closely related to KHI performance. Therefore, TSP efficiency can be further improved by improvements such as grafting hydrophilic groups or the use of microcapsules." On page 2720, the authors conclude that "TSP is poorly soluble in room temperature water but appears to solubilize rapidly in boiling water. This information helps operators solubilize the polymer in a hotter fluid during injection. Since the objective is to prevent hydrate formation, the injection of additional hotter fluid would contribute to hydrate prevention techniques that should be encapsulated."

[0014] However, it should be considered that Effendi et al. are referring to a specific technical field, as is evident from the abstract, for example ("In offshore systems, hydrocarbon fluids are typically produced in deeper parts of the ocean, and long pipelines are used to deliver the fluids over long distances. Subsequently, these practices tend to expose the water-containing hydrocarbon fluids before treatment to lower temperatures and higher pressures that favor hydrate formation. One solution to this problem is to introduce hydrate inhibitors, preferably low-dose hydrate inhibitors (LDHIs). More versatile kinetic hydrate inhibitors (KHIs) of LDHIs can be further optimized in terms of cost and their biodegradability. [...] The results show that TSPs can delay hydrate formation at high subcooling levels. TSPs remain relatively economical and biodegradable while performing well at low concentrations at high subcooling levels").

[0015] In fact, the solutions proposed and described by Effendi et al., namely boiling TSP in water and / or using microcapsule technology, cannot be borrowed for the pharmaceutical field, where the requirements are more stringent and TSP should be processed without negatively affecting its inherent properties. Indeed, high temperatures, especially when exposed to high temperatures for extended periods, lead to the depolymerization of the TSP mucopolysaccharide chain. This degradation renders TSP unsuitable for pharmaceutical formulations because the degradation products will make the pharmaceutical preparation highly unstable and ineffective. Furthermore, the encapsulation method may only be suitable for TSP with low molecular weight, which is not applicable to pharmaceutical applications.

[0016] Based on our experience, in order to maintain its physicochemical properties and make it usable for further preparations, dried TSP, such as freeze-dried TSP, needs to be redissolved in water at room temperature, resulting in immersion of the dried TSP in water for several hours, i.e., at least 6 hours. In fact, as the dried TSP becomes purer and its molecular weight increases, the immersion time required to regenerate properly rehydrated TSP increases.

[0017] Therefore, it is an object of the present invention to improve the overall processability and economic convenience of TSP in the context of pharmaceutical manufacturing while simultaneously achieving good stability over time and higher compatibility with other pharmaceutical components.

Summary of the Invention

[0018] The above object is achieved by the sulfated tamarind seed polysaccharide (TSP) described in claim 1.

[0019] In a further aspect, the present invention relates to a pharmaceutical composition comprising the sulfated TSP.

[0020] In a further aspect, the present invention relates to a biomaterial comprising the sulfated TSP.

[0021] In another aspect, the present invention relates to the cosmetic use of the sulfated TSP.

[0022] In a further aspect, the present invention relates to a process for the preparation of the sulfated TSP.

[0023] The features and advantages of the present invention are provided by way of non-limiting examples and will become apparent from the following detailed description of the embodiments illustrated by the drawings attached hereto.

Brief Description of the Drawings

[0024] [Figure 1] Figure 1 shows the structure of the octasaccharide repeating unit of unprocessed tamarind seed polysaccharide (briefly "TSP"). [Figure 2] Figure 2 shows the 1H NMR spectrum of unprocessed TSP. [Figure 3] Figure 3 shows the HSQC-DEPT spectrum of unprocessed TSP. [Figure 4] Figure 4 shows the HSQC-DEPT spectra of unprocessed TSP and the sulfated TSP of the present invention according to Example 1. [Figure 5]Figure 5 shows a comparison between the 13C-NMR spectra of raw TSP according to Example 1 and the sulfated TSP of the present invention. [Figure 6] Figure 6 shows the viscosity value as a function of shear rate of the sulfated TSP of the present invention according to Example 1. [Figure 7] Figure 7 shows the HSQC-DEPT spectra of the unprocessed TSP according to Example 2 and the sulfated TSP of the present invention. [Figure 8] Figure 8 shows a comparison between the 13C-NMR spectra of raw TSP according to Example 2 and the sulfated TSP of the present invention. [Figure 9] Figure 9 shows the viscosity variation as a function of shear rate for the sulfated TSP of the present invention according to Example 2. [Figure 10] Figure 10 shows the HSQC-DEPT spectra of the unprocessed TSP according to Example 3 and the sulfated TSP of the present invention. [Figure 11] Figure 11 shows a comparison between the 13C-NMR spectra of raw TSP according to Example 3 and the sulfated TSP of the present invention. [Figure 12] Figure 12 shows the viscosity variation as a function of shear rate for the sulfated TSP of the present invention according to Example 3. [Modes for carrying out the invention]

[0025] The present invention therefore relates to a sulfated tamarind seed polysaccharide (TSP) having a weight-average molecular weight of 600 to 1,500 kDa and a degree of sulfate (DS) of 1 to 10 per octase repeating unit, wherein the DS is determined by electrical conductivity titration and calculated according to the following formula:

number

[0026] Specifically, the degree of substitution (DS) represents the average number of sulfone groups linked to the octase repeating unit of the raw TSP and is calculated by the above formula, where "1207 g / mol" is the weight of the average repeating unit of the TSP and "80 g / mol" is the weight difference between the -OS3H group and the -OH group.

