A method for extracting, purifying and standardizing active components of proteoglycans from cartilage of marine bony fish

Abstract

The present application relates to the technical fields of cosmetic active ingredients, high-value utilization of marine biological resources, separation and purification of sugar complexes and topical skin delivery, and discloses an extraction, purification and standardized preparation method of active components of marine cartilage-derived proteoglycans, comprising the following steps: S1: making 3-8 mm particles from by-product cartilage obtained from edible marine cartilage processing, adding an extraction solution containing 10-30 mmol / L phosphate and 0.8-1.2 mol / L sodium chloride, pH 6.6-7.2, at a ratio of wet cartilage mass (kg) to extraction solution volume (L) of 1:6-1:12, extracting at 2-10 DEG C for 4-10 h to obtain a crude extract. The extraction, purification and standardized preparation method of the active components of marine cartilage-derived proteoglycans excludes exogenous proteases, glycosaminoglycan-degrading enzymes, guanidines, ethylenediaminetetraacetic acid and organic alcohol precipitation during the extraction to drying process, and is suitable for converting edible marine cartilage processing by-products into cosmetic active ingredients.

Description

Technical Field

[0001] This invention relates to the fields of cosmetic active ingredients, high-value utilization of marine biological resources, separation and purification of glycocomplexes and local skin delivery technology, specifically a method for the extraction, purification and standardized preparation of proteoglycan active components derived from the cartilage of marine bony fish. Background Technology

[0002] Proteoglycans are glycocomplexes formed by the covalent linkage of a core protein with one or more glycosaminoglycan side chains. Cartilaginous proteoglycans typically have a high charge density, large hydration volume, and a structural basis for interacting with extracellular matrix components and growth factors. Various types of proteoglycans in skin tissue participate in the organization, hydration, viscoelasticity, and maintenance of the cell signaling environment of the extracellular matrix. Non-patent literature has reported that water-soluble proteoglycans derived from salmon nasal cartilage can promote the proliferation of normal human skin fibroblasts in vitro and are accompanied by Erk1 / 2 phosphorylation. The above studies provide a technical basis for the use of proteoglycans in moisturizing, matrix support, and fine line appearance management in cosmetic care.

[0003] Multiple routes have been developed for the preparation of cartilage-type proteoglycans. Patent publication number CN1439663A discloses a route of acidic extraction combined with salt-saturated ethanol precipitation and dialysis. Patent publication number WO2012099216A1 discloses the treatment of frozen fish cartilage in high-temperature water to prepare large quantities of high molecular weight proteoglycans. Patent publication number JP7197891B2 discloses the extraction of proteoglycans or glycosaminoglycans from biological raw materials using a 0.5 to 5 mol / L magnesium sulfate solution. Patent publication number JP7295572B2 discloses a route of extraction in an acidic solution with a pH not higher than 5.5 and in the presence of a low proportion of protease, while removing lipids and substances with a molecular weight not higher than 50 kDa.

[0004] Regarding molecular weight and purification window, patent publication number EP4520838A1 discloses the extraction and purification of proteoglycans from animal tissues, the separation of fractions conforming to a predetermined peak molecular weight, and a proteoglycan composition with a peak molecular weight of 400 to 1200 kDa; patent publication number WO2025163949A1 discloses the selective precipitation of cartilage-derived proteoglycans with a specific concentration of alcohol in the presence of salt; regarding skin applications, patent publication number KR101808597B1 discloses moisturizing liposomes containing proteoglycans, lecithin, or phosphatidylcholine and having a particle size of 100 to 400 nm; patent publication number KR102259668B1 discloses a route for preparing nanoliposomes by microfluidic mixing of proteoglycans and glycolic acid and mixing them with an emulsion at 65 to 70 °C.

[0005] The above technologies address issues related to extraction efficiency, purification, single molecular weight control, or delivery, but the following areas for improvement remain: First, acid, protease, high temperature, guanidine salt or alcohol precipitation routes may increase the complexity of core protein breakage, glycan alteration, solvent removal and batch fluctuation control. Second, using only total sugar, total protein, or a single molecular weight peak as quality indicators makes it difficult to simultaneously reflect species origin, core protein, glycan composition, and low molecular weight free matter. Third, when single-form proteoglycans are directly added to the continuous phase of cosmetics, it is difficult to simultaneously address the skin's surface film formation and water retention, as well as localized deposition in the lower part of the stratum corneum and hair follicle openings. Fourth, existing routes have not formed a complete integrated technical solution for the independent manufacturing, standardization, and formulation space allocation of H / M dual molecular weight fractions; Therefore, a method for the extraction, purification and standardized preparation of active components of proteoglycans derived from the cartilage of marine bony fish is proposed. Summary of the Invention

[0006] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for the extraction, purification, and standardized preparation of proteoglycan active components derived from marine bony fish cartilage. This method utilizes cartilage, a byproduct of marine bony fish processing, as raw material. During extraction, clarification, membrane purification, membrane fractionation, and drying, no exogenous proteases, glycosaminoglycan degrading enzymes, guanidine salts, or EDTA are added, and organic alcohol precipitation is avoided. This results in the separation of a high-molecular-weight H component and a medium-molecular-weight M component. Furthermore, through a dual-component raw material and a dual-space formulation structure for local skin application, the H component is primarily configured in the continuous aqueous phase, while the M component is co-dispersed with a phospholipid layered carrier, thus forming a dual-space formulation structure for differentiated local retention evaluation. This method overcomes the problems of existing technologies, such as the susceptibility of core protein-glycan structures to damage due to extraction methods involving acids, proteases, high temperatures, guanidine salts, or organic alcohol precipitation; the difficulty in reflecting the source and structural integrity with single-molecular-weight proteoglycans; the inability of single-molecular-weight proteoglycans to simultaneously address differentiated delivery to the skin surface and the lower stratum corneum; and the lack of an integrated H / M dual-molecular-weight fractionation preparation and formulation space configuration scheme.

