Preparation method and application of small molecule olea europaea sugar peptide

Abstract

This application relates to the field of small molecule reagents for cosmetics, and particularly to a method for preparing small molecule olive glycopeptides and their applications. The preparation method includes: crushing olive pomace to obtain olive pomace powder; extracting the olive pomace powder with an organic solvent to obtain a solid residue; drying the solid residue to obtain a dried solid residue; extracting the dried solid residue with distilled water in multiple rounds to obtain an extract; performing a first solid-liquid separation and concentration of the extract to obtain a concentrate; purifying the concentrate with ethanol to obtain a crude polysaccharide powder; enzymatically hydrolyzing a complex enzyme system, a buffer solution, and the crude polysaccharide to obtain an enzymatic hydrolysis product; inactivating the enzyme in the enzymatic hydrolysis product to obtain an enzyme inactivation reaction product; performing a second solid-liquid separation of the enzyme inactivation reaction product to obtain a crude olive glycopeptide; and further purifying the crude olive glycopeptide to obtain the final olive glycopeptide. This preparation method improves the utilization rate of olive glycopeptides from four dimensions: raw material utilization, extraction yield, component activity, and application value.

Description

Technical Field

[0001] This application relates to the field of small molecule reagents for cosmetics, and in particular to a method for preparing a small molecule olive glycopeptide and its application. Background Technology

[0002] Olive glycopeptides are high-molecular-weight natural polysaccharides extracted from olive pomace, possessing various biological activities such as moisturizing, anti-oxidation, and anti-inflammation.

[0003] However, due to their large molecular weight and poor water solubility, olive glycopeptides can easily lead to unstable viscosity in formulations such as face creams and serums, potentially causing layering or precipitation during long-term storage. Furthermore, in existing technologies, these olive glycopeptides are mostly added as adjuncts, with concentrations below 1%, making it difficult to fully utilize their multiple synergistic effects and thus limiting their overall effectiveness and utilization rate. Summary of the Invention

[0004] This application provides a method for preparing small molecule olive glycopeptides and their applications, in order to solve the following technical problem: how to improve the utilization rate of olive glycopeptides.

[0005] In a first aspect, embodiments of this application provide a method for preparing small molecule olive glycopeptides, the preparation method comprising: The olive pomace is crushed to obtain olive pomace powder; The olive pomace powder was extracted using an organic solvent to obtain a solid residue. The solid slag is dried to obtain dried solid slag; The distilled water and the dried solid residue were subjected to multiple extractions to obtain the extract; The extract was subjected to a first solid-liquid separation and concentration process to obtain a concentrated solution. The concentrate was first purified using ethanol to obtain crude polysaccharide powder. The complex enzyme system, buffer solution, and crude polysaccharide powder were subjected to enzymatic hydrolysis to obtain the enzymatic hydrolysis product. The enzyme hydrolysis product is subjected to enzyme inactivation to obtain an enzyme inactivation reaction product. The enzyme inactivation reaction product was subjected to a second solid-liquid separation to obtain crude olive glycopeptide; The crude olive glycopeptide was subjected to a second purification to obtain olive glycopeptide.

[0006] Optionally, the complex enzyme system is a mixture of cellulase, pectinase, and hemicellulase, wherein the mass m1 of the cellulase, the mass m2 of the pectinase, and the mass m3 of the hemicellulase satisfy: m1:m2:m3 = (1 to 3):(1 to 2):(0.5 to 1.0); and / or The mass m4 of the complex enzyme system, the mass m5 of the crude polysaccharide powder, and the volume V1 of the buffer solution satisfy: (m4 + m5):V1 = 1:(15 to 40); and / or The amount of the complex enzyme system added is 3 wU / g to 5 wU / g.

[0007] Optionally, the enzymatic hydrolysis temperature is 45°C to 50°C, and the enzymatic hydrolysis time is 2 hours to 3 hours.

[0008] Optionally, the mass m6 of the dried solid residue and the volume V2 of the distilled water satisfy: m6:V2 = 1:(15 to 40), where if m6 is in g, then V2 is in mL; and / or The number of extractions in the multiple rounds is 1 to 3, the temperature of the multiple extractions is 70°C to 100°C, and the extraction time for each extraction is 1 hour to 4 hours.

[0009] Optionally, the enzyme inactivation is performed by water bath heating at a temperature of 90°C to 100°C for a time of 10 to 15 minutes; and / or The second solid-liquid separation includes high-speed centrifugation and filtration; when the second solid-liquid separation is a high-speed separation, the high-speed centrifugation speed is 4000 rpm to 8000 rpm, and the high-speed centrifugation time is 10 min to 20 min.

[0010] Secondly, embodiments of this application provide an olive glycopeptide, which is prepared by the preparation method described in the first aspect; the weight-average molecular weight of the olive glycopeptide is 4226 Da to 4246 Da.

[0011] Optionally, the olive glycopeptide comprises a monosaccharide component and an amino acid component; by mass fraction, the monosaccharide component comprises: mannose: 10% to 12%, rhamnose: 19% to 20%, glucuronic acid: 3% to 4%, galacturonic acid: 7% to 9%, glucose: 2% to 3%, galactose: 28% to 31%, and arabinose: 18% to 21%; and / or The amino acid composition, by mass fraction, includes: aspartic acid: 5% to 7%, glutamic acid: 6% to 7%, serine: 8% to 10%, glycine: 7% to 9%, histidine: 4% to 6%, arginine: 5% to 7%, threonine: 5% to 7%, alanine: 10% to 12%, proline: 5% to 6%, cysteine: 3% to 5%, tyrosine: 7% to 9%, valine: 6% to 8%, and phenylalanine: 5% to 7%.

[0012] Thirdly, embodiments of this application provide an application of a small molecule olive glycopeptide, the application including: using the olive glycopeptide described in the second aspect in cosmetics; the cosmetics include at least one of hydrogel masks, serums, and creams.

[0013] Optionally, when the cosmetic product includes a hydrogel mask, the raw materials of the hydrogel mask, by mass fraction, include: olive glycopeptide: 20% to 25%, sodium alginate: 65% to 70%, and purslane extract: 5% to 15%; and / or When the cosmetic is a serum, the ingredients of the serum, by mass fraction, include: olive glycopeptide: 1% to 5%, sodium hyaluronate: 0.1% to 1.0%, glycerin: 3% to 8%, niacinamide: 1% to 3%, vitamin E acetate: 0.5% to 2.0%, PEG-40 hydrogenated castor oil: 2-8%, purslane extract: 0.5% to 2%, centella asiatica extract: 0.5% to 2%, dipotassium glycyrrhizate: 0.1% to 0.5%, preservative: 0.3% to 0.8%, and deionized water: 75% to 95%; and / or In the case of a face cream, the ingredients of the face cream, by mass fraction, include: olive glycopeptide: 1% to 5%, ceramide complex: 1% to 3%, squalane: 3% to 8%, purslane extract: 0.5% to 2%, centella asiatica extract: 0.5% to 2%, vitamin E acetate: 0.5% to 2%, emulsifier: 3% to 5%, moisturizer: 5% to 10%, preservative: 0.3% to 0.8%, and deionized water: 60% to 86%.

[0014] Optionally, when the cosmetic product includes a hydrogel mask, the method for preparing the hydrogel mask includes: Sodium alginate and deionized water were stirred and dissolved to obtain a sodium alginate solution; Olive glycopeptides and deionized water were stirred and dissolved to obtain an olive glycopeptide solution; The purslane extract and the sodium alginate solution were stirred and dissolved to obtain a hydrogel mixture; The olive glycopeptide solution and the hydrogel mixture are stirred and mixed to obtain a mixed solution; A calcium chloride solution is added to the mixed solution to initiate a gelation reaction, resulting in a hydrogel mask; and / or When the cosmetic is a serum, the method for preparing the serum includes: Sodium hyaluronate and some deionized water are mixed to obtain a sodium hyaluronate solution; Glycerin, nicotinamide, dipotassium glycyrrhizate, and the sodium hyaluronate solution were mixed and sterilized sequentially to obtain the first liquid phase; Vitamin E acetate, a nonionic surfactant, and a portion of the deionized water were mixed to obtain a solution. The olive glycopeptide and the remaining deionized water are mixed to obtain an olive glycopeptide solution; The purslane extract, centella asiatica extract, preservative, and the olive glycopeptide solution were mixed to obtain a second liquid phase. The first liquid phase, the enrichment solution, and the second liquid phase are mixed and emulsified uniformly in sequence to obtain the crude essence. Phenoxyethanol and the crude essence were mixed and the pH was adjusted to obtain the essence; and / or When the cosmetic is a face cream, the method for preparing the face cream includes: Squalane, vitamin E acetate, and emulsifier were mixed and heated to melt in sequence to obtain the oil phase component; The humectant and preservative were dissolved in deionized water and heated to obtain the aqueous phase component. The oil phase component and the aqueous phase component are homogenized and emulsified to obtain a mixed emulsion; Olive glycopeptide, purslane extract, centella asiatica extract, ceramide complex and the mixed emulsion were stirred and mixed to obtain a mixed emulsion product; The mixed emulsion products were sequentially subjected to pH adjustment and filling to obtain a face cream.

