Human ginseng galacturonic acid oligosaccharide as well as preparation method and application thereof

Abstract

The invention discloses a human ginseng galacturonic acid oligosaccharide as well as a preparation method and application thereof, and belongs to the technical field of biochemical engineering. The monosaccharide composition of the inventor of the galacturonic acid oligosaccharide comprises galacturonic acid GalA of which the mass percentage content is greater than or equal to 90%; the molecular weight of the human ginseng galacturonic acid oligosaccharide is lower than 3.0 kDa; the polymerization degree of the human ginseng galacturonic acid oligosaccharide is lower than 15; the human ginseng galacturonic acid oligosaccharide is composed of alpha-D-GalpA residues, and a glucosidic bond connection mode is alpha-1, 4-GalpA. Biological experiments show that the inventor of the invention has good antioxidant property, moisturizing effect and cutin-removing and acne-removing activity, can achieve repairing and relieving effects by promoting cell healing, promoting expression of barrier-related proteins FLG and LOR and inhibiting secretion of inflammatory factors TNF-alpha, IL-6 and IL-1alpha, and can be used for preparing the anti-acne cream. The method has a wide application prospect in cosmetics such as beauty skin care products and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to ginseng galacturonic acid oligosaccharide and a preparation method and application thereof. Background Art

[0002] Ginseng is a plant of the Araliaceae family ( Panax ginseng The dried root of Ginseng Root (Ginseng Root) is a precious Chinese herbal medicine, known as the "king of herbs". It has shown extremely high biological activity and unique medicinal value in skin care such as anti-oxidation, anti-aging, sun protection and whitening, moisturizing and anti-inflammatory. Modern pharmaceutical research shows that ginsenosides and ginseng polysaccharides are the main active ingredients of ginseng and the material basis for its efficacy. So far, ginsenosides and ginseng polysaccharides have been widely studied and applied, while research on ginseng active oligosaccharides has been less reported.

[0003] Oligosaccharides are generally defined as carbohydrate compounds composed of 2-10 monosaccharide molecules connected by glycosidic bonds, which can be obtained from natural polysaccharides through physical, chemical or enzymatic treatment. In recent years, with the deepening of research on natural oligosaccharides, their biological significance and application prospects have been continuously recognized, and more and more oligosaccharides have been developed and applied in the fields of medicine, drugs, health foods, cosmetics, etc.

[0004] Among the existing ginseng extract preparation processes, the extraction processes of polysaccharides and saponins are mainly used. For example, patent publication CN115531430A discloses a method for preparing a mixture of ginseng sugar and ginsenosides, and patent publication CN104911233A discloses a method for preparing ginseng sugar with hypoglycemic function. However, there are no reports on the preparation method of ginseng oligosaccharides mainly composed of galacturonic acid oligosaccharides and their application in skin care products. Therefore, the development of the preparation method of ginseng galacturonic acid oligosaccharides and their application in anti-oxidation, moisturizing, exfoliating, acne removal and soothing and repairing skin care products has important research significance and application value. Summary of the invention

[0005] The purpose of the present invention is to provide a ginseng galacturonic acid oligosaccharide and a preparation method thereof and application thereof in the preparation of cosmetics. The method is simple and easy to operate, has the characteristics of high efficiency, environmental protection, few by-products and high purity, is easy to industrialize and has broad application prospects in antioxidant, moisturizing, exfoliating, acne-removing and soothing and repairing cosmetics.

[0006] In a first aspect, the present invention provides a method for preparing ginseng galacturonic acid oligosaccharide, characterized in that it comprises the following steps: S1. adding weak acid to ginseng medicinal materials for extraction to obtain ginseng polysaccharide extract; S2. Add a complex enzyme composed of glucoamylase and amylase to the ginseng polysaccharide extract for enzymatic hydrolysis to obtain a ginseng polysaccharide enzymatic hydrolysate; Separate the polysaccharide components in the ginseng polysaccharide enzymatic hydrolysate that are not enzymatically hydrolyzed by the complex enzyme; S3. Add pectinase to the polysaccharide components not enzymatically hydrolyzed by the complex enzyme for enzymatic hydrolysis to obtain a ginseng pectin enzymatic hydrolysate; Separate the oligosaccharides enzymatically hydrolyzed by the pectinase in the ginseng pectin enzymatic hydrolysate; Obtain the ginseng galacturonic acid oligosaccharide through freeze-drying; The monosaccharide composition of the ginseng galacturonic acid oligosaccharide includes galacturonic acid GalA with a mass percentage content of ≥90%; The molecular weight of the ginseng galacturonic acid oligosaccharide is lower than 3.0 kDa; The degree of polymerization of the ginseng galacturonic acid oligosaccharide is lower than 15; The ginseng galacturonic acid oligosaccharide is composed of α-D-GalpA residues, and the glycosidic bond linkage is α-1,4-GalpA.

[0007] As an example, the content of GalA in the monosaccharide composition of the ginseng galacturonic acid oligosaccharide is 92.4%; the high-performance gel permeation chromatogram of the ginseng galacturonic acid oligosaccharide contains chromatographic peaks at 1.8 kDa / 1.4 kDa and 0.6 kDa; the degree of polymerization of the ginseng galacturonic acid oligosaccharide is 1-8, where the degree of polymerization represents the number of galacturonic acid units; the glycosidic linkage of the galacturonic acid oligosaccharide is α-1,4-GalpA, and the ginseng galacturonic acid oligosaccharide contains methyl-esterified galacturonic acid.

[0008] In the above preparation method of the ginseng galacturonic acid oligosaccharide, the ginseng is any one of wild ginseng, forest ginseng and garden ginseng; the ginseng medicinal material is ginseng slices, such as the slice thickness is 2-3 cm and dry cut; In step S1, the material-liquid ratio for extraction is 1 g: (10-20) mL, such as 1 g: 15 mL; The pH value of the extraction system composed of the ginseng medicinal material and the weak acid is 4.0-5.0, such as 4.5; the weak acid can be any organic acid that provides the above pH value range, such as citric acid, acetic acid, oxalic acid, pectic acid, malic acid, lactic acid, etc.; In step S1, the extraction temperature is 80-100 °C, and the extraction is carried out 2-4 times, 2-4 h each time. The extraction liquids are combined, such as extracting 3 times at a slightly boiling state, 2 h each time.

[0009] In the above-mentioned preparation method of ginseng galacturonic acid oligosaccharide, the enzymatic activity of the saccharifying enzyme in the compound enzyme (liquid enzyme) is 200,000 to 260,000 U / ml, such as 260,000 U / ml, and the enzymatic activity of the amylase is 10,000 to 20,000 U / ml, such as 20,000 U / ml; The ratio of the ginseng medicinal material to the complex enzyme is 1kg: (12-16)mL, such as 1kg: 16mL; The present invention has no special limitation on the sources of amylase and saccharifying enzyme in the composite enzyme composed of amylase and saccharifying enzyme, and conventional commercial products can be used; The enzymatic hydrolysis temperature in step S2 is 50° C., and the enzymatic hydrolysis is carried out under stirring conditions of 40 to 60 rpm (e.g., 50 rpm), and the enzymatic hydrolysis is performed 2 to 3 times (e.g., 2 times), each time for 2 to 3 hours (e.g., 2 hours); the amylase and the saccharifying enzyme can act synergistically at 50° C. and the same weakly acidic pH conditions to efficiently catalyze the complete hydrolysis of ginseng starch into low molecular weight glucose; After the enzymolysis in step S2, the following step is further included: heating the reaction solution after enzymolysis to 100° C. to inactivate the enzyme.