[0007] (II) Technical Solution To achieve the goal of obtaining a high-molecular-weight H component and a medium-molecular-weight M component byproduct from marine bony fish cartilage processing, without adding exogenous proteases, glycosaminoglycan degrading enzymes, guanidine salts, or ethylenediaminetetraacetic acid during extraction, clarification, membrane purification, membrane fractionation, and drying, and without using organic alcohol precipitation; further, through a dual-component raw material and local skin dual-space formulation structure, the H component is mainly configured in the continuous aqueous phase, and the M component is co-dispersed with a phospholipid layered carrier, thereby forming a dual-space formulation structure for differentiated local retention evaluation, this invention provides the following technical solution: a method for the extraction, purification, and standardized preparation of proteoglycan active components derived from marine bony fish cartilage, comprising the following steps: S1: Processed cartilage from edible marine bony fish with confirmed source is made into 3 to 8 mm particles. The wet cartilage mass (kg) to the extraction liquid volume (L) ratio is 1:6 to 1:12. An extraction solution containing 10 to 30 mmol / L phosphate and 0.8 to 1.2 mol / L sodium chloride, with a pH of 6.6 to 7.2, is added and extracted at 2 to 10 °C for 4 to 10 h to obtain a crude extract. S2: The crude extract is subjected to solid-liquid separation and clarification filtration with a pore size of no more than 0.45 μm, and then concentrated and constant-volume liquid exchange is performed using a tangential flow membrane with a nominal molecular weight cutoff of 50 kDa, so that the conductivity of the feed liquid reaches 4 to 9 mS / cm. S3: Pass the feed solution from step S2 through a quaternary ammonium strong anion exchange membrane, elute with a buffer solution containing 0.45 to 0.95 mol / L sodium chloride and with a pH of 6.7 to 7.3, and collect the proteoglycan enrichment fraction; S4: After the enrichment fraction is replaced, it is first fractionated by a tangential flow membrane with a nominal molecular weight cutoff of 500kDa. The 500kDa cutoff liquid is used as candidate fraction H. Then, the 500kDa permeate is fractionated by a tangential flow membrane with a nominal molecular weight cutoff of 50kDa. The 50kDa cutoff liquid is used as candidate fraction M. S5: Form one or more fractional collectors from the H candidate fraction and the M candidate fraction and perform size exclusion chromatography detection. Combine the H fractional collectors with an apparent molecular weight of 1.25 to 1.90 MDa at the peak to form the H fraction, and combine the M fractional collectors with an apparent molecular weight of 80 to 220 kDa at the peak to form the M fraction. Ensure that the peak area of ​​the region less than 30 kDa in the H fraction and the M fraction is no more than 5.0% of their respective total peak area. Return the boundary fractional collectors located outside the target window to the adjacent membrane fractionation step for processing. S6: Add trehalose and ectoine to H component and M component respectively, and dry them to obtain H powder and M powder; In particular, no exogenous proteases, glycosaminoglycan degrading enzymes, guanidine salts or ethylenediaminetetraacetic acid are added during the extraction, clarification, membrane purification, membrane fractionation and drying processes, and no organic alcohol precipitation is used.

[0008] Preferably, the marine bony fish is selected from one of the following: golden pomfret, grouper, cobia, and sea bass, and the cartilage is selected from one of the following: head cartilage, fin base cartilage, operculum connecting cartilage, and intervertebral cartilage.

[0009] Preferably, in step S2, the constant-volume buffer exchange is performed using 5 to 8 times the retention volume of the membrane system in phosphate buffer; in step S3, the loading throughput of the strong anion exchange membrane is 50 to 180 L·m⁻²·h⁻¹, and the membrane is washed with buffer containing 0.20 to 0.35 mol / L sodium chloride, and the fraction obtained by elution with buffer containing 0.55 to 0.85 mol / L sodium chloride is collected.

[0010] Preferably, the H component and the M component are replaced with 5 to 8 times the retention volume of the membrane system with purified water; the mass ratio of trehalose to proteoglycan active solids is 0.50:1 to 1.50:1, and the mass ratio of ectoine to proteoglycan active solids is 0.05:1 to 0.25:1.

[0011] Preferably, the H component accounts for 55% to 70% of the total mass of the active solids of the H and M components, and the M component accounts for 30% to 45%. The compound forms an H main peak and an M main peak in the same size exclusion chromatography-differential refractive index detection spectrum, respectively. The sum of the peak areas of the H main peak and the M main peak accounts for no less than 90% of the total peak area. The response intensity of the valley value between the two main peaks is no higher than 70% of the peak height of the lower main peak, and the peak area of ​​the region less than 30 kDa accounts for no more than 5.0% of the total peak area.