[0015] The technical solutions provided in this application have the following advantages compared with the prior art: This application provides a method for preparing small-molecule olive glycopeptides. The method first uses olive processing pomace as raw material, achieving high-value utilization of agricultural waste to fully extract polysaccharide and amino acid components from the byproducts, improving raw material utilization from the source and avoiding waste of resources. Furthermore, this preparation method only requires organic solvent extraction for impurity removal, multiple rounds of distilled water extraction, and solid-liquid separation and concentration to maximize the enrichment of effective polysaccharide components in the olive pomace, reduce the loss of active substances, and improve the extraction yield of olive glycopeptides. Simultaneously, crude polysaccharides from the olive pomace are obtained through preliminary purification with ethanol, and then controlled degradation is performed using a composite enzyme system to obtain olive glycopeptides within a specific molecular weight range. This solves the problem of unclear activity of natural polysaccharides, ensures stable glycopeptide activity, improves effective utilization during application, and facilitates quality control. The olive glycopeptide obtained by this preparation method is derived from food processing byproducts, has extremely high safety and no side effects. Moreover, the olive glycopeptide has multiple skin care effects such as moisturizing, anti-inflammatory and antioxidant properties. It can be directly applied to cosmetics, is suitable for a variety of skin problems, and can also be combined with purslane extract for synergistic effects, broadening the application scenarios and ultimately greatly increasing its actual usage rate. Attached Figure Description

[0016] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A schematic flowchart of a method for preparing small molecule olive glycopeptides provided in this application embodiment; Figure 2 A schematic diagram of the preparation method of the hydrogel mask provided in the embodiments of this application; Figure 3 A schematic diagram of the preparation method of the essence provided in the embodiments of this application; Figure 4 A schematic diagram of the preparation method of face cream provided in the embodiments of this application; Figure 5 This is a high-performance liquid chromatography (HPLC) result of the monosaccharide component in olive glycopeptide provided in the embodiments of this application; Figure 6 This is a high-performance liquid chromatography (HPLC) result of the amino acid components in olive glycopeptides provided in the embodiments of this application; Figure 7 Differential refractive index and ultraviolet detection results of olive glycopeptides provided in the embodiments of this application; Figure 8 The Disease Activity Index (DAI) score results of the experimental animals provided in the embodiments of this application; Figure 9 Comparative images of macroscopic morphology of colon tissue from experimental animals provided in embodiments of this application; Figure 10 H&E-stained pathological section of colon tissue from experimental animals provided in this application embodiment, wherein, Figure 10 a is a pathological section of colon tissue from the normal control group stained with H&E. Figure 10 b is an H&E-stained pathological section of the colon tissue from the model control group. Figure 10 c is a pathological section of colon tissue from the positive control group stained with H&E. Figure 10 d is a pathological section of colon tissue stained with H&E from the high-dose group. Figure 10 e is a pathological section of colon tissue stained with H&E from the medium-dose group. Figure 10 f is a pathological section of colon tissue stained with H&E in the low-dose group; Figure 11This is a comparison chart of the expression levels of anti-inflammatory factors in experimental animals provided in the embodiments of this application, wherein, Figure 11 Figure a shows a comparison of TNF-γ expression levels in experimental animals. Figure 11 b is a comparison of IL-6 expression levels in experimental animals. Figure 11 c is a comparison of IL-1β expression levels in experimental animals. Figure 11 d is a comparison of TNF-α expression levels in experimental animals. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0020] The range descriptions used in this application, such as numerical ranges and proportional ranges, include all possible sub-ranges and single numerical values ​​within that range. For example, the range descriptions of "1 to 6" or "1~6" cover all sub-ranges (such as 1 to 3, 2 to 5, etc.) and single numbers (such as 1, 2, 3, 4, 5, 6) between 1 and 6. Unless otherwise specified, the terms "comprising" and others used herein mean "including but not limited to"; relational terms such as "first" and "second" are used only to distinguish different entities or operations and do not imply an actual order or relationship; "and / or" indicates that multiple situations can exist individually or simultaneously; expressions such as "at least one," "multiple," and "at least one" refer to any combination of the corresponding objects, including combinations of single or multiple objects. The proportional relationships involved in this document, such as mass ratios and molar ratios, should be understood as the correspondence between the first and second terms of a proportional formula, according to the order of description. The raw materials, reagents, instruments, and equipment used herein can all be obtained by purchasing from the market or by existing methods.

[0021] It should be noted that this olive glycopeptide is mostly used alone or in combination with simple ingredients. It lacks systematic combination with barrier repair ingredients such as ceramides and squalane, or with anti-inflammatory ingredients such as purslane extract and centella asiatica extract, which limits the scope of application of olive glycopeptide.

[0022] Based on the aforementioned deficiencies in the prior art, the embodiments of this application provide the following technical solutions: Figure 1 An exemplary schematic diagram of a method for preparing small molecule olive glycopeptides provided in an embodiment of this application is shown. like Figure 1As shown in the embodiments of this application, a method for preparing small molecule olive glycopeptides is provided, the preparation method comprising: S1. Crush the olive pomace to obtain olive pomace powder; S2. Extract the olive pomace powder using an organic solvent to obtain a solid residue; S3. Dry the solid slag to obtain dried solid slag; S4. The distilled water and the dried solid residue are subjected to multiple extractions to obtain the extract; S5. The extract is subjected to a first solid-liquid separation and concentration sequentially to obtain a concentrated solution; S6. The concentrate is first purified using ethanol to obtain crude polysaccharide powder; S7. The complex enzyme system, buffer solution and crude polysaccharide powder are subjected to enzymatic hydrolysis to obtain the enzymatic hydrolysis product; S8. Inactivate the enzyme in the enzymatic hydrolysis product to obtain an enzyme inactivation reaction product; S9. The enzyme inactivation reaction product is subjected to a second solid-liquid separation to obtain crude olive glycopeptide; S10. The crude olive glycopeptide is subjected to a second purification to obtain olive glycopeptide.

[0023] It should be noted that this extraction can be carried out by soaking or rinsing with an organic solvent. The organic solvent can be n-hexane or ethyl acetate.

[0024] It should be noted that the first solid-liquid separation can be performed by centrifugation or filtration. The goal of this concentration is to concentrate the filtrate obtained from the first solid-liquid separation to 1 / 4 to 1 / 5 of its original volume. This first purification can be performed using 3 to 5 times the volume of high-concentration (volume fraction above 95%) ethanol. The process following this first purification also includes: standing overnight at low temperature (below 4°C), collecting the precipitate by centrifugation or filtration, washing and dehydrating with anhydrous ethanol and acetone, and finally dehydrating by vacuum drying or hot air drying.

[0025] It should be noted that the second solid-liquid separation can be carried out by centrifugation or filtration. When the second solid-liquid separation is carried out by centrifugation, the rotation speed can be 4000 rpm to 8000 rpm, and the separation time can be 10 min to 20 min.

[0026] It should be noted that the second purification process can be dialysis, concentration, and lyophilization.

[0027] It should be noted that the buffer solution can be a phosphate buffer or a combination buffer of acetic acid and sodium acetate.

[0028] It should be noted that the method for preparing small molecule olive glycopeptides provided in this application improves the utilization rate of olive glycopeptides from four dimensions: raw material utilization, extraction yield, component activity, and application value. The specific mechanism is as follows: 1. Turning waste into treasure, improving raw material utilization from the source: This preparation method uses olive processing waste (olive pomace) as the starting material. After crushing and grinding, it directly utilizes agricultural waste, realizing the high-value transformation of olive processing by-products. It no longer relies on high-priced raw materials such as fresh fruit, which not only solves the problem of raw material source, but also makes full use of the polysaccharide components in the waste pomace, greatly improving the overall utilization rate of raw materials from the source.