[0010] In the above-mentioned preparation method of ginseng galacturonic acid oligosaccharide, the following steps are further included before the separation step in step S2: the ginseng polysaccharide enzymatic hydrolyzate is concentrated and centrifuged in sequence, and the supernatant is collected; wherein the concentration is concentrated to 1 / 2 to 1 / 4 of the original solution, such as 1 / 3 or 1 / 4; as an example, the centrifugation is centrifuged at 4000 rpm for 15 min to remove insoluble substances; In step S2, the separation includes the following steps: adding ethanol to the ginseng polysaccharide hydrolyzate for alcohol precipitation, collecting the alcohol precipitation precipitate, wherein the final volume fraction of ethanol in the ginseng polysaccharide hydrolyzate in the alcohol precipitation step is 40% to 60%, such as 40%, 50%, or 60%, within which the ginseng polysaccharide components such as ginseng pectin that are not hydrolyzed by the composite enzyme can be separated from the ginseng starch hydrolyzed oligosaccharides. As an example, in the alcohol precipitation step, the precipitate is collected by centrifugation. The alcohol precipitation time in the alcohol precipitation step can be specifically 12 hours of standing.

[0011] It is understandable that the separation in step S2 may also be carried out by other separation methods, such as replacing the above-mentioned alcohol precipitation separation method with a membrane filtration separation method. That is, the separation in step S2 includes the following steps: filtering the ginseng polysaccharide hydrolyzate through a membrane and collecting the retentate. The membrane filtration used may be a ceramic membrane with a specification of 0.2 μm, 0.4 μm or 0.5 μm, or an organic membrane filtration with a specification of 3 kDa, 5 kDa or 10 kDa.

[0012] In the above-mentioned method for preparing ginseng galacturonic acid oligosaccharide, in step S3, the enzyme activity of the pectinase (liquid enzyme) is 20,000 to 30,000 U / ml, such as 30,000 U / ml; The ratio of the ginseng medicinal material to the pectinase is 1kg: (1.0-1.4)mL, such as 1kg: 1.4mL; The present invention has no special limitation on the source of pectinase, and conventional commercial products can be used; The enzymatic hydrolysis temperature in step S3 is 50° C., and the enzymatic hydrolysis is carried out under stirring conditions of 40 to 60 rpm (such as 50 rpm), and the enzymatic hydrolysis is performed 1 to 2 times (such as 1 time), each time for 4 to 6 hours (such as 6 hours); as an example, in the enzymatic hydrolysis step, the alcohol precipitate is redissolved in water for the enzymatic hydrolysis.

[0013] After the enzymolysis in step S3, the following steps are further included: heating the enzymolysis-completed reaction solution to 100° C. to inactivate the enzyme; In the above-mentioned preparation method of ginseng galacturonic acid oligosaccharide, the following steps are further included before the separation step in step S3: the ginseng pectin enzymatic hydrolyzate is concentrated and centrifuged in sequence, and the supernatant is collected; wherein the concentration is concentrated to 1 / 2 to 1 / 4 of the original solution, such as 1 / 2 or 1 / 3; as an example, the centrifugation is centrifuged at 4000 rpm for 15 min; In step S3, the separation comprises the following steps: adding ethanol to the ginseng pectin enzymatic hydrolysate for alcohol precipitation, collecting the supernatant, wherein the final volume fraction of ethanol in the ginseng pectin enzymatic hydrolysate in the alcohol precipitation step is 50% to 70%, such as 50%, 60% or 70%, within which the ginseng polysaccharide components not hydrolyzed by pectinase and the ginseng galacturonic acid oligosaccharides produced after being hydrolyzed by pectinase can be separated. The alcohol precipitation time in the alcohol precipitation step can be specifically 12 hours.

[0014] It is understandable that the separation in step S3 may also be carried out by other separation methods, such as replacing the above-mentioned alcohol precipitation separation method with a membrane filtration separation method. That is, the separation in step S3 includes the following steps: filtering the ginseng pectin enzymatic hydrolyzate through a membrane and collecting the filtrate. The membrane filtration used may be a ceramic membrane with a specification of 0.2 μm, 0.4 μm or 0.5 μm, or an organic membrane filtration with a specification of 3 kDa, 5 kDa or 10 kDa.

[0015] In a second aspect, the present invention provides ginseng galacturonic acid oligosaccharide obtained by any of the preparation methods described above.

[0016] In a third aspect, the present invention provides the use of ginseng galacturonic acid oligosaccharide obtained by any of the preparation methods described above in any of the following A1)-A3): A1) Antioxidant in vitro; A2) Preparation of cosmetics for anti-oxidation; A3) Preparation of cosmetics for anti-oxidative stress-induced skin damage.

[0017] As an example, the antioxidant property is manifested in the scavenging effect on DPPH free radicals, the scavenging effect on ABTS free radicals and the scavenging effect on hydroxyl free radicals; the antioxidant stress property is manifested in reducing H 2 O 2 Induced cellular ROS levels and relief of H 2 O 2 The induced cell mitochondrial membrane potential level is reduced.

[0018] In a fourth aspect, the present invention provides the use of ginseng galacturonic acid oligosaccharide obtained by any of the preparation methods described above in any of the following B1)-B2): B1) Skin moisturizing; B2) Preparation of skin moisturizing cosmetics.

[0019] As an example, the moisturizing property and hygroscopic property of the ginseng galacturonic acid oligosaccharide of the present invention are both greater than that of sodium alginate. The skin moisturizing effect is reflected in increasing the content of AQP3 and FLG in keratinocytes.

[0020] In a fifth aspect, the present invention provides the use of ginseng galacturonic acid oligosaccharide obtained by any of the preparation methods described above in any of the following C1)-C5): C1) External exfoliation; C2) Preparation of cosmetic products for in vitro exfoliation; C3) In vitro inhibition of Propionibacterium acnes for non-therapeutic purposes; C4) preparing cosmetics for inhibiting Propionibacterium acnes; C5) Preparation of cosmetics for improving acne.

[0021] As an example, the in vitro exfoliation is manifested in the exfoliation of HaCaT cells; the inhibition of Propionibacterium acnes and the treatment or relief of acne is manifested in the inhibitory effect of ginseng galacturonic acid oligosaccharide on Propionibacterium acnes at a test concentration of 10 mg / mL.

[0022] In a fifth aspect, the present invention provides the use of ginseng galacturonic acid oligosaccharide obtained by any of the preparation methods described above in any of the following items D1) to D3): D1) Promotes healing of human immortalized keratinocytes in vitro; D2) Preparation of cosmetics for promoting the healing of human immortalized keratinocytes; D3) Preparation of cosmetics for maintaining the skin barrier or repairing skin damage.

[0023] As an example, the in vitro promotion of the healing of human immortalized keratinocytes is embodied in promoting the cell healing of HaCaT cells; the maintenance of the skin barrier or the repair of skin damage is embodied in promoting the content of FLG protein and LOR protein.

[0024] In a sixth aspect, the present invention provides the use of ginseng galacturonic acid oligosaccharide obtained by any of the preparation methods described above in any of the following E1)-E2): E1) Preparation of anti-inflammatory cosmetics; E2) Preparation of cosmetic products for skin soothing or repairing skin inflammation.

[0025] As an example, the skin soothing or skin inflammation repairing is manifested in inhibiting the secretion of macrophage RAW264.7 inflammatory factors; the macrophage RAW264.7 inflammatory factors are TNF-α, IL-6 and IL-1α.

[0026] In the above-mentioned applications, the cosmetics may be skin care cosmetics.

[0027] The present invention has the following beneficial effects: (1) The method for preparing ginseng galacturonic acid oligosaccharide of the present invention can obtain high-purity ginseng galacturonic acid oligosaccharide, which has low separation cost, simple process and can be prepared on a large scale compared with the prior art.

