Purification of fermented dendrobium polysaccharide and its application in skin barrier repair

Abstract

The application belongs to the technical field of natural products, and discloses purified fermentation dendrobium polysaccharide and application thereof in skin barrier repair. The application provides a fermentation dendrobium polysaccharide DOP-1A, which comprises mannose, arabinose, gluconic acid, glucose, galactose and fucose; and a fermentation dendrobium polysaccharide DOP-2A, which comprises mannose and glucose. The fermentation dendrobium polysaccharide with high purity and uniform structure is obtained by using a fermentation dendrobium powder extraction solution, alcohol precipitation and column chromatography purification. Research proves that the fermentation dendrobium polysaccharide obtained by separation and purification has the effects of skin moisturizing and skin barrier repair, and also has anti-inflammatory activity. The extraction and separation method provided by the application can efficiently extract and purify polysaccharide from fermentation dendrobium, is suitable for the fields of skin barrier repair and anti-inflammatory, and has good application prospect and value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural products. More specifically, it relates to the purification of fermented dendrobium polysaccharides and their application in skin barrier repair. BACKGROUND

[0002] The Dendrobium genus belongs to the Orchidaceae family, among which Dendrobium officinale is widely studied for its rich medicinal value. Dendrobium polysaccharides, as one of the main active ingredients in Dendrobium officinale, have been proven to have various biological activities. With the increasing awareness of health and the growing demand for natural products, the market demand for dendrobium polysaccharides is increasing.

[0003] Despite the significant pharmacological effects of dendrobium polysaccharides, their extraction and purification processes still face challenges. Traditional extraction methods are often inefficient and difficult to remove impurities such as proteins and pigments. In recent years, studies have shown that fermenting dendrobium through microbial methods can increase the extraction amount of polysaccharides. Microorganisms secrete a large amount of extracellular enzymes such as proteases, cellulases, hemicellulases, glycosidases, amylases, and pectinases during growth and metabolism, which can cause plant cell rupture, increase cell interstitial space, accelerate the dissolution of effective components of traditional Chinese medicine, and effectively improve the extraction rate of effective components of traditional Chinese medicine. For example, the fermented dendrobium polysaccharides prepared in Chinese patent CN114231575B, but there is still room for improvement in terms of purity and biological activity. In addition, existing purification techniques are often costly and complex, which is not conducive to large-scale production and commercial application. SUMMARY

[0004] The present application aims to provide an improved method for extracting and purifying fermented dendrobium polysaccharides, which produces a high-purity, structurally uniform component of fermented dendrobium polysaccharides that can inhibit inflammatory responses in skin cells and be applied to repair skin barriers.

[0005] The first object of the present application is to provide fermented dendrobium polysaccharides DOP-1A and DOP-2A.

[0006] The second object of the present application is to provide a method for separating and purifying fermented dendrobium polysaccharides.

[0007] The third object of the present application is to provide the application of the above-mentioned fermented dendrobium polysaccharides.

[0008] The above-mentioned objects of the present application are achieved by the following technical solutions:

[0009] The present application provides a kind of fermented dendrobium polysaccharide DOP-1A, which is composed of mannose, arabinose, gluconic acid, glucose, galactose and fucose;The content of mannose is 60-80%, the content of arabinose is 0-5%, the content of gluconic acid is 1-10%, the content of glucose is 5-15%, the content of galactose is 0-5%, and the content of fucose is 5-15%;The main chain of the fermented dendrobium polysaccharide is composed of β-1,4-D-Manp, β-1,4-D-Glcp and β-1-D-Arap residues, and the branch is mainly composed of β-1,3-D-Manp residues and O-acetyl connected to O-2 of 1,4-D-Manp;The molecular weight distribution of the fermented dendrobium polysaccharide DOP-1A is 100-500 kDa.

[0010] As known in the art, due to the complexity of polysaccharide structure, the content of monosaccharide composition and the molecular weight distribution results determined using different batches of polysaccharide samples may differ.

[0011] As a detectable result, the molecular weight distribution of the fermented dendrobium polysaccharide DOP-1A is 100-300 kDa.

[0012] As a detectable result, the molecular weight distribution of the fermented dendrobium polysaccharide DOP-1A is 150-200 kDa.

[0013] In the embodiment of the present application, the monosaccharide composition determination result of the fermented dendrobium polysaccharide DOP-1A is as follows: the content of mannose is 73.25%, the content of arabinose is 0.96%, the content of gluconic acid is 5.9%, the content of glucose is 10.53%, the content of galactose is 0.91%, and the content of fucose is 8.46%.