[0029] 2. Multi-stage extraction and enrichment to improve the yield of active ingredients: This preparation method obtains a solid residue through organic solvent extraction to remove impurities, followed by multiple rounds of distilled water extraction, combined with two solid-liquid separations, concentration, and initial purification with ethanol. This maximizes the enrichment of effective polysaccharide components in olive pomace, reduces the loss of active ingredients, improves the extraction yield of olive glycopeptides, avoids the waste of effective substances in raw materials, and enhances the utilization rate of the preparation process.

[0030] 3. Controllable enzymatic hydrolysis and purification improve the effective utilization rate of active ingredients: This preparation method uses a complex enzyme system to controllably degrade (enzymatically hydrolyze) crude polysaccharide powder, precisely obtaining glycopeptides within a specific molecular weight range. After enzyme inactivation, secondary solid-liquid separation, and secondary purification, high-purity olive glycopeptides are obtained. This method solves the problems of wide molecular weight distribution and unclear active ingredients in natural polysaccharides, ensuring stable glycopeptide activity and controllable quality. It allows the effective ingredients to precisely exert their anti-inflammatory and skin-care effects, eliminates the loss of ineffective ingredients, and improves the effective utilization rate during application.

[0031] 4. High security + multi-functional adaptation, expanding application scenarios and increasing usage rate: The raw materials used in this preparation method are food processing by-products. The preparation process is mild and does not involve the addition of toxic substances, resulting in olive glycopeptides with extremely high safety and no side effects. At the same time, the olive glycopeptides prepared by this method have multiple effects such as moisturizing, anti-inflammatory and soothing, anti-oxidation, and skin barrier repair. They can be directly used in cosmetics and are suitable for sensitive, dry, and barrier-damaged skin types. They can also be combined with purslane extract for synergistic effects, comprehensively expanding the scope of application in terms of application scenarios, skin type compatibility, and compounding value, ultimately greatly improving the actual utilization rate of olive glycopeptides.

[0032] In some optional embodiments, the complex enzyme system is a mixture of cellulase, pectinase, and hemicellulase, wherein the mass m1 of the cellulase, the mass m2 of the pectinase, and the mass m3 of the hemicellulase satisfy: m1:m2:m3 = (1 to 3):(1 to 2):(0.5 to 1.0); and / or The mass m4 of the complex enzyme system, the mass m5 of the crude polysaccharide powder, and the volume V1 of the buffer solution satisfy: (m4 + m5):V1 = 1:(15 to 40); and / or The amount of the complex enzyme system added is 3 wU / g to 5 wU / g.

[0033] In these embodiments, a complex enzyme system of cellulase, pectinase, and hemicellulase in a mass ratio of (1 to 3):(1 to 2):(0.5 to 1.0) is used. This complex enzyme system can fully decompose the glycopeptide components in the crude polysaccharide powder, which is beneficial for subsequent second solid-liquid separation and second purification to obtain high-purity olive glycopeptides. Furthermore, using a complex enzyme system, crude polysaccharide powder, and buffer in a mass-to-volume ratio of 1:(15 to 40) allows for a gentler enzymatic hydrolysis process, which is beneficial for subsequent second solid-liquid separation and second purification to obtain high-purity olive glycopeptides. Additionally, adding a complex enzyme system at a concentration of 3 wU / g to 5 wU / g can fully decompose the glycopeptide components in the crude polysaccharide powder in the buffer solution, which is beneficial for subsequent second solid-liquid separation and second purification to obtain high-purity olive glycopeptides.

[0034] The mass m1 of the cellulase can be 1, 1.5, 2.0, 2.5 or 3.0.

[0035] The mass m2 of this pectinase can be 1, 1.1, 1.2, 1.3, 1.4, 1.5 or 2.0.

[0036] The mass m3 of the hemicellulase can be 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0.

[0037] The volume V1 of the buffer solution can be 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40.

[0038] The amount of this complex enzyme system can be 3 wU / g, 3.1 wU / g, 3.2 wU / g, 3.3 wU / g, 3.4 wU / g, 3.5 wU / g, 4.0 wU / g, 4.5 wU / g, or 5.0 wU / g.

[0039] In some alternative embodiments, the enzymatic hydrolysis temperature is 45°C to 50°C, and the enzymatic hydrolysis time is 2 hours to 3 hours.

[0040] In these embodiments, a temperature of 45°C to 50°C and a time of 2 to 3 hours allow the complex enzyme system to fully decompose the glycopeptide components in the crude polysaccharide powder in the buffer solution.

[0041] The enzymatic hydrolysis temperature can be 45℃, 46℃, 47℃, 48℃, 49℃ or 50℃.

[0042] The enzymatic hydrolysis time can be 2.0h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, or 3.0h.

[0043] In some optional embodiments, the mass m6 of the dried solid residue and the volume V2 of the distilled water satisfy: m6:V2 = 1:(15 to 40), where the unit of m6 is g, and the unit of V2 is mL; and / or The number of extractions in the multiple rounds is 1 to 3, the temperature of the multiple extractions is 70°C to 100°C, and the extraction time for each extraction is 1 hour to 4 hours.

[0044] In these embodiments, using a dry solid residue and distilled water at a mass-to-volume ratio of 1:(15 to 40) allows for sufficient multiple extractions, which is beneficial for extracting hydrophilic oleuropeins from the dry solid residue and for their subsequent applications. Furthermore, multiple extractions of 1 to 3 times at a temperature of 70°C to 100°C and a single extraction time of 1 to 4 hours can also extract hydrophilic oleuropeins from the dry solid residue, further facilitating their subsequent applications.

[0045] The volume V2 of the distilled water can be 15, 20, 25, 30, 35 or 40.

[0046] The number of extractions in this multi-round process can be 1, 2, or 3.

[0047] The temperature for this multi-round extraction can be 70℃, 75℃, 80℃, 85℃, 90℃, 95℃ or 100℃.

[0048] The extraction time for each round of extraction can be 1 hour, 1.5 hours, 2.0 hours, 2.5 hours, 3.0 hours, 3.5 hours, or 4.0 hours.

[0049] In some optional embodiments, the enzyme inactivation is performed by water bath heating at a temperature of 90°C to 100°C for a time of 10 to 15 minutes; and / or The second solid-liquid separation includes high-speed centrifugation and filtration; when the second solid-liquid separation is a high-speed separation, the high-speed centrifugation speed is 4000 rpm to 8000 rpm, and the high-speed centrifugation time is 10 min to 20 min.

[0050] In these embodiments, enzyme inactivation at a temperature of 90°C to 100°C and a time of 10 min to 15 min can fully deactivate the complex enzyme system in the enzymatic hydrolysis product without affecting the function of olive glycopeptides, which is beneficial to obtaining high-purity olive glycopeptide products in the future.

[0051] The enzyme inactivation temperature can be 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃ or 100℃.

[0052] The enzyme inactivation time can be 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min.

[0053] Based on a general inventive concept, embodiments of this application provide an olive glycopeptide, which is prepared by the aforementioned preparation method; the weight-average molecular weight of the olive glycopeptide is 4226 Da to 4246 Da.

[0054] The olive glycopeptide is prepared based on the above-described preparation method. The specific steps of the preparation method can be referred to the above embodiments. Since the olive glycopeptide adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0055] It should be noted that the weight-average molecular weight of this olive glycopeptide can be 4226 Da, 4231 Da, 4236 Da, 4241 Da, or 4246 Da.

[0056] In some optional embodiments, the olive glycopeptide comprises a monosaccharide component and an amino acid component; by mass fraction, the monosaccharide component comprises: mannose: 10% to 12%, rhamnose: 19% to 20%, glucuronic acid: 3% to 4%, galacturonic acid: 7% to 9%, glucose: 2% to 3%, galactose: 28% to 31%, and arabinose: 18% to 21%; and / or The amino acid composition, by mass fraction, includes: aspartic acid: 5% to 7%, glutamic acid: 6% to 7%, serine: 8% to 10%, glycine: 7% to 9%, histidine: 4% to 6%, arginine: 5% to 7%, threonine: 5% to 7%, alanine: 10% to 12%, proline: 5% to 6%, cysteine: 3% to 5%, tyrosine: 7% to 9%, valine: 6% to 8%, and phenylalanine: 5% to 7%.