[0012] Preferably, the proportion of proteoglycan active solids in H powder and M powder to the mass of the corresponding anhydrous powder is not less than 35%; on a dry basis, the content of sulfated glycosaminoglycan is 35% to 70%, the protein content is 8% to 25%, the mass ratio of sulfated glycosaminoglycan to protein is 1.5 to 6.0, and the hydroxyproline content is not higher than 2.0%; the moisture content of H powder and M powder is not higher than 8.0%.

[0013] A standardized proteoglycan two-component raw material includes separately packaged H component and M component, wherein the H component and M component are prepared by extraction, purification and standardized preparation method of proteoglycan active components derived from marine bony fish cartilage, the peak apparent molecular weight of the H component is 1.25 to 1.90 MDa, the peak apparent molecular weight of the M component is 80 to 220 kDa, and the H component and M component meet the composition range of claim 6.

[0014] A method for preparing a cosmetic composition, comprising a standardized proteoglycan two-component raw material, comprising the following steps: Step 1: Hydrogenated lecithin, phytosterols and ceramides are used to form a blank layered carrier at 65 to 75°C, and the blank layered carrier is emulsified and granulated before the addition of proteoglycan active components; Step 2: Cool the blank layered carrier to 25 to 35°C, add component M and let it stand for hydration for 20 to 60 minutes; if further granulation is required, extrude 1 to 3 times using a membrane with a pore size of 200 to 400 nm, or homogenize once using a pressure not exceeding 15 MPa. Step 3: After completing all high-shear, homogenization, microfluidization, ultrasonication and membrane extrusion operations, add component H at a temperature not exceeding 35°C and mix at a low speed to keep component H in the continuous aqueous phase.

[0015] A cosmetic composition, based on a method for preparing the cosmetic composition, comprises the following steps: the total active solids of component H and component M account for 0.010% to 0.150% of the total mass of the cosmetic composition, wherein component H accounts for 55% to 70% of the total mass of the active solids of component H and component M, and component M accounts for 30% to 45%; the mass ratio of hydrogenated lecithin, phytosterols, and ceramides is 100:(5 to 30):(1 to 10); the Z-average hydrated particle size of the layered carrier, measured by dynamic light scattering, is 100 to 450 nm, and the polydispersity index is not higher than 0.35; component H is located in a continuous aqueous phase, and component M is co-dispersed with the layered carrier; the cosmetic composition is a firming essence, anti-wrinkle cream, eye cream, or mask essence, with a pH of 5.2 to 6.3.

[0016] (III) Beneficial Effects Compared with existing technologies, this invention provides a method for the extraction, purification, and standardized preparation of proteoglycan active components derived from the cartilage of marine bony fish, which has the following beneficial effects: 1. The extraction, purification, and standardized preparation method of proteoglycan active components derived from marine bony fish cartilage eliminates exogenous proteases, glycosaminoglycan degrading enzymes, guanidine salts, ethylenediaminetetraacetic acid, and organic alcohol precipitates through the extraction to drying process. The process has clear differences and is suitable for converting by-products of edible marine bony fish processing into cosmetic active ingredients.

[0017] 2. The extraction, purification and standardized preparation method of the active components of proteoglycan derived from the cartilage of marine bony fish. The H component and the M component were obtained from the same batch of enriched materials by sequential membrane fractionation and size exclusion chromatography. They have the same source but different molecular weights and functional orientations. The influence of the difference in natural molecular weight distribution on the consistency of the formulation can be reduced by fixing the mass ratio of H / M active solids.

[0018] 3. The extraction, purification and standardized preparation method of proteoglycan active components derived from marine bony fish cartilage, and the standardized system for mutual verification of source sequence, core protein marker peptide, glycodisaccharide composition and size exclusion chromatographic bimodal fingerprint formation, can distinguish proteoglycans, free glycosaminoglycans, collagen contamination and excessively low molecular weight products.

[0019] 4. The extraction, purification and standardized preparation method of proteoglycan active components derived from the cartilage of marine bony fish. The dual-space formulation configures the high molecular weight H component in a continuous aqueous phase and disperses the medium molecular weight M component with a layered carrier, which can be used to evaluate and achieve differentiated local retention.

[0020] 5. The extraction, purification, and standardized preparation method of the active proteoglycan component derived from marine bony fish cartilage is described with key parameters in terms of detectable process windows and release indicators, which facilitates subsequent scale-up, third-party testing, raw material filing data organization, and consistency maintenance in PCT applications. Attached Figure Description

[0021] Figure 1 A flowchart illustrating the extraction, purification, fractionation, standardization, and application of active components of proteoglycans; Figure 2 This is a schematic diagram for determining the bimodal size exclusion chromatographic quality fingerprint formed by component H and component M. Figure 3 A schematic diagram of the H / M dual-space configuration and local skin retention; Figure 4 A multi-dimensional quality control chain for source, structure, sequence, molecular weight, stability, and formulation release. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1 This embodiment describes the steps for species identification and pretreatment of raw materials to ensure traceability of origin and compliance with subsequent extraction requirements.