[0028] (2) Biological experiments show that the ginseng galacturonic acid oligosaccharide of the present invention has good antioxidant properties, moisturizing effect and exfoliating and anti-acne activity. It can achieve repair and soothing effects by promoting cell healing, promoting the expression of barrier-related proteins FLG and LOR, and inhibiting the secretion of inflammatory factors TNF-α, IL-6 and IL-1α. It can be used in cosmetics such as skin care and beauty, and has broad application prospects in beauty and skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 High performance gel permeation chromatography for molecular weight analysis of ginseng galacturonic acid oligosaccharide in the present invention; Figure 2 The HPLC chromatogram of the monosaccharide composition analysis of ginseng galacturonic acid oligosaccharide in the present invention; Figure 3 is the ion chromatogram of ginseng galacturonic acid oligosaccharide in the present invention; Figure 4 The ginseng galacturonic acid oligosaccharide of the present invention 13 C NMR spectrum; Figure 5 This is a diagram showing the scavenging effect of ginseng galacturonic acid oligosaccharide on DPPH free radicals in the present invention; Figure 6This is a diagram showing the scavenging effect of ginseng galacturonic acid oligosaccharide on ABTS free radicals in the present invention; Figure 7 This is a diagram showing the scavenging effect of ginseng galacturonic acid oligosaccharide on hydroxyl radicals in the present invention; Figure 8 H in the present invention 2 O 2 Diagram of the model of induced oxidative stress; Fig. 9 This is a graph showing the effect of ginseng galacturonic acid oligosaccharide on HaCaT cell viability in the present invention; Fig.10 The ginseng galacturonic acid oligosaccharide of the present invention is 2 O 2 The results of the effects of induced ROS levels on HaCaT cells; Fig.11 The ginseng galacturonic acid oligosaccharide of the present invention is 2 O 2 The results of the effects of induced mitochondrial membrane potential on HaCaT cells; Fig.12 The results of measuring the moisturizing ability of ginseng galacturonic acid oligosaccharide in the present invention; Fig.13 The hygroscopicity test result of ginseng galacturonic acid oligosaccharide in the present invention; Fig.14 The effect of ginseng galacturonic acid oligosaccharide on the content of hydration-related proteins in keratinocytes in the present invention: AQP3 immunofluorescence staining results; Fig.15 The effect of ginseng galacturonic acid oligosaccharide on the content of hydration-related proteins in keratinocytes in the present invention: AQP3 relative integrated optical density (IOD) value bar graph; Fig.16 The effect of ginseng galacturonic acid oligosaccharide on the content of hydration-related proteins in keratinocytes: FLG immunofluorescence staining results; Fig.17 The effect of ginseng galacturonic acid oligosaccharide on the content of hydration-related proteins in keratinocytes in the present invention: FLG relative integrated optical density (IOD) value bar graph; Fig.18 The results of the cell exfoliation experiment of ginseng galacturonic acid oligosaccharide in the present invention; Fig.19 The results of the antibacterial experiment of ginseng galacturonic acid oligosaccharide on Propionibacterium acnes in the present invention; Fig. 20 The results of the effect of ginseng galacturonic acid oligosaccharide on the scratch healing of HaCaT cells in the present invention; Fig.21The results of the effect of ginseng galacturonic acid oligosaccharide on the expression level of FLG protein in HaCaT cells under UVB stimulation: FLG protein immunofluorescence staining results; Fig. 22 The results of the effect of ginseng galacturonic acid oligosaccharide on the expression level of FLG protein in HaCaT cells under UVB stimulation are as follows: relative integrated optical density (IOD) value; Fig.23 The results of the effect of ginseng galacturonic acid oligosaccharide on the expression level of LOR protein in HaCaT cells under UVB stimulation: LOR protein immunofluorescence staining results; Fig.24 The results of the effect of ginseng galacturonic acid oligosaccharide on the expression level of LOR protein in HaCaT cells under UVB stimulation in the present invention are: relative integrated optical density (IOD) value; Fig.25 The results are as follows: the effect of ginseng galacturonic acid oligosaccharide on the secretion of inflammatory factor TNF-α by RAW264.7 cells under LPS stimulation; Fig.26 The results of the effect of ginseng galacturonic acid oligosaccharide on the secretion of inflammatory factor IL-6 by RAW264.7 cells under LPS stimulation; Fig. 27 These are the results of the effect of ginseng galacturonic acid oligosaccharide on the secretion of inflammatory factor IL-1α by RAW264.7 cells under LPS stimulation. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0031] The methods used in the following examples, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0032] The ginseng in the following examples was purchased from Tonghua, Jilin Province. The ginseng species was garden ginseng, and the slice thickness was 2-3 cm, and the ginseng was dry cut.

[0033] In the complex enzyme composed of amylase and saccharifying enzyme used in the following examples, the enzymatic activity of saccharifying enzyme is 260,000 U / ml, and the enzymatic activity of amylase is 20,000 U / ml. The enzymatic activity of saccharifying enzyme is defined as follows: 1 ml of enzyme solution hydrolyzes soluble starch to produce 1 mg of glucose in 1 hour at 40°C and pH 4.6, which is one enzyme activity unit, expressed in U / ml; the enzymatic activity of amylase is defined as follows: 1 ml of enzyme solution hydrolyzes 1 g of soluble starch in 1 hour at 60°C and pH 6.0, which is one enzyme activity unit, expressed in U / ml.

[0034] The enzyme activity of pectinase is 30,000 U / ml, and its enzyme activity is defined as follows: 1 ml of enzyme solution decomposes pectin solution to produce 1 mg of galacturonic acid in 1 hour under the conditions of 50°C and pH 3.5. This is one enzyme activity unit, expressed in U / ml.

[0035] Example 1: Preparation of ginseng galacturonic acid oligosaccharide This embodiment provides a method for preparing ginseng galacturonic acid oligosaccharide, and the specific steps are as follows: (1) Take 200 g of ginseng slices and add 4 L of pectin acid aqueous solution with a pH value of 4.5. Extract three times at a slightly boiling state, each time for 2 h. After combining the extracts, the ginseng polysaccharide extract was obtained.

[0036] (2) The ginseng polysaccharide extract was transferred to a reactor, and 3.2 mL of a compound enzyme consisting of amylase and saccharifying enzyme was added. The mixture was hydrolyzed at 50°C for 2 h. During the hydrolysis, low-speed stirring was maintained at 50 rpm. The hydrolysis was then repeated once. After the hydrolysis was completed, the reaction system was heated to 100°C to inactivate the enzymes, and the ginseng polysaccharide hydrolyzate was obtained. After the ginseng polysaccharide hydrolyzate was concentrated to 4 L under reduced pressure, the insoluble matter was removed by centrifugation at 4000 rpm for 15 min. 95% ethanol was added to a final concentration of 60%, the mixture was allowed to stand for 12 h, and the precipitate was collected by centrifugation.

[0037] (3) The above precipitate was dissolved in 6 L of distilled water, transferred to a reactor, and 280 μL of pectinase was added. The mixture was hydrolyzed at 50°C for 6 h, and low-speed stirring was maintained at 50 rpm during the hydrolysis. After the hydrolysis was completed, the reaction system was heated to 100°C to inactivate the enzyme, and ginseng pectin hydrolysate was obtained. After the ginseng pectin hydrolysate was concentrated to about 2 L under reduced pressure, it was centrifuged at 4000 rpm for 15 min. 95% ethanol was added to a final concentration of 60%, and the mixture was allowed to stand for 12 h. The supernatant was collected by centrifugation. After the supernatant was concentrated and freeze-dried, 2.64 g of ginseng galacturonic acid oligosaccharide was obtained.

[0038] Take an appropriate amount of 2 mg of the above-mentioned ginseng galacturonic acid oligosaccharide, and perform molecular weight distribution analysis using high performance gel permeation chromatography (HPGPC) (the sample solvent is 0.2 M NaCl, Shimadzu LC-20Ai liquid chromatography in Japan, RID-20A refractive index detector, TSK-gel G2500 PWxl chromatographic column, the mobile phase is 0.2 M NaCl aqueous solution, the flow rate is 0.5 mL / min, and the injection volume is 20 μL). As Figure 1 shown, its molecular weight is less than 3.0 kDa.

[0039] Take another appropriate amount of 2 mg of this ginseng galacturonic acid oligosaccharide. After PMP derivatization treatment, perform monosaccharide composition analysis using high performance liquid chromatography (HPLC) (Shimadzu LC-20A liquid chromatography in Japan, SPD-20A ultraviolet detector, the chromatographic column is COSMOSIL 5C18-PAQ, acetonitrile is used as mobile phase A, and sodium hydrogen phosphate-sodium dihydrogen phosphate aqueous solution is used as mobile phase B, and isocratic elution is carried out according to the ratio of A:B = 18.8:81.2. The column temperature is 35 °C, the flow rate is 1.0 mL / min, the detection wavelength is 245 nm, and the injection volume is 10 μL). As Figure 2 shown, the content of GalA in its monosaccharide composition is 92.4%.

[0040] Take another 1 mg of this ginseng galacturonic acid oligosaccharide, and use a high performance ion chromatography system equipped with a Carbo Pac PA200 chromatographic column (the sample solvent is water, Thermo Fisher ICS 6000 ion chromatograph in the United States, pulsed amperometric detector, anion exchange chromatographic column Carbo Pac PA200, and detection is carried out at 35 °C, the elution flow rate is 0.5 mL / min, and the injection volume is 25 μL. The ion chromatography elution program is: -6 to 0 min, 100 mM NaOH, 100 mM NaAc isocratic elution to equilibrate the chromatographic column; 0 to 20 min, 100 mM NaOH isocratic elution, and NaAc is linearly gradient eluted from 100 mM to 500 mM; 20 to 30 min, the NaOH concentration is increased to 200 mM, and the NaAc concentration is increased to 700 mM for isocratic elution to clean the chromatographic column.) to separate and identify the oligosaccharide. As Figure 3 shown, the degree of polymerization of ginseng galacturonic acid oligosaccharide is 1-8.

[0041] Take another 15 mg of this ginseng galacturonic acid oligosaccharide, add D 2 O (99.8%) and dissolve it, and analyze it by a nuclear magnetic resonance spectrometer. Its 13C NMR spectrum is as Figure 4As shown in the figure, the characteristic absorption peak of (→4)-α-GalpA-(1→) can be clearly observed, indicating that the connection mode of galacturonic acid oligosaccharide is α-1,4-GalpA. At the same time, there is also a characteristic absorption peak of methylated galacturonic acid.

[0042] Example 2: Preparation of ginseng galacturonic acid oligosaccharide This embodiment provides a method for preparing ginseng galacturonic acid oligosaccharide, and the specific steps are as follows: (1) Take 200 g of ginseng slices and add 3 L of citric acid solution with a pH of 4.5. Extract three times at a slight boiling point, each time for 2 h, and combine the extracts.

[0043] (2) The ginseng polysaccharide extract was transferred to a reactor, and 3.2 mL of a compound enzyme consisting of amylase and saccharifying enzyme was added. The mixture was hydrolyzed at 50°C for 2 h. During the hydrolysis, low-speed stirring was maintained at 50 rpm. The hydrolysis was then repeated once. After the hydrolysis was completed, the reaction system was heated to 100°C to inactivate the enzymes, and the ginseng polysaccharide hydrolyzate was obtained. After the ginseng polysaccharide hydrolyzate was concentrated to 4.5 L under reduced pressure, the insoluble matter was removed by centrifugation at 4000 rpm for 15 min. 95% ethanol was added to a final concentration of 50%, the mixture was allowed to stand for 12 h, and the precipitate was collected by centrifugation.

[0044] (3) The above precipitate was dissolved in 6 L of distilled water, transferred to a reactor, and 280 μL of pectinase was added. The mixture was hydrolyzed at 50°C for 6 h, and low-speed stirring was maintained at 50 rpm during the hydrolysis. After the hydrolysis was completed, the reaction system was heated to 100°C to inactivate the enzyme, and ginseng pectin hydrolysate was obtained. After the ginseng pectin hydrolysate was concentrated to about 2 L under reduced pressure, it was centrifuged at 4000 rpm for 15 min. 95% ethanol was added to a final concentration of 70%, and the mixture was allowed to stand for 12 h. The supernatant was collected by centrifugation. After the supernatant was concentrated and freeze-dried, 2.23 g of ginseng galacturonic acid oligosaccharide was obtained.

[0045] According to HPLC detection, the content of GalA in the monosaccharide composition of the ginseng galacturonic acid oligosaccharide obtained in this example is 92.9%, and the test conditions are the same as those in Example 1.

[0046] Example 3: Preparation of ginseng galacturonic acid oligosaccharide This embodiment provides a method for preparing ginseng galacturonic acid oligosaccharide, and the specific steps are as follows: (1) Take 200 g of ginseng slices and add 4 L of oxalic acid aqueous solution with a pH value of 4.5. Extract three times at a slight boiling point, each time for 2 h, and combine the extracts.

[0047] (2) The ginseng polysaccharide extract was transferred to a reactor, and 3.2 mL of a compound enzyme consisting of amylase and saccharifying enzyme was added. The mixture was hydrolyzed at 50°C for 2 h. During the hydrolysis, low-speed stirring was maintained at 50 rpm. The hydrolysis was then repeated once. After the hydrolysis was completed, the reaction system was heated to 100°C to inactivate the enzymes, and the ginseng polysaccharide hydrolyzate was obtained. After the ginseng polysaccharide hydrolyzate was concentrated to 3 L under reduced pressure, the insoluble matter was removed by centrifugation at 4000 rpm for 15 min. 95% ethanol was added to a final concentration of 40%, the mixture was allowed to stand for 12 h, and the precipitate was collected by centrifugation.

[0048] (3) The above precipitate was dissolved in 6 L of distilled water, transferred to a reactor, and 280 μL of pectinase was added. The mixture was hydrolyzed at 50°C for 6 h, and low-speed stirring was maintained at 50 rpm during the hydrolysis. After the hydrolysis was completed, the reaction system was heated to 100°C to inactivate the enzyme, and ginseng pectin hydrolysate was obtained. After the ginseng pectin hydrolysate was concentrated to about 2 L under reduced pressure, it was centrifuged at 4000 rpm for 15 min. 95% ethanol was added to a final concentration of 50%, and the mixture was allowed to stand for 12 h. The supernatant was collected by centrifugation. After the supernatant was concentrated and freeze-dried, 2.51 g of ginseng galacturonic acid oligosaccharide was obtained.

[0049] HPLC detection showed that the content of GalA in the monosaccharide composition of the ginseng galacturonic acid oligosaccharide obtained in this example was 93.7%.

[0050] Comparative Example 1: Preparation of ginseng galacturonic acid oligosaccharide (1) Take 200 g of ginseng slices and add 4 L of pectin acid aqueous solution with a pH value of 4.5. Extract three times at a slightly boiling state, each time for 2 h. After combining the extracts, reduce the pressure and concentrate the extracts to 4 L. Centrifuge at 4000 rpm for 15 min to remove insoluble matter. Add 95% ethanol to a final concentration of 60%, let stand for 12 h, and collect the precipitate by centrifugation.

[0051] (2) The above precipitate was dissolved in 6 L of distilled water, transferred to a reactor, and 280 μL of pectinase was added. The mixture was hydrolyzed at 50°C for 6 h, and low-speed stirring was maintained at 50 rpm during the hydrolysis. After the hydrolysis was completed, the reaction system was heated to 100°C to inactivate the enzyme, and ginseng pectin hydrolysate was obtained. After the ginseng pectin hydrolysate was concentrated to about 2 L under reduced pressure, it was centrifuged at 4000 rpm for 15 min. 95% ethanol was added to a final concentration of 60%, and the mixture was allowed to stand for 12 h. The supernatant was collected by centrifugation. After the supernatant was concentrated and freeze-dried, 1.32 g of ginseng galacturonic acid oligosaccharide was obtained.

[0052] Compared with Examples 1-3, Comparative Example 1 omits the step of enzymatic hydrolysis with a composite enzyme of amylase and saccharifying enzyme, and the yield of ginseng galacturonic acid oligosaccharides in Comparative Example 1 is significantly reduced. This indicates that in Examples 1-3, the composite enzyme effectively degrades the straight-chain starch structure in ginseng polysaccharides, achieving the purpose of purifying ginseng pectin. In the subsequent pectinase enzymatic hydrolysis system, in Examples 1-3, compared with Comparative Example 1, the ginseng pectin concentration (substrate concentration) in the reaction system is greater, the enzymatic reaction rate is faster, the enzymatic hydrolysis efficiency is better, and the oligosaccharide yield is higher.

[0053] Example 4: In vitro antioxidant activity test of ginseng galacturonic acid oligosaccharide (1) Detection of scavenging effect on DPPH free radicals Add 50 μL of different concentrations (0.5, 1.0, 2.0, 5.0, 10.0 mg / mL) of ginseng galacturonic acid oligosaccharide solution and Vc solution prepared in distilled water in Example 1 to a 2 mL EP tube, and set 4 parallels for each concentration; add 200 μL of 0.004% DPPH solution to 3 of the parallel groups under light-proof conditions, and add 200 μL of anhydrous methanol to the other parallel group. React at 37°C in the dark for 1 h, centrifuge at 12000 rpm for 3 min, take 180 μL of supernatant and add it to a 96-well plate, and measure the absorbance of each reaction solution at 510 nm. The absorbance measured by an equal volume of distilled water instead of the sample solution is A0; the absorbance measured by the sample solution is A1; the absorbance measured by an equal volume of anhydrous methanol instead of DPPH solution is A2, and ascorbic acid (Vc) is used as a positive control.

[0054] DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0] × 100% (1) The experimental results are as follows Figure 5 As shown by Figure 5 It can be seen that the ginseng galacturonic acid oligosaccharide prepared in Example 1 has the ability to scavenge DPPH free radicals within the concentration range of 0.1 mg / mL-10 mg / mL, and has a certain concentration dependence.

[0055] (2) Detection of scavenging effect on ABTS free radicals Add 50 μL of different concentrations (0.5, 1.0, 2.0, 5.0, 10.0 mg / mL) of ginseng galacturonic acid oligosaccharide solution and Vc solution prepared in distilled water in Example 1 to a 2 mL EP tube, and set 4 parallels for each concentration; add 500 μL of ABTS working solution to 3 of the parallel groups under light-proof conditions, and add 500 μL of 0.1 M pH=7.4 PBS solution to the other parallel group. React at 37°C in the dark for 30 min, centrifuge at 12000 rpm for 3 min, take 180 μL of supernatant and add it to a 96-well plate, and measure the absorbance of each reaction solution at 734 nm. The absorbance measured by an equal volume of distilled water instead of the sample solution is A0; the absorbance measured by the sample solution is A1; the absorbance measured by an equal volume of PBS solution instead of the ABTS working solution is A2, and ascorbic acid (Vc) is used as a positive control.

[0056] ABTS free radical scavenging rate (%) = [1-(A1-A2) / A0] × 100% (2) The experimental results are as follows Figure 6 As shown by Figure 6 It can be seen that the ginseng galacturonic acid oligosaccharide prepared in Example 1 has the ability to scavenge ABTS free radicals within the concentration range of 0.1 mg / mL-10 mg / mL, and has a certain concentration dependence.

[0057] (3) Detection of scavenging effect on hydroxyl free radicals 50 μL of different concentrations (0.5, 1.0, 2.0, 5.0, 10.0 mg / mL) of ginseng galacturonic acid oligosaccharide solution and Vc solution prepared in distilled water in Example 1 were added to 2 mL EP tubes, and 4 parallels were set for each concentration; 100 μL of 8.8 mM H 2 O 2 solution, add 100 μL of distilled water to the other set of parallel tubes. Then add 100 μL of 9 mM FeSO 4 Solution and 100 μL 9 mM salicylic acid solution. The reaction was carried out at 25°C in the dark for 30 min. After centrifugation at 12000 rpm for 3 min, 180 μL of the supernatant was added to a 96-well plate and the absorbance of each reaction solution was measured at 510 nm. The absorbance measured by replacing the sample solution with an equal volume of distilled water was A0; the absorbance measured by the sample solution was A1; the absorbance measured by replacing the H 2 O 2 The absorbance of the solution was measured as A2, and ascorbic acid (Vc) was used as a positive control.

[0058] Hydroxyl free radical scavenging rate (%) = [1-(A1-A2) / A0] × 100% (3) The experimental results are as follows Figure 7 As shown by Figure 7 It can be seen that the ginseng galacturonic acid oligosaccharide prepared in Example 1 has the ability to scavenge hydroxyl free radicals within the concentration range of 0.1 mg / mL-10 mg / mL, and has a certain concentration dependence.

[0059] The above results show that the ginseng galacturonic acid oligosaccharide prepared by the present invention has certain in vitro antioxidant activity.

[0060] Example 5: Effect of ginseng galacturonic acid oligosaccharide on H 2 O 2 Inducing oxidative damage to cells (1) MTT assay to detect different concentrations of H 2 O 2 Effects on HaCaT cell viability This embodiment uses H 2 O 2 The oxidative stress model was induced in human immortalized keratinocytes (HaCaT cells). The construction process was as follows: HaCaT cells in the logarithmic phase were seeded into 96-well cell culture plates (at a concentration of 2.0 × 10 4 cells / well) and cultured for 24 h. Different concentrations (100 μM-1000 μM) of H 2 O 2 Co-culture for 4 hours, set 4 replicate wells for each concentration. Then add 100 μL MTT and incubate for 4 hours in the dark. The resulting precipitate is dissolved in 50 μL SDS-HCl and the absorbance at 570 nm is detected by a microplate reader. The absorbance of the blank solution is A 空白 ; The absorbance of the sample is A 样品 .

[0061] Cell survival rate (%) = [A 样品 / A 空白 ]×100% (4) The experimental results are as follows Figure 8 As shown in the test results, compared with the blank control group, 2 O 2 The viability of HaCaT cells decreased in a dose-dependent manner under the action of 800 μM H 2 O 2 After 4 h of injury, cell viability decreased significantly. In subsequent studies, 800 μM H 2 O 2 For modeling conditions.

[0062] (2) MTT assay to detect the effects of different concentrations of ginseng galacturonic acid oligosaccharide on HaCaT cell viability HaCaT cells in the logarithmic phase were seeded into 96-well cell culture plates (at a concentration of 2.0 × 10 4 cells / well), after culturing for 24 h, different concentrations (12.5 μg / mL-5000 μg / mL) of cell culture medium prepared with oligosaccharides obtained in Example 1 were added to the experimental group and cultured for 24 h, with 4 replicate wells for each concentration. Then 100 μL of MTT was added and incubated in the dark for 4 h. The resulting precipitate was dissolved in 50 μL of SDS-HCl, and the absorbance at 570 nm was detected by an enzyme marker. Ascorbic acid (Vc) was used as a positive control. The absorbance of the blank solution was A 空白 ; The absorbance of the sample is A 样品 .

[0063] Cell survival rate (%) = [A 样品 / A 空白 ]×100% (5) The experimental results are as follows Fig. 9 As shown, the test results show that compared with the blank control group, after incubation for 24 h with 5000 μg / mL of the oligosaccharide obtained in Example 1, the viability of HaCaT cells was significantly reduced, and there was no significant change in the viability of HaCaT cells after incubation for 24 h with the oligosaccharides obtained in Example 1 at other concentrations. This indicates that ginseng galacturonic acid oligosaccharides have no toxicity to HaCaT cells within the concentration range of 12.5 μg / mL-2500 μg / mL.

[0064] (3) Detection of intracellular ROS levels HaCaT cells in the logarithmic phase were collected and seeded into 24-well cell culture plates (at a concentration of 2.0 × 10 4 cells / well), and incubated for 24 h; in the presence of the oligosaccharides obtained in Example 1, 800 μM H 2 O 2 HaCaT cells were induced for 4 h, and DCFH-DA probe was added according to the instructions and incubated for 30 min. After incubation, the dye was washed with PBS, and then its fluorescence intensity was detected by flow cytometry.

[0065] like Fig.10 As shown, compared with the untreated control group, the 2 O 2 After 4 h of treatment on HaCaT cells, the fluorescence intensity of ROS was significantly enhanced. Under the action of oligosaccharides (12.5 μg / mL-200 μg / mL) obtained in Example 1, H 2 O 2The fluorescence intensity of induced ROS was best with 12.5 μg / mL ginseng oligogalacturonic acid.

[0066] The above results show that ginseng galacturonic acid oligosaccharide can effectively reduce H 2 O 2 Induced cellular ROS levels.

[0067] (4) Detection of intracellular mitochondrial membrane potential level HaCaT cells in the logarithmic phase were collected and inoculated into a 24-well cell culture plate (at a concentration of 2.0×104 cells / well). After incubation for 24 h, 800 μM H 2 O 2 HaCaT cells were induced for 4 h, and Rhodamine 123 probe was added according to the instructions and incubated for 30 min. After incubation, the dye was washed with PBS, and then its fluorescence intensity was detected by flow cytometry.

[0068] like Fig.11 As shown, compared with the untreated control group, the 2 O 2 After 4 h of treatment on HaCaT cells, the fluorescence intensity of mitochondrial membrane potential was significantly reduced. Under the action of oligosaccharides (12.5 μg / mL-1000 μg / mL) obtained in Example 1, H 2 O 2 The induced mitochondrial membrane potential fluorescence intensity decreased.

[0069] The above results show that ginseng galacturonic acid oligosaccharide can effectively alleviate H 2 O 2 The induced cell mitochondrial membrane potential level is reduced.

[0070] The above experimental results confirmed the effect of ginseng galacturonic acid oligosaccharide on H 2 O 2 The induced oxidative stress injury in HaCaT cells was protected.

[0071] Example 6: Determination of Moisture-Retention and Hygroscopicity of Ginseng Galacturonic Acid Oligosaccharide (1) Determination of moisturizing properties Take a desiccator, place the color-changing silica gel at the bottom of the desiccator, then weigh 0.2 g of the ginseng galacturonic acid oligosaccharide sample and sodium alginate sample prepared in Example 1, dissolve them in 1 mL of distilled water, and place them in a vial. Place the vial in the desiccator, set up a repeat group, take out the vial at 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, and 48 h, weigh it accurately, and calculate the moisture retention rate of the sample by the following formula. After the experiment, the sample that has absorbed moisture is dissolved again and freeze-dried for recovery. The mass of the vial is m0; the mass of the sample plus the vial is m1; the mass of the sample plus the vial after each time period is mx.

[0072] Moisture retention rate (%) = 100% - [(m1 - mx) / (m1 - m0)] × 100% (6) from Fig.12 The results show that as time goes by, the moisture retention rates of ginseng galacturonic acid oligosaccharide and sodium alginate prepared in Example 1 continue to decrease, and the rate of decrease of sodium alginate is greater than that of the oligosaccharide sample prepared in Example 1. The moisture retention performance is arranged from high to low as follows: ginseng galacturonic acid oligosaccharide prepared in Example 1> sodium alginate.

[0073] The above results show that ginseng galacturonic acid oligosaccharide has good moisturizing properties.

[0074] (2) Determination of hygroscopicity Take a desiccator, use saturated ammonium sulfate solution (relative humidity 81%) at the bottom to maintain relatively stable humidity conditions inside, then weigh 0.5 g of each of the ginseng galacturonic acid oligosaccharide sample and sodium alginate sample prepared in Example 1, and place them in a culture dish. Place the culture dish in the desiccator, set up a repeat group, take out the culture dish at 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, and 48 h, weigh it accurately, and calculate the moisture absorption rate of the sample by the following formula. After the experiment, the moisture-absorbed sample was re-dissolved and freeze-dried for recovery. The mass (gram) of the sample before the moisture absorption experiment is Wn; the mass (gram) of the sample after the moisture absorption experiment is W0.

[0075] Moisture absorption rate (%) = [(Wn-W0) / W0] × 100% (7) from Fig.13 It can be seen that the hygroscopicity of the ginseng galacturonic acid oligosaccharides and sodium alginate prepared in Example 1 in an environment with a relative humidity of 81% is in the following order: ginseng galacturonic acid oligosaccharides obtained in Example 1 > sodium alginate, and within the first 24 h, the hygroscopicity of the oligosaccharides obtained in Example 1 increases rapidly with time, and after 24 h, the growth rate of the hygroscopicity gradually slows down.

[0076] The above results show that ginseng galacturonic acid oligosaccharide has good hygroscopic properties.

[0077] (3) Moisturizing efficacy test - determination of keratinocyte hydration-related protein content Human immortalized keratinocytes (HaCaT) were collected at logarithmic phase and 1 × 10 5 Cells were seeded into 24-well plates at a seeding density of 100 cells / well and incubated in an incubator (37°C, 5% CO 2 ) and incubate overnight. When the cell plating rate in the 24-well plate reaches 40%~60%, the original cell culture medium is aspirated and the blank control group and the ginseng galacturonic acid oligosaccharide experimental group obtained in Example 1 are divided into groups for drug administration. The concentration gradient of ginseng galacturonic acid oligosaccharide is set to 0.063%, 0.125%, and 0.250% (m / V). 1.0 mL of sample working solution was added to each well of the experimental group, and 3 replicate wells were set for each concentration of working solution. 1.0 mL of cell culture solution was added to each well of the blank control group (BC group), and 3 replicate wells were set. Place in an incubator (37°C, 5% CO 2 ) Continue to culture for 24 h. Discard the supernatant, rinse the cells 3 times with PBS, and perform immunofluorescence staining. The main steps are: fixation, blocking, adding primary antibody, adding secondary antibody, DAPI counterstaining, and then taking pictures using a fluorescence microscope. Result analysis The fluorescence intensity of aquaporin (AQP3) and filaggrin (FLG) was quantitatively analyzed using Image Pro Plus software.

[0078] like Figure 14-17 As shown, according to the results of AQP3 immunofluorescence staining of the samples, based on the HaCaT cell model, the fluorescence intensity of aquaporin (AQP3) and filaggrin (FLG) was significantly enhanced at concentrations of 0.063%, 0.125% and 0.250% (m / V) of ginseng galacturonic acid oligosaccharide, and was statistically different from that of the BC group (p<0.05).

[0079] The above results indicate that ginseng galacturonic acid oligosaccharide can increase the contents of AQP3 and FLG in keratinocytes and has a moisturizing effect.

[0080] Example 7: Determination of the exfoliating and anti-acne activity of ginseng galacturonic acid oligosaccharide (1) In vitro exfoliation activity analysis HaCaT cells in the logarithmic phase were collected and seeded into 24-well cell culture plates (at a concentration of 2.0 × 10 4 cells / well), and incubated for 24 h; solutions of different concentrations (1 mg / mL-5 mg / mL) prepared with the oligosaccharides obtained in Example 1 were added to the cells and cultured for 40 min, the culture medium in the culture wells was collected, and the number of detached cells was calculated.

[0081] The results are as follows Fig.18 After different concentrations of ginseng galacturonic acid oligosaccharides (prepared in Example 1) were co-cultured with cells for 40 min, the cell stripping results showed that with the increase of concentration, the cell stripping ability of the oligosaccharide samples gradually increased.

[0082] The above results indicate that ginseng galacturonic acid oligosaccharide has certain exfoliating activity.

[0083] (2) Propionibacterium acnes MIC minimum inhibitory rate test The C. acnes bacteria in the logarithmic growth phase were diluted with liquid culture medium to make the concentration of the added bacteria about 4.5×10 5 CFU / mL, 180 μL of liquid culture medium containing bacteria was added to the first column of the 96-well polystyrene plate to be tested, 100 μL of liquid culture medium containing bacteria was added to each well of the 2nd to 9th columns to be tested, and 20 μL of the corresponding drug was added to each well of the first column, so that the initial concentration of ginseng galacturonic acid oligosaccharide was 20 mg / mL. 5 μL of clindamycin or erythromycin was added to the antibiotic wells in the first column, so that the initial concentration of antibiotics was 0.5 μg / mL, and the mixture was gently blown and mixed with a spray gun, and 100 μL of liquid culture medium in the first column well was pipetted into the second column well, and 100 μL of liquid culture medium in the second column well was pipetted into the third column well, and so on, and continuous multiple dilution was performed, and a total of 9 concentrations were diluted, and 100 μL of the mixed solution in the 9th column well was pipetted and discarded. At the same time, the 10th column was set as the positive control (containing culture medium and bacterial solution), and the 11th column was set as the negative control (blank culture medium), and each bacteria had 1 plate. The minimum drug concentration of the wells that can be seen to be clear by the naked eye is used as the MIC to judge the MIC value of the corresponding drug. OD experimental group represents the OD value of the experimental group; OD negative control group represents the OD value of the negative control group; OD solvent control group represents the OD value of the solvent control group; OD positive control group represents the OD value of the positive control group.

[0084] Bacterial inhibition rate (%) = [1 - (OD experimental group - OD negative control group - OD solvent control group) / (OD positive control group - OD solvent control group)] × 100% (8) The results are as follows Fig.19 As shown, ginseng galacturonic acid oligosaccharide (prepared in Example 1) has a good inhibitory effect on Propionibacterium acnes at a test concentration of 10 mg / mL.

[0085] The above results indicate that ginseng galacturonic acid oligosaccharide has a certain anti-acne effect.

[0086] Example 8: Determination of the repairing effect of ginseng galacturonic acid oligosaccharide (1) Determination of cell healing effects HaCaT cells in the logarithmic phase were collected and seeded into 24-well cell culture plates (at a concentration of 2.0 × 105 cells / well), incubated for 24 h, and then scratched the 24-well plate with a 200 μL pipette tip, washed the cells 3 times with PBS, and removed the scratched cells; the ginseng galacturonic acid oligosaccharide sample obtained in Example 1 was added to the cell suspension of each experimental group to make its concentration in the system 0.31 mg / mL, 0.63 mg / mL and 1.25 mg / mL, and the culture medium containing 10% FBS was added to the cell suspension of the positive control group (PC), and the culture medium without FBS was added to the cell suspension of the blank control group (BC), and placed in an incubator (37 o C, 5% CO 2 ) were cultured for 24 h, with 3 parallels in each group. The migrated cells of each group were photographed using an inverted microscope, and the average scratch area was calculated using Image Pro Plus software.

[0087] Healing rate (%) = (initial scratch area - current scratch area) / initial scratch area × 100% (9) like Fig. 20 As shown, based on the human immortalized keratinocyte (HaCaT cell) model, after being treated with ginseng galacturonic acid oligosaccharides obtained in Example 1 at concentrations of 0.31 mg / mL, 0.63 mg / mL and 1.25 mg / mL for 24 h, the cell healing rate of HaCaT cells was significantly improved, and there was a statistical difference compared with the BC group ( P <0.01).

[0088] The above results indicate that ginseng galacturonic acid oligosaccharide can promote cell healing of HaCaT cells and has a repairing effect.

[0089] (2) In vitro experiments to promote barrier-related proteins FLG and LOR Place the sterile cell slide into the bottom of a 24-well plate and inoculate the cells into the 24-well cell culture plate (at a concentration of 1.0 × 10 5 cells / well) and incubated in an incubator (37 o C, 5% CO 2 ) and incubated overnight. According to the test scheme in Table 1, the experiment set up a blank control group (BC), a negative control group (NC), a positive control group (PC) and a sample group. Multiple concentration gradients were set for the sample group. Using cell culture medium, different concentrations of the test substance working solution were prepared according to the concentrations listed in the test scheme (Table 1). When the cell plating rate in the 24-well plate reached 40%~60%, irradiation was carried out. The total dose received by the NC group, PC group and sample group was 300 mJ / cm 2 At the same time, the blank control group was placed in a UVB radiation dose of 0 J / cm 2After irradiation, the cell culture medium was discarded and the drugs were administered in groups. 1.0 mL of working solution was added to each well of the PC group and the sample group, and 3 replicate wells were set for each concentration. 1.0 mL of cell culture medium was added to each well of the BC group and the NC group, and 3 replicate wells were set. In the incubator (37 o C, 5% CO 2 ) for 24 h. After the incubation, the supernatant was discarded and the cells were rinsed with PBS three times. Then the conventional immunofluorescence staining process was performed. After fixation, blocking, adding primary antibody, adding secondary antibody, DAPI counterstaining, and sealing, the cells were photographed using a fluorescence microscope. The fluorescence intensity of filaggrin (FLG) and loricrin (LOR) was quantitatively analyzed using Image ProPlus software.

[0090]

[0091] Test results such as Figure 21-24 shown.

[0092] After the samples acted on HaCaT cells, the results of FLG protein immunofluorescence staining were as follows: Fig.21 As shown (green fluorescence represents FLG, blue fluorescence represents cell nucleus), the relative integrated optical density (IOD) values ​​are as follows Fig. 22 As shown. Compared with the BC group, the FLG content in the NC group decreased significantly (P < 0.01), indicating that the UVB-stimulated model was successful. Compared with the NC group, the FLG content of the ginseng galacturonic acid oligosaccharide obtained in Example 1 was significantly increased at the administration concentrations of 0.63 mg / mL, 1.25 mg / mL and 2.50 mg / mL (P < 0.01), indicating that ginseng galacturonic acid oligosaccharide can significantly promote the content of FLG protein.

[0093] After the samples acted on HaCaT cells, the results of LOR protein immunofluorescence staining were as follows: Fig.23 As shown in (green fluorescence represents LOR, blue fluorescence represents cell nucleus), the relative IOD values ​​are as follows Fig.24 As shown. Compared with the BC group, the LOR content in the NC group decreased significantly (P < 0.01), indicating that the UVB-stimulated model was successful. Compared with the BC group, the LOR content of the ginseng galacturonic acid oligosaccharide obtained in Example 1 was significantly increased at the administration concentrations of 0.63 mg / mL, 1.25 mg / mL and 2.50 mg / mL (P < 0.01), indicating that ginseng galacturonic acid oligosaccharide can significantly promote the content of LOR protein.

[0094] The above results indicate that ginseng galacturonic acid oligosaccharide can significantly promote the content of cell barrier-related proteins FLG and LOR, and has good cell barrier repair effect.

[0095] Example 9: Determination of the soothing effect of the ginseng galacturonic acid oligosaccharide of the present invention.

[0096] (1) MTT assay to detect the effects of different concentrations of ginseng oligogalacturonic acid on the viability of macrophage RAW264.7 cells RAW264.7 cells in the logarithmic phase were seeded into 96-well cell culture plates (at a concentration of 1.0 × 10 4 Cells / well) were cultured for 24 h, and then different concentrations (0.08 mg / mL-10.00 mg / mL) of cell culture fluid prepared with oligosaccharides obtained in Example 1 were added to the experimental group for co-culture for 24 h, with 3 replicate wells set for each concentration. Then 100 μL of MTT was added and incubated in the dark for 4 h. The resulting precipitate was dissolved in 100 μL of DMSO, and the absorbance at 490 nm was detected by an ELISA instrument. The absorbance of the blank solution was A 空白 ; The absorbance of the sample is A 样品 .

[0097] Cell survival rate (%) = [A 样品 / A 空白 ]×100% (10) The test results showed that ginseng galacturonic acid oligosaccharide had no toxicity to RAW264.7 cells within the concentration range of 0.08 mg / mL-0.63 mg / mL.

[0098] (2) In vitro inhibition of TNF-α, IL-6 and IL-1α activity Cells were seeded into 24-well cell culture plates (at a concentration of 1.0 × 10 5 cells / well) and incubated in an incubator (37 o C, 5% CO 2 ) and incubate overnight. Prepare the test substance working solution according to the concentration listed in the test scheme (Table 2). When the cell plating rate in the 24-well plate reaches 40%~60%, add 100 μL of 10×LPS working solution to the 24-well plate according to the test scheme (Table 2), shake the 24-well plate left and right to mix the drug, and add the sample at the same time. The final concentration of LPS is 1 μg / mL, and 3 replicates are set for each group. After the administration is completed, place it in an incubator (37 o C, 5% CO 2 ) Continue to culture for 24 h. After the incubation, collect the cell culture supernatant in an EP tube. Detect the TNF-α content according to the operating instructions of the Mouse TNF-α ELISA kit. Detect the IL-6 content according to the operating instructions of the Mouse IL-6 ELISA kit. Detect the IL-1α content according to the operating instructions of the Mouse IL-1α ELISA kit.

[0099]

[0100] The results are as follows Figure 25-27 As shown. Based on the LPS-stimulated macrophage RAW264.7 model, when the concentrations of ginseng galacturonic acid oligosaccharide obtained in Example 1 were 0.16 mg / mL, 0.31 mg / mL and 0.63 mg / mL, the secretion of inflammatory factors TNF-α and IL-1α in macrophage RAW264.7 cells was significantly reduced, and there was a statistical difference compared with the NC group ( P <0.05); When the concentration of ginseng galacturonic acid oligosaccharide obtained in Example 1 was 0.31 mg / mL and 0.63 mg / mL, the secretion of inflammatory factor IL-6 by macrophage RAW264.7 was significantly reduced, and there was a statistical difference compared with the NC group ( P <0.01).

[0101] The above results indicate that ginseng galacturonic acid oligosaccharide can inhibit the secretion of inflammatory factors TNF-α, IL-6 and IL-1α in macrophages RAW264.7, and has a soothing effect.

[0102] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, can implement the present invention in a wider range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. A method for preparing ginseng galacturonic acid oligosaccharide, characterized in that: The steps include: S1. adding weak acid to ginseng medicinal materials for extraction to obtain ginseng polysaccharide extract; S2, adding a complex enzyme consisting of saccharifying enzyme and amylase to the ginseng polysaccharide extract for enzymolysis to obtain a ginseng polysaccharide enzymolysis solution; Separating the polysaccharide components that have not been hydrolyzed by the composite enzyme from the ginseng polysaccharide hydrolyzate; S3, adding pectinase to the polysaccharide component that has not been hydrolyzed by the composite enzyme for hydrolysis to obtain ginseng pectin hydrolyzate; Separating oligosaccharides hydrolyzed by the pectinase in the ginseng pectin hydrolyzate; The ginseng galacturonic acid oligosaccharide is obtained by freeze-drying; The monosaccharide composition of the ginseng galacturonic acid oligosaccharide includes galacturonic acid GalA with a mass percentage of ≥90%; the molecular weight of the ginseng galacturonic acid oligosaccharide is lower than 3.0 kDa; the degree of polymerization of the ginseng galacturonic acid oligosaccharide is lower than 15; the ginseng galacturonic acid oligosaccharide is composed of α-D-GalpA residues, and the glycosidic bond connection method is α-1,4-GalpA.

2. The method for preparing ginseng galacturonic acid oligosaccharide according to claim 1, characterized in that: The ginseng is any one of wild ginseng, forest ginseng and garden ginseng; The ginseng medicinal material is ginseng slices; The material-liquid ratio of the extraction in step S1 is 1 g: (10-20) mL; The pH value of the extraction system composed of the ginseng medicinal material and the weak acid is 4.0 to 5.0; The weak acid is an organic acid; The extraction temperature in step S1 is 80-100° C., and the extraction is performed 2-4 times, each time for 2-4 hours, and the extracts are combined.

3. The method for preparing ginseng galacturonic acid oligosaccharide according to any one of claims 1 to 2, characterized in that: The activity of the saccharifying enzyme in the complex enzyme is 200,000 to 260,000 U / ml, and the activity of the amylase is 10,000 to 20,000 U / ml; The ratio of the ginseng medicinal material to the complex enzyme is 1kg: (12-16) mL; The enzymatic hydrolysis in step S2 is carried out at a temperature of 50° C. and at a stirring condition of 40 to 60 rpm for 1 to 3 times, each time for 2 to 3 hours; After the enzymolysis in step S2, the following step is further included: heating the reaction solution after enzymolysis to 100° C. to inactivate the enzyme.

4. The method for preparing ginseng galacturonic acid oligosaccharide according to any one of claims 1 to 2, characterized in that: Before the separation step in step S2, the following steps are also included: sequentially concentrating and centrifuging the ginseng polysaccharide hydrolyzate to collect the supernatant; wherein the concentration is concentrated to 1 / 2 to 1 / 4 of the original solution; The separation in step S2 includes the following steps: adding ethanol to the ginseng polysaccharide hydrolyzate for alcohol precipitation, and collecting the alcohol precipitate. In the alcohol precipitation step, the final volume fraction of ethanol in the ginseng polysaccharide hydrolyzate is 40% to 60%.

5. The method for preparing ginseng galacturonic acid oligosaccharide according to any one of claims 1 to 2, characterized in that: In step S3, the enzyme activity of the pectinase is 20,000 to 30,000 U / ml; The ratio of the ginseng medicinal material to the pectinase is 1 kg: (1.0-1.4) mL; The enzymatic hydrolysis in step S3 is carried out at a temperature of 50° C. and at a stirring condition of 40 to 60 rpm for 1 to 2 times, each time for 4 to 6 hours; After the enzymolysis in step S3, the following step is further included: heating the reaction solution after enzymolysis to 100° C. to inactivate the enzyme.

6. The method for preparing ginseng galacturonic acid oligosaccharide according to any one of claims 1 to 2, characterized in that: Before the separation step in step S3, the following steps are also included: sequentially concentrating and centrifuging the ginseng pectin enzymatic hydrolyzate to collect the supernatant; wherein the concentration is concentrated to 1 / 2 to 1 / 4 of the original solution; The separation in step S3 includes the following steps: adding ethanol to the ginseng pectin enzymatic hydrolysate for alcohol precipitation, and collecting the supernatant. In the alcohol precipitation step, the final volume fraction of ethanol in the ginseng pectin enzymatic hydrolysate is 50% to 70%.

7. Ginseng galacturonic acid oligosaccharide obtained by the preparation method according to any one of claims 1 to 6.

8. Use of ginseng galacturonic acid oligosaccharide obtained by the preparation method according to any one of claims 1 to 6 in any one of the following items A1) to A3): A1) Antioxidant in vitro; A2) Preparation of cosmetics for anti-oxidation; A3) Preparation of cosmetics for anti-oxidative stress-induced skin damage.

9. Use of ginseng galacturonic acid oligosaccharide obtained by the preparation method according to any one of claims 1 to 6 in any one of the following items B1) to B2): B1) Skin moisturizing; B2) Preparation of skin moisturizing cosmetics.

10. Use of ginseng galacturonic acid oligosaccharide obtained by the preparation method according to any one of claims 1 to 6 in any one of the following items C1) to C5): C1) External exfoliation; C2) Preparation of cosmetic products for in vitro exfoliation; C3) In vitro inhibition of Propionibacterium acnes for non-therapeutic purposes; C4) preparing cosmetics for inhibiting Propionibacterium acnes; C5) Preparation of cosmetics for improving acne.

11. Use of ginseng galacturonic acid oligosaccharide obtained by the preparation method according to any one of claims 1 to 6 in any one of the following items D1) to D3): D1) Promotes healing of human immortalized keratinocytes in vitro; D2) Preparation of cosmetics for promoting the healing of human immortalized keratinocytes; D3) Preparation of cosmetics for maintaining the skin barrier or repairing skin damage.

12. Use of ginseng galacturonic acid oligosaccharide obtained by the preparation method according to any one of claims 1 to 6 in any one of the following E1) to E2): E1) Preparation of anti-inflammatory cosmetics; E2) Preparation of cosmetic products for skin soothing or repairing skin inflammation.

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