[0057] In these embodiments, the presence of 10% to 12% mannose, 19% to 20% rhamnose, 3% to 4% glucuronic acid, 7% to 9% galacturonic acid, 2% to 3% glucose, 28% to 31% galactose, and 18% to 21% arabinose by mass can enrich the olive glycopeptide with a large number of anti-inflammatory and antibacterial polysaccharide components and improve the antioxidant properties of the olive glycopeptide. In addition, the amino acid composition of the olive glycopeptide, consisting of 5% to 7% aspartic acid, 6% to 7% glutamic acid, 8% to 10% serine, 7% to 9% glycine, 4% to 6% histidine, 5% to 7% arginine, 5% to 7% threonine, 10% to 12% alanine, 5% to 6% proline, 3% to 5% cysteine, 7% to 9% tyrosine, 6% to 8% valine, and 5% to 7% phenylalanine, makes the olive glycopeptide rich in a large number of effective peptides, which is beneficial to enhancing the anti-inflammatory, antibacterial, and antioxidant properties of the olive glycopeptide.

[0058] Based on a general inventive concept, embodiments of this application provide an application of a small molecule olive glycopeptide, the application including: using the olive glycopeptide in cosmetics; the cosmetics including at least one of hydrogel masks, serums, and creams.

[0059] This application is based on the above-mentioned olive glycopeptide. The specific composition of the olive glycopeptide can be referred to in the above embodiments. Since the olive glycopeptide adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0060] It should be noted that, in addition to hydrogel masks, serums, and creams, this cosmetic product can also be a hydrogel repair product, a repair ointment, or other reagents containing this olive glycopeptide.

[0061] In some alternative embodiments, where the cosmetic includes a hydrogel mask, the raw materials of the hydrogel mask, by mass fraction, include: olive glycopeptide: 20% to 25%, sodium alginate: 65% to 70%, and purslane extract: 5% to 15%; and / or When the cosmetic is a serum, the ingredients of the serum, by mass fraction, include: olive glycopeptide: 1% to 5%, sodium hyaluronate: 0.1% to 1.0%, glycerin: 3% to 8%, niacinamide: 1% to 3%, vitamin E acetate: 0.5% to 2.0%, purslane extract: 0.5% to 2%, centella asiatica extract: 0.5% to 2%, dipotassium glycyrrhizate: 0.1% to 0.5%, preservative: 0.3% to 0.8%, and deionized water: 75% to 95%; and / or In the case of a face cream, the ingredients of the face cream, by mass fraction, include: olive glycopeptide: 1% to 5%, ceramide complex: 1% to 3%, squalane: 3% to 8%, purslane extract: 0.5% to 2%, centella asiatica extract: 0.5% to 2%, vitamin E acetate: 0.5% to 2%, emulsifier: 3% to 5%, moisturizer: 5% to 10%, preservative: 0.3% to 0.8%, and deionized water: 60% to 86%.

[0062] In these embodiments, using 20% ​​to 25% by mass of olive glycopeptide, 65% to 70% by mass of sodium alginate, and 5% to 15% by mass of purslane extract as a hydrogel mask can alleviate the expression of inflammatory factors through the anti-inflammatory and bactericidal effects of olive glycopeptide and purslane extract. In addition, 1% to 5% by mass of olive glycopeptide, 0.1% to 1.0% by mass of sodium hyaluronate, 3% to 8% by mass of glycerin, 1% to 3% by mass of niacinamide, 0.5% to 2.0% by mass of vitamin E acetate, 0.5% to 2% by mass of purslane extract, 0.5% to 2% by mass of centella asiatica extract, 0.1% to 0.5% by mass of dipotassium glycyrrhizate, 0.3% to 0.8% by mass of preservatives, and 75% to 95% by mass of deionized water can achieve anti-inflammatory and repairing effects on the skin through the synergistic effect of olive glycopeptide and other components. In addition, a face cream containing 1% to 5% olive glycopeptide, 1% to 3% ceramide complex, 3% to 8% squalane, 0.5% to 2% purslane extract, 0.5% to 2% centella asiatica extract, 0.5% to 2% vitamin E acetate, 3% to 5% emulsifier, 5% to 10% moisturizer, 0.3% to 0.8% preservative, and 60% to 86% deionized water can achieve anti-inflammatory and repairing effects on the skin through the synergistic effect of olive glycopeptide and other components.

[0063] In cosmetic products, including hydrogel masks, the mass fraction of the olive glycopeptide can be 20%, 21%, 22%, 23%, 24%, or 25%.

[0064] The mass fraction of sodium alginate can be 65%, 66%, 67%, 68%, 69%, or 70%.

[0065] The mass fraction of the purslane extract can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%.

[0066] When the cosmetic is an essence, the mass fraction of the olive glycopeptide can be 1%, 2%, 3%, 4%, or 5%.

[0067] The mass fraction of sodium hyaluronate can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or 1.0%.

[0068] The glycerin can be present in parts by weight of 3%, 4%, 5%, 6%, 7%, or 8%.

[0069] The mass fraction of nicotinamide can be 1%, 1.5%, 2.0%, 2.5%, or 3%.

[0070] The mass fraction of this vitamin E acetate can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, or 2.0%.

[0071] The mass fraction of the purslane extract can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, or 2.0%.

[0072] The mass fraction of the Centella asiatica extract can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, or 2.0%.

[0073] The mass fraction of the dipotassium glycyrrhizate can be 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%.

[0074] The preservative can be present in parts by weight of 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, or 0.8%.

[0075] The mass fraction of the deionized water can be 75%, 80%, 85%, 90%, or 95%.

[0076] When the cosmetic product is a face cream, the mass fraction of the olive glycopeptide can be 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5%.

[0077] The mass fraction of the ceramide complex can be 1%, 1.5%, 2.0%, 2.5%, or 3.0%.

[0078] The squalane can be present in parts by mass of 3%, 4%, 5%, 6%, 7%, or 8%.

[0079] The mass fraction of the purslane extract can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, or 2.0%.

[0080] The mass fraction of the Centella asiatica extract can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, or 2.0%.

[0081] The mass fraction of this vitamin E acetate can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, or 2.0%.

[0082] The emulsifier can be present in parts by weight of 3%, 3.5%, 4.0%, 4.5%, or 5%.

[0083] The humectant can be present in quantities of 5%, 6%, 7%, 8%, 9%, or 10% by weight.

[0084] The preservative can be present in parts by weight of 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, or 0.8%.

[0085] The mass fraction of the deionized water can be 60%, 65%, 70%, 75%, 80%, 85%, or 86%.

[0086] It should be noted that the total flavonoid content in this purslane extract is ≥5%.

[0087] It should be noted that, in the case of a hydrogel mask, this hydrogel mask forms a three-dimensional network structure through coordination bonds and hydrogen bonds between the active groups in olive glycopeptides and segments of sodium alginate. Simultaneously, this three-dimensional network structure can load 1% to 3% of purslane extract, achieving controlled sustained release of active ingredients. This hydrogel mask possesses excellent mechanical properties, self-healing ability, and biocompatibility, and can sustainably release antibacterial and anti-inflammatory active ingredients, making it suitable for sensitive skin care and the treatment of inflammatory skin problems.

[0088] It should be noted that, in the case of a face cream, the ceramide complex may be at least one of ceramide NP, ceramide AP, and ceramide EOP. The emulsifier may be at least one of olive oil esters, cetearyl alcohol, and stearate PEG-100. The moisturizer may be at least one of glycerin, butylene glycol, and sodium hyaluronate.

