Method for preparing ultra-small molecular zinc hyaluronate

Abstract

The invention provides a method for preparing ultra-small molecular zinc hyaluronate, and relates to the field of hyaluronate. The method comprises the following steps: carrying out enzymolysis on macromolecular sodium hyaluronate by using leech hyaluronidase to obtain ultra-small molecular sodium hyaluronate; the method comprises the following steps: adsorbing ultra-small molecular sodium hyaluronate by using anion exchange resin, eluting by using a zinc salt solution, and reacting the obtained eluent to obtain ultra-small molecular zinc hyaluronate; the average molecular weight of the ultra-small molecular sodium hyaluronate is 600 Da to 1000 Da. The whole process is simple, the obtained ultra-small molecular zinc hyaluronate is high in zinc content, free of sodium ion residues and high in preparation yield, an organic solvent is not needed in the whole production process, and the method is suitable for industrial production.

Description

Technical Field

[0001] The present application relates to the field of hyaluronates, and in particular to a method for preparing ultra-small molecule zinc hyaluronate. Background Art

[0002] Hyaluronic acid (HA) is a glycosaminoglycan composed of disaccharide units of D-glucuronic acid and N-acetylglucosamine, with excellent moisturizing and repairing properties. Zinc, as a cofactor, significantly affects cell growth and differentiation. Its physiological functions in the skin are mainly reflected in wound repair and healing, anti-inflammatory, and sebum suppression. Zinc hyaluronate (HA-Zn) is the zinc salt of hyaluronic acid. It is produced by ion exchange between zinc ions and sodium ions on the carboxyl group of sodium hyaluronate. It combines the biological activities of hyaluronic acid and zinc ions, and has excellent wound repair, antibacterial, antioxidant, and anti-inflammatory effects. It has good application prospects in the field of skin care products.

[0003] The efficacy of HA-Zn with the same molecular weight but different content or the same content but different molecular weight shows differences. At present, the research on the preparation process of hyaluronate zinc mainly focuses on small molecule hyaluronate zinc (molecular weight 5kDa~10kDa), medium molecule hyaluronate zinc (molecular weight 500kDa~700kDa) and high molecular weight hyaluronate zinc (molecular weight 1000kDa~1300kDa). There is no research involving the preparation process of ultra-small molecule hyaluronate zinc (molecular weight <2kDa). Therefore, the development of the preparation process of ultra-small molecule hyaluronate zinc and the research on its skin care efficacy are novel, which will further broaden the hyaluronate zinc product system and improve the application value of hyaluronic acid products.

[0004] Currently, there are no studies addressing the preparation process of ultra-small molecule zinc hyaluronate. This may be due to the following two reasons: 1. Difficulty in obtaining raw materials: High-purity ultra-small molecule sodium hyaluronate (primarily composed of hyaluronic acid disaccharide (HA2), hyaluronic acid tetrasaccharide (HA4), and hyaluronic acid hexasaccharide (HA6)) is unavailable; 2. Difficulty in purification: Theoretically, ultra-small molecule sodium hyaluronate has a low degree of polymerization, making it difficult to efficiently precipitate in organic solvents. If ultra-small molecule zinc hyaluronate is prepared using a conventional zinc salt replacement system and organic solvent precipitation, the ultra-small molecule zinc hyaluronate may contain high levels of residual sodium ions, low zinc content, and high organic solvent consumption. Summary of the Invention

[0005] The purpose of this application is to provide a method for preparing ultra-small molecule zinc hyaluronate to solve the above problems.

[0006] To achieve the above objectives, the present application provides a method for preparing ultra-small molecule zinc hyaluronate, comprising:

[0007] Leech hyaluronidase is used to enzymatically hydrolyze the macromolecular sodium hyaluronate to obtain ultra-small molecular sodium hyaluronate;

[0008] Adsorbing the ultra-small molecule sodium hyaluronate using an anion exchange resin, eluting with a zinc salt solution, and reacting the obtained eluate to obtain ultra-small molecule zinc hyaluronate;

[0009] The average molecular weight of the ultra-small molecule sodium hyaluronate is 600Da-1000Da.

[0010] Optionally, the method for preparing ultra-small molecule zinc hyaluronate satisfies at least one of the following conditions:

[0011] A. The dosage of the leech hyaluronidase is 1 mU / g(ha)-3 mU / g(ha);

[0012] B. The temperature of the enzymatic hydrolysis is 38-40°C and the time is 6-12 hours.

[0013] Optionally, a first purification is performed after the enzymatic hydrolysis;

[0014] The first purification includes microfiltration and ultrafiltration performed sequentially.

[0015] Optionally, the method for preparing ultra-small molecule zinc hyaluronate satisfies at least one of the following conditions:

[0016] A. The filtration pore size of the microfiltration is 100nm-200nm;

[0017] B. The molecular weight cut-off of the ultrafiltration is greater than or equal to 5000Da.

[0018] Optionally, the method for preparing ultra-small molecule zinc hyaluronate satisfies at least one of the following conditions:

[0019] A. the anion exchange resin comprises a styrene-based strong anion exchange resin;

[0020] B. the anion exchange resin comprises a chloride-type anion exchange resin;

[0021] C. The anion exchange resin includes quaternary ammonium groups.

[0022] Optionally, the method for preparing ultra-small molecule zinc hyaluronate satisfies at least one of the following conditions:

[0023] A. the pH value of the zinc salt solution is 5.0-5.5;

[0024] B. the molar ratio of zinc ions in the zinc salt solution to carboxyl groups of the ultra-small molecule sodium hyaluronate is 0.6-1.2:1;

[0025] C. The reaction temperature is 40°C-60°C and the reaction time is 1h-6h;

[0026] D. Pre-elution is performed using a pre-elution solution between the adsorption and the elution;

[0027] The pre-wash solution includes deionized water.

[0028] Optionally, a second purification is performed after the reaction;

[0029] The second purification includes desalting and concentration performed sequentially.