[0014] In the embodiment of the present application, the molecular weight distribution of the fermented dendrobium polysaccharide DOP-1A has a peak value of 184.2 kDa.

[0015] The present application also provides a kind of fermented dendrobium polysaccharide DOP-2A, which is composed of mannose and glucose;The content of mannose is 55-75%, and the content of glucose is 25-45%;The main chain of the fermented dendrobium polysaccharide is composed of β-1,4-D-Manp and β-1,4-D-Glcp residues, and the branch is mainly composed of β-1,3-D-Manp residues and O-acetyl connected to O-2 of 1,4-D-Manp;The molecular weight distribution of the fermented dendrobium polysaccharide DOP-2A is 3-100 kDa.

[0016] As a detectable result, the molecular weight distribution of the fermented dendrobium polysaccharide DOP-2A is 3-80 kDa.

[0017] As a detectable result, the molecular weight distribution of the fermented dendrobium polysaccharide DOP-2A is 3-50 kDa.

[0018] In the embodiment of the present application, the monosaccharide composition determination result of the fermented dendrobium polysaccharide DOP-2A is that the content of mannose is 66.73%, and the content of glucose is 33.27%.

[0019] In the embodiment of the present application, the molecular weight distribution of the fermented dendrobium polysaccharide DOP-2A has peak values at 39.36 kDa and 7.89 kDa.

[0020] Specifically, the fermented dendrobium polysaccharide DOP-1A and the fermented dendrobium polysaccharide DOP-2A are obtained by alcohol precipitation and column chromatography separation from the fermented dendrobium powder extract.

[0021] The present application provides a separation and purification method of fermented dendrobium polysaccharide, comprising the following steps:

[0022] (1) The supernatant of the fermented dendrobium powder extract is taken, and ethanol is added to a volume fraction of 15-25%, and ice bathed for 0.5-2 h, and then centrifuged to obtain supernatant A; ethanol is added to supernatant A to a volume fraction of 35-45%, and ice bathed for 0.5-2 h, and then centrifuged to obtain supernatant B; ethanol is added to supernatant B to a volume fraction of 55-65%, and ice bathed for 0.5-2 h, and then centrifuged to obtain supernatant C and precipitate DOP-1;

[0023] (2) The precipitate DOP-1 is dissolved using a mobile phase, separated by column chromatography, eluted at a flow rate of 0.2-0.3 mL / min, and the eluate collected at an elution time of 234-426 min is removed and dried to obtain the fermented dendrobium polysaccharide DOP-1A.

[0024] Specifically, the fermented dendrobium powder is obtained by inoculating Lactobacillus reuteri into dendrobium, fermenting, ultrasonic extraction, solid-liquid separation, protein removal, alcohol precipitation, and freeze-drying.

[0025] Preferably, the Lactobacillus reuteri is Lactobacillus reuteri CCFM8631 (Lactobacillus reuteri), which was preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 7, 2017, at the address of No. 1, Beichen West Road, Haidian District, Beijing, China, and the preservation number is CGMCC NO. 14394.

[0026] As an alternative embodiment, the Dendrobium officinale powder is dried to a constant weight in step (1), and then the Dendrobium officinale powder is mixed with hot water at a temperature of 30-60°C at a ratio of 1:(9-11) (w / v), and stirred for 0.5-1.5 hours to obtain a Dendrobium officinale powder extract.

[0027] As an alternative embodiment, the Dendrobium officinale powder extract is centrifuged at 10000-14000 r / min after standing for 3-4 hours in step (1) to obtain a supernatant (preferably at 12000 r / min).

[0028] As an alternative embodiment, the supernatant is sequentially added with ethanol to an ethanol volume fraction of 20%, 40%, 60%, and 80% in step (1).

[0029] As an alternative embodiment, the precipitate DOP-1 is dissolved using a mobile phase and then loaded at a flow rate of 0.3-0.5 mL / min in step (2).

[0030] As an alternative embodiment, the precipitate DOP-1 is dissolved using a mobile phase and then loaded at a flow rate of 0.4 mL / min in step (2).

[0031] As an alternative embodiment, the elution is performed at a flow rate of 0.25 mL / min in step (2).

[0032] As an alternative embodiment, the mobile phase is ammonium bicarbonate and the chromatography column filler is Sepharose S-400HR in step (2).