[0089] Figure 2 An exemplary schematic diagram of the preparation method of the hydrogel mask provided in the embodiments of this application is shown; Figure 3 An exemplary schematic diagram of the preparation method of the essence provided in the embodiments of this application is shown; Figure 4 An exemplary schematic diagram of the preparation method of face cream provided in the embodiments of this application is shown; In some alternative embodiments, where the cosmetic includes a hydrogel mask, such as Figure 2 As shown, the preparation method of the hydrogel mask includes: S1. Dissolve sodium alginate and deionized water by stirring to obtain a sodium alginate solution; S2. Dissolve olive glycopeptides in deionized water by stirring to obtain an olive glycopeptide solution; S3. Stir and dissolve the purslane extract and the sodium alginate solution to obtain a hydrogel mixture; S4. Stir and mix the olive glycopeptide solution and the hydrogel mixture to obtain a mixed solution; S5. Add calcium chloride solution to the mixed solution to carry out a gelation reaction, thereby obtaining a hydrogel mask; and / or In the case where the cosmetic product is a serum, such as Figure 3 As shown, the preparation method of the serum includes: S1. Mix sodium hyaluronate and a portion of deionized water to obtain a sodium hyaluronate solution; S2. Glycerin, nicotinamide, dipotassium glycyrrhizate and the sodium hyaluronate solution are mixed and sterilized sequentially to obtain the first liquid phase; S3. Mix vitamin E acetate, nonionic surfactant, and a portion of the deionized water to obtain a solution; S4. Mix the olive glycopeptide with the remaining deionized water to obtain an olive glycopeptide solution; S5. The purslane extract, centella asiatica extract, preservative and the olive glycopeptide solution are mixed to obtain the second liquid phase; S6. The first liquid phase, the enrichment solution and the second liquid phase are mixed and emulsified uniformly in sequence to obtain the crude essence; S7. Mix phenoxyethanol and the crude essence, and adjust the pH to obtain the essence; and / or In the case where the cosmetic product is a face cream, such as Figure 4 As shown, the preparation method of the face cream includes: S1. Squalane, vitamin E acetate and emulsifier are mixed and heated to melt in sequence to obtain the oil phase component; S2. Dissolve the humectant and preservative in deionized water and heat to mix them to obtain the aqueous phase component; S3. The oil phase component and the aqueous phase component are homogenized and emulsified to obtain a mixed emulsion; S4. The olive glycopeptide, purslane extract, centella asiatica extract, ceramide complex and the mixed emulsion are stirred and mixed to obtain a mixed emulsion product; S5. The mixed emulsion product is subjected to pH adjustment and filling in sequence to obtain face cream.

[0090] In these embodiments, when the cosmetics include hydrogel masks, serums, or creams, mixing different raw materials can form a hydrogel mask, serum, or cream product with uniform composition.

[0091] The present application is further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national / industry standards; if there is no corresponding national / industry standard, they are performed according to general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0092] Example 1

[0093] like Figure 1 As shown, a method for preparing a small molecule olive glycopeptide includes: S1. Crush the olive pomace to obtain olive pomace powder; S2. Extract the olive pomace powder using an organic solvent to obtain a solid residue; S3. Dry the solid slag to obtain dried solid slag; S4. The distilled water and dried solid residue were subjected to multiple extractions to obtain the extract; S5 sequentially performs the first solid-liquid separation and concentration on the extract to obtain a concentrated solution; S6. The concentrate is purified using ethanol to obtain crude polysaccharide powder; S7. The complex enzyme system, buffer solution and crude polysaccharide powder are enzymatically hydrolyzed to obtain the enzymatic hydrolysis product; S8. Inactivate the enzyme in the enzymatic hydrolysis product to obtain the enzyme inactivation reaction product; S9. The enzyme inactivation reaction product is subjected to a second solid-liquid separation to obtain crude olive glycopeptide; S10. The crude olive glycopeptide is subjected to a second purification to obtain olive glycopeptide.

[0094] The complex enzyme system is a mixture of cellulase, pectinase and hemicellulase, and the mass m1 of cellulase, the mass m2 of pectinase and the mass m3 of hemicellulase satisfy the following: m1:m2:m3=2:1.5:0.75; The mass m4 of the complex enzyme system, the mass m5 of the crude polysaccharide powder, and the volume V1 of the buffer solution satisfy: (m4+m5):V1=1:25; The amount of the complex enzyme system added was 4 wU / g.

[0095] The enzymatic hydrolysis temperature was 50℃, and the hydrolysis time was 2.5h.

[0096] The mass m6 of the dried solid residue and the volume V2 of the distilled water satisfy the following: m6:V2=1:20. If the unit of m6 is g, then the unit of V2 is mL. The number of extractions was 1 to 3 times, the extraction temperature was 85℃, and the extraction time for each extraction was 2.5 hours.

[0097] Enzyme inactivation was carried out by water bath heating at a temperature of 95°C for 14 minutes. The second solid-liquid separation includes high-speed centrifugation and filtration; in the case of high-speed separation, the high-speed centrifugation speed is 6000 rpm and the high-speed centrifugation time is 15 min.

[0098] Example 2

[0099] Based on the olive glycopeptide obtained in Example 1, the following experiments were further conducted: An olive glycopeptide, prepared by the method of Example 1, has a weight-average molecular weight of 4226 Da to 4246 Da.

[0100] The following experiments were conducted on the olive glycopeptides obtained by the preparation method: I. Molecular weight determination of olive glycopeptides: High performance liquid chromatography was used for detection.

[0101] 1. Chromatographic conditions: Column: YMC-PACK-Diol-60; Mobile phase: 0.7% sodium sulfate (v / v); Flow rate: 0.7 mL / min; Detector: RID detector; Column temperature: 35℃; Run time: 20 min.

[0102] 2. Sample preparation: Prepare a detection solution of olive glycopeptide with water to a mass concentration of 5 mg / mL, and then filter the prepared detection solution through a filter membrane with a pore size of 0.45 μm.

[0103] II. Determination of Monosaccharide Composition: 1. Hydrolysis of crude olive glycopeptides: The crude olive glycopeptide was prepared into a sample solution with a mass concentration of 6 mg / 3 mL using a 4 mol / L trifluoroacetic acid solution. The sample solution was then placed into ampoules in groups of 3 mL, purged with nitrogen, and sealed. The solution was then hydrolyzed at 105 °C for 2 h. The pH was then adjusted to neutral using a 6 mol / L sodium hydroxide solution. 0.5 mL of the neutral hydrolysate was then taken and 0.5 mL of a 0.25 mol / L sodium hydroxide solution and 0.5 mL of a 0.4 mol / L 1-phenyl-3-methyl-5-pyrazolone (PMP)-methanol solution were added sequentially and mixed to obtain the hydrolysate mixture.

[0104] The hydrolysis mixture was reacted in an oven at 70°C for 90 min. The dried product was then placed in an ice water bath for 10 min and allowed to return to room temperature. 0.5 mL of 0.3 mol / L hydrochloric acid solution was added and mixed to obtain the acidified product. Finally, PMP in the acidified product was extracted three times with dichloromethane. The first extraction was performed overnight, and the extraction time for the next two extractions was 2 to 3 hours.

[0105] 2. High Performance Liquid Chromatography (HPLC) Detection Procedure: (1) Chromatographic column: ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: potassium dihydrogen phosphate with a molar concentration of 0.05 mol / L (volume ratio of potassium dihydrogen phosphate to acetonitrile is 19:81); flow rate: 1 mL / min; detector: DAD ultraviolet detection wavelength 250 nm; column temperature oven: 40 ℃; running time: 60 min.

[0106] The results are as follows Figure 5 As shown, the components of the oleuropein determined to be monosaccharide components were found. Figure 5 In the sample, peak 1 is mannose, peak 2 is rhamnose, peak 3 is glucuronic acid, peak 4 is galacturonic acid, peak 5 is glucose, peak 6 is galactose, and peak 7 is arabinose. By mass fraction, the monosaccharide components include: mannose: 10% to 12%, rhamnose: 19% to 20%, glucuronic acid: 3% to 4%, galacturonic acid: 7% to 9%, glucose: 2% to 3%, galactose: 28% to 31%, and arabinose: 18% to 21%.

[0107] III. Amino acid composition analysis: Weigh 30 mg of olive glycopeptide and add it to 30 mL of 1 mol / L trifluoroacetic acid (TFA) solution to obtain the test sample. Dispense the test sample into 5 mL ampoules and hydrolyze it at 100 °C for 24 h under nitrogen protection to obtain a fully hydrolyzed sample. Remove the TFA by rotary evaporation of the hydrolyzed sample with methanol, then dissolve it in 1 mL of water for later use as the pure solution for testing. Take 900 μL of the standard solution and the pure solution for testing, add 450 μL of derivatizing reagent to each, and react in a 45 °C oven for 1 h. Then, add the reactants to 3 mL of n-hexane and extract three times with thorough shaking. Collect the lower extract, filter it through a 0.45 μm filter membrane, and then dilute it halfway before injection.

[0108] The preparation of the derivatizing reagents includes: Solution A: Use a pipette to take 0.12 mL of phenyl isothiocyanate and add it to 0.988 mL of acetonitrile in a brown vial and mix well.

[0109] Solution B: Using a pipette, pipette 1.35 mL of triethylamine and add it to 6.75 mL of acetonitrile in a brown vial. Mix well.

[0110] Mix solution A and solution B at a volume ratio of 1:1.