[0030] Optionally, the method for preparing ultra-small molecule zinc hyaluronate satisfies at least one of the following conditions:

[0031] A. The desalination comprises electrodialysis desalination and / or dialysis desalination;

[0032] B. The concentration comprises nanofiltration.

[0033] Optionally, spray drying is further performed after the concentration;

[0034] The spray drying temperature is 120°C-140°C.

[0035] Optionally, the zinc mass content of the ultra-small molecule zinc hyaluronate is greater than or equal to 7%.

[0036] Compared with the prior art, the advantages of this application include:

[0037] The present application provides a method for preparing ultra-small molecule zinc hyaluronate. Leech hyaluronidase can improve the specificity of cleaving glycosidic bonds, converting the β-1,3 glycosidic bonds in the macromolecular sodium hyaluronate molecular chain into a saturated oligosaccharide series with glucuronic acid at the reducing end. The final products are hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide and hyaluronic acid disaccharide, thereby obtaining a saturated small molecule sodium hyaluronate with a concentrated molecular weight distribution and high purity. There is no need to use organic solvents such as ethanol and acetone, avoiding tedious steps such as multiple alcohol precipitation, desalting and washing. A one-step strong anion exchange resin is used to adsorb the hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide and hyaluronic acid disaccharide in the saturated small molecule sodium hyaluronate. Sodium ions cannot be adsorbed and exchanged with the strong anion exchange resin, so the sodium ions can be completely removed. The ultra-small molecule hyaluronic acid is then eluted with a zinc salt solution to dissociate the ultra-small molecule hyaluronic acid from the resin to obtain an ultra-small molecule zinc hyaluronate mixed solution. The elution process is simple, the yield is high, and the amount of wastewater generated is small. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope of the present application.

[0039] Figure 1 This is the liquid phase spectrum of the ultra-small molecule sodium hyaluronate prepared in Example 1;

[0040] Figure 2 This is a test diagram for IL-6 expression in macrophages stimulated by LPS;

[0041] Figure 3 This is a test chart showing the effect of zinc hyaluronate on collagen secretion in HFF-1. DETAILED DESCRIPTION

[0042] As used herein:

[0043] "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0044] The conjunction "consisting of" excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed, excluding materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim, rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0045] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed alone. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including the range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range.

[0046] In these examples, parts and percentages are by mass unless otherwise indicated.

[0047] "Parts by mass" refers to the basic unit of measurement used to express the mass ratio of multiple components. One part can represent any unit of mass, such as 1g or 2.689g. If we say that the mass of component A is a parts and the mass of component B is b parts, this means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, we could say that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number representing a multiplication factor). It's important to note that, unlike parts by mass, the sum of the mass of all components is not limited to 100 parts.

[0048] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0049] It is important to note that the main methods for preparing o-HAs are physical degradation, chemical degradation, and enzymatic degradation. Physical methods primarily involve heating and irradiation; the o-HAs produced by these methods are less stable. Chemical methods often utilize hydrolysis and oxidative degradation, which may introduce chemical reagents that can affect the properties of o-HAs and product quality control. Enzymatic degradation, on the other hand, uses specific enzymes to break the glycosidic bonds in HA to produce small-molecule hyaluronic acid.

[0050] It should be noted that hyaluronidase (HAase) is a general term for a class of enzymes that are widely distributed in nature and can degrade hyaluronic acid (HA). Based on the source, structure and mechanism of action of HAase, HAase can be divided into three categories: endo-β-N-acetylglucosaminidase (EC 3.2.1.35, mainly found in mammals and the venom of bees, snakes, spiders, etc.), endo-β-glucuronidase (EC 3.2.1.36, mainly found in leeches) and HA lyase (EC 4.2.2.1, mainly found in bacteria, bacteriophages and fungi);

[0051] Endo-β-N-acetylglucosaminidase belongs to the hydrolase family and has not only hydrolase glycosidase activity but also transglycosidase activity. Bovine testicular HAase is a representative example and has good substrate specificity (it has no activity against the polysaccharides heparin and chitin, but can hydrolyze chondroitin). HA lyase belongs to bacterial lyase and the final product of HA degradation is a disaccharide containing an unsaturated bond. In comparison, endo-β-glucuronidase HAases from leeches is the first HAase gene sequence cloned from leeches and was isolated using P. pastoris. It was successfully expressed in the GS115 expression system and purified through fermentation to obtain highly pure HAases. The use of leech-derived HAase can improve the specificity of cleaving glycosidic bonds, breaking down the β-1,3 glycosidic bonds within the hyaluronic acid (HA) molecular chain to produce a series of saturated oligosaccharides with glucuronic acid at the reducing end. The final products are hyaluronan tetrasaccharide, hyaluronan hexasaccharide, and hyaluronan disaccharide, resulting in a saturated small-molecule sodium hyaluronate with a concentrated molecular weight distribution and high purity.

[0052] Sodium hyaluronate is a linear polysaccharide composed of hyaluronic acid disaccharide (HA2) as the base unit. A single disaccharide contains a carboxyl group, which combines with sodium ions to form sodium hyaluronate. Zinc ions are replaced by sodium ions on the carboxyl group to form zinc hyaluronate.

[0053] Based on this, the present application provides a method for preparing ultra-small molecule zinc hyaluronate, comprising:

[0054] Leech hyaluronidase is used to enzymatically hydrolyze the macromolecular sodium hyaluronate to obtain ultra-small molecular sodium hyaluronate;

[0055] It should be noted that leech hyaluronidase can decompose the β-1,3 glycosidic bonds within the molecular chain of macromolecular sodium hyaluronate HA to generate a series of saturated oligosaccharides with glucuronic acid as the reducing end. In addition, leech hyaluronidase can enzymatically hydrolyze macromolecular sodium hyaluronate under mild conditions. The resulting ultra-small molecule sodium hyaluronate has a low molecular weight, a uniform product distribution, and a stable structure. The enzymatic hydrolysis process is loss-free, thus avoiding the uncertainty of the β-1,3 glycosidic bond breakage site during the acid hydrolysis of sodium hyaluronate, and also avoiding the effects of excessive acid on the structure and stability of hyaluronic acid. Combined with impurity removal steps such as microfiltration and ultrafiltration, the overall preparation yield and purity are high.