[0027] As described above and shown in Figure 1, TSP is composed of sugar units containing α-D-xylopyranose, β-D-galactopyranose, and glucose, linked by glycosidic bonds to form a branched polymer.

[0028] The selected DS range of 1 to 10, combined with the weight-average molecular weight, was observed to provide a favorable balance between the degree of sulfatedness and the protection of the starting TSP backbone. In fact, when a degree of sulfatedness higher than 10 was assigned, unwanted depolymerization of the TSP was observed, while a degree of sulfatedness lower than 1 had no significant effect on the TSP properties.

[0029] Furthermore, it was found that, surprisingly, all that was required to redissolve and make it available for further preparations was immersion of the dried sulfated TSP in water for just 3 hours at room temperature. This means that once sulfated to the degree shown above, TSP becomes more processable and stable over time, and is more compatible with other pharmaceutical ingredients.

[0030] Preferably, the sulfated tamarind seed polysaccharide has a degree of sulfate (DS) of 1 to 5 per octase repeating unit.

[0031] The molecular weight of sulfated TSP is another important parameter that affects its properties and biological activity. The range shown above means that the sulfated process is carried out successfully without degradation or depolymerization of the starting TSP.

[0032] Preferably, the sulfated tamarind seed polysaccharide has a weight-average molecular weight of 700 to 1,100 kDa.

[0033] Preferably, the sulfated TSP of the present invention has a viscosity of 20 to 60 mPa*s at a shear rate of 10 1 / s and a temperature of 20°C. The corresponding starting TSP has a viscosity of 80 mPa*s or more, and therefore it should be noted that the sulfated TSP clearly shows a decrease in viscosity, making it more processable.

[0034] Preferably, the sulfated TSP of the present invention has a zeta potential of -20.00 to -50.00 mV as measured by dynamic light scattering (DLS). The corresponding starting TSP has a zeta potential of -0.36 mV or higher (i.e., a neutral polymer), and therefore, it should be noted that, clearly, the sulfated TSP is an electropolymer, making it more compatible with water-soluble (or polar solvent-soluble) components.

[0035] In some embodiments, sulfated TSP is converted into a salt with a heavy metal or active pharmaceutical ingredient.

[0036] Preferably, the heavy metal is a metallic element selected from groups 4, 5, and 6 of the periodic table.

[0037] More preferably, the heavy metal is silver, cobalt, iron, copper, zinc, arsenic, strontium, zirconium, antimony, gold, cesium, tungsten, selenium, platinum, ruthenium, bismuth, tin, titanium, or mercury.

[0038] Preferably, the active pharmaceutical ingredient is selected from the group consisting of antibiotics, anti-infective agents, antibacterial agents, antiviral agents, cell division inhibitors, antitumor agents, anti-inflammatory agents, wound treatment agents, anesthetics, anticholinergic agents, adrenergic agents, antithrombotic agents, anticoagulants, hemostatic agents, fibrinolytic agents, thrombolytic agents, proteins, protein fragments, peptides, and polynucleotides.

[0039] In another embodiment, the present invention relates to a pharmaceutical composition comprising the above-mentioned sulfated tamarind seed polysaccharide and a pharmaceutically acceptable excipient.

[0040] The term "excipient" refers to a compound or mixture of compounds suitable for pharmaceutical use. For example, excipients used in pharmaceutical-grade formulations generally should not cause harmful reactions in the target substance and should not significantly impair the effectiveness of the sulfated TSP contained therein. Suitable excipients include acidifiers, acidity regulators, anticaking agents, antioxidants, bulking agents, resistive agents, gelling agents, glazing agents, modified starches, metal ion chelating agents, thickeners, sweeteners, diluents, de-agglomerates, lubricants, dyes, binders, lubricants, stabilizers, adsorbents, humectants, fragrances, film-forming substances, emulsifiers, wetting agents, release inhibitors, and mixtures thereof.Preferably, the excipients are olive oil, mineral oil, liquid paraffin, white petrolatum, polyoxyethylene, emulsifying wax, stearyl alcohol, isostearyl alcohol, cetyl stearyl alcohol, stearic acid, glyceryl stearate, sodium lauryl sarcosinate, glycerin, diethylene glycol monoethyl ether, polyethylene glycol, polyethylene glycol, polyethylene glycol stearate, Carbopol, carbomer, poloxamer 407, macrogol 400, purified bentonite, myristyl propionate, dimethicone, titanium dioxide, anionic, cationic, and nonionic surfactants, water, potassium sorbate, sodium benzoate, ε-polylysine, sucralose, maltodextrin, citric acid, sodium carbonate, calcium carbonate, magnesium carbonate, magnesium stearate, natural starch, partially hydrolyzed starch, modified starch, lactose, calcium phosphate, calcium carbonate, calcium sulfate, polyvinylpyrrolidone, silica, colloidal russet Rica, precipitated silica, magnesium silicate, aluminum silicate, sodium lauryl sulfate, magnesium lauryl sulfate, methacrylate copolymer, sodium dehydroacetate, xanthan gum, guar gum, tara gum, carob gum, fenugreek gum, gum arabic, alginic acid, sodium alginate, propylene glycol alginate, croscarmellose sodium, polyvinylpolypyrrolidone, glyceryl behenate, indigo carmine, cellulose, modified cellulose, calcium carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, ethylcellulose, gelatin, hydroxyethylcellulose, hydroxypropylcellulose, polydextrose, carrageenan, methylcellulose, sucrose, sucrose ester, sorbitol, xylitol, dextrose, maltitol, tragacanth gum, pectin, agar, carboxypolymethylene, hydroxypropylmethylcellulose, tragacanth gum, mannitol, or mixtures thereof.