[0024] (1) Raw material selection: Select head cartilage, fin base cartilage, gill cover connecting cartilage or intervertebral cartilage separated within 4 hours after processing of edible marine bony fish. The raw materials are transported under refrigeration at 0 to 4℃. If they cannot be processed on the same day, they are frozen and stored at no higher than -20℃.

[0025] (2) Species confirmation: Three parts are randomly selected from each batch of raw materials, and about 50 mg of tissue is taken from each part to extract DNA. The mitochondrial COI barcode fragment is amplified, and the sequencing results are compared with the confirmed reference sequence. The species consistency is preferably not less than 98%, and the original peak diagram, reference sequence version and batch traceability record are retained.

[0026] (3) Washing and chopping: Rinse quickly with purified water at 2 to 8°C to remove visible blood, muscle and fat. Chop the cartilage into 3 to 8 mm particles. If necessary, use low-speed centrifugation or skimming to remove free fat. Do not use organic solvents for degreasing.

[0027] (4) Raw material release: There is no putrid odor, and the volatile basic nitrogen, microorganisms and contaminants meet the enterprise's internal control and target market requirements for the raw materials to be used. Moisture and ash content are recorded as input conversion parameters.

[0028] Example 2 This embodiment uses 10 kg of wet cartilage as an example to specifically illustrate the operation of low-temperature extraction and charge purification through an ion exchange membrane.

[0029] (1) Prepare 80L of extraction solution: 20mmol / L sodium phosphate buffer system, 1.0mol / L sodium chloride, adjust pH to 6.9±0.1 and pre-cool to 4 to 6℃.

[0030] (2) Add 10 kg of pretreated wet cartilage to a jacketed mixing tank, add the extract, and stir for 6 h at 4 to 8 °C and 80 to 160 r / min. The stirring paddle speed should be limited to the point that it can form an overall circulation without producing obvious foam.

[0031] (3) Centrifuge at 3000 to 8000×g for 20 to 30 minutes or equivalent solid-liquid separation, collect the supernatant, and pass it through 5μm and 0.45μm filter units in sequence. Replace the filter material when the filtration pressure difference is close to the upper limit of the equipment to avoid high pressure shearing for a long time.

[0032] (4) The clarified solution is concentrated to about 10 to 15 L by tangential flow membrane with 50 kDa, and constant volume liquid exchange is performed with 5 to 8 times the retention volume of buffer solution containing 20 mmol / L phosphate, 50 mmol / L sodium chloride, and pH 7.0, so that the conductivity is 4 to 9 mS / cm, the membrane process temperature is maintained at 4 to 12 °C, and the transmembrane pressure is preferably 0.05 to 0.20 MPa.

[0033] (5) Pass the feed solution after the solution exchange through a quaternary ammonium strong anion exchange membrane. Before loading the sample, treat the membrane unit with an equilibration solution of 20 mmol / L phosphate, 50 mmol / L sodium chloride, and pH 7.0. The loading throughput is 50 to 180 L·m⁻²·h⁻¹. Wash with 3 to 5 membrane volumes of equilibration solution.

[0034] (6) The fractions were eluted sequentially with 20 mmol / L phosphate buffer containing 0.30 mol / L, 0.55 mol / L and 0.85 mol / L sodium chloride, and collected separately. The fractions were screened for proteoglycan enrichment by DMMB colorimetry, 280 nm absorption, hydroxyproline and size exclusion chromatography. The fractions of 0.55 to 0.85 mol / L that met the requirements of sulfated glycosaminoglycan enrichment and collagen-related signal control were combined.

[0035] Example 3 This embodiment illustrates the sequential classification using 500kDa and 50kDa tangential flow films, as well as the subsequent stabilization and drying processes.

[0036] (1) The combined fraction obtained in Example 2 was first replaced with a 50kDa membrane to 20mmol / L phosphate, 50mmol / L sodium chloride, pH 7.0, so that the conductivity was 4 to 9mS / cm.

[0037] (2) The feed solution is passed through a 500kDa tangential flow membrane, and the temperature is controlled at 4 to 12℃ and the transmembrane pressure is controlled at 0.03 to 0.15Mpa. During the membrane concentration and liquid exchange process, one or more H-segment collects are formed according to the treatment time or concentration factor. The 500kDa permeate is used as the feed for the M component.

[0038] (3) Pass the 500kDa permeate through a 50kDa tangential flow membrane. During membrane concentration and liquid exchange, one or more M-segmented collections are formed according to the treatment time or concentration factor. Permeate with less than 50kDa is not included in the active component.

[0039] (4) The H-fractional and M-fractional samples were replaced with 5 to 8 times the retention volume of purified water to ensure that the conductivity was not higher than 3.0 mS / cm. They were then screened by size exclusion chromatography-differential refractive index detection. Only the H-fractional samples with an apparent molecular weight at the peak of 1.25 to 1.90 MDa and a peak area of ​​less than 30 kDa that was not higher than 5.0% were merged into the H-fraction. Only the M-fractional samples with an apparent molecular weight at the peak of 80 to 220 kDa and a peak area of ​​less than 30 kDa that was not higher than 5.0% were merged into the M-fraction. Boundary fractional samples located outside the target window were not considered qualified by changing the concentration factor, but were sent back to the adjacent membrane fractionation step or processed separately.