[0056] Adsorbing the ultra-small molecule sodium hyaluronate using an anion exchange resin, eluting with a zinc salt solution, and reacting the obtained eluate to obtain ultra-small molecule zinc hyaluronate;

[0057] It should be noted that the most commonly used classification of ion exchange resins is based on the resin ion exchange functional group classification, including strong acid cationic ion exchange resins, weak acid cationic ion exchange resins, strong basic anionic ion exchange resins and weak basic anionic ion exchange resins and other types. The anion exchange resin used in this application is preferably a strong basic anion exchange resin. Due to the low molecular weight of small molecule hyaluronic acid, anion resins have extremely high adsorption and purification capabilities for it, while the use of weak anion resins or cationic resins will significantly reduce the adsorption exchange capacity or purification quality.

[0058] It is also important to note that through optimization of the elution process, ZN-HA2, ZN-HA4, and ZN-HA6 monomers can be separated;

[0059] The average molecular weight of the ultra-small molecule sodium hyaluronate is 600Da-1000Da.

[0060] Optionally, the average molecular weight of ultra-small molecule sodium hyaluronate can be 600Da, 700Da, 800Da, 900Da, 1000Da or any value between 600Da and 1000Da.

[0061] It should be noted that the ultra-low average molecular weight (600Da-1000Da) results in an extremely low degree of polymerization, making it impossible to efficiently replace sodium ions with zinc ions in the aqueous or organic phase, resulting in a high concentration of sodium ions remaining in the small molecule zinc hyaluronate, affecting the quality of the small molecule zinc hyaluronate.

[0062] In some embodiments, the method for preparing ultra-small molecule zinc hyaluronate satisfies at least one of the following conditions:

[0063] A. The dosage of the leech hyaluronidase is 1 mU / g(ha)-3 mU / g(ha);

[0064] Optionally, the dosage of leech hyaluronidase can be 1 mU / g(ha), 2 mU / g(ha), 3 mU / g(ha) or any value between 1 mU / g(ha) and 3 mU / g(ha);

[0065] B. The temperature of the enzymatic hydrolysis is 38-40°C and the time is 6-12 hours.

[0066] Optionally, the enzymatic hydrolysis temperature can be 38, 39, 40, 41, 42 or any value between 38-40°C, and the enzymatic hydrolysis time can be 6h, 7h, 8h, 9h, 10h, 11h, 12h or any value between 6-12h.

[0067] In some embodiments, the enzymatic hydrolysis is followed by a first purification;

[0068] The first purification includes microfiltration and ultrafiltration performed sequentially.

[0069] It should be noted that ceramic membranes are used for microfiltration to remove large particles of insoluble impurities;

[0070] It is important to note that ultrafiltration removes leech hyaluronidase.

[0071] In some embodiments, the method for preparing ultra-small molecule zinc hyaluronate satisfies at least one of the following conditions:

[0072] A. The filtration pore size of the microfiltration is 100nm-200nm;

[0073] Optionally, the filtration pore size of the microfiltration can be 100 nm, 150 nm, 200 nm, or any value between 100 nm and 200 nm;

[0074] B. The molecular weight cut-off of the ultrafiltration is greater than or equal to 5000Da.

[0075] Optionally, the molecular weight cut-off of ultrafiltration can be 5000Da, 6000Da, 7000Da, 8000Da, 9000Da, 10000Da or any value greater than or equal to 5000Da.

[0076] In some embodiments, the method for preparing ultra-small molecule zinc hyaluronate satisfies at least one of the following conditions:

[0077] A. the anion exchange resin comprises a styrene-based strong anion exchange resin;

[0078] B. the anion exchange resin comprises a chloride-type anion exchange resin;

[0079] In some embodiments, the anion exchange resin is activated to the hydroxide form using sodium hydroxide and then replaced to the chloride form using hydrochloric acid;

[0080] C. The anion exchange resin includes quaternary ammonium groups.

[0081] It should be noted that the anion exchange resin with quaternary ammonium groups efficiently dissociates in water, making itself positively charged, and can combine with the carboxyl groups (negatively charged) of small molecule sodium hyaluronate, thereby efficiently and high-quality adsorption of small molecule hyaluronic acid without adsorbing sodium ions in small molecule sodium hyaluronate, thereby efficiently separating hyaluronic acid from sodium ions.

[0082] In some embodiments, the method for preparing ultra-small molecule zinc hyaluronate satisfies at least one of the following conditions:

[0083] A. the pH value of the zinc salt solution is 5.0-5.5;

[0084] Optionally, the pH value of the zinc salt solution may be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, or any value between 5.0 and 5.5;

[0085] It should be noted that when the pH value of the zinc salt solution is 5.0-5.5, the zinc salt can be fully dissolved and is clear and transparent, making it easy to carry out the elution process. If the pH of the zinc salt solution is lower than 5.0, the stability of the eluted and dissociated hyaluronic acid oligosaccharides will be reduced, and the impurities in the elution collection liquid will increase significantly. If the pH of the zinc salt solution is higher than 5.5, the zinc salt will be turbid and cannot effectively dissociate the hyaluronic acid oligosaccharides adsorbed on the resin.