[0041] In another embodiment, the present invention relates to a biomaterial comprising the above-mentioned sulfated tamarind seed polysaccharide and a natural, semi-synthetic, or synthetic polymer, wherein the natural polymer is selected from the group consisting of collagen, co-precipitates of collagen and glycosaminoglycans, cellulose, and polysaccharides in gel form selected from the group consisting of chitin, chitosan, pectin, pectic acid, agarose, xanthan gum, guerine, alginic acid, alginate, polymannan, polyglycan, starch, and natural gum, wherein the semi-synthetic polymer is an aldehyde, aldehyde precursor, dicarboxylic acid, dicarb The collagen is crosslinked with a crosslinking agent selected from the group consisting of hydroxyl halides, diamines, cellulose derivatives, hyaluronic acid, chitin, chitosan, guerlain, xanthan gum, pectin, pectic acid, polyglycan, polymannan, agar, agarose, natural gum, and glycosaminoglycan. Here, the synthetic polymer is selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid copolymer, polylactic acid derivative, polyglycolic acid copolymer, polyglycolic acid derivative, polydioxane, polyphosphazene, polysulfone resin, polyurethane, and PTFE.

[0042] In a further embodiment, the present invention relates to cosmetic applications of the above-mentioned sulfated tamarind seed polysaccharide as a thickening agent, gelling agent, stabilizer, humectant, solubilizer, and / or smoothing agent.

[0043] When the sulfated TSP of the present invention is used in cosmetic products, 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, where the composition is preferably a suspension or dissolved in one or more suitable excipients.

[0044] Examples of suitable cosmetically acceptable excipients include mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, stearyl alcohol, isostearyl alcohol, cetyl stearyl alcohol, stearic acid, glyceryl stearate, sodium lauryl sarcosinate, glycerin, diethylene glycol monoethyl ether, polyethylene glycol, polyethylene glycol stearate, starch, Carbopol, carbomer, methylparaben, poloxamer 407, macrogol 400, purified bentonite, propylparaben, myristyl propionate, dimethicone, titanium dioxide, anionic, cationic, and nonionic surfactants, water, and mixtures thereof.

[0045] The sulfurized TSP of the present invention i) A step of providing a suspension of TSP in a tertiary amine solvent, ii) Adding a sulfur trioxide complex with an organic radical selected from the group consisting of pyridine, DMF, trimethylamine, dioxane, N,N-dimethylaniline, B',B-dichlorodiethyl ether, and mixtures thereof, under stirring at a temperature of 15-60°C, thereby obtaining a dispersion of sulfated TSP. iii) Add water to the dispersion to obtain a homogeneous mixture and adjust the pH to 6-7. iv) The step of adding an alcohol-based solvent to the mixture in order to promote the precipitation of sulfated TSP, v) A step of separating and purifying sulfated TSP, It can be prepared according to a process that includes

[0046] This preparation process makes it possible to obtain the sulfated TSP of the present invention in a yield of at least 90%.

[0047] In step i), the TSP is preferably suspended in a tertiary amine solvent. This enhances the nucleophilicity of the hydroxyl groups on the TSP backbone.

[0048] Preferably, the tertiary amine solvent is selected from DMF (dimethylformamide), pyridine, trimethylamine, picoline, N,N-dimethylaniline, quinoline, and mixtures thereof.

[0049] In a preferred embodiment, the tertiary amine solvent is DMF, pyridine, trimethylamine, or a mixture thereof.

[0050] Preferably, the weight-average molecular weight of the (raw) TSP in step i) is 450-750 kDa, more preferably 550-700 kDa. In fact, TSPs with lower molecular weights tend to depolymerize, and as a result, the sulfation reaction may further accelerate degradation, but TSPs with higher molecular weights are, in principle, less suitable for pharmaceutical applications.

[0051] In step ii), the sulfur trioxide complex is added to the suspension of TSP from step i).

[0052] Specifically, the sulfur trioxide is complexed with an organic radical selected from the group consisting of pyridine, DMF, trimethylamine, dioxane, N,N-dimethylaniline, B',B-dichlorodiethyl ether, and mixtures thereof.

[0053] The complex acts as a sulfating agent; that is, the sulfate group migrates to the TSP, releasing an organic radical as the corresponding neutral compound. This can then be used as a solvent when carrying out the process.