[0111] The results are as follows Figure 6 As shown, the components of the oleuropein obtained by measurement include amino acid components. Figure 6 The percentages of aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, cysteine, tyrosine, valine, phenylalanine, tryptophan, and lysine were 4.043, 4.93, 10.30, 11.26, 12.15, 14.26, 15.45, 16.52, 17.75, 26.75, 28.23, 30.47, 57.66, 58.62, and 60.01, respectively. Calculations show that the amino acid composition includes: aspartic acid: 5% to 7%, glutamic acid: 6% to 7%, serine: 8% to 10%, glycine: 7% to 9%, histidine: 4% to 6%, arginine: 5% to 7%, threonine: 5% to 7%, alanine: 10% to 12%, proline: 5% to 6%, cysteine: 3% to 5%, tyrosine: 7% to 9%, valine: 6% to 8%, and phenylalanine: 5% to 7%.

[0112] Meanwhile, the results of the RID detector and the DAD ultraviolet detection are as follows: Figure 8 As shown, the olive glycopeptide is a covalently bound homogeneous product, rather than a physical blend, and the olive glycopeptide has high purity with no obvious impurities such as free sugars or free peptides.

[0113] Example 3

[0114] Based on the olive glycopeptide obtained in Example 1, the following experiments were further conducted: I. Animal modeling and drug administration.

[0115] Sixty healthy male C57BL / 6 mice aged 6 to 8 weeks, with a weight range of 18g to 22g, were selected and randomly divided into 6 groups of 10 mice each. (1) Normal control group: normal drinking water, and oral administration of physiological saline; (2) Model control group: Induction with sodium dextran sulfate (DSS) followed by gavage administration of physiological saline; (3) Low-dose group (olive glycopeptide-L): DSS induction, followed by gavage treatment with olive glycopeptide (the amount of olive glycopeptide administered was designed at a ratio of 100 mg / kg / d). (3) Medium dose group (olive glycopeptide-M): DSS induction, followed by oral administration of olive glycopeptide (the amount of olive glycopeptide administered was designed at a ratio of 200 mg / kg / d). (4) High-dose group (olive glycopeptide-H): DSS induction, followed by oral administration of olive glycopeptide (the amount of olive glycopeptide administered was designed at a ratio of 400 mg / kg / d). (5) Positive control group: DSS induction, followed by gavage treatment with 5-aminosalicylic acid (5-ASA) (the amount of 5-aminosalicylic acid administered was designed at a ratio of 100 mg / kg / d).

[0116] The ulcerative colitis model was induced by free drinking of a 3% DSS solution (molecular weight 36,000 to 50,000) for 7 days. All treatment groups began receiving the medication via gavage at the time of model establishment, continuing for 14 days.

[0117] II. Observation Indicators and Methods.

[0118] (1) Disease Activity Index (DAI) score: Daily changes in mouse body weight, fecal bleeding, and fecal characteristics were recorded and scored according to a standard scoring system. Results are as follows: Figure 8 As shown.

[0119] (2) Histopathological examination of colon tissue: After the experiment, colon tissue was taken and the length of the colon was measured. The results are as follows: Figure 9 As shown. The colon tissue was then fixed with 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin and eosin (HE), and the histopathological changes were observed under a light microscope. The results are as follows. Figure 10 As shown in Table 1. Simultaneously, scores are awarded according to the standard scoring system shown in Table 1.

[0120] Table 1 Standard Scoring System

[0121] (3) Detection of inflammatory factors: Blood was collected from the eyeballs of mice, and the peripheral blood was centrifuged to obtain serum. The levels of pro-inflammatory factors such as TNF-γ, IL-6, IL-1β, and TNF-α were detected by ELISA. The results are as follows: Figure 11 As shown.

[0122] Depend on Figure 8 The results showed that the DAI score in the model group was significantly higher than that in the normal control group, indicating that the ulcerative colitis model was successfully established. Compared with the model group, the DAI scores of the low, medium, and high dose groups of olive glycopeptide (100 mg / kg, 200 mg / kg, and 400 mg / kg) decreased in a dose-dependent manner. The high-dose olive glycopeptide group showed a significant decrease in DAI score, indicating a significant reduction in disease severity. The positive control group (5-ASA, 100 mg / kg) showed a significantly lower DAI score compared to the model group. These results indicate that olive glycopeptide can effectively improve the clinical symptoms of DSS-induced ulcerative colitis in mice.

[0123] Depend on Figure 9 It can be seen that the colon of mice in the DSS model group was significantly shortened, indicating that the model was successful. The colon length of the other treatment groups was shortened to varying degrees compared with the blank group. Among them, the colon length of the high-dose olive glycopeptide group (olive glycopeptide-H) was comparable to that of the positive control group (5-ASA), indicating that the olive glycopeptide provided in this application has an anti-inflammatory effect.

[0124] Depend on Figure 10 It was found that the colon of mice in the blank control group had an intact tissue structure, with a large number of goblet cells, intact crypts, and neatly arranged intestinal glands in the mucosal layer, without inflammatory cell infiltration. In the DSS model group, the intestinal glands and tissue cells in the colonic mucosa of mice were severely damaged, with a significant reduction or even disappearance of crypts and goblet cells, and abscesses, connective tissue hyperplasia, and a large number of inflammatory cell infiltrations, mainly macrophages and neutrophils. The damage extended to the submucosa and muscularis propria, indicating that the DSS colitis model was successfully induced. Different concentrations of oleuropein significantly reduced inflammatory cell infiltration in colitis mice and resulted in a relatively intact colonic structure with varying degrees of mucosal recovery. Although inflammatory cells were still present in the mucosal and muscular layers, their presence was significantly reduced. The high-dose oleuropein group showed the best effect, followed by the medium-dose and low-dose groups. This indicates that oleuropein has a certain protective effect against DSS-induced colitis and can reduce the damage to the colonic tissue structure caused by colitis in mice.

[0125] Depend on Figure 11 The results of the detection of inflammatory factors in mouse colon tissue are as follows: Compared with the normal control group, the gene expression levels of pro-inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in the colon tissue of the model group were significantly upregulated, indicating that DSS-induced ulcerative colitis is accompanied by a significant inflammatory response. Compared with the model group, the expression levels of pro-inflammatory factors in each dose group of olive glycopeptide (100 mg / kg, 200 mg / kg, 400 mg / kg) decreased in a dose-dependent manner, with the high-dose olive glycopeptide-H group (400 mg / kg) showing a significant downregulation of IL-1β, IL-6, IFN-γ, and TNF-α. The positive control group (5-ASA, 100 mg / kg) also showed a significant anti-inflammatory effect, with a significant decrease in the expression levels of the four pro-inflammatory factors. These results indicate that olive glycopeptide can effectively alleviate the DSS-induced colonic inflammatory response in mice by inhibiting the production of pro-inflammatory factors, thus exerting a protective effect.

[0126] Example 4

[0127] Based on the olive glycopeptide obtained in Example 1, the following experiments were further conducted: An application of a small molecule olive glycopeptide, the application of which includes: using the olive glycopeptide in cosmetics; the cosmetic being a hydrogel mask.

[0128] like Figure 2 As shown, the preparation method of hydrogel masks includes: S1. Dissolve 1.0 g of sodium alginate (weight average molecular weight 120,000 Da) and 99 mL of deionized water at room temperature by stirring to obtain a sodium alginate solution with a mass concentration of 1%. S2. Dissolve 0.33g of olive glycopeptide (weight average molecular weight 120,000 Da) in 33mL of deionized water by stirring to obtain a 1% olive glycopeptide solution. S4. Dissolve 0.15g of purslane extract (total flavonoid content ≥5%) in sodium alginate solution by stirring to obtain a hydrogel mixture; S5. Slowly add the olive glycopeptide solution to the hydrogel mixture and mix with magnetic stirring to obtain a mixed solution; S6. Under stirring conditions, slowly add 5 mL of 0.2 mol / L calcium chloride solution (containing a total of 0.1 g of calcium chloride) to the mixed solution, and continue stirring to carry out the gelation reaction for 4 h to obtain a hydrogel; S7. Transfer the hydrogel to a mold and allow it to solidify at room temperature to obtain a hydrogel mask.

[0129] The prepared hydrogel mask was subjected to the following experiments: (1) The mass ratio of olive glycopeptide and sodium alginate in the above preparation method was changed, and the effect of different mass ratios on the performance of hydrogel mask was determined. The results are shown in Table 2.

[0130] Table 2. Effects of different mass ratios of olive glycopeptides and sodium alginate on the performance of hydrogel masks.