[0040] (5) Based on the active solids of proteoglycan, add 0.50 to 1.50 times the mass of trehalose and 0.05 to 0.25 times the mass of ectoine to the H component and the M component respectively, adjust the solids of the solution to 1.5% to 3.5%, freeze at a temperature not higher than -35°C and then freeze dry, and the secondary drying temperature not higher than 25°C.

[0041] (6) H powder and M powder are placed in moisture-proof, light-proof, and low-adsorption packaging respectively. They can be reconstituted separately when used, or they can be mixed in a ratio of 55% to 70% of the total mass of active solids of the two components and 30% to 45% of the M component to form a bimodal standardized raw material.

[0042] Example 4 This embodiment specifies multidimensional indicators and their detection methods for quality control of H powder and M powder, ensuring consistency between product batches.

[0043] The preferred conditions for size exclusion chromatography-refractive index detection are as follows: a size exclusion column suitable for the analysis of water-soluble polymers is used; the mobile phase is 50 mmol / L phosphate and 0.20 mol / L sodium chloride, pH 7.0; the flow rate is 0.5 mL / min; the column temperature is 30–40 °C; and refractive index detection is performed. A log(Mp)-retention time curve is established using a validated pullulan standard capable of encompassing the retention times of components H and M, without extrapolation of the curve range. If the conventional standard cannot stably encompass component H, size exclusion chromatography-multi-angle laser light scattering is used to confirm the method and establish a transfer relationship with the routine size exclusion chromatography-refractive index detection method. Sample concentration, injection volume, column type, system delay volume, and integration rule are fixed during method transfer.

[0044] The preferred method for fingerprinting core protein marker peptides is as follows: In the analysis and detection, the major glycan chains are first removed with chondroitinase ABC and a suitable keratinase, followed by trypsin digestion, and high-resolution LC-MS / MS is used for collection; marker peptides with good source specificity are selected from the G1, G2 or other conserved regions of agglutinin, and a database of retention time, parent ion, major fragment ion and spectral similarity is established; glycosaminoglycans are only used for analysis and detection and are not involved in the extraction, purification, fractionation or drying processes.

[0045] Example 5 This embodiment lists the compound specifications and applicable scenarios for different H / M active solids mass ratios.

[0046] The liquid concentrate can be made by redissolving H powder and M powder separately in an aqueous phase with a pH of 5.5 to 6.2, with an active solids concentration of 0.5% to 2.0% for each; add 0.2% to 1.0% of 1,2-hexanediol, 0.1% to 0.5% of p-hydroxyacetophenone, or a preservative system that meets the requirements of the target market relative to the total mass of the concentrate; H liquid and M liquid are packaged separately and added according to the active solids during formulation production.

[0047] Example 6 This embodiment provides a specific method for loading component M into a pre-prepared phospholipid layered carrier, emphasizing loading at low temperature to avoid degradation of the active ingredient.

[0048] (1) Hydrogenated lecithin, phytosterol and ceramide are added to squalane or caprylic / capric triglyceride in a mass ratio of 100: (5 to 30): (1 to 10) and dispersed uniformly at 65 to 75°C.

[0049] (2) The lipid phase is added to the isothermal aqueous phase, and a blank layered dispersion is formed by high shear. Pre-emulsification and granulation are completed without the addition of proteoglycan active components, and then cooled to 25 to 35°C.

[0050] (3) Add component M slowly to the blank layered dispersion in an aqueous solution of 0.1% to 1.0% and let it stand at 25 to 35°C for 20 to 60 minutes to hydrate. If further adjustment of particle size is required, extrude it through a membrane with a pore size of 200 to 400 nm 1 to 3 times, or homogenize it once under a pressure not higher than 15 MPa. Do not use multiple high-pressure micro-jet or long-term ultrasonic treatment.

[0051] (4) After the addition of component M, the average hydrated particle size and PDI were measured, and the co-distribution of glycosaminoglycans and phospholipids after size separation, ultrafiltration retention or equivalent methods were used to confirm that component M and the layered carrier were co-dispersed. After all the granulation operations were completed, the continuous aqueous phase containing component H was added and mixed at low speed. After the addition of component H, high shear, high pressure homogenization, microfluidization, ultrasonication or membrane extrusion were no longer performed.

[0052] Example 7 This embodiment provides a firming serum formula with H / M dual-space configuration and its preparation points.

[0053] Preparation method: First, a blank layered dispersion was prepared using squalane, hydrogenated lecithin, phytosterols and ceramides, and pre-emulsification and granulation were completed without the addition of active components; after cooling to 30±5℃, component M was added and hydrated according to Example 6; the thickener was fully hydrated in the main aqueous phase, and polyol, betaine, trehalose, ectoine and preservative synergistic system were added; the layered dispersion containing component M was added to the main aqueous phase, and after completing all granulation operations, component H was added and mixed at low speed, and the pH was adjusted to 5.5 to 6.2.

[0054] The formula composition is shown in Table 3.

[0055] Table 3 Firming Essence Formula Example 8 This embodiment provides an anti-wrinkle face cream formula containing an H / M dual-space configuration.