[0054] Step ii) is carried out under stirring at a temperature of 15–60°C to obtain a dispersion of sulfated TSP. Preferably, the temperature is room temperature, i.e., 20–25°C. However, if a higher temperature is selected, the resulting dispersion of sulfated TSP is cooled to room temperature before carrying out step iii).

[0055] Preferably, in step ii), the molar ratio of the complex and TSP is 1:1 to 1:10, more preferably 1:1 to 1:5. It has been experimentally observed that as the ratio between the sulfating agent and TSP increases, more unwanted TSP depolymerization occurs. Therefore, these ranges provide a better balance between the degree of sulfation and the protection of the starting TSP backbone.

[0056] In step iii), water is added to the dispersion to obtain a homogeneous mixture and adjust the pH to neutral, i.e., 6-7.

[0057] pH can be adjusted by adding inorganic bases such as NaOH, KOH, or NH4OH.

[0058] In step iv), an alcoholic solvent is added to the homogeneous mixture obtained in step iii) to promote the precipitation of sulfated TSP.

[0059] Preferably, the alcohol-based solvent is methyl alcohol, ethyl alcohol, propyl alcohol, or a mixture thereof, and is optionally diluted with water.

[0060] In step v), the precipitated sulfated TSP is separated and purified.

[0061] Preferably, separation is carried out by filtration, centrifugation, or a combination thereof.

[0062] Preferably, the separated sulfated TSP is then purified by dissolution in water, neutralization to pH 6-7, and reprecipitation by adding an alcoholic solvent, which may be the same as in step iv). Purification allows for further removal of solvent residue, neutral compounds derived from the complex, unreacted reagents, and inorganic salts.

[0063] To determine the complete removal of sodium sulfate, a barium test was used. Simply put, an aqueous solution of barium chloride (10% w / v) was added to the aqueous solution of the sulfuric acid product. If a precipitate formed, it indicated that barium sulfate (which is almost insoluble in water, 2.5 × 10⁻⁶) was present. -3 (g / L), sodium sulfate was present in the solution. If the solution remains clear, it indicates that the inorganic salt (sodium sulfate) has been completely removed.

[0064] In a preferred embodiment, the sulfated TSP obtained from step v) is then freeze-dried for long-term storage.

[0065] In the most preferred embodiment, the preparation process is carried out as follows:

[0066] Tamarind seed polysaccharide (TSP-500 mg) was suspended in dry dimethylformamide (DMF, 50 ml) in a three-necked flask at a selected temperature and stirred overnight at room temperature. Then, sulfur trioxide-pyridine complexes were added in different molar / residue ratios of SO3-py / polysaccharide (1:1, 1:2, and 1:4). The resulting dispersions were vigorously stirred for 24 hours at different temperatures (room temperature or 50°C). After dilution with water and cooling to room temperature, a homogeneous mixture was obtained, the pH of which was adjusted to 6-7 with NaOH solution (1N), and then alcohol (EtOH / H2O, 70% v / v) was added to precipitate the sulfated TSP recovered by centrifugation. Then, the solid sulfated TSP is dissolved in water and, after careful neutralization with a sodium hydroxide solution, is re-precipitation with 70% v / v ethanol to remove DMF, pyridine, and excess sulfating agents, as well as the possible decomposition products of the latter (Na2SO4), before freeze-drying.

[0067] The kinetics of TSP sulfation were studied at 50°C by varying the reaction time in step ii) to 15 minutes, 24 hours, and 48 hours. It was observed that a reaction time of 24–48 hours was preferable because the sulfated TSP obtained in this way is advantageously protected from chemical depolymerization that occurs as a result of prolonged exposure to acidic conditions.

[0068] Furthermore, the effect of temperature on the reaction was studied by testing step ii) at different temperatures of 25, 50, 60, and 95°C. It was observed that temperatures of 15–60°C are preferred, as this was found to minimize the risk of depolymerization of the TSP backbone. Although we do not wish to be constrained by theory, this temperature range is thought to allow for a regular and uniform distribution of sulfate groups on the TSP chain, particularly in high molecular weight TSPs. More preferably, the temperature in step ii) is about 50°C.

[0069] In a preferred embodiment, step ii) is carried out at a temperature of 15 to 60°C for 24 to 48 hours.

[0070] Finally, the effectiveness of the sulfating agents was evaluated by testing different complexes, namely sulfur trioxide pyridine complex (abbreviated as "SO3*Py"), sulfur trioxide trimethylamine complex (abbreviated as "SO3*Me3N"), and sulfur trioxide N,N'dimethylformamide complex (abbreviated as "SO3*DMF"), all of which showed sufficient performance in sulfating TSP.

[0071] It should also be understood that all combinations of preferred embodiments of the sulfated TSPs reported above, as well as products containing them, their preparations, and uses, are disclosed herein and should be considered equally preferred.

[0072] It should also be understood that all preferred combinations of the sulfated TSP, preparation process, and uses of the present invention disclosed above are described herein.

[0073] Below are examples of the present invention provided for illustrative purposes. [Examples]

[0074] material The samples and reagents used in the following examples are listed in Tables 1 and 2 below, respectively.