[0086] B. the molar ratio of zinc ions in the zinc salt solution to carboxyl groups of the ultra-small molecule sodium hyaluronate is 0.6-1.2:1;

[0087] Optionally, the molar ratio of zinc ions in the zinc salt solution to the carboxyl groups of ultra-small molecule sodium hyaluronate can be 0.6:1, 0.9:1, 1.2:1, or any value between 0.6 and 1.2:1;

[0088] C. The reaction temperature is 40°C-60°C and the reaction time is 1h-6h;

[0089] Optionally, the reaction temperature may be 40°C, 50°C, 60°C or any value between 40°C and 60°C, and the reaction time may be 1h, 2h, 3h, 4h, 5h, 6h or any value between 1h and 6h;

[0090] It should be noted that the reaction can promote the full combination of zinc ions and hyaluronic acid carboxyl groups. This is because the hyaluronic acid carboxyl group is an electron-donating group, which easily forms a complex with zinc ions within a certain temperature range, and a coordination reaction occurs. As the temperature rises, the collision frequency between hyaluronic acid and zinc ions increases, which is conducive to the combination of hyaluronic acid carboxyl groups and zinc ions. However, too high a temperature will cause the hyaluronic acid and zinc ions to move too fast and cannot combine with each other. High temperature will cause changes in the groups between or within the hyaluronic acid molecules, thereby hindering its coordination reaction with zinc ions.

[0091] D. Pre-elution is performed using a pre-elution solution between the adsorption and the elution;

[0092] The pre-wash solution includes deionized water.

[0093] It should be noted that pre-elution is performed to remove residual sodium ions between anion exchange resins.

[0094] In some embodiments, the reaction is followed by a second purification step;

[0095] The second purification includes desalting and concentration performed sequentially.

[0096] In some embodiments, the method for preparing ultra-small molecule zinc hyaluronate satisfies at least one of the following conditions:

[0097] A. The desalination comprises electrodialysis desalination and / or dialysis desalination;

[0098] It should be noted that desalination is to remove free zinc ions and free chloride ions that are not bound to the carboxyl groups of hyaluronic acid, thereby improving the purity of ultra-small molecule zinc hyaluronate. Among them, electrodialysis desalination is more suitable for industrial production.

[0099] B. The concentration comprises nanofiltration.

[0100] It should be noted that the desalted material is treated with a nanofiltration membrane, which retains ultra-small molecule zinc hyaluronate, while water and chloride ions can pass through the nanofiltration membrane. The material is concentrated in the early stage, and then dialyzed and desalted with small amounts of water multiple times in the later stage to further improve the purity of ultra-small molecule zinc hyaluronate. The nanofiltration yield is 95%.

[0101] It should be noted that electrodialysis combined with nanofiltration membrane can remove free zinc ions and chloride ions from small molecule zinc hyaluronate with high quality. At the same time, nanofiltration can efficiently concentrate small molecule zinc hyaluronate, which is beneficial for subsequent spray drying and powdering.

[0102] In some embodiments, the concentration is followed by spray drying;

[0103] The spray drying temperature is 120°C-140°C.

[0104] Optionally, the spray drying temperature may be 120°C, 130°C, 140°C or any value between 120°C and 140°C.

[0105] In some embodiments, the zinc content of the ultra-small molecule zinc hyaluronate is greater than or equal to 7% by weight.

[0106] It should be noted that the process route provided in this application is simple, has a high yield, produces little wastewater, does not require organic solvents, is environmentally friendly, and is easy to scale up industrially.

[0107] It should also be noted that the present application is to purify ultra-small molecule sodium hyaluronate by adsorption through a strong anion exchange resin. The dissociation of the quaternary ammonium group on the resin in water makes it positively charged, thereby adsorbing and binding with the ultra-small molecule hyaluronic acid carboxyl group (negatively charged), while the sodium ions do not carry a negative charge and cannot be adsorbed and bound to the resin, thereby separating the ultra-small molecule hyaluronic acid from the sodium ions. A small amount of pure water is used to remove the sodium ions remaining between the resins, and then a certain concentration of zinc chloride (chloride ions act as an elution) is used to completely decompose the ultra-small molecule hyaluronic acid from the resin, thereby forming an ultra-small molecule hyaluronic acid zinc mixture, and then reacting to organically combine the zinc ions with the hyaluronic acid carboxyl group, and then removing the free zinc ions and chloride ions by electrodialysis treatment, and concentrating through an organic nanofiltration membrane. The ultra-small molecule hyaluronic acid zinc is retained by the nanofiltration membrane, and impurities such as water and a small amount of zinc chloride pass through the nanofiltration membrane. The nanofiltration is concentrated and desalted in a small amount in the early stage, and a small amount of water is added for dialysis desalination in the later stage, thereby improving the quality of the ultra-small molecule zinc hyaluronate.

[0108] The embodiments of the present application will be described in detail below in conjunction with specific examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, the conditions are carried out according to conventional conditions or manufacturer recommendations. The reagents or instruments used are not specified by the manufacturer and are conventional products that can be purchased commercially.

[0109] The high-purity leech hyaluronidase used in the examples was from Amphiprion.

[0110] Example 1

[0111] This embodiment provides a method for preparing ultra-small molecule zinc hyaluronate, which specifically includes:

[0112] 1) Add 1 L of pure water to a 2 L glass beaker and heat to 38°C. Stir at 400 rpm / min and slowly add 20 g of macromolecular HA (molecular weight: 1300 kDa). Stir at high speed until the HA is completely dissolved (no particles are present).

[0113] 2) Add 50 mL of high-purity leech hyaluronidase (HAase dosage: 2.0 mU / g(HA), HAase activity: 0.8 mU / mL) to the dissolved macromolecular HA solution. Control the reaction temperature at 38-40°C. After 8 h, terminate the reaction. Microfiltration is performed using a ceramic membrane (pore size of 100 nm) to remove macromolecular impurities, and 5K ultrafiltration is used to remove HAase protein to obtain high-purity ultra-small molecule sodium hyaluronate.