[0056] Preparation method: The conventional oil phase and the main aqueous phase are heated and emulsified separately to form a cream matrix; a blank layered carrier containing hydrogenated lecithin, phytosterols and ceramides is prepared as an independent pre-dispersion and pre-granulated; after the cream matrix is ​​cooled to below 35°C, component M is added to the layered dispersion and hydrated, and then the dispersion is added to the cream matrix; after all granulation operations are completed, component H is added, and the mixture is mixed uniformly at low speed, and the pH is adjusted to 5.4 to 6.1.

[0057] The formula composition is shown in Table 4.

[0058] Table 4 Anti-wrinkle face cream formulas Example 9 This embodiment provides a gentle eye cream formula.

[0059] Preparation method: No fragrance or volatile essential oil is added to the eye cream; the emulsion matrix and blank layered carrier are prepared and granulated separately; after cooling to below 35°C, component M is added to the layered carrier and hydrated, then mixed with the emulsion matrix, panthenol and ectoine; finally, component H is added, mixed at low speed and the pH is adjusted to 5.5 to 6.2.

[0060] The formula composition is shown in Table 5.

[0061] Table 5 Eye Cream Formulas Example 10 This embodiment provides a facial mask essence formula with good fluidity.

[0062] Preparation method: First, prepare a low-oil phase blank layered dispersion containing hydrogenated lecithin, phytosterols and ceramides and complete granulation. After cooling, add component M and hydrate. Hydrate hydroxyethyl cellulose and sclerotium gum in the main aqueous phase, and add polyol, betaine, trehalose, ectoine and preservative synergistic system. Add the layered dispersion containing component M, and after completing all granulation operations, add component H and adjust the pH to 5.4 to 6.2.

[0063] The formula composition is shown in Table 6.

[0064] Table 6 Mask Essence Formula Example 11 This embodiment provides a method for evaluating the stability of H / M dual-space formulations under storage and freeze-thaw conditions.

[0065] Example 12 This embodiment uses a Franz diffusion cell and pig ear skin to evaluate the effect of H / M dual-space configuration on local retention distribution.

[0066] (1) Use pig ear skin or human skin with a complete barrier, with consistent thickness, place it in the Franz diffusion cell, record the test area, maintain the leakage conditions with body fluid, and control the skin surface temperature to 32±1℃.

[0067] (2) Set up blank matrix group, ungraded proteoglycan continuous phase group, M component only layered carrier group and H / M dual space group; the total active solids of each proteoglycan group are kept consistent, and the application amount is 2.0±0.2mg / cm².

[0068] (3) After 6 hours of action, collect the skin surface residue, continuous tape peeling material and cyanoacrylate hair follicle peeling material in sequence, and then collect the remaining active epidermis, dermis and receptor fluid for mass balance; H component and M component can be labeled with low degree of substitution fluorescence, or quantified by size grading combined with DMMB, marker peptide or equivalent method; the peak shift of size exclusion chromatography before and after labeling is not higher than 10%.

[0069] (4) The main indicators are the recovery ratio of H component in the skin surface and outer stratum corneum tape, and the recovery ratio of M component in the middle and lower stratum corneum tape and hair follicle stripping material; the recovery amount in active epidermis, dermis and receptor fluid is used as the delivery boundary and quality balance indicator; each group has at least 6 parallel skin units, and the barrier integrity and quality balance exclusion criteria are set in advance.

[0070] (5) The criteria for determining local retention are: relative to the ungraded proteoglycan continuous phase group, the H component of the H / M dual space group is more distributed on the skin surface and the outer side of the stratum corneum, and the M component is more distributed in the middle and lower part of the stratum corneum or the hair follicle opening; the present invention does not require proteoglycan to enter the active epidermis, dermis or systemic circulation as a necessary condition.

[0071] Example 13 This embodiment evaluates the effect of the H / M component on the expression of matrix-related genes in skin fibroblasts through in vitro cell experiments.

[0072] (1) Use normal human skin fibroblasts, preferably passaged 3 to 8 times; set up unirradiated blank group, UVB model group, vector control group, ungraded PG group, H group group, M group group and H / M combination group.

[0073] (2) The sample concentration can be set in the range of 100, 400 and 800 μg / mL proteoglycan active solids, and the non-cytotoxic window should be confirmed by cell viability test first; at least 3 technical replicates should be set for each condition, and each condition should be independently repeated no less than 3 times.

[0074] (3) Evaluation indicators include cell viability, mRNA or protein expression of COL1A1, ELN, HAS2, and MMP1, as well as Erk1 / 2 phosphorylation; for the UVB model, UVB dose, irradiation distance, culture time after irradiation, and solvent control should be recorded simultaneously.

[0075] (4) Statistical analysis uses predetermined main indicators and multiple comparison methods, and reports the mean, dispersion, sample size, effect size and confidence interval; matrix-related indicators are interpreted only when cell viability meets predetermined thresholds.

[0076] Example 14 This embodiment provides a design framework for human clinical evaluation trials to verify the actual efficacy of a product.

[0077] (1) A firming essence containing H / M dual space raw materials can be selected as a representative formula. A randomized, double-blind, parallel control design with the same subject's left and right faces or parallel controls is adopted, and a control without proteoglycans but with the same matrix is ​​set up.