[0075] [Table 1]

[0076] [Table 2]

[0077] device The apparatus used in the following examples is reported below. • Heating plates: IKA RCT Basic and IKA C-MAG HS7, • Thermo Shaker: PSC32, PHMT, Grant Bio, Vortex: Velp Scientifica. • Zetasizer Nano ZS (Malvern Panalytical) • Rheometer: Modular compact rheometer MCR92, Anton Paar. • HP-SEC-TDA system: Model Viscotek-TDA302 • NMR: Bruker Avance 500 Neo instrument with 5mm cryoprobe. • 888Titrando System

[0078] method The zeta potential (Zp) of molecules in the test samples was evaluated by dynamic light scattering (DLS) techniques using the following parameters.

[0079] Zeta potential Material: Polystyrene latex; Dispersant: Water; Cuvette: Disposable polystyrene cuvette; Temperature: 40℃; Measurements: 3; Runs: 10; Delay between measurements: 10s; Measurement type: General purpose. Temperature: 40℃.

[0080] The sample was solubilized in ionized water at a concentration of 1 mg / ml.

[0081] Rheometer Characterization The rheological properties of the samples were studied at a temperature of 20°C using a modular compact rheometer MCR92 (Anton Paar GmbH, Graz, Austria) with the measurement system DG26.7 (double gap shape). Viscosity measurements were performed in rotation mode, and the results were measured over 1-1000 seconds. -1 The range was investigated using a logarithmic ramp, and 10 points were measured per decade.

[0082] The sample was solubilized in deionized water at a concentration of 10 mg / ml.

[0083] HP-SEC-TDA The detector included refractive index, right-angle and low-angle light scattering, and viscometer detectors. It was used under the following conditions: • Columns: 2 columns TSKGelGPWXL 13μm, 7.8mm ID × 30cm L, Tosoh Bioscience. ·Mobile phase: 0.3M AcONa+NaN30.05%, pH~8.1 • Injection volume: 100 μl, ·Temperature: 40℃, ·Flow rate: 0.6ml / min.

[0084] The system was calibrated using a certified pullulan standard (PolyCAL-Pullulan STD-Malvern Panalytical) with a molecular weight, polydispersity index, and intrinsic viscosity.

[0085] The sample was solubilized at a concentration of 1 mg / mL in 0.3 M AcONa + NaN3 0.05% (pH ~ 8.1).

[0086] NMR The HSQC-DEPT experiment was performed using a Bruker Avance 500 Neo instrument equipped with a 5 mm cryoprepole.

[0087] Measurement parameters: • Pulse sequence: hsqcedetgpsisp2.2, • Number of scans (NS): 24 • Pulse delay (D1): 2s ·Temperature: 313K, • 1J(C,H): 133Hz. ·Sweep width (SW): 7.9ppm (F2), 160ppm (F1). • Irradiation frequency: (O1): 4.7 ppm (F2), 80 ppm (F1) ·Time domain (TD): 2048 (F2), 320 (F1).

[0088] Processing parameters: • Spectral size (SI): 1024 (F2), 1024 (F1) • Window Multiplication: QSINE shifted in both directions.

[0089] 1 The H spectrum was measured under the following conditions. • Pulse program: zgcppr.ricb • Irradiation frequency (O1): 2353.60Hz • Spectral width (SW): 18 ppm ·Time domain (TD): 32768 • Pulse delay (D1): 12s • Number of scans (NS): 16 ·Temperature: 313K

[0090] 13 The C spectrum was measured under the following conditions. • Pulse program: zggppgse.t1.ez • Irradiation frequency (O1): 12576.11Hz • Spectral width (SW): 294 ppm ·Time domain (TD): 32768 • Pulse delay (D1): 1s • Number of scans (NS): 10000 ·Temperature: 313K

[0091] The sample was solubilized in deuterium oxide (D2O) at a concentration of 8-10 mg / ml.

[0092] Titrator Electrical conductivity titration was performed using the 888Titrando system. The following conditions were used: • Titrate: NaOH 0.1N • Volume increment: 0.150 mL • Stopping volume: 10 mL • Stirring speed: 4

[0093] Approximately 150 mg of each sample was solubilized in the correct amount of deionized water to obtain a homogeneous solution. Then, the solution was subjected to ion exchange resin (Amberlite IR-120 (H + It was converted to its acid form using )) and then titrated by adding 0.1N sodium hydroxide solution.

[0094] Figure 1 shows the structure of the octase repeating unit of raw TSP, and Figure 2 shows the structure of raw TSP. 1 Figure 3 shows the 1H NMR spectrum and the HSQC-DEPT spectrum of the raw TSP.