[0033] The application also provides a method for separating and purifying Dendrobium officinale polysaccharides, comprising the following steps:

[0034] S1. The supernatant C is added with ethanol to an ethanol volume fraction of 75-85%, and then ice-bath for 0.5-2 hours, and then centrifuged to obtain a precipitate DOP-2;

[0035] S2. The precipitate DOP-2 is dissolved using a mobile phase, and then separated by column chromatography, and then eluted at a flow rate of 2-4 mL / min after standing for adsorption for 20-40 minutes, and then the eluate collected at an elution time of 12-48 minutes is removed, desalted, and dried to obtain Dendrobium officinale polysaccharides DOP-2A.

[0036] As an alternative embodiment, the precipitate DOP-2 is dissolved using a mobile phase and then loaded at a flow rate of 2-4 mL / min in step S2.

[0037] As an alternative embodiment, the precipitate DOP-2 is dissolved using a mobile phase and then loaded at a flow rate of 3 mL / min in step S2.

[0038] As an alternative embodiment, the elution in step S2 is eluted at a flow rate of 3 mL / min.

[0039] As an alternative embodiment, the mobile phase in step S2 is water, and the chromatographic column filler is Q-sepharose Fast Flow.

[0040] As an alternative embodiment, the standing adsorption in step S2 is 30 min.

[0041] As an alternative embodiment, the method for removing the eluent in the above separation and purification method is rotary evaporation.

[0042] As an alternative embodiment, the method for removing salt in the above separation and purification method is dialysis, and the specific method is dialysis in distilled water using a 3500 Da dialysis bag.

[0043] In addition, the present application provides a product for repairing skin barrier, containing the above-mentioned fermented dendrobium polysaccharide DOP-1A and / or the above-mentioned fermented dendrobium polysaccharide DOP-2A.

[0044] The present application research proves that the dendrobium polysaccharide obtained by separation and purification has the effects of moisturizing skin and repairing skin barrier, and also has anti-inflammatory activity, and therefore the following application schemes are claimed:

[0045] The application of the above-mentioned fermented dendrobium polysaccharide DOP-1A or the above-mentioned fermented dendrobium polysaccharide DOP-2A in preparing a skin anti-inflammatory product.

[0046] The application of the above-mentioned fermented dendrobium polysaccharide DOP-1A or the above-mentioned fermented dendrobium polysaccharide DOP-2A in preparing a skin barrier repair product.

[0047] The application of the above-mentioned fermented dendrobium polysaccharide DOP-1A or the above-mentioned fermented dendrobium polysaccharide DOP-2A in preparing a skin moisturizing product.

[0048] The present application has the following beneficial effects:

[0049] The preparation method of the fermented dendrobium polysaccharide provided by the present application takes the dendrobium fermented powder after fermentation by Lactobacillus reuteri as raw material, and prepares the fermented dendrobium polysaccharide component with high purity and uniform structure through fractional alcohol precipitation and column chromatography purification technology. Research proves that the fermented dendrobium polysaccharide obtained by separation and purification has the effects of moisturizing skin and repairing skin barrier, and also has anti-inflammatory activity. The extraction and separation method provided by the present application can efficiently extract and purify polysaccharides from fermented dendrobium, is suitable for skin barrier repair and anti-inflammatory fields, and has good application prospect and value. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 Molecular weight distribution chart of polysaccharide DOP-1A.

[0051] Figure 2 Molecular weight distribution chart of polysaccharide DOP-2A.

[0052] Figure 3 Infrared spectrum analysis results of polysaccharides DOP-1A and DOP-2A.

[0053] Figure 4 Chemical structure of polysaccharides DOP-1A and DOP-2A 1 H-NMR chart.

[0054] Figure 5 Chemical structure of polysaccharides DOP-1A and DOP-2A

[0055] Figure 6 Effects of different polysaccharides on the viability of LPS pretreated HaCaT cells.

[0056] Figure 7 Effects of different polysaccharides on the expression of FLG, AQP3, CAMP / LL37 proteins in LPS pretreated HaCaT.

[0057] Figure 8 Effects of different polysaccharides on the expression of TRPV4 protein in LPS pretreated HaCaT.

[0058] Figure 9 Effects of different polysaccharides on the expression of MCP-1 protein in LPS pretreated RAW264.7.