[0078] (2) Subjects are healthy adults with concerns about dryness, decreased elasticity or fine lines; those with active skin diseases, recent cosmetic treatments or use of drugs that affect the evaluation are excluded.

[0079] (3) The main observation indicators may be the stratum corneum water content, transepidermal water loss, skin elasticity parameters and fine lines around the eyes; the secondary indicators may be self-assessment of tightness, skin feel and tolerance.

[0080] (4) The evaluation period can be immediate, 2 weeks, 4 weeks and 8 weeks; the ambient temperature and humidity and the subject's equilibration time should be controlled before the instrument measurement; all subject data, informed consent and ethical requirements should be implemented in accordance with applicable norms.

[0081] In summary, the extraction, purification, and standardized preparation method of proteoglycan active components derived from marine bony fish cartilage effectively eliminates exogenous proteases, glycosaminoglycan degrading enzymes, guanidine salts, EDTA, and organic alcohol precipitates during the extraction to drying process. The process exhibits clear differences and is suitable for converting by-products of edible marine bony fish processing into cosmetic active ingredients.

[0082] Furthermore, the extraction, purification, and standardized preparation method of the active components of proteoglycans derived from marine bony fish cartilage allows the H and M components to be obtained from the same batch of enriched materials through sequential membrane fractionation and size exclusion chromatography. Although they have the same source, their molecular weights and functional orientations are different. The impact of differences in natural molecular weight distribution on the consistency of the formulation can be reduced by fixing the mass ratio of H / M active solids.

[0083] Furthermore, the extraction, purification, and standardized preparation method of the active components of proteoglycan derived from marine bony fish cartilage, along with the standardized system that verifies the source sequence, core protein marker peptides, glycodisaccharide composition, and size exclusion chromatographic bimodal fingerprint, can distinguish between proteoglycans, free glycosaminoglycans, collagen contamination, and excessively low molecular weight products.

[0084] Furthermore, the extraction, purification, and standardized preparation method of the active components of proteoglycans derived from the cartilage of marine bony fish, with its dual-space formulation that places the high molecular weight H component in a continuous aqueous phase and disperses the medium molecular weight M component with a layered carrier, can be used to evaluate and achieve differentiated local retention.

[0085] Furthermore, the extraction, purification, and standardized preparation method of proteoglycan active components derived from marine bony fish cartilage uses detectable process windows and release indicators for key parameters, facilitating subsequent scale-up, third-party testing, raw material filing, and consistency maintenance in PCT applications. This method solves the problems in existing technologies, such as the easy destruction of the core protein-glycan structure due to extraction methods using acids, proteases, high temperatures, guanidine salts, or organic alcohol precipitation; the difficulty in reflecting the source and structural integrity with single quality control indicators; the inability of single molecular weight proteoglycans to simultaneously address the differentiated delivery to the skin surface and the lower part of the stratum corneum; and the lack of an integrated preparation and formulation space configuration scheme for H / M dual molecular weight fractions.

[0086] The relevant modules involved in this system are all hardware system modules or functional modules that combine computer software programs or protocols with hardware in the prior art. The computer software programs or protocols involved in these functional modules are technologies known to those skilled in the art and are not improvements to this system. The improvement of this system lies in the interaction or connection between the modules, that is, in improving the overall structure of the system to solve the corresponding technical problems that this system aims to address.

[0087] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for the extraction, purification, and standardized preparation of proteoglycan active components derived from the cartilage of marine bony fish, characterized in that, Includes the following steps: S1: Processed cartilage from edible marine bony fish with confirmed source is made into 3 to 8 mm particles. The wet cartilage mass (kg) to the extraction liquid volume (L) ratio is 1:6 to 1:

12. An extraction solution containing 10 to 30 mmol / L phosphate and 0.8 to 1.2 mol / L sodium chloride, with a pH of 6.6 to 7.2, is added and extracted at 2 to 10 °C for 4 to 10 h to obtain a crude extract. S2: The crude extract is subjected to solid-liquid separation and clarification filtration with a pore size of no more than 0.45 μm, and then concentrated and constant-volume liquid exchange is performed using a tangential flow membrane with a nominal molecular weight cutoff of 50 kDa, so that the conductivity of the feed liquid reaches 4 to 9 mS / cm. S3: Pass the feed solution from step S2 through a quaternary ammonium strong anion exchange membrane, elute with a buffer solution containing 0.45 to 0.95 mol / L sodium chloride and with a pH of 6.7 to 7.3, and collect the proteoglycan enrichment fraction; S4: After the enrichment fraction is replaced, it is first fractionated by a tangential flow membrane with a nominal molecular weight cutoff of 500kDa. The 500kDa cutoff liquid is used as candidate fraction H. Then, the 500kDa permeate is fractionated by a tangential flow membrane with a nominal molecular weight cutoff of 50kDa. The 50kDa cutoff liquid is used as candidate fraction M. S5: Form one or more fractional collectors from the H candidate fraction and the M candidate fraction and perform size exclusion chromatography detection. Combine the H fractional collectors with an apparent molecular weight of 1.25 to 1.90 MDa at the peak to form the H fraction, and combine the M fractional collectors with an apparent molecular weight of 80 to 220 kDa at the peak to form the M fraction. Ensure that the peak area of ​​the region less than 30 kDa in the H fraction and the M fraction is no more than 5.0% of their respective total peak area. Return the boundary fractional collectors located outside the target window to the adjacent membrane fractionation step for processing. S6: Add trehalose and ectoine to H component and M component respectively, and dry them to obtain H powder and M powder; In particular, no exogenous proteases, glycosaminoglycan degrading enzymes, guanidine salts or ethylenediaminetetraacetic acid are added during the extraction, clarification, membrane purification, membrane fractionation and drying processes, and no organic alcohol precipitation is used.