[0114] The high-purity ultra-small molecule sodium hyaluronate was subjected to liquid chromatography analysis. The separation chromatographic conditions were as follows: chromatographic column: YMC-Pack Polyamine II chromatographic column (250 mm × 4.6 mm, 5 μm) (YMC, Japan); mobile phase: 0.08 mol / L sodium dihydrogen phosphate: acetonitrile (90:10), flow rate: 1 mL / min, column temperature: 25°C, UV detector: 210 nm, injection volume: 5 μL. The liquid chromatography is shown in FIG. Figure 1 As shown;

[0115] 3) 1500 mL of the ultrafiltration and dialysis mixture was adsorbed on an ion exchange resin (strong anionic, activated to H+ form, and washed with water to a neutral pH). After the material was completely adsorbed, it was pre-eluted with 1000 mL of pure water, and then eluted with 1500 mL of 0.06 M zinc chloride (pH adjusted to 5.2 with hydrochloric acid), and 1500 mL of the zinc chloride elution solution was collected;

[0116] 4) The eluted solution was reacted at 50°C for 2 hours, and then free zinc ions and chloride ions were removed by electrodialysis. The material was then concentrated and desalted by nanofiltration (membrane pore size 250 Da). When the solid content was about 6%, the material was dialyzed with water in small amounts multiple times until there was no significant decrease in the conductivity of the material. The dialysis was then stopped. The concentrate was then sterilized by passing it through a 0.22 μm membrane and spray dried at 130°C.

[0117] 5) After complete drying, 20.2 g of white ultra-small molecule zinc hyaluronate powder was obtained, with a zinc content of 8.3%, a sodium content of 0%, and a molecular weight of 635 Da.

[0118] Example 2

[0119] This embodiment provides a method for preparing ultra-small molecule zinc hyaluronate, which specifically includes:

[0120] 1) Add 1 L of pure water to a 2 L glass beaker and heat to 38°C. Stir at 400 rpm / min and slowly add 20 g of macromolecular HA (molecular weight: 1300 kDa). Stir at high speed until the HA is completely dissolved (no particles are present).

[0121] 2) Add 50 mL of high-purity leech hyaluronidase (HAase dosage: 1.0 mU / g(HA), HAase activity: 0.8 mU / mL) to the dissolved macromolecular HA solution. Control the reaction temperature at 38-40°C. After 9 h, terminate the reaction. Microfiltration is performed using a ceramic membrane (pore size of 100 nm) to remove macromolecular impurities, and 5K ultrafiltration is performed to remove HAase protein to obtain high-purity ultra-small molecule sodium hyaluronate.

[0122] 3) 1500 mL of the ultrafiltration and dialysis mixture was adsorbed on an ion exchange resin (strong anionic, activated to H+ form, and washed with water to a neutral pH). After the material was completely adsorbed, it was pre-eluted with 1000 mL of pure water, and then eluted with 1500 mL of 0.06 M zinc chloride (pH adjusted to 5.1 with hydrochloric acid), and 1500 mL of the zinc chloride elution solution was collected;

[0123] 4) The eluted solution was reacted at 40°C for 1 hour, and then free zinc ions and chloride ions were removed by electrodialysis. The material was then concentrated and desalted by nanofiltration (membrane pore size 250 Da). When the solid content was about 6%, the material was dialyzed with water in small amounts multiple times until there was no significant decrease in the conductivity of the material. The dialysis was then stopped. The concentrate was then sterilized by passing it through a 0.22 μm membrane and spray dried at 140°C.

[0124] 5) After complete drying, 20.7 g of white ultra-small molecule zinc hyaluronate powder was obtained, with a zinc content of 7.7%, a sodium content of 0%, and a molecular weight of 835 Da.

[0125] Example 3

[0126] This embodiment provides a method for preparing ultra-small molecule zinc hyaluronate, which specifically includes:

[0127] 1) Add 1 L of pure water to a 2 L glass beaker and heat to 38°C. Stir at 400 rpm / min and slowly add 20 g of macromolecular HA (molecular weight: 1300 kDa). Stir at high speed until the HA is completely dissolved (no particles are present).

[0128] 2) Add 50 mL of high-purity leech hyaluronidase (HAase dosage: 3.0 mU / g(HA), HAase activity: 0.8 mU / mL) to the dissolved macromolecular HA solution. Control the reaction temperature at 38-40°C. After 12 h, terminate the reaction. Microfilter the macromolecular impurities using a ceramic membrane (pore size 200 nm) and ultrafilter the HAase protein using 5K to obtain high-purity ultra-small molecule sodium hyaluronate.

[0129] 3) 1500 mL of the ultrafiltration and dialysis mixture was adsorbed on an ion exchange resin (strong anionic, activated to H+ form, and washed with water to a neutral pH). After the material was completely adsorbed, it was pre-eluted with 1000 mL of pure water, and then eluted with 1500 mL of 0.06 M zinc chloride (pH adjusted to 5.5 with hydrochloric acid), and 1500 mL of the zinc chloride elution solution was collected;

[0130] 4) The eluted solution was reacted at 60°C for 3 hours, and then free zinc ions and chloride ions were removed by electrodialysis. The material was then concentrated and desalted by nanofiltration (membrane pore size 250 Da). When the solid content was about 6%, the material was dialyzed with water in small amounts multiple times until there was no significant decrease in the conductivity of the material. The dialysis was then stopped. The concentrate was then sterilized by passing it through a 0.22 μm membrane and spray dried at 140°C.

[0131] 5) After complete drying, 19.8 g of white ultra-small molecule zinc hyaluronate powder was obtained, with a zinc content of 8.5%, a sodium content of 0%, and a molecular weight of 615 Da.

[0132] Example 4

[0133] This embodiment provides a method for preparing ultra-small molecule zinc hyaluronate, which specifically includes:

[0134] 1) Add 1 L of pure water to a 2 L glass beaker and heat to 38°C. Stir at 400 rpm / min and slowly add 20 g of macromolecular HA (molecular weight: 1300 kDa). Stir at high speed until the HA is completely dissolved (no particles are present).

[0135] 2) Add 50 mL of high-purity leech hyaluronidase (HAase dosage: 3.0 mU / g(ha), HAase activity: 0.8 mU / mL) to the dissolved macromolecular HA solution. Control the reaction temperature at 38-40°C. After 10 h, terminate the reaction. Microfiltration is performed using a ceramic membrane (pore size of 200 nm) to remove macromolecular impurities, and 5K ultrafiltration is used to remove HAase protein to obtain high-purity ultra-small molecule sodium hyaluronate.