[0131] As shown in Table 2, the hydrogel mask exhibits the best overall performance with a mass ratio of 1:3 of olive glycopeptide and sodium alginate. It also has moderate mechanical strength, high encapsulation rate, and good release control.

[0132] (2) Study on the release performance of purslane extract: Hydrogel samples were prepared by using olive glycopeptide and sodium alginate at a mass ratio of 1:3. The release behavior of purslane extract in the hydrogel samples was determined by the dialysis bag method: the hydrogel samples were placed in a dialysis bag and immersed in phosphate buffer solution with a pH of 7.4, and kept at a constant temperature of 37°C with shaking. Samples were taken periodically to determine the mass concentration of purslane extract in the release medium, and the cumulative release rate was calculated using the following formula: , In the formula, Cn is the mass concentration of purslane extract in the release medium at the nth sampling time, in mg / mL; V0 represents the total volume of the released medium, in mL. Ci is the mass concentration at the i-th sampling (i=1, 2, ..., n-1), and the unit is mg / mL; Vs represents the volume of each sample taken, in mL; m_total represents the total loading of purslane extract in the hydrogel sample, in mg. n represents the number of samples.

[0133] The results are shown in Table 3.

[0134] Table 3. Cumulative release rate of purslane extract in the release medium of hydrogel samples

[0135] As shown in Table 3, when the mass ratio of olive glycopeptide to sodium alginate is 1:3, the purslane extract in the prepared hydrogel sample can be released for a long time, which meets the requirements for use in hydrogel masks. That is, by mass fraction, the raw materials of the hydrogel mask include: olive glycopeptide: 23%, sodium alginate: 67%, and purslane extract: 10%.

[0136] Example 5

[0137] Based on the olive glycopeptide obtained in Example 1, the following experiments were further conducted: An application of a small molecule olive glycopeptide, the application of which includes: using the olive glycopeptide in cosmetics; the cosmetic being an essence.

[0138] By mass fraction, the ingredients of the serum include: olive glycopeptide: 5%, sodium hyaluronate: 0.5%, glycerin: 5%, niacinamide: 1%, vitamin E acetate: 1%, purslane extract: 1.5%, centella asiatica extract: 1.5%, dipotassium glycyrrhizate: 0.2%, preservative: 0.3%, and deionized water: 84%.

[0139] like Figure 3 As shown, the preparation method of this serum is as follows: S1. Take about 60% of the total volume of deionized water into a jacketed heating reactor. Slowly add sodium hyaluronate under stirring at 200 rpm to 300 rpm and stir until dissolved without particles to obtain a sodium hyaluronate solution. S2. Add glycerol, nicotinamide and dipotassium glycyrrhizate to the sodium hyaluronate solution and stir to dissolve. Then turn on the heat and raise the temperature to 80°C to 85°C. Sterilize by stirring at this temperature for 10 minutes. Then start cooling to obtain the first liquid phase. S3. Weigh out vitamin E acetate in a small beaker and add PEG-40 hydrogenated castor oil in a volume of 2 to 4 times that of vitamin E acetate. Stir slowly at room temperature until completely transparent and without streaks. At the same time, while stirring, slowly add a small amount (about 10 times the volume of vitamin E acetate) of deionized water (40°C to 50°C) to the above mixture and stir to obtain a clear and transparent solution. S4. Take another 20% of the total deionized water volume (reserved from the total water volume) into a clean container, control the water bath temperature at 40℃ to 45℃, add olive glycopeptide under stirring at 400rpm to 600rpm, dissolve until clear, and obtain olive glycopeptide solution. S5. Add purslane extract, centella asiatica extract and preservative (specifically phenoxyethanol) to the olive glycopeptide solution, stir to dissolve evenly, keep warm for later use, and obtain the second liquid phase; S6. After the first liquid phase cools down to 45°C to 50°C, the enrichment solution and the second liquid phase are pumped into the reaction vessel under stirring, mixed evenly, and then the homogenizing emulsifier is turned on. The speed of homogenizing emulsification is adjusted to 1500 rpm to 2000 rpm, and homogenized for 3 min to 5 min until the system is uniform and fine, and the crude essence is obtained. S7. Turn on the cooling water to cool the crude essence to below 40℃, then add phenoxyethanol, stir to dissolve, test the pH of the solution, and slowly adjust the pH of the solution to 5.5 to 6.5 (target 6.0) using a 10% citric acid solution. Then turn on vacuum degassing at -0.06MPa to -0.08MPa and a rotation speed of 100rpm to 200rpm for 10 to 15 minutes. Finally, turn off vacuum degassing, filter the product through a 200-mesh filter cloth, and fill it into a brown glass bottle to obtain the essence.

[0140] Example 6

[0141] Based on the olive glycopeptide obtained in Example 1, the following experiments were further conducted: An application of a small molecule olive glycopeptide, the application of which includes: using the olive glycopeptide in cosmetics; the cosmetic being a face cream.

[0142] By mass fraction, the ingredients of this face cream include: olive glycopeptide: 3%, ceramide complex (ceramide MP to ceramide AP mass ratio of 1:1): 2%, squalane: 5%, purslane extract: 1%, centella asiatica extract: 1%, vitamin E acetate: 1%, emulsifier (olive oil ester to cetearyl alcohol mass ratio of 2:1): 3%, moisturizer (glycerin to butylene glycol mass ratio of 5:3): 8%, preservative (phenoxyethanol): 0.5%, and deionized water: 75.5%.

[0143] like Figure 4 As shown, the preparation method of this face cream is as follows: S1. Squalane, vitamin E acetate and emulsifier are mixed sequentially and heated to 75°C to melt, thus obtaining the oil phase component; S2. Dissolve the humectant and preservative in deionized water and heat the mixture at 75°C to obtain the aqueous phase component; S3. Slowly add the oil phase component to the aqueous phase component and homogenize and emulsify using a homogenizer at 3000 rpm for 5 min to obtain a mixed emulsion; S4. Cool the mixed emulsion to below 45°C, then add olive glycopeptide, purslane extract, centella asiatica extract, and ceramide complex and stir to obtain the mixed emulsion product; S5. The mixed emulsion products are subjected to pH adjustment (using citric acid to adjust the pH value to 6.0) and filling (after the temperature of the mixed emulsion products drops below 40°C) to obtain the face cream.

[0144] The specific procedure for conducting safety tests on face cream products is as follows: Skin irritation test (skin irritation-free test): 1) Testing method: Conducted in accordance with the "Cosmetic Safety Technical Specifications".

[0145] The results showed that the face cream product was non-irritating (pH value was 6).

[0146] In summary, the method for preparing small-molecule olive glycopeptides provided in this application uses food processing by-products as raw materials. The preparation process is mild and free of toxic substances, resulting in olive glycopeptides with extremely high safety and no side effects. Furthermore, the olive glycopeptides prepared by this method possess multiple effects, including moisturizing, anti-inflammatory and soothing, antioxidant, and skin barrier repair. They can be directly used in cosmetics, suitable for sensitive, dry, and barrier-damaged skin types. They can also be combined with purslane extract for synergistic effects, comprehensively expanding their application scope in terms of application scenarios, skin type compatibility, and compounding value, ultimately significantly increasing the actual utilization rate of olive glycopeptides.

[0147] In addition, the method for preparing small molecule olive glycopeptides provided in this application embodiment also has the following effects: (1) This preparation method is the first to use the glycopeptide component extracted from olive processing waste (olive pomace) in cosmetics, realizing the high-value utilization of agricultural waste, with low cost and sustainable source; (2) This preparation method obtains olive glycopeptides with a specific molecular weight range through controlled degradation technology, which solves the problems of wide molecular weight distribution and unclear active ingredients of natural polysaccharides, and is conducive to the quality control of olive glycopeptides. (3) Through a rigorous DSS-induced acute colitis animal model, it was confirmed that the olive glycopeptide prepared by this method can significantly improve the disease activity index, reverse colon length shortening, effectively inhibit colonic tissue pathological damage, and regulate immune balance by downregulating pro-inflammatory factors (TNF-α, IL-1β, IFN-γ, and IL-6). The anti-inflammatory effect of the olive glycopeptide is comparable to that of the positive control drug 5-aminosalicylic acid; (4) The olive glycopeptide obtained by this preparation method is derived from food processing by-products, has extremely high safety, no significant toxic side effects, and has good clinical application prospects; (5) The olive glycopeptide obtained by this preparation method can be used in cosmetics: Olive glycopeptide exhibits excellent multi-effect skin care properties due to its natural molecular structure. Olive glycopeptide forms a multi-hydrogen bond network with water molecules through hydroxyl groups, which can not only achieve instant and long-lasting moisturization, but also form a breathable protective film on the skin surface to reduce transepidermal water loss. The excellent anti-inflammatory and soothing ability of olive glycopeptide can significantly inhibit the expression of key inflammatory factors such as TNF-α and IL-6, and quickly relieve sensitive symptoms such as skin redness and stinging. At the same time, olive glycopeptide can be combined with other drugs to form a composition with strong antioxidant activity. This composition can effectively remove free radicals, reduce oxidative damage, and delay skin photoaging. Moreover, when olive glycopeptide is used in face cream, it can work synergistically with ingredients such as ceramide and squalane in the face cream formula to repair the skin barrier, enhance the integrity of the stratum corneum, and improve skin tolerance. Therefore, the face cream product formed by olive glycopeptide is especially suitable for daily care and repair of sensitive, dry and barrier-damaged skin. (6) The olive glycopeptide can be used in hydrogel masks, serums and creams containing purslane extract. Through the obvious anti-inflammatory effects of olive glycopeptide and purslane extract, it can effectively reduce the expression of inflammatory factors such as TNF-α and IL-6, and relieve skin redness, stinging and itching. At the same time, the olive glycopeptide and purslane extract and other components are harmless to the human body.