2. The method for extraction, purification, and standardized preparation of proteoglycan active components derived from marine bony fish cartilage according to claim 1, characterized in that, The marine bony fish is selected from one of the following: golden pomfret, grouper, cobia, and sea bass. The cartilage is selected from one of the following: head cartilage, fin base cartilage, operculum connecting cartilage, and intervertebral cartilage.

3. The method for extraction, purification, and standardized preparation of proteoglycan active components derived from marine bony fish cartilage according to claim 1, characterized in that, In step S2, the constant-volume buffer exchange is performed using 5 to 8 times the retention volume of the membrane system in phosphate buffer. In step S3, the loading throughput of the strong anion exchange membrane is 50 to 180 L·m⁻²·h⁻¹, and the membrane is washed with buffer containing 0.20 to 0.35 mol / L sodium chloride. The fraction obtained by elution with buffer containing 0.55 to 0.85 mol / L sodium chloride is collected.

4. The method for extraction, purification, and standardized preparation of proteoglycan active components derived from marine bony fish cartilage according to claim 1, characterized in that, The H and M components are replaced with 5 to 8 times the retention volume of purified water in the membrane system; the mass ratio of trehalose to proteoglycan active solids is 0.50:1 to 1.50:1, and the mass ratio of ectoine to proteoglycan active solids is 0.05:1 to 0.25:

1.

5. The method for extraction, purification, and standardized preparation of proteoglycan active components derived from marine bony fish cartilage according to claim 1, characterized in that, The H component is formulated in a ratio of 55% to 70% of the total mass of active solids of the H and M components, and the M component is formulated in a ratio of 30% to 45%. The formulated compound forms an H main peak and an M main peak in the same size exclusion chromatography-differential refractive index detection spectrum. The sum of the peak areas of the H main peak and the M main peak accounts for no less than 90% of the total peak area. The response intensity of the valley value between the two main peaks is no higher than 70% of the peak height of the lower main peak, and the peak area of ​​the region less than 30 kDa accounts for no more than 5.0% of the total peak area.

6. The method for extraction, purification, and standardized preparation of proteoglycan active components derived from marine bony fish cartilage according to claim 1, characterized in that, The active proteoglycan solids in the H powder and M powder account for no less than 35% of the mass of the corresponding anhydrous powder; on a dry basis, the content of sulfated glycosaminoglycan is 35% to 70%, the protein content is 8% to 25%, the mass ratio of sulfated glycosaminoglycan to protein is 1.5 to 6.0, and the hydroxyproline content is no higher than 2.0%; the moisture content of the H powder and M powder is no higher than 8.0%.

7. A standardized proteoglycan two-component raw material, characterized in that, The product comprises separately packaged components H and M, which are prepared by the extraction, purification, and standardized preparation method of the active proteoglycan derived from marine bony fish cartilage according to any one of claims 1 to 4. The apparent molecular weight at the peak of component H is 1.25 to 1.90 MDa, and the apparent molecular weight at the peak of component M is 80 to 220 kDa. Furthermore, components H and M satisfy the composition range described in claim 6.

8. A method for preparing a cosmetic composition, characterized in that, Including the standardized proteoglycan bicomponent raw material as described in claim 7, the steps are as follows: Step 1: Hydrogenated lecithin, phytosterols and ceramides are used to form a blank layered carrier at 65 to 75°C, and the blank layered carrier is emulsified and granulated before the addition of proteoglycan active components; Step 2: Cool the blank layered carrier to 25 to 35°C, add component M and let it stand for hydration for 20 to 60 minutes; if further granulation is required, extrude 1 to 3 times using a membrane with a pore size of 200 to 400 nm, or homogenize once using a pressure not exceeding 15 MPa. Step 3: After completing all high-shear, homogenization, microfluidization, ultrasonication and membrane extrusion operations, add component H at a temperature not exceeding 35°C and mix at a low speed to keep component H in the continuous aqueous phase.

9. A cosmetic composition, characterized in that, The preparation method of the cosmetic composition according to claim 8 includes the following steps: the total active solids of component H and component M account for 0.010% to 0.150% of the total mass of the cosmetic composition, wherein component H accounts for 55% to 70% of the total mass of the active solids of component H and component M, and component M accounts for 30% to 45%; the mass ratio of hydrogenated lecithin, phytosterol and ceramide is 100:(5 to 30):(1 to 10); the Z-average hydrated particle size of the layered carrier, measured by dynamic light scattering, is 100 to 450 nm, and the polydispersity index is not higher than 0.35; component H is located in the continuous aqueous phase, and component M is co-dispersed with the layered carrier; the cosmetic composition is a firming essence, anti-wrinkle cream, eye cream or mask essence, with a pH of 5.2 to 6.3.

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