[0095] Example 1 Tamarind seed polysaccharide (500 mg; 0.41 mmol; 1 eq.) was suspended in dry dimethylformamide (DMF, 50 ml), and the mixture was stirred overnight at room temperature. Then, 1.38 g (8.7 mmol; 1 eq.) of sulfur trioxide-pyridine complex (SO3-Py) was added. The resulting finely dispersed suspension was vigorously stirred at atmospheric pressure for 24 h at room temperature. After dilution with water (10 ml) and cooling to room temperature, a homogeneous mixture was obtained, the solution pH was adjusted to 6 - 7 with NaOH solution (1 N), and then precipitated with alcohol (EtOH / H2O, 70% v / v). Sulfated TSP was recovered by centrifugation. The solid was dissolved in water (40 ml), carefully neutralized with sodium hydroxide solution, and precipitated again with 70% v / v ethanol to remove DMF, pyridine, and the sulfate excess of the sulfating agent, as well as possible decomposition products thereof (Na2SO4) before lyophilization. 595 mg of sulfated TSP (briefly referred to as "P7351") was obtained, meaning a yield of approximately 95%. Barium chloride test: negative

[0096] The achievement of the sulfated sample (TSPS-P7351) was 13 confirmed by 13C-NMR spectra and conductometric titration. The results showed that TSP was sulfated to give the sulfated polysaccharide P7351 with a degree of substitution DS (モル) = 3.16

[0097] Then, 5.31 mg of P7351 was solubilized in 0.6 mL of D2O for NMR studies.[[ID=**13**]] [[ID=**14**]]

[0098] [[ID=**15**]] [[ID=**16**]]In Figures 4 and 5, the HSQC-DEPT and[[ID=**17**]] 13The 1C spectra are being compared. Due to the presence of sulfate groups, the signal of sample P7351 is shifted compared to that of P7300. Carbons directly bonded to the sulfone group may shift to lower field positions, while others indirectly bonded to the sulfone group will shift to higher field positions. The decrease in peak intensity at δ69.97 ppm indicates that the hydroxyl group on C-6 of galactose and unsubstituted backbone glucose residues is partially substituted by the sulfone group.

[0099] The degree of substitution (DS) was determined by electrical conductivity titration as follows.

[0100] 140 mg of P7351 is used with ion exchange resin (Amberlite IR-120 (H + The free acid was converted using )), and then titrated by adding 0.1N sodium hydroxide solution.

[0101] The calculated number of sulfate groups on the repeating polysaccharide unit is 3.16, which corresponds to 15.1% of the total substituted hydroxyl groups (21) in the repeating unit of the TSP shown in Figure 1.

[0102] The zeta potentials of P7300 and P7351 were also determined by dynamic light scattering as follows.

[0103] 5 mg of P7300 was solubilized in 10 mL of deionized water, and 3 mg of P7351 was dissolved in 3 mL of deionized water. TSP is a neutral polysaccharide, and its Zp value is indeed -0.356 mV. P7351, on the other hand, has a Zp value of -38.2 mV due to the negative charge of its sulfate group.

[0104] The molecular weights of P7300 and P7351 were evaluated by HP-SEC-TDA as follows.

[0105] Both samples were solubilized in 5 mL of 0.3 M AcONa + 30.05% NaN (pH ~8.1). The molecular weight of P7300 is 613 kDa, and the molecular weight of P7351 is 965 kDa. The increase in molecular weight of derivative P7351 is attributable to the presence of sulfate groups.

[0106] To determine the viscosity of the solution, 100 mg each of P7300 and P7351 were solubilized in 10 mL of deionized water. The viscosity variation as a function of shear rate was measured for both P7300 and P7351 using a double-gap configuration. Shear rate (γ) range: 1 s -1 ~100s -1 The viscosity curves for both products are reported at a temperature of 20°C, as shown in Figure 6.

[0107] Unprocessed TPS P7300 exhibits a higher viscosity than sulfated P7351. At a shear rate of 10 1 / s, P7300 has a viscosity of 86.4 mPa·s, while P7351 has a viscosity of 42.7 mPa·s. This difference can be attributed to the presence of charged groups, namely sulfate groups, which affect the structural density of TPS.

[0108] Example 2 Tamarind seed polysaccharide (500 mg; 0.41 mmol; 1 eq.) was suspended in dry dimethylformamide (DMF, 50 ml), and the mixture was stirred overnight at room temperature. Then, 1.38 g (8.7 mmol; 1 eq.) of sulfur trioxide-pyridine complex (SO3-Py) was added. The resulting finely dispersed suspension was vigorously stirred at atmospheric pressure at 50°C for 24 hours. After dilution with water (10 ml) and cooling to room temperature, a homogeneous mixture was obtained. The solution pH was adjusted to 6-7 with NaOH solution (1N), and then precipitated with alcohol (EtOH / H2O, 70% v / v). Sulfated TSP was recovered by centrifugation. The solid was dissolved in water (40 ml), and after careful neutralization with sodium hydroxide solution, it was again precipitated with ethanol (70% v / v) to remove DMF, pyridine, and excess sulfating agent, as well as any possible decomposition products of the latter (Na2SO4), before lyophilization. 557 mg of sulfated TSP (simply referred to as "P7352") was obtained, representing a yield of approximately 94%. Barium chloride test: Negative

[0109] The content of linked ester sulfate groups in the P7352 sample 13 This was confirmed by 13C-NMR spectroscopy and electrical conductivity titration. The analysis showed that TSP was sulfated, and the degree of substitution was DS. (モル) This demonstrated that it yields the sulfated polysaccharide P7352 having a ratio of =2.29.