[0148] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed in this application.

Claims

1. A method for preparing small molecule olive glycopeptides, characterized in that, The preparation method includes: The olive pomace is crushed to obtain olive pomace powder; The olive pomace powder was extracted using an organic solvent to obtain a solid residue. The solid slag is dried to obtain dried solid slag; The distilled water and the dried solid residue were subjected to multiple extractions to obtain the extract; The extract was subjected to a first solid-liquid separation and concentration process to obtain a concentrated solution. The concentrate was first purified using ethanol to obtain crude polysaccharide powder. The complex enzyme system, buffer solution, and crude polysaccharide powder were subjected to enzymatic hydrolysis to obtain the enzymatic hydrolysis product. The enzyme hydrolysis product is subjected to enzyme inactivation to obtain an enzyme inactivation reaction product. The enzyme inactivation reaction product was subjected to a second solid-liquid separation to obtain crude olive glycopeptide; The crude olive glycopeptide was subjected to a second purification to obtain olive glycopeptide.

2. The preparation method according to claim 1, characterized in that, The composite enzyme system is a mixture of cellulase, pectinase, and hemicellulase, wherein the mass m1 of the cellulase, the mass m2 of the pectinase, and the mass m3 of the hemicellulase satisfy the following conditions: m1:m2:m3 = (1 to 3):(1 to 2):(0.5 to 1.0); and / or The mass m4 of the complex enzyme system, the mass m5 of the crude polysaccharide powder, and the volume V1 of the buffer solution satisfy: (m4 + m5):V1 = 1:(15 to 40); and / or The amount of the complex enzyme system added is 3 wU / g to 5 wU / g.

3. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis temperature is 45°C to 50°C, and the enzymatic hydrolysis time is 2 hours to 3 hours.

4. The preparation method according to claim 1, characterized in that, The mass m6 of the dried solid residue and the volume V2 of the distilled water satisfy: m6:V2 = 1:(15 to 40), where m6 is in g and V2 is in mL; and / or The number of extractions in the multiple rounds is 1 to 3, the temperature of the multiple extractions is 70°C to 100°C, and the extraction time for each extraction is 1 hour to 4 hours.

5. The preparation method according to claim 1, characterized in that, The enzyme inactivation is performed by water bath heating at a temperature of 90°C to 100°C for a time of 10 to 15 minutes; and / or The second solid-liquid separation includes high-speed centrifugation and filtration; when the second solid-liquid separation is a high-speed separation, the high-speed centrifugation speed is 4000 rpm to 8000 rpm, and the high-speed centrifugation time is 10 min to 20 min.

6. An olive glycopeptide, characterized in that, The olive glycopeptide is prepared by the preparation method according to any one of claims 1 to 5; the weight-average molecular weight of the olive glycopeptide is 4226 Da to 4246 Da.

7. The olive glycopeptide according to claim 6, characterized in that, The olive glycopeptide comprises monosaccharide components and amino acid components; by mass fraction, the monosaccharide components include: mannose: 10% to 12%, rhamnose: 19% to 20%, glucuronic acid: 3% to 4%, galacturonic acid: 7% to 9%, glucose: 2% to 3%, galactose: 28% to 31%, and arabinose: 18% to 21%; and / or The amino acid composition, by mass fraction, includes: aspartic acid: 5% to 7%, glutamic acid: 6% to 7%, serine: 8% to 10%, glycine: 7% to 9%, histidine: 4% to 6%, arginine: 5% to 7%, threonine: 5% to 7%, alanine: 10% to 12%, proline: 5% to 6%, cysteine: 3% to 5%, tyrosine: 7% to 9%, valine: 6% to 8%, and phenylalanine: 5% to 7%.

8. An application of a small molecule oleuropein, characterized in that, The application includes: using the olive glycopeptide of claim 6 or 7 in cosmetics; the cosmetics include at least one of hydrogel masks, serums and creams.

9. The application according to claim 8, characterized in that, In the case where the cosmetic product includes a hydrogel mask, the raw materials of the hydrogel mask, by mass fraction, include: olive glycopeptide: 20% to 25%, sodium alginate: 65% to 70%, and purslane extract: 5% to 15%; and / or When the cosmetic is a serum, the ingredients of the serum, by mass fraction, include: olive glycopeptide: 1% to 5%, sodium hyaluronate: 0.1% to 1.0%, glycerin: 3% to 8%, niacinamide: 1% to 3%, vitamin E acetate: 0.5% to 2.0%, purslane extract: 0.5% to 2%, centella asiatica extract: 0.5% to 2%, dipotassium glycyrrhizate: 0.1% to 0.5%, preservative: 0.3% to 0.8%, and deionized water: 75% to 95%; and / or In the case of a face cream, the ingredients of the face cream, by mass fraction, include: olive glycopeptide: 1% to 5%, ceramide complex: 1% to 3%, squalane: 3% to 8%, purslane extract: 0.5% to 2%, centella asiatica extract: 0.5% to 2%, vitamin E acetate: 0.5% to 2%, emulsifier: 3% to 5%, moisturizer: 5% to 10%, preservative: 0.3% to 0.8%, and deionized water: 60% to 86%.

10. The application according to claim 8, characterized in that, In the case where the cosmetic product includes a hydrogel mask, the method for preparing the hydrogel mask includes: Sodium alginate and deionized water were stirred and dissolved to obtain a sodium alginate solution; Olive glycopeptides and deionized water were stirred and dissolved to obtain an olive glycopeptide solution; The purslane extract and the sodium alginate solution were stirred and dissolved to obtain a hydrogel mixture; The olive glycopeptide solution and the hydrogel mixture are stirred and mixed to obtain a mixed solution; A calcium chloride solution is added to the mixed solution to initiate a gelation reaction, resulting in a hydrogel mask; and / or When the cosmetic is a serum, the method for preparing the serum includes: Sodium hyaluronate and some deionized water are mixed to obtain a sodium hyaluronate solution; Glycerin, nicotinamide, dipotassium glycyrrhizate, and the sodium hyaluronate solution were mixed and sterilized sequentially to obtain the first liquid phase; Vitamin E acetate, a nonionic surfactant, and a portion of the deionized water were mixed to obtain a solution. The olive glycopeptide and the remaining deionized water are mixed to obtain an olive glycopeptide solution; The purslane extract, centella asiatica extract, preservative, and the olive glycopeptide solution were mixed to obtain a second liquid phase. The first liquid phase, the enrichment solution, and the second liquid phase are mixed and emulsified uniformly in sequence to obtain the crude essence. Phenoxyethanol and the crude essence were mixed and the pH was adjusted to obtain the essence; and / or When the cosmetic is a face cream, the method for preparing the face cream includes: Squalane, vitamin E acetate, and emulsifier were mixed and heated to melt in sequence to obtain the oil phase component; The humectant and preservative were dissolved in deionized water and heated to obtain the aqueous phase component. The oil phase component and the aqueous phase component are homogenized and emulsified to obtain a mixed emulsion; Olive glycopeptide, purslane extract, centella asiatica extract, ceramide complex and the mixed emulsion were stirred and mixed to obtain a mixed emulsion product; The mixed emulsion products were sequentially subjected to pH adjustment and filling to obtain a face cream.

Images