[0136] 3) 1500 mL of the ultrafiltration and dialysis mixture was adsorbed on an ion exchange resin (strong anionic, activated to H+ form, and washed with water to a neutral pH). After the material was completely adsorbed, it was pre-eluted with 1000 mL of pure water, and then eluted with 1500 mL of 0.06 M zinc chloride (pH adjusted to 5.0 with hydrochloric acid), and 1500 mL of the zinc chloride elution solution was collected;

[0137] 4) The eluted solution was reacted at 40°C for 1 hour, and then free zinc ions and chloride ions were removed by electrodialysis. The material was then concentrated and desalted by nanofiltration (membrane pore size 250 Da). When the solid content was about 6%, the material was dialyzed with water in small amounts multiple times until there was no significant decrease in the conductivity of the material. The dialysis was then stopped. The concentrate was then sterilized by passing it through a 0.22 μm membrane and spray dried at 140°C.

[0138] 5) After complete drying, 19.5 g of white ultra-small molecule zinc hyaluronate powder was obtained, with a zinc content of 7.6%, a sodium content of 0%, and a molecular weight of 722 Da.

[0139] Example 5

[0140] This embodiment provides a method for preparing ultra-small molecule zinc hyaluronate, which specifically includes:

[0141] 1) Add 1 L of pure water to a 2 L glass beaker and heat to 38°C. Stir at 400 rpm / min and slowly add 20 g of macromolecular HA (molecular weight: 1300 kDa). Stir at high speed until the HA is completely dissolved (no particles are present).

[0142] 2) Add 50 mL of high-purity leech hyaluronidase (HAase dosage: 2.0 mU / mL, HAase activity: 0.8 mU / mL) to the dissolved macromolecular HA solution. Control the reaction temperature at 38-40°C. After 6 h, terminate the reaction. Microfilter the macromolecular impurities using a ceramic membrane (pore size 100 nm) and ultrafilter the HAase protein using 5K to obtain high-purity ultra-small molecule sodium hyaluronate.

[0143] 3) 1500 mL of the ultrafiltration and dialysis mixture was adsorbed on an ion exchange resin (strong anionic, activated to H+ form, and washed with water to a neutral pH). After the material was completely adsorbed, it was pre-eluted with 1000 mL of pure water, and then eluted with 1500 mL of 0.06 M zinc chloride (pH adjusted to 5.2 with hydrochloric acid), and 1500 mL of the zinc chloride elution solution was collected;

[0144] 4) The eluted solution was reacted at 50°C for 3 hours, and then free zinc ions and chloride ions were removed by electrodialysis. The material was then concentrated and desalted by nanofiltration (membrane pore size 250 Da). When the solid content was about 6%, the material was dialyzed with water in small amounts multiple times until there was no significant decrease in the conductivity of the material. The dialysis was then stopped. The concentrate was then sterilized by passing it through a 0.22 μm membrane and spray dried at 130°C.

[0145] 5) After complete drying, 19.6 g of white ultra-small molecule zinc hyaluronate powder was obtained, with a zinc content of 6.9%, a sodium content of 0%, and a molecular weight of 692 Da.

[0146] Comparative Example 1

[0147] This comparative example provides a method for preparing zinc hyaluronate, which differs from Example 1 in that 0.01 M zinc chloride is used.

[0148] After complete drying, 15.3 g of white zinc hyaluronate powder was obtained, with a zinc content of 4.2%, a sodium content of 0, and a molecular weight of 713 Da.

[0149] Comparative Example 2

[0150] This comparative example provides a method for preparing zinc hyaluronate, which differs from Example 1 in that the enzyme dosage is 0.2 MU / g HA.

[0151] After complete drying, 14.9 g of zinc hyaluronate powder was obtained, with a zinc content of 4.7%, a sodium content of 0%, and a molecular weight of 2513 Da.

[0152] Comparative Example 3

[0153] This comparative example provides a method for preparing zinc hyaluronate, which differs from Example 1 in that no heating reaction is performed.

[0154] After complete drying, 15.9 g of zinc hyaluronate powder was obtained, with a zinc content of 3.6%, a sodium content of 0, and a molecular weight of 793 Da.

[0155] Comparative Example 4

[0156] This comparative example provides a method for preparing zinc hyaluronate, which differs from Example 1 in that no electrodialysis is performed.

[0157] After complete drying, 16.9 g of zinc hyaluronate powder was obtained, with a zinc content of 12.6%, a sodium content of 0.29%, and a molecular weight of 763 Da.

[0158] The yields of the white ultra-small molecule zinc hyaluronate or zinc hyaluronate prepared in the above examples and comparative examples are shown in Table 1.

[0159] Table 1 Yield

[0160] Test components Yield Example 1 93.4％ Example 2 95.7％ Example 3 91.6％ Example 4 90.2％ Example 5 90.6％ Comparative Example 1 70.8％ Comparative Example 2 68.9％ Comparative Example 3 73.5％ Comparative Example 4 78.2％

[0161] analyze:

[0162] The above experiments show that by controlling the amount of hyaluronidase added in the enzymatic hydrolysis reaction, the zinc salt concentration, pH and elution conditions in the elution process, the temperature and time of the complexation between hyaluronic acid and zinc salt, etc., the molecular weight, purity, zinc content and yield of small molecule zinc hyaluronate can be effectively controlled, thereby facilitating the stable preparation of small molecule zinc hyaluronate and ensuring its quality.

[0163] Cell efficacy verification experiment:

[0164] Anti-inflammatory: Inflammation is caused by a variety of stimuli, including injury, pathogens, and their products. It is the body's response to pathogenic factors and their damaging effects. However, persistent or excessive inflammatory responses can cause irreversible damage. LPS, a component of the cell wall of Gram-negative bacteria and a bacterial endotoxin, can induce the release of inflammatory mediators, causing strong inflammatory responses and various diseases.