[0110] Then, 6.5 mg of P7352 was solubilized in 0.6 mL of D2O for NMR studies.

[0111] In Figures 7 and 8, HSQC-DEPT and P7351 are shown. 13 We are comparing C spectra.

[0112] Sulfated sample P7352 13 The 1C-NMR spectrum shows the presence of a new signal at δ=69.93, indicating substitution of the -CH2OH group after the sulfation reaction.

[0113] The degree of substitution (DS) was determined by electrical conductivity titration as follows.

[0114] 150 mg of P7352 is used with ion exchange resin (Amberlite IR-120 (H + The free acid was converted using )), and then titrated by adding 0.1N sodium hydroxide solution.

[0115] The calculated number of sulfate groups on the repeating polysaccharide unit is 2.3, which corresponds to 11% of the total substituted hydroxyl groups (21) in the repeating unit of the TSP shown in Figure 1.

[0116] The zeta potential of P7352 was also determined by dynamic light scattering as follows.

[0117] 3 mg of P7352 was dissolved in 3 mL of deionized water. Due to the negative charge of the sulfate group, P7352 has a Zp value of -37.8 mV.

[0118] The molecular weight of P7352 was evaluated by HP-SEC-TDA as follows.

[0119] A 5 mg sample was solubilized in 5 mL of 0.3 M AcONa + 30.05% NaN (pH ~ 8.1). The molecular weight of P7352 is 804 kDa. The increase in molecular weight of derivative P7352 is attributable to the presence of sulfate groups.

[0120] To determine the viscosity of the solution, 100 mg of P7352 was solubilized in 10 mL of deionized water. The viscosity variation as a function of shear rate was measured for P7352 using a double-gap shape. Shear rate (γ) range 1 s -1 ~100s -1 The viscosity curve for this product is reported at a temperature of 20°C, as shown in Figure 9.

[0121] P7352 has a viscosity of 31.2 mPa·s at a shear rate of 10 1 / s.

[0122] Example 3 Tamarind seed polysaccharide (500 mg; 0.41 mmol; 1 eq.) was suspended in dry dimethylformamide (DMF, 50 ml), and the mixture was stirred overnight at room temperature. Then, 1.33 g (8.7 mmol; 1 eq.) of sulfur trioxide-N,N'dimethylformamide complex (SO3-DMF) was added. The resulting finely dispersed suspension was vigorously stirred at room temperature and atmospheric pressure for 24 hours. After dilution with water (10 ml) and cooling to room temperature, a homogeneous mixture was obtained. The solution pH was adjusted to 6-7 with NaOH solution (1N), and then precipitated with alcohol (EtOH / H2O, 70% v / v). Sulfated TSP was recovered by centrifugation. The solid was dissolved in water (40 ml), carefully neutralized with sodium hydroxide solution, and then re-precipitation with 70% v / v ethanol to remove DMF, pyridine, and excess sulfating agents, as well as any possible decomposition products of the latter (Na2SO4), before lyophilization. 515 mg of sulfated TSP (briefly referred to as "P7353") was obtained, representing a yield of approximately 94%. Barium chloride test: negative.

[0123] The content of linked ester sulfate groups in the P7353 sample 13 This was confirmed by 13C-NMR spectroscopy and electrical conductivity titration. The results showed that TSP was sulfated, and the degree of substitution was DS. (モル) This demonstrated that it yields the sulfated polysaccharide P7353 having a ratio of =1.27.

[0124] 5.34 mg of P7353 was solubilized in 0.6 mL of D2O for NMR studies.

[0125] Figures 10 and 11 compare the HSQC-DEPT spectra of P7300 and P7352.

[0126] Due to the presence of sulfate groups, the signal from sample P7353 is shifted compared to that of P7300.

[0127] The degree of substitution (DS) was determined by electrical conductivity titration as follows.

[0128] 150 mg of P7353 was converted to its free acid using an ion exchange resin (Amberlite IR-120(H+)), and then titrated by adding 0.1 N sodium hydroxide solution.

[0129] The calculated number of sulfate groups on the repeating polysaccharide unit is 1.2, which corresponds to 5.7% of the total substituted hydroxyl groups (21) in the repeating unit of the TSP shown in Figure 1.

[0130] The zeta potential of P7353 was also determined by dynamic light scattering as follows.

[0131] 3 mg of P7353 was dissolved in 3 mL of deionized water. Due to the negative charge of the sulfate group, P7353 has a Zp value of -24.6 mV.

[0132] The molecular weight of P7353 was evaluated by HP-SEC-TDA as follows.

[0133] A 5 mg sample was solubilized in 5 mL of 0.3 M AcONa + 30.05% NaN (pH ~ 8.1). The molecular weight of P7353 is 804 kDa. The increase in molecular weight of derivative P7353 is attributable to the presence of sulfate groups.

[0134] To determine the viscosity of the solution, 100 mg of P7353 was solubilized in 10 mL of deionized water. The viscosity variation as a function of shear rate was measured for P7353 using a double-gap shape. Shear rate (γ) range 1 s -1 ~100s -1 The viscosity curve for this product is reported at a temperature of 20°C, as shown in Figure 12.