[0059] Figure 10 Flow cytometry detection results of ROS release of LPS pretreated RAW264.7 by different polysaccharides. DETAILED DESCRIPTION

[0060] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0061] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0062] In the following examples, the dendrobium fermentation powder (Lactobacillus reuteri CCFM8631 was inoculated into dendrobium, fermented, ultrasonic extracted, solid-liquid separated, deproteinized, alcohol precipitated, and then freeze-dried to obtain dendrobium polysaccharide powder), i.e. the original mixed crude polysaccharide (referred to as MIX for short), was provided by Infinite Extreme Company. The preparation method of the dendrobium fermentation powder is specifically referred to the patent CN114231575B.

[0063] CCK8 kit, brand: Biyun Tian, product number: C0038.

[0064] FLG ELISA kit, brand: ABclonal, catalog number: RK09147.

[0065] AQP3 ELISA kit, brand: ABclonal, catalog number: RK00932.

[0066] TRPV4 ELISA kit, brand: abbexa, catalog number: abx383968.

[0067] CAMP / LL-37 ELISA kit, brand: Fuyuebio, catalog number: FY-EH3984.

[0068] DEME medium, brand: Sivler, catalog number: G4511.

[0069] The references for analyzing the structure of polysaccharides in Dendrobium officinale in Example 5 are as follows:

[0070] Chen W H, Wu J J, Li X F, et al. Isolation, structural properties, bioactivities of polysaccharides from Dendrobium officinale Kimura et. Migo: A review[J]. International journal of biological macromolecules, 184: 1000-1013 [2024-11-12].

[0071] Liu B. Systematic analysis of chemical structure of polysaccharides from Dendrobium officinale (cultivated) and the structure-activity relationship of anti-gastric cancer activity[D]. Hefei University of Technology, 2019.

[0072] Wang H N, Fan J, Yu K Z, et al. Structural analysis of polysaccharides from Dendrobium officinale, research progress of biological activity, and analysis of differences in polysaccharide composition among different species[J]. Chinese Herbal Drugs, 2023, 54: 5044-5056.

[0073] Zhang Y. Isolation, purification, and structural characterization of polysaccharides from Dendrobium candidum and their protective effect on ethanol-induced gastric mucosal damage[D]. Jiangnan University, 2019.

[0074] Liu, Z., Guo, S., Lin, Y., et al. Research Progress on Structure-activity Relationship of Dendrobium Polysaccharides [J]. Modern Food Science and Technology, 2021, 37(1): 31.

[0075] Luo, Z., Jiang, Y., Yu, X., et al. Research Progress on Structure Characteristics, Biological Activity, Structure-activity Relationship and Product Development of Dendrobium Polysaccharides [J]. Food Industry Science and Technology, 2024, 45(15): 1-14.

[0076] Gao, Y., Hu, X., Wang, Y., et al. Primary Structure Analysis of Dendrobium Polysaccharides [J]. Chemical Journal of Chinese Universities, 2018.

[0077] Luo, Q., Tang, Z., Zhang, X., et al. Isolation and Purification of Dendrobium Polysaccharides and Its Structure Research [J]. Journal of Guangxi University: Natural Science Edition, 2016, 41(6): 7.

[0078] Li, X., Bai, Q., Sun, T., et al. Structure Characterization and Antioxidant Activity of Dendrobium Polysaccharides [J]. Food Industry Science and Technology, 2024(2).

[0079] Zhao, X., Lu, Y., Kang, X., et al. Extraction, Structure Characterization and in Vitro Antioxidant Activity of Dendrobium Polysaccharides from Yandang Mountain [J]. Zhejiang Journal of Agricultural Sciences, 2024(6).

[0080] Example 1 Fractionation and purification of water-extracted polysaccharides from Dendrobium fermentation powder

[0081] I. Fractionation and purification of polysaccharides

[0082] (1) Take 10 g of Dendrobium fermentation powder, dry at 40°C, add 100 mL of double distilled water, adjust the pH to neutral, stir at 45°C for 1 h, obtain the extract, let the extract stand for 3-4 h, centrifuge at 12000 r / min to obtain the supernatant, add ethanol to a volume fraction of 20%, ice bath for 1 h, centrifuge at 12000 r / min to obtain the supernatant, add ethanol to a volume fraction of 40%, ice bath for 1 h, centrifuge at 12000 r / min to obtain the supernatant, add ethanol to a volume fraction of 60%, ice bath for 1 h, centrifuge at 12000 r / min to obtain the supernatant and precipitate DOP-1; add ethanol to a volume fraction of 80% to the supernatant, ice bath for 1 h, centrifuge at 12000 r / min to obtain the precipitate DOP-