[0165] The most important immune cell involved in the inflammatory response is the monocyte-macrophage system. Macrophages will undergo polarization and participate in and regulate the inflammatory response by secreting inflammatory factors and expressing inflammatory receptors. Interleukin-6 (IL-6) is a major inflammatory cytokine secreted by macrophages. It can be rapidly produced when stimulated by infectious factors and tissue damage, and aggravates the inflammatory response by regulating intracellular signal transduction. It is considered an important sign of inflammation in the body.

[0166] The basic principle of the enzyme-linked immunosorbent assay (ELISA) is to immobilize a certain concentration of antigen or antibody on the surface of a polystyrene microplate by physical adsorption. The specimen to be tested is then added, and the depth of the color developed by the enzyme label indirectly reflects the presence or amount of the antigen or antibody to be tested. Therefore, by detecting the effect of the test substance on IL-6 production through ELISA, the anti-inflammatory effect of the test substance can be reflected.

[0167] Specific test method:

[0168] 1) Cell seeding: 4000 Ana-1 cells were seeded into a 96-well cell culture plate and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0169] 2) Liquid preparation: Prepare the test substance working solution according to the experimental design (Table 2), wherein samples 1-6 are all from the ultra-small molecule zinc hyaluronate prepared in Example 1;

[0170] Table 2 Anti-inflammatory experimental design

[0171]

[0172] 3) Induction of inflammatory response: When the cell plating rate in the 96-well plate reaches 80%-90%, dilute the 1 mg / mL LPS solution to 1 μg / mL and add 50 μL / well to the plate. Add the same amount of culture medium to the blank control group and incubate in a 37°C, 5% CO2 incubator for 4 h.

[0173] 4) Administration: According to the test protocol, the drugs were administered in groups, with three replicate wells per group. After administration, the 96-well plate was placed in an incubator (37°C, 5% CO2) and incubated for 24 hours.

[0174] 5) Detection: After drug addition, the cells were cultured in an incubator (37° C., 5% CO 2 ) for 24 h, the culture medium was collected, and the IL-6 content in the culture medium was detected using a kit.

[0175] 6) Statistical analysis of results: GraphPad Prism 8.0 software was used for data statistical analysis and graphing. Measurement data were expressed as x±s. Differences between groups were analyzed using one-way ANOVA. P < 0.05 was considered statistically significant, P < 0.05 was considered a significant difference, and P < 0.01 was considered a very significant difference.

[0176] Test results: Based on the above experimental method, the cell supernatant was collected and the IL-6 content was detected. The test results are shown in Table 3. The change trend is as follows Figure 2 shown.

[0177] Table 3 Summary of IL-6 content data

[0178] Group IL-6 release (pg / mL) P-value Efficacy evaluation Blank control (BC) 0.2 0.0001 / Negative control (NC) 784.0 / / 2.5ug / mL HA-Zn 686.4 0.0001 Has a significant inhibitory effect 5ug / mL HA-Zn 535.4 0.0001 Has a significant inhibitory effect 10ug / mL HA-Zn 478.8 0.0001 Has a significant inhibitory effect 20ug / mL HA-Zn 514.9 0.0001 Has a significant inhibitory effect 40ug / mL HA-Zn 427.9 0.0001 Has a significant inhibitory effect 80ug / mL HA-Zn 413.6 0.0001 Has a significant inhibitory effect

[0179] analyze:

[0180] Compared with the negative group, the IL-6 content in the positive group increased significantly, indicating that the stimulation conditions of this experiment were effective. Ultra-small molecule zinc hyaluronate can inhibit the upregulation of IL-6 stimulated by LPS at different concentrations. Therefore, it has certain anti-inflammatory effects, and the anti-inflammatory ability gradually increases with the increase of zinc hyaluronate concentration.

[0181] Anti-aging: The primary manifestation of epidermal aging at the normal cellular level is apoptosis. Dermal fibroblasts are a major cellular component of the human dermis. Their gradual decline in proliferation and function is a primary driver of skin aging. Aging skin is associated with decreased elastin and collagen production, as well as increased wrinkling. The expression of COL1α1 in human fibroblasts (HFF-1) directly reflects skin condition.

[0182] Therefore, the anti-aging effect of the target substance can be reflected by detecting the effect of the target substance on the expression level of COL1α1 in human fibroblasts (HFF-1).

[0183] Testing Process

[0184] 1) Cell seeding: 10,000 HFF cells were seeded into a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0185] 2) Dilution and loading of test samples: Using complete culture medium as the diluent, prepare sample working solutions of varying concentrations according to the test sample concentrations (as shown in Table 4), at 100 μL / well. Add equal volumes of complete culture medium to the negative and positive control groups, incubate in a 37°C, 5% CO2 incubator for 24 h, and observe cell morphology under a microscope.

[0186] Table 4 Anti-aging test design

[0187]

[0188] 3) Take the supernatant from the well plate and detect the collagen content by ELISA. The operation steps are as follows:

[0189] A. Calculate the required enzyme label strips in advance, take out the kit 30 minutes before the experiment, and return it to room temperature;

[0190] B. Serial dilution of standard: dilute the standard with standard & sample diluent in series to 500, 250, 125, 62.5, and 0 ng / mL;

[0191] C. Collect the cell culture supernatant into a sterile centrifuge tube and centrifuge (4°C, 1000×g, 20 min). Take the supernatant and dilute it to an appropriate concentration as the test sample.

[0192] D. Add 100 μL of standard working solution and test sample (diluted 1-fold) to each reaction well. Set up two replicate wells for each group and incubate at 37°C for 90 min.

[0193] E. Discard the liquid, spin dry, add 100 μL of biotin-labeled antibody working solution to each reaction well, and incubate in a 37°C incubator for 60 minutes;

[0194] F. Discard the liquid, spin dry, add 300 μL of washing solution to each reaction well, soak for 1-2 minutes, and spin dry. Repeat 4 times;

[0195] G. Add 100 μL of HRP-labeled streptavidin working solution to each reaction well and incubate in a 37°C incubator for 30 min.