[0135] P7353 has a viscosity of 26 mPa·s at a shear rate of 10 1 / s.

Claims

1. A sulfated tamarind seed polysaccharide (TSP) having a weight-average molecular weight of 600 to 1,500 kDa and a degree of sulfate (DS) of 1 to 10 per octose repeating unit, wherein the DS is determined by electrical conductivity titration and calculated according to the following formula: [Math 1] In the formula, NaOH is the titrant. Sulfated tamarind seed polysaccharide (TSP).

2. The sulfated tamarind seed polysaccharide according to claim 1, having a weight-average molecular weight of 700 to 1,100 kDa and a degree of sulfate (DS) of 1 to 5 per octose repeating unit.

3. The sulfated tamarind seed polysaccharide according to claim 1 or 2, having a viscosity of 20 to 60 mPa*s at a shear rate of 10 1 / s and a temperature of 20°C.

4. A sulfated tamarind seed polysaccharide according to any one of claims 1 to 3, having a zeta potential of -20.00 to -50.00 mV as measured by dynamic light scattering (DLS).

5. A sulfated tamarind seed polysaccharide according to any one of claims 1 to 4, which is salted with a heavy metal or an active pharmaceutical ingredient.

6. The sulfated tamarind seed polysaccharide according to claim 5, wherein the heavy metal is a metallic element selected from groups 4, 5, and 6 of the periodic table.

7. The sulfated tamarind seed polysaccharide according to claim 6, wherein the heavy metal is silver, cobalt, iron, copper, zinc, arsenic, strontium, zirconium, antimony, gold, cesium, tungsten, selenium, platinum, ruthenium, bismuth, tin, titanium, or mercury.

8. The sulfated tamarind seed polysaccharide according to claim 5, wherein the active pharmaceutical ingredient is selected from the group consisting of antibiotics, anti-infective agents, antibacterial agents, antiviral agents, cell division inhibitors, antitumor agents, anti-inflammatory agents, wound treatment agents, anesthetics, anticholinergic agents, adrenergic agents, antithrombotic agents, anticoagulants, hemostatic agents, fibrinolytic agents, thrombolytic agents, proteins, protein fragments, peptides, and polynucleotides.

9. A pharmaceutical composition comprising a sulfated tamarind seed polysaccharide according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient.

10. A biomaterial comprising a sulfated tamarind seed polysaccharide according to any one of claims 1 to 8 and a natural, semi-synthetic, or synthetic polymer, wherein the natural polymer is selected from the group consisting of collagen, co-precipitates of collagen and glycosaminoglycans, cellulose, and polysaccharides in gel form selected from the group consisting of chitin, chitosan, pectin, pectic acid, agarose, xanthan gum, guerine, alginic acid, alginate, polymannan, polyglycan, starch, and natural gum, wherein the semi-synthetic polymer is an aldehyde, an aldehyde precursor, a dicarboxylic acid, or a dicarbon Collagen crosslinked with a crosslinking agent selected from the group consisting of acid halides, diamines, cellulose derivatives, hyaluronic acid, chitin, chitosan, guerlain, xanthan gum, pectin, pectic acid, polyglycan, polymannan, agar, agarose, natural gum, and glycosaminoglycan, wherein the synthetic polymer is a biomaterial selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid copolymer, polylactic acid derivative, polyglycolic acid copolymer, polyglycolic acid derivative, polydioxane, polyphosphazene, polysulfone resin, polyurethane, and PTFE.

11. The cosmetic use of sulfated tamarind seed polysaccharide according to any one of claims 1 to 4, as a thickener, gelling agent, stabilizer, moisturizer, solubilizer, and / or smoothing agent in topical cosmetic products.

12. A process for preparing sulfated tamarind seed polysaccharide according to any one of claims 1 to 4, wherein the process comprises: i) A step of providing a suspension of TSP in a tertiary amine solvent, ii) Adding a sulfur trioxide complex with an organic radical selected from the group consisting of pyridine, DMF, trimethylamine, dioxane, N,N-dimethylaniline, B',B-dichlorodiethyl ether, and mixtures thereof, under stirring at a temperature of 15 to 60°C, thereby obtaining a dispersion of sulfated TSP. iii) Add water to the dispersion to obtain a homogeneous mixture and adjust the pH to 6-7. iv) The step of adding an alcohol-based solvent to the mixture in order to promote the precipitation of the sulfated TSP, v) A step of separating and purifying the sulfated TSP, A process that includes this.

13. The process according to claim 12, wherein in step i), the tertiary amine solvent is selected from DMF (dimethylformamide), pyridine, trimethylamine, picoline, N,N-dimethylaniline, quinoline, and mixtures thereof.

14. The process according to claim 12 or 13, wherein in step ii), the molar ratio of the complex and TSP is 1:1 to 1:10, preferably 1:1 to 1:

5.

15. The process according to any one of claims 12 to 14, wherein in step iv), the alcohol-based solvent is methyl alcohol, ethyl alcohol, propyl alcohol, or a mixture thereof, and is optionally diluted with water.

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