Ginseng galacturonic acid oligosaccharide and its preparation method and application

Abstract

The present invention discloses a ginseng galacturonic acid oligosaccharide and its preparation method and application, which belong to the field of biochemical technology. The monosaccharide composition of the ginseng galacturonic acid oligosaccharide of the present invention 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 mode is α-1,4-GalpA. 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, and can achieve repairing 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, and has broad application prospects in cosmetics such as beauty and skin care products.

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 C. acuta Meyer is a precious Chinese medicinal herb, known as the "King of Herbs." It exhibits high bioactivity and unique medicinal value in skin care, including antioxidant, anti-aging, sun protection, whitening, moisturizing, and anti-inflammatory properties. Modern pharmaceutical research indicates that ginsenosides and ginseng polysaccharides are the primary active components of ginseng and form the basis for its therapeutic effects. While ginsenosides and polysaccharides have been extensively studied and applied, research on ginseng's active oligosaccharides is relatively limited.

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

[0004] Existing ginseng extract preparation processes primarily focus on the extraction of polysaccharides and saponins. 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 properties. However, there are no reports on the preparation of ginseng oligosaccharides primarily composed of galacturonic acid oligosaccharides or their use in skin care products. Therefore, the development of methods for preparing ginseng galacturonic acid oligosaccharides and their use in skin care products with antioxidant, moisturizing, exfoliating, acne-removing, and soothing properties has significant research significance and application value. Summary of the Invention

[0005] The present invention aims to provide a ginseng galacturonic acid oligosaccharide, a preparation method thereof, and application thereof in the preparation of cosmetics. The method is simple and easy to carry out, 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:

[0007] S1. adding a weak acid to ginseng medicinal materials for extraction to obtain a ginseng polysaccharide extract;

[0008] S2, adding a complex enzyme consisting of saccharifying enzyme and amylase to the ginseng polysaccharide extract for enzymatic hydrolysis to obtain a ginseng polysaccharide enzymatic hydrolyzate;

[0009] separating the polysaccharide components that have not been hydrolyzed by the complex enzyme from the ginseng polysaccharide hydrolyzate;

[0010] S3, adding pectinase to the polysaccharide component that has not been enzymatically hydrolyzed by the composite enzyme to perform enzymatic hydrolysis to obtain a ginseng pectin enzymatic hydrolyzate;

[0011] separating oligosaccharides hydrolyzed by the pectinase from the ginseng pectin hydrolyzate;

[0012] The ginseng galacturonic acid oligosaccharide is obtained by freeze-drying;

[0013] The ginseng galacturonic acid oligosaccharide monosaccharide composition includes galacturonic acid GalA with a mass percentage of ≥90%;

[0014] The molecular weight of the ginseng galacturonic acid oligosaccharide is less than 3.0 kDa;

[0015] The polymerization degree of the ginseng galacturonic acid oligosaccharide is less than 15;

[0016] The ginseng galacturonic acid oligosaccharide is composed of α-D-GalpA residues, and the glycosidic bond connection mode is α-1,4-GalpA.

[0017] 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 of 1.8 kDa / 1.4 kDa and 0.6 kDa; the degree of polymerization of the ginseng galacturonic acid oligosaccharide is 1-8, wherein the degree of polymerization represents the number of galacturonic acid units; the glycosidic linkage mode of the galacturonic acid oligosaccharide is α-1,4-GalpA, and the ginseng galacturonic acid oligosaccharide contains methylated galacturonic acid.

[0018] In the above-mentioned preparation method of 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 slices with a thickness of 2 to 3 cm, which are dry-cut;

[0019] The material-liquid ratio of the extraction in step S1 is 1 g: (10-20) mL, such as 1 g: 15 mL;

[0020] The pH value of the extraction system composed of the ginseng medicinal material and the weak acid is 4.0 to 5.0, such as 4.5; the weak acid can provide any organic acid in the above pH range, such as citric acid, acetic acid, oxalic acid, pectin acid, malic acid, lactic acid, etc.;

[0021] The extraction temperature in step S1 is 80-100° C., and the extraction is performed 2-4 times, each time for 2-4 hours. The extracts are combined, and the extraction is performed 3 times under a slightly boiling state, each time for 2 hours.

[0022] In the above-mentioned method for preparing ginseng galacturonic acid oligosaccharide, the enzymatic activity of the saccharifying enzyme in the complex 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;

[0023] The ratio of the ginseng medicinal material to the complex enzyme is 1 kg: (12-16) mL, such as 1 kg: 16 mL;

[0024] The present invention has no particular limitation on the sources of amylase and saccharifying enzyme in the complex enzyme composed of amylase and saccharifying enzyme, and conventional commercially available products can be used;

[0025] The enzymatic hydrolysis in step S2 is carried out at a temperature of 50° C., under stirring conditions of 40 to 60 rpm (e.g., 50 rpm), for 2 to 3 times (e.g., 2 times), each time for 2 to 3 hours (e.g., 2 hours); amylase and saccharifying enzyme can synergistically act at 50° C. and the same weakly acidic pH conditions to efficiently catalyze the complete hydrolysis of ginseng starch into low molecular weight glucose;

[0026] After the enzymatic hydrolysis in step S2, the following step is further included: heating the reaction solution after the enzymatic hydrolysis to 100° C. to inactivate the enzyme.

[0027] In the above-mentioned method for preparing ginseng galacturonic acid oligosaccharides, the following steps are further included before the separation step in step S2: the ginseng polysaccharide enzymatic hydrolyzate is concentrated and centrifuged in sequence to collect the supernatant; 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 minutes to remove insoluble matter;

[0028] The separation in step S2 includes the following steps: adding ethanol to the ginseng polysaccharide hydrolyzate for alcohol precipitation, and collecting the alcohol precipitate. 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 this range, ginseng polysaccharide components such as ginseng pectin that are not hydrolyzed by the complex enzyme can be separated from 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 specifically be 12 hours.

[0029] It is understood that the separation in step S2 may also employ other separation methods, such as replacing the aforementioned alcohol precipitation separation method with membrane filtration separation. Specifically, the separation in step S2 includes the following steps: subjecting the ginseng polysaccharide enzymatic hydrolyzate to membrane filtration and collecting the retentate. The membrane filtration employed 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.

[0030] 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;

[0031] The ratio of the ginseng medicinal material to the pectinase is 1 kg: (1.0-1.4) mL, such as 1 kg: 1.4 mL;

[0032] The present invention has no particular limitation on the source of pectinase, and conventional commercial products can be used;

[0033] 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 (e.g., 50 rpm), and the enzymatic hydrolysis is performed 1 to 2 times (e.g., 1 time), each time for 4 to 6 hours (e.g., 6 hours). As an example, in the enzymatic hydrolysis step, the alcohol precipitate is redissolved in water for the enzymatic hydrolysis.

[0034] After the enzymatic hydrolysis in step S3, the following steps are further included: heating the enzymatically hydrolyzed reaction solution to 100° C. to inactivate the enzyme;

[0035] In the above-mentioned method for preparing ginseng galacturonic acid oligosaccharides, the following steps are further included before the separation step in step S3: concentrating and centrifuging the ginseng pectin hydrolyzate in sequence, and collecting the supernatant; 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;

[0036] The separation in step S3 includes the following steps: adding ethanol to the ginseng pectin enzymatic hydrolyzate for alcohol precipitation, and collecting the supernatant. The final volume fraction of ethanol in the ginseng pectin enzymatic hydrolyzate in the alcohol precipitation step is 50% to 70%, such as 50%, 60% or 70%. Within this range, the ginseng polysaccharide components not hydrolyzed by pectinase and the ginseng galacturonic acid oligosaccharides produced after pectinase hydrolysis can be separated. The alcohol precipitation time in the alcohol precipitation step can specifically be 12 hours.

[0037] It is understood that the separation in step S3 may also employ other separation methods, such as replacing the aforementioned alcohol precipitation separation method with a membrane filtration separation method. Specifically, the separation in step S3 includes the following steps: subjecting the ginseng pectin enzymatic hydrolyzate to membrane filtration and collecting the filtrate. The membrane filtration employed 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.

[0038] In a second aspect, the present invention provides ginseng galacturonic acid oligosaccharide obtained by any of the preparation methods described above.

[0039] 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 items A1) to A3):

[0040] A1) Antioxidant in vitro;

[0041] A2) Preparation of cosmetics for antioxidant purposes;

[0042] A3) Preparation of cosmetics for protecting against oxidative stress-induced skin damage.

[0043] As an example, the antioxidant effect 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 resistance is manifested in reducing the cellular ROS level induced by H2O2 and alleviating the decrease in the cellular mitochondrial membrane potential level induced by H2O2.

[0044] 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 items B1) to B2):

[0045] B1) Skin moisturizing;

[0046] B2) Preparation of skin moisturizing cosmetics.

[0047] As an example, the moisturizing and hygroscopic properties of the ginseng galacturonic acid oligosaccharide of the present invention are both greater than those of sodium alginate. The skin moisturizing effect is manifested in increasing the content of AQP3 and FLG in keratinocytes.

[0048] 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 C1) to C5):

[0049] C1) External exfoliation;

[0050] C2) Preparation of cosmetic products for in vitro exfoliation;

[0051] C3) In vitro inhibition of Propionibacterium acnes for non-therapeutic purposes;

[0052] C4) preparing cosmetics for inhibiting Propionibacterium acnes;

[0053] C5) Preparation of cosmetics for improving acne.

[0054] 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.

[0055] 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):

[0056] D1) Promotes healing of human immortalized keratinocytes in vitro;

[0057] D2) Preparation of cosmetics for promoting the healing of human immortalized keratinocytes;

[0058] D3) Preparation of cosmetics for maintaining the skin barrier or repairing skin damage.

[0059] As an example, the in vitro promotion of human immortalized keratinocyte healing is embodied in promoting the cell healing of HaCaT cells; the maintenance of the skin barrier or repair of skin damage is embodied in promoting the content of FLG protein and LOR protein.

[0060] 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):

[0061] E1) Preparation of anti-inflammatory cosmetics;

[0062] E2) Preparation of cosmetics for skin soothing or repairing skin inflammation.

[0063] 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.

[0064] In the above-mentioned applications, the cosmetics may be skin care and beauty cosmetics.

[0065] The present invention has the following beneficial effects:

[0066] (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 existing technology.

[0067] (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

[0068] Figure 1 High performance gel permeation chromatography for molecular weight analysis of ginseng galacturonic acid oligosaccharide in the present invention;

[0069] Figure 2 This is a high performance liquid chromatogram of the monosaccharide composition analysis of ginseng galacturonic acid oligosaccharide in the present invention;

[0070] Figure 3 is the ion chromatogram of ginseng galacturonic acid oligosaccharide in the present invention;

[0071] Figure 4 The ginseng galacturonic acid oligosaccharide of the present invention 13 C NMR spectrum;

[0072] Figure 5 This is a diagram showing the scavenging effect of ginseng galacturonic acid oligosaccharide on DPPH free radicals in the present invention;

[0073] Figure 6 This is a diagram showing the scavenging effect of ginseng galacturonic acid oligosaccharide on ABTS free radicals in the present invention;

[0074] Figure 7 This is a diagram showing the scavenging effect of ginseng galacturonic acid oligosaccharide on hydroxyl radicals in the present invention;

[0075] Figure 8 This is a diagram of the H2O2-induced oxidative stress model of the present invention;

[0076] Figure 9 This is a graph showing the effect of ginseng galacturonic acid oligosaccharide on HaCaT cell viability;

[0077] Figure 10 The results show the effect of ginseng galacturonic acid oligosaccharide on the ROS level of HaCaT cells induced by H2O2;

[0078] Figure 11 The results show the effect of ginseng galacturonic acid oligosaccharide on the mitochondrial membrane potential of HaCaT cells induced by H2O2.

[0079] Figure 12 The moisturizing ability test results of ginseng galacturonic acid oligosaccharide in the present invention;

[0080] Figure 13 The results of the hygroscopicity test of ginseng galacturonic acid oligosaccharide in the present invention are as follows;

[0081] Figure 14 Effects of ginseng galacturonic acid oligosaccharide of the present invention on the content of hydration-related proteins in keratinocytes: AQP3 immunofluorescence staining results;

[0082] Figure 15 Effects of ginseng galacturonic acid oligosaccharide of the present invention on the content of hydration-related proteins in keratinocytes: AQP3 relative integrated optical density (IOD) value bar graph;

[0083] Figure 16 Effects of ginseng galacturonic acid oligosaccharide of the present invention on the content of hydration-related proteins in keratinocytes: FLG immunofluorescence staining results;

[0084] Figure 17 Effects of ginseng galacturonic acid oligosaccharide of the present invention on the content of hydration-related proteins in keratinocytes: FLG relative integrated optical density (IOD) value histogram;

[0085] Figure 18 The results of the cell exfoliation experiment of ginseng galacturonic acid oligosaccharide in the present invention are as follows;

[0086] Figure 19 The results of the antibacterial experiment of ginseng galacturonic acid oligosaccharide on Propionibacterium acnes in the present invention are as follows;

[0087] Figure 20 This is the effect of ginseng galacturonic acid oligosaccharide on HaCaT cell scratch healing;

[0088] Figure 21 The 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;

[0089] Figure 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;

[0090] Figure 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;

[0091] Figure 24 The results of the effect of ginseng galacturonic acid oligosaccharide on the expression level of LOR protein in HaCaT cells under UVB stimulation are as follows: relative integrated optical density (IOD) value;

[0092] Figure 25 This is the effect of ginseng galacturonic acid oligosaccharide on the secretion of inflammatory factor TNF-α by RAW264.7 cells under LPS stimulation;

[0093] Figure 26 This is the effect of ginseng galacturonic acid oligosaccharide on the secretion of inflammatory factor IL-6 by RAW264.7 cells under LPS stimulation;

[0094] Figure 27 These are the results of the effect of ginseng galacturonic acid oligosaccharide in the present invention on the secretion of inflammatory factor IL-1α by RAW264.7 cells under LPS stimulation. DETAILED DESCRIPTION

[0095] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0096] Unless otherwise specified, the methods used in the following examples are all conventional methods and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0097] The ginseng in the following examples was purchased from Tonghua, Jilin Province. The ginseng species was garden ginseng, and the slices had a thickness of 2-3 cm and were dry-cut.

[0098] 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.

[0099] The enzymatic activity of pectinase is 30,000 U / ml, and its enzymatic activity is defined as follows: 1 ml of enzyme solution decomposes pectin solution to produce 1 mg of galacturonic acid in 1 hour at 50°C and pH 3.5. This is one enzyme activity unit, expressed in U / ml.

[0100] Example 1: Preparation of Ginseng Galacturonic Acid Oligosaccharide

[0101] This embodiment provides a method for preparing ginseng galacturonic acid oligosaccharide, and the specific steps are as follows:

[0102] (1) Take 200 g of ginseng slices and add 4 L of pectin aqueous solution with a pH of 4.5. Extract three times at a low boiling point, each time for 2 h. After combining the extracts, the ginseng polysaccharide extract was obtained.

[0103] (2) Transfer the ginseng polysaccharide extract to a reactor, add 3.2 mL of a complex enzyme consisting of amylase and saccharifying enzyme, and perform enzymatic hydrolysis at 50°C for 2 h. Maintain low-speed stirring at 50 rpm during the hydrolysis process; then repeat the enzymatic hydrolysis once. After the enzymatic hydrolysis is completed, heat the reaction system to 100°C to inactivate the enzymes to obtain the ginseng polysaccharide hydrolyzate. Concentrate the ginseng polysaccharide hydrolyzate to 4 L under reduced pressure and centrifuge at 4000 rpm for 15 min to remove insoluble matter. Add 95% ethanol to a final concentration of 60%, let it stand for 12 h, and collect the precipitate by centrifugation.

[0104] (3) The above precipitate was dissolved in 6 L of distilled water and transferred to a reactor. 280 μL of pectinase was added and enzymatic hydrolysis was carried out at 50°C for 6 h. During the enzymatic hydrolysis, low-speed stirring was maintained at 50 rpm. After the enzymatic hydrolysis was completed, the reaction system was heated to 100°C to inactivate the enzyme to obtain ginseng pectin enzymatic hydrolysate. The ginseng pectin enzymatic hydrolysate was concentrated under reduced pressure to about 2 L and 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.

[0105] Take 2 mg of the above-mentioned ginseng galacturonic acid oligosaccharide and perform molecular weight distribution analysis using high performance gel permeation chromatography (HPGPC) (sample solvent is 0.2 M NaCl, Japan Shimadzu LC-20Ai liquid chromatography, RID-20A differential refractive index detector, TSK-gel G2500 PWxl column, mobile phase is 0.2 M NaCl aqueous solution, flow rate is 0.5 mL / min, injection volume is 20 μL), as shown in FIG. Figure 1 As shown, its molecular weight is less than 3.0 kDa.

[0106] Another 2 mg of ginseng galacturonic acid oligosaccharide was taken, and after PMP derivatization treatment, the monosaccharide composition was analyzed by high performance liquid chromatography (HPLC) (Shimadzu LC-20A liquid chromatography, SPD-20A UV detector, chromatographic column COSMOSIL 5C18-PAQ, acetonitrile as mobile phase A, disodium hydrogen phosphate-sodium dihydrogen phosphate aqueous solution as mobile phase B, isocratic elution according to the ratio of A:B = 18.8:81.2. The column temperature was 35 ° C, the flow rate was 1.0 mL / min, the detection wavelength was 245 nm, and the injection volume was 10 μL). Figure 2 As shown, the content of GalA in its monosaccharide composition is 92.4%.

[0107] Another 1 mg of ginseng galacturonic acid oligosaccharide was taken and separated and identified using a high-performance ion chromatography system equipped with a Carbo Pac PA200 column (the sample solvent was water, the Thermo Fisher Scientific ICS 6000 ion chromatograph, a pulsed amperometric detector, an anion exchange column Carbo Pac PA200, detection was performed at 35 °C, the elution flow rate was 0.5 mL / min, and the injection volume was 25 μL. The ion chromatography elution program was as follows: from -6 to 0 min, 100 mM NaOH and 100 mM NaAc were used for isocratic elution to equilibrate the column; from 0 to 20 min, 100 mM NaOH was used for isocratic elution, and NaAc was increased from 100 mM to 500 mM for linear gradient elution; from 20 to 30 min, the NaOH concentration was increased to 200 mM, and the NaAc concentration was increased to 700 mM for isocratic elution to clean the column.) Figure 3 As shown, the degree of polymerization of ginseng galacturonic acid oligosaccharide is 1-8.

[0108] Another 15 mg of ginseng galacturonic acid oligosaccharide was taken and dissolved in D2O (99.8%). The 13C NMR spectrum was analyzed by nuclear magnetic resonance spectrometer. Figure 4 As 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, the characteristic absorption peak of methylated galacturonic acid is also present.

[0109] Example 2: Preparation of Ginseng Galacturonic Acid Oligosaccharide

[0110] This embodiment provides a method for preparing ginseng galacturonic acid oligosaccharide, and the specific steps are as follows:

[0111] (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 low boiling temperature for 2 h each time and combine the extracts.

[0112] (2) Transfer the ginseng polysaccharide extract to a reactor, add 3.2 mL of a complex enzyme consisting of amylase and saccharifying enzyme, and perform enzymatic hydrolysis at 50°C for 2 h. Maintain low-speed stirring at 50 rpm during the hydrolysis process; then repeat the enzymatic hydrolysis once. After the enzymatic hydrolysis is completed, heat the reaction system to 100°C to inactivate the enzymes to obtain the ginseng polysaccharide hydrolyzate. Concentrate the ginseng polysaccharide hydrolyzate to 4.5 L under reduced pressure and centrifuge at 4000 rpm for 15 min to remove insoluble matter. Add 95% ethanol to a final concentration of 50%, let it stand for 12 h, and collect the precipitate by centrifugation.

[0113] (3) The above precipitate was dissolved in 6 L of distilled water and transferred to a reactor. 280 μL of pectinase was added and the mixture was enzymatically hydrolyzed at 50°C for 6 h. During the enzymatic hydrolysis, low-speed stirring was maintained at 50 rpm. After the enzymatic hydrolysis was completed, the reaction system was heated to 100°C to inactivate the enzyme, and ginseng pectin enzymatic hydrolyzate was obtained. The ginseng pectin enzymatic hydrolyzate was concentrated under reduced pressure to about 2 L and 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.

[0114] HPLC analysis showed that the content of GalA in the monosaccharide composition of the ginseng galacturonic acid oligosaccharide obtained in this example was 92.9%, and the test conditions were the same as those in Example 1.

[0115] Example 3: Preparation of Ginseng Galacturonic Acid Oligosaccharide

[0116] This embodiment provides a method for preparing ginseng galacturonic acid oligosaccharide, and the specific steps are as follows:

[0117] (1) Take 200 g of ginseng slices and add 4 L of oxalic acid solution with a pH of 4.5. Extract at a low boiling temperature three times for 2 h each time and combine the extracts.

[0118] (2) Transfer the ginseng polysaccharide extract to a reactor, add 3.2 mL of a complex enzyme consisting of amylase and saccharifying enzyme, and perform enzymatic hydrolysis at 50°C for 2 h. Maintain low-speed stirring at 50 rpm during the hydrolysis process; then repeat the enzymatic hydrolysis once. After the enzymatic hydrolysis is completed, heat the reaction system to 100°C to inactivate the enzymes to obtain the ginseng polysaccharide hydrolyzate. Concentrate the ginseng polysaccharide hydrolyzate to 3 L under reduced pressure and centrifuge at 4000 rpm for 15 min to remove insoluble matter. Add 95% ethanol to a final concentration of 40%, let it stand for 12 h, and collect the precipitate by centrifugation.

[0119] (3) The above precipitate was dissolved in 6 L of distilled water and transferred to a reactor. 280 μL of pectinase was added and enzymatic hydrolysis was carried out at 50°C for 6 h. During the enzymatic hydrolysis, low-speed stirring was maintained at 50 rpm. After the enzymatic hydrolysis was completed, the reaction system was heated to 100°C to inactivate the enzyme to obtain ginseng pectin enzymatic hydrolysate. The ginseng pectin enzymatic hydrolysate was concentrated under reduced pressure to about 2 L and 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.

[0120] HPLC analysis showed that the content of GalA in the monosaccharide composition of the ginseng galacturonic acid oligosaccharide obtained in this example was 93.7%.

[0121] Comparative Example 1: Preparation of Ginseng Galacturonic Acid Oligosaccharide

[0122] (1) Take 200 g of ginseng slices and add 4 L of pectin aqueous solution with a pH of 4.5. Extract at a slight boiling point three times for 2 h each time. Combine the extracts and concentrate under reduced pressure 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.

[0123] (2) The above precipitate was dissolved in 6 L of distilled water and transferred to a reactor. 280 μL of pectinase was added and enzymatic hydrolysis was carried out at 50°C for 6 h. During the enzymatic hydrolysis, low-speed stirring was maintained at 50 rpm. After the enzymatic hydrolysis was completed, the reaction system was heated to 100°C to inactivate the enzyme to obtain ginseng pectin enzymatic hydrolysate. The ginseng pectin enzymatic hydrolysate was concentrated under reduced pressure to about 2 L and 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.

[0124] Compared with Examples 1-3, Comparative Example 1 omitted the enzymatic hydrolysis step using a combination of amylase and saccharifying enzymes. Consequently, the yield of ginseng galacturonic acid oligosaccharides in Comparative Example 1 was significantly reduced. This indicates that in Examples 1-3, the combination of enzymes effectively degraded the amylose structure in the ginseng polysaccharides, achieving the goal of purifying ginseng pectin. Furthermore, in the subsequent pectinase hydrolysis system, Examples 1-3 achieved a higher ginseng pectin concentration (substrate concentration) in the reaction system, faster enzymatic reaction rates, improved enzymatic hydrolysis efficiency, and a higher oligosaccharide yield compared to Comparative Example 1.

[0125] Example 4: In vitro antioxidant activity assay of ginseng galacturonic acid oligosaccharides

[0126] (1) Detection of scavenging effect on DPPH free radicals

[0127] To a 2 mL EP tube, add 50 μL of the ginseng galacturonic acid oligosaccharide solution obtained in Example 1 and a vitamin C solution at different concentrations (0.5, 1.0, 2.0, 5.0, and 10.0 mg / mL) prepared in distilled water. Set up four replicates for each concentration. Add 200 μL of 0.004% DPPH solution to three replicates in the dark, and 200 μL of anhydrous methanol to the other replicate. Incubate at 37°C in the dark for 1 hour. After centrifugation at 12,000 rpm for 3 minutes, 180 μL of the supernatant was transferred 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 designated A0; the absorbance measured by replacing the sample solution with an equal volume of distilled water was designated A1; and the absorbance measured by replacing the DPPH solution with an equal volume of anhydrous methanol was designated A2. Ascorbic acid (Vc) was used as a positive control.

[0128] DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0] × 100% (1)

[0129] 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 in the concentration range of 0.1 mg / mL-10 mg / mL, and has a certain concentration dependence.

[0130] (2) Detection of ABTS free radical scavenging effect

[0131] To a 2 mL EP tube, add 50 μL of the ginseng galacturonic acid oligosaccharide solution and vitamin C solution prepared in distilled water at different concentrations (0.5, 1.0, 2.0, 5.0, and 10.0 mg / mL) obtained in Example 1. Set up four replicates for each concentration. Under dark conditions, add 500 μL of ABTS working solution to three replicates, and add 500 μL of 0.1 M PBS (pH 7.4) to another replicate. Incubate at 37°C in the dark for 30 minutes. After centrifugation at 12,000 rpm for 3 minutes, 180 μL of the supernatant was transferred to a 96-well plate, and the absorbance of each reaction solution was measured at 734 nm. The absorbance measured by replacing the sample solution with an equal volume of distilled water was designated A0; the absorbance measured by the sample solution was designated A1; and the absorbance measured by replacing the ABTS working solution with an equal volume of PBS was designated A2. Ascorbic acid (Vc) was used as a positive control.

[0132] ABTS free radical scavenging rate (%) = [1-(A1-A2) / A0] × 100% (2)

[0133] 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 in the concentration range of 0.1 mg / mL-10 mg / mL, and has a certain concentration dependence.

[0134] (3) Detection of scavenging effect on hydroxyl free radicals

[0135] To a 2 mL EP tube, add 50 μL of the ginseng galacturonic acid oligosaccharide solution obtained in Example 1 and Vc solution, prepared in distilled water at different concentrations (0.5, 1.0, 2.0, 5.0, and 10.0 mg / mL). Set up four replicates for each concentration. Add 100 μL of 8.8 mM H₂O₂ solution to three replicates, and 100 μL of distilled water to the other replicate. Then, add 100 μL of 9 mM FeSO₄ solution and 100 μL of 9 mM salicylic acid solution to each replicate, all in the dark. Incubate at 25°C in the dark for 30 minutes. After centrifugation at 12,000 rpm for 3 minutes, aliquot 180 μL of the supernatant and transfer to a 96-well plate. The absorbance of each reaction solution is measured at 510 nm. The absorbance measured when an equal volume of distilled water replaces the sample solution is A0; the absorbance measured when the sample solution is A1; the absorbance measured when an equal volume of distilled water replaces the H2O2 solution is A2, and ascorbic acid (Vc) is used as a positive control.

[0136] Hydroxyl radical scavenging rate (%) = [1-(A1-A2) / A0] × 100% (3)

[0137] 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 in the concentration range of 0.1 mg / mL-10 mg / mL, and has a certain concentration dependence.

[0138] The above results show that the ginseng galacturonic acid oligosaccharide prepared by the present invention has certain in vitro antioxidant activity.

[0139] Example 5: Effects of ginseng galacturonic acid oligosaccharide on H2O2-induced oxidative damage in cells

[0140] (1) MTT assay to detect the effect of different concentrations of H2O2 on HaCaT cell viability

[0141] In this example, H2O2 was used to induce an oxidative stress model in human immortalized keratinocytes (HaCaT cells). The construction process was as follows:

[0142] HaCaT cells in the logarithmic phase were seeded into 96-well cell culture plates (at a concentration of 2.0 × 10 4 Cells were cultured for 24 h. Different concentrations of H2O2 (100 μM-1000 μM) were added to the cells and incubated for 4 h. Four replicate wells were set for each concentration. 100 μL of MTT was then 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 measured using a microplate reader. The absorbance of the blank solution was A空白 The absorbance of the sample is A 样品 .

[0143] Cell survival rate (%) = [A 样品 / A 空白 ]×100% (4)

[0144] The experimental results are as follows Figure 8 As shown in the results, compared with the blank control group, the HaCaT cell viability decreased in a dose-dependent manner under the action of H2O2. After 4 hours of damage with 800 μM H2O2, the cell viability was significantly reduced. In subsequent studies, 800 μM H2O2 was selected as the modeling condition.

[0145] (2) MTT assay to detect the effects of different concentrations of ginseng galacturonic acid oligosaccharide on HaCaT cell viability

[0146] 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 the oligosaccharide obtained in Example 1 were added to the experimental group and cultured for 24 h. Four replicate wells were 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 50 μL of SDS-HCl, and the absorbance at 570 nm was measured using a microplate reader. 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 .

[0147] Cell survival rate (%) = [A 样品 / A 空白 ]×100% (5)

[0148] The experimental results are as follows Figure 9 As shown in the results, compared with the blank control group, the viability of HaCaT cells was significantly reduced after incubation for 24 hours with 5000 μg / mL of the oligosaccharide obtained in Example 1, while the viability of HaCaT cells did not change significantly after incubation for 24 hours 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.

[0149] (3) Detection of intracellular ROS levels

[0150] HaCaT cells in the logarithmic phase were collected and seeded into 24-well cell culture plates (at a concentration of 2.0 × 10 4cells / well) and incubated for 24 hours. HaCaT cells were induced with 800 μM H₂O₂ for 4 hours in the presence of the oligosaccharides obtained in Example 1. The DCFH-DA probe was added according to the manufacturer's instructions and incubated for 30 minutes. After incubation, the dye was washed with PBS, and the fluorescence intensity was measured by flow cytometry.

[0151] like Figure 10 As shown, compared with the untreated control group, the ROS fluorescence intensity of HaCaT cells was significantly enhanced after 4 hours of treatment with 800 μM H2O2. The oligosaccharides obtained in Example 1 (12.5 μg / mL-200 μg / mL) significantly reduced the ROS fluorescence intensity induced by H2O2, with 12.5 μg / mL ginseng oligogalacturonic acid having the best effect.

[0152] The above results indicate that ginseng galacturonic acid oligosaccharide can effectively reduce the level of cellular ROS induced by H2O2.

[0153] (4) Detection of intracellular mitochondrial membrane potential level

[0154] HaCaT cells in the logarithmic phase were harvested and seeded into 24-well cell culture plates (at a concentration of 2.0 × 10 cells / well). After incubation for 24 hours, the cells were induced with 800 μM H₂O₂ for 4 hours in the presence of the oligosaccharides obtained in Example 1. Rhodamine 123 probe was added according to the manufacturer's instructions and incubated for 30 minutes. After incubation, the dye was washed off with PBS, and the fluorescence intensity was measured by flow cytometry.

[0155] like Figure 11 As shown, compared with the untreated control group, the fluorescence intensity of mitochondrial membrane potential was significantly reduced after 4 hours of treatment with 800 μM H2O2 in HaCaT cells. Under the action of the oligosaccharides obtained in Example 1 (12.5 μg / mL-1000 μg / mL), the decrease in mitochondrial membrane potential fluorescence intensity induced by H2O2 was significantly alleviated.

[0156] The above results indicate that ginseng galacturonic acid oligosaccharide can effectively alleviate the decrease in cell mitochondrial membrane potential induced by H2O2.

[0157] The above experimental results confirmed that ginseng galacturonic acid oligosaccharide has a protective effect on H2O2-induced oxidative stress damage in HaCaT cells.

[0158] Example 6: Determination of Moisture-Retention and Hygroscopicity of Ginseng Galacturonic Acid Oligosaccharide

[0159] (1) Moisturizing performance measurement

[0160] Take a desiccator and place color-changing silica gel at the bottom of the desiccator. Then weigh 0.2 g each 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 for a replicate period of 2, 4, 6, 8, 12, 24, 36, and 48 hours. Accurately weigh the vials and calculate the moisture retention rate of the samples using the following formula. After the experiment, re-dissolve the moisture-absorbed samples and freeze-dry them for recovery. The mass of the vial is m0; the mass of the sample plus the vial is m1; and the mass of the sample plus the vial after each time period is mx.

[0161] Moisture retention rate (%) = 100% - [(m1 - mx) / (m1 - m0)] × 100% (6)

[0162] from Figure 12 The results show that as time goes by, the moisturizing rates of the 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 moisturizing performance is arranged from high to low as follows: ginseng galacturonic acid oligosaccharide prepared in Example 1 > sodium alginate.

[0163] The above results show that ginseng galacturonic acid oligosaccharide has good moisturizing properties.

[0164] (2) Determination of hygroscopicity

[0165] Take a desiccator and maintain a relatively stable humidity inside with a saturated ammonium sulfate solution (relative humidity 81%). Then weigh 0.5 g each of the ginseng galacturonic acid oligosaccharide sample and sodium alginate sample prepared in Example 1 and place them in a Petri dish. Place the Petri dish in the desiccator for a replicate set. Remove the Petri dish after 2, 4, 6, 8, 12, 24, 36, and 48 hours, accurately weigh the sample, and calculate the moisture absorption rate using the following formula. After the experiment, re-dissolve the sample that has absorbed moisture and freeze-dry it for recovery. The sample mass (g) before the moisture absorption experiment is Wn; the sample mass (g) after the moisture absorption experiment is W0.

[0166] Moisture absorption rate (%) = [(Wn-W0) / W0] × 100% (7)

[0167] from Figure 13 It can be seen that the hygroscopicity of the ginseng galacturonic acid oligosaccharide and sodium alginate prepared in Example 1 in an environment with a relative humidity of 81% is in the following order: ginseng galacturonic acid oligosaccharide obtained in Example 1 > sodium alginate, and within the first 24 h, the moisture absorption rate of the oligosaccharide obtained in Example 1 increases rapidly with time, and after 24 h, the growth rate of the moisture absorption rate gradually slows down.

[0168] The above results show that ginseng galacturonic acid oligosaccharide has good hygroscopic properties.

[0169] (3) Moisturizing efficacy test - determination of keratinocyte hydration-related protein content

[0170] Human immortalized keratinocytes (HaCaT) in the logarithmic phase were collected and 1×10 5 Cells were seeded into 24-well plates at a seeding density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2). When the cell plating rate in the 24-well plates reached 40%-60%, the original cell culture medium was aspirated and the cells were divided into groups for dosing, as in the blank control group and the experimental group of ginseng galacturonic acid oligosaccharides obtained in Example 1. The concentration gradient of ginseng galacturonic acid oligosaccharides was set at 0.063%, 0.125%, and 0.250% (m / v). For the experimental groups, 1.0 mL of sample working solution was added to each well, with three replicates for each concentration. For the blank control group (BC group), 1.0 mL of cell culture medium was added to each well, with three replicates. The plates were incubated in an incubator (37°C, 5% CO2) for an additional 24 hours. The supernatant was discarded, and the cells were rinsed three times with PBS before immunofluorescence staining. The main steps were: fixation, blocking, addition of primary and secondary antibodies, counterstaining with DAPI, and imaging using a fluorescence microscope. Results Analysis The fluorescence intensity of aquaporin (AQP3) and filaggrin (FLG) was quantitatively analyzed using Image Pro Plus software.

[0171] like Figure 14-17 As shown in the figure, according to the AQP3 immunofluorescence staining results 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 there was a statistical difference compared with the BC group (p<0.05).

[0172] The above results indicate that ginseng galacturonic acid oligosaccharide can increase the contents of AQP3 and FLG in keratinocytes and has moisturizing effect.

[0173] Example 7: Determination of the Exfoliating and Anti-acne Activity of Ginseng Galacturonic Acid Oligosaccharide

[0174] (1) In vitro exfoliation activity analysis

[0175] HaCaT cells in the logarithmic phase were collected and seeded into 24-well cell culture plates (at a concentration of 2.0 × 10 4cells / well), and incubated for 24 h; solutions of different concentrations (1 mg / mL-5 mg / mL) of the oligosaccharides prepared 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.

[0176] The results are as follows Figure 18 After co-culturing cells with different concentrations of ginseng galacturonic acid oligosaccharides (prepared in Example 1) for 40 min, the cell exfoliation results showed that the cell exfoliation ability of the oligosaccharide samples gradually increased with increasing concentration.

[0177] The above results indicate that ginseng galacturonic acid oligosaccharide has certain exfoliating activity.

[0178] (2) Propionibacterium acnes MIC minimum inhibitory rate test

[0179] C. acnes bacteria in the logarithmic growth phase were diluted with liquid culture medium to make the concentration of the added bacteria approximately 4.5×10 5 To determine the CFU / mL, 180 μL of culture medium containing bacteria was added to the first well of a 96-well polystyrene plate. 100 μL of culture medium was added to each well of the second to ninth wells. 20 μL of the corresponding drug was added to each well of the first well, giving an initial concentration of 20 mg / mL for each ginseng galacturonic acid oligosaccharide. 5 μL of clindamycin or erythromycin was added to the antibiotic wells in the first well, giving an initial antibiotic concentration of 0.5 μg / mL. Mix thoroughly by gently pipetting with a pipette. Pipette 100 μL of culture medium from the first well into the second well. Mix thoroughly, then pipette 100 μL of culture medium from the second well into the third well. Repeat this process for a total of nine serial dilutions. Pipette 100 μL of the mixture from the ninth well and discard. A positive control (containing culture medium and bacteria) was established in the tenth well, and a negative control (blank culture medium) was established in the eleventh well. Each bacterial strain was plated separately. The MIC value of the corresponding drug was determined using the minimum drug concentration in the wells that were visibly clear. 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; and OD positive control group represents the OD value of the positive control group.

[0180] 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)

[0181] The results are as follows Figure 19 As shown in FIG. 1 , ginseng galacturonic acid oligosaccharide (prepared in Example 1) had a good inhibitory effect on Propionibacterium acnes at a test concentration of 10 mg / mL.

[0182] The above results indicate that ginseng galacturonic acid oligosaccharide has certain anti-acne effect.

[0183] Example 8: Determination of the Repairing Efficacy of Ginseng Galacturonic Acid Oligosaccharide

[0184] (1) Determination of cell healing effects

[0185] HaCaT cells in the logarithmic phase were collected and seeded into 24-well cell culture plates (at a concentration of 2.0 × 10 5 cells / well), incubated for 24 h, and then scratched the 24-well plate with a 200 μL pipette tip. The cells were washed three times with PBS to remove 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 the concentration in the system 0.31 mg / mL, 0.63 mg / mL and 1.25 mg / mL. The cell suspension of the positive control group (PC) was added with a culture medium containing 10% FBS, and the cell suspension of the blank control group (BC) was added with a culture medium without FBS. The plates were placed in an incubator (37 o The cells were cultured at 4 °C, 5% CO₂ for 24 h, with three replicates per group. Migrating cells were photographed using an inverted microscope, and the average scratch area was calculated using Image Pro Plus software.

[0186] Healing rate (%) = (initial scratch area - current scratch area) / initial scratch area × 100% (9)

[0187] like Figure 20 As shown, based on the human immortalized keratinocyte (HaCaT cell) model, after treatment with ginseng galacturonic acid oligosaccharide 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).

[0188] The above results indicate that ginseng galacturonic acid oligosaccharide can promote cell healing of HaCaT cells and has a repair effect.

[0189] (2) In vitro experiments on promoting barrier-related proteins FLG and LOR

[0190] Place the sterile cell slides 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), in an incubator (37 oC, 5% CO2) 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 test substance working solutions 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 2 After irradiation, the cell culture medium was discarded and the cells were dosed in groups. 1.0 mL of working solution was added to each well of the PC group and the sample group, and 3 replicates 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 replicates were set. o Culture was continued at 4°C (5% CO2) for 24 hours. After incubation, the supernatant was discarded and the cells were rinsed three times with PBS. Standard immunofluorescence staining procedures were then performed, including fixation, blocking, addition of primary and secondary antibodies, counterstaining with DAPI, and mounting. Images were taken using a fluorescence microscope. The fluorescence intensities of filaggrin (FLG) and loricrin (LOR) were quantified using Image ProPlus software.

[0191]

[0192] The test results are as follows Figure 21-24 shown.

[0193] After the sample acted on HaCaT cells, the immunofluorescence staining results of FLG protein were as follows: Figure 21 As shown (green fluorescence represents FLG, blue fluorescence represents cell nucleus), the relative integrated optical density (IOD) values are as follows Figure 22 Compared with the BC group, the FLG content in the NC group was significantly decreased (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.

[0194] After the sample acted on HaCaT cells, the results of LOR protein immunofluorescence staining were as follows: Figure 23 As shown (green fluorescence represents LOR, blue fluorescence represents cell nucleus), the relative IOD values are as follows Figure 24Compared with the BC group, the LOR content in the NC group was significantly decreased (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.

[0195] 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.

[0196] Example 9: Determination of the soothing effect of the ginseng galacturonic acid oligosaccharide of the present invention.

[0197] (1) MTT assay to detect the effects of different concentrations of ginseng oligogalacturonic acid on the viability of macrophage RAW264.7 cells

[0198] 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 medium prepared with the oligosaccharide obtained in Example 1 were added to the experimental group and cultured for 24 h. Three replicate wells were 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 measured using a microplate reader. The absorbance of the blank solution was A 空白 The absorbance of the sample is A 样品 .

[0199] Cell survival rate (%) = [A 样品 / A 空白 ]×100% (10)

[0200] 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.

[0201] (2) In vitro inhibition of TNF-α, IL-6 and IL-1α activity

[0202] Cells were seeded into 24-well cell culture plates (at a concentration of 1.0 × 10 5 cells / well), in an incubator (37 oC, 5% CO2) and incubate overnight. Prepare the test substance working solution according to the concentration listed in the test protocol (Table 2). When the cell plating rate in the 24-well plate reaches 40%-60%, add 100 μL of 10× LPS working solution to each 24-well plate according to the test protocol (Table 2). Shake the 24-well plate left and right to mix the drug. At the same time, add the sample. The final LPS concentration is 1 μg / mL. Set up 3 replicates for each group. After administration, place the plate in an incubator (37 o Culture the cells at 4°C, 5% CO₂ for 24 h. After incubation, collect the cell culture supernatant into an EP tube. Detect TNF-α levels according to the instructions of the Mouse TNF-α ELISA kit. Detect IL-6 levels according to the instructions of the Mouse IL-6 ELISA kit. Detect IL-1α levels according to the instructions of the Mouse IL-1α ELISA kit.

[0203]

[0204] The results are as follows Figures 25-27 As shown. Based on the LPS-stimulated macrophage RAW264.7 model, the secretion of inflammatory factors TNF-α and IL-1α by macrophage RAW264.7 cells was significantly reduced at the concentrations of 0.16 mg / mL, 0.31 mg / mL, and 0.63 mg / mL by ginseng galacturonic acid oligosaccharide obtained in Example 1, and there was a statistically significant 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).

[0205] The above results indicate that ginseng galacturonic acid oligosaccharide can inhibit the secretion of inflammatory factors TNF-α, IL-6 and IL-1α in macrophage RAW264.7 cells, and has a soothing effect.

[0206] 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, concentration 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 variation, purposes or improvements of 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 a weak acid to ginseng medicinal materials for extraction to obtain a ginseng polysaccharide extract; The pH value of the extraction system composed of the ginseng medicinal material and the weak acid is 4.0 to 5.0; S2, adding a complex enzyme consisting of saccharifying enzyme and amylase to the ginseng polysaccharide extract for enzymatic hydrolysis to obtain a ginseng polysaccharide enzymatic hydrolyzate; separating the polysaccharide components that have not been hydrolyzed by the complex enzyme from the ginseng polysaccharide hydrolyzate; The separation in step S2 comprises the following steps: adding ethanol to the ginseng polysaccharide hydrolyzate for alcohol precipitation, and collecting the alcohol precipitate, wherein the final volume fraction of ethanol in the ginseng polysaccharide hydrolyzate in the alcohol precipitation step is 40% to 60%; S3, adding pectinase to the polysaccharide component that has not been enzymatically hydrolyzed by the composite enzyme to perform enzymatic hydrolysis to obtain a ginseng pectin enzymatic hydrolyzate; separating oligosaccharides hydrolyzed by the pectinase from the ginseng pectin hydrolyzate; The separation in step S3 comprises the following steps: adding ethanol to the ginseng pectin enzymatic hydrolysate for alcohol precipitation, and 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%; 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, wherein: 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 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 enzymatic activity of the saccharifying enzyme in the complex enzyme is 200,000 to 260,000 U / ml, and the enzymatic activity of the amylase is 10,000 to 20,000 U / ml; The ratio of the ginseng medicinal material to the complex enzyme is 1 kg: (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 enzymatic hydrolysis in step S2, the following step is further included: heating the reaction solution after the enzymatic hydrolysis 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 step is also included: concentrating and centrifuging the ginseng polysaccharide hydrolyzate in sequence, and collecting the supernatant; wherein the concentration is concentrated to 1 / 2 to 1 / 4 of the original solution.

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 pectinase activity 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 enzymatic hydrolysis in step S3, the following step is further included: heating the reaction solution after the enzymatic hydrolysis 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: concentrating and centrifuging the ginseng pectin hydrolysate in sequence, and collecting the supernatant; wherein the concentration is concentrated to 1 / 2 to 1 / 4 of the original solution.

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

8. Use of the 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 antioxidant purposes; A3) Preparation of cosmetics for protecting against 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 the 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 the 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 E1) to E2): E1) Preparation of anti-inflammatory cosmetics; E2) Preparation of cosmetics for skin soothing or repairing skin inflammation.

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