[0196] H. Add 300 μL of washing solution to each reaction well, spin dry the washing solution after 30 seconds, and repeat 4 times;

[0197] I. Add 90 μL of color developing reagent to each reaction well and incubate at 37°C in the dark for approximately 15 minutes.

[0198] J. Add 50 μL of stop solution to each reaction well and immediately measure the OD value at 450 nm using a microplate reader;

[0199] K. Calculate the standard curve regression equation (R² > 0.99) based on the known concentration of the standard and the measured OD value. Substitute the OD value of the sample well into the calculated concentration of the measured sample, and then multiply by the dilution factor to obtain the actual concentration of the original sample.

[0200] 4) Statistical analysis: GraphPad Prism 8.0 software was used for data statistical analysis and graphs were drawn. Measurement data were expressed as x±s. Differences between groups were analyzed by one-way ANOVA. P < 0.05 was considered statistically significant, P < 0.05 was considered a significant difference, and P < 0.01 was considered a very significant difference.

[0201] Test results: The collagen content of each group was detected by ELISA. According to the experimental results, the standard curve y = 0.0006x + 0.0915, R2 = 0.9934 (the horizontal axis x is the collagen content of the sample, the vertical axis y = absorbance), and the collagen content was inferred from the absorbance of the standard curve. The change trend is shown in Table 5. The effect of HA-Zn on the collagen secretion in HFF-1 is shown in Table 5. Figure 3 shown.

[0202] Table 5. Summary of collagen content

[0203] Group Collagen content (ng / ml) P-value Efficacy evaluation Blank control (BC) 3500 0.0001 / 2.5ug / mL HA-Zn 3865.4 0.0001 Has a significant promoting effect 5ug / mL HA-Zn 4035.4 0.0001 Has a significant promoting effect 10ug / mL HA-Zn 4678.8 0.0001 Has a significant promoting effect 20ug / mL HA-Zn 4514.9 0.0001 Has a significant promoting effect 40ug / mL HA-Zn 4787.9 0.0001 Has a significant promoting effect 80ug / mL HA-Zn 3513.6 0.0234 Has a promoting effect

[0204] analyze:

[0205] According to the results of ELISA detection of collagen content, HA-Zn has a significant promoting effect on the secretion of collagen in human fibroblasts within a certain concentration range (2.5-40ug / ml). When the concentration reaches 80ug / mL, its promoting effect on collagen secretion decreases moderately.

[0206] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

[0207] Furthermore, those skilled in the art will appreciate that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are intended to be within the scope of this application and to form distinct embodiments. For example, in the claims above, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is intended solely to enhance understanding of the overall background of this application and should not be construed as an admission or any implication that such information constitutes prior art known to those skilled in the art.

Claims

1. A method for preparing ultra-small molecule zinc hyaluronate, characterized in that: include: Leech hyaluronidase is used to enzymatically hydrolyze the macromolecular sodium hyaluronate to obtain ultra-small molecular sodium hyaluronate; Adsorbing the ultra-small molecule sodium hyaluronate using an anion exchange resin, eluting with a zinc salt solution, and reacting the obtained eluate to obtain ultra-small molecule zinc hyaluronate; The average molecular weight of the ultra-small molecule sodium hyaluronate is 600Da-1000Da.

2. The method for preparing ultra-small molecule zinc hyaluronate according to claim 1, wherein: At least one of the following conditions is met: A. The dosage of the leech hyaluronidase is 1 mU / g(ha)-3 mU / g(ha); B. The temperature of the enzymatic hydrolysis is 38-40°C and the time is 6-12 hours.

3. The method for preparing ultra-small molecule zinc hyaluronate according to claim 1, wherein: After the enzymatic hydrolysis, a first purification is performed; The first purification includes microfiltration and ultrafiltration performed sequentially.

4. The method for preparing ultra-small molecule zinc hyaluronate according to claim 3, wherein: At least one of the following conditions is met: A. The filtration pore size of the microfiltration is 100nm-200nm; B. The molecular weight cut-off of the ultrafiltration is greater than or equal to 5000Da.

5. The method for preparing ultra-small molecule zinc hyaluronate according to claim 1, wherein: At least one of the following conditions is met: A. the anion exchange resin comprises a styrene-based strong anion exchange resin; B. the anion exchange resin comprises a chloride-type anion exchange resin; C. The anion exchange resin includes quaternary ammonium groups.

6. The method for preparing ultra-small molecule zinc hyaluronate according to claim 1, characterized in that: At least one of the following conditions is met: A. the pH value of the zinc salt solution is 5.0-5.5; B. the molar ratio of zinc ions in the zinc salt solution to carboxyl groups of the ultra-small molecule sodium hyaluronate is 0.6-1.2:1; C. The reaction temperature is 40°C-60°C and the reaction time is 1h-6h; D. Pre-elution is performed using a pre-elution solution between the adsorption and the elution; The pre-wash solution includes deionized water.

7. The method for preparing ultra-small molecule zinc hyaluronate according to claim 1, characterized in that: After carrying out the reaction, a second purification is also carried out; The second purification includes desalting and concentration performed sequentially.

8. The method for preparing ultra-small molecule zinc hyaluronate according to claim 7, characterized in that: At least one of the following conditions is met: A. The desalination comprises electrodialysis desalination and / or dialysis desalination; B. The concentration comprises nanofiltration.

9. The method for preparing ultra-small molecule zinc hyaluronate according to claim 7, characterized in that: After the concentration, spray drying is also performed; The spray drying temperature is 120°C-140°C.

10. The method for preparing ultra-small molecule zinc hyaluronate according to any one of claims 1 to 9, characterized in that: The zinc content of the ultra-small molecule zinc hyaluronate is greater than or equal to 7% by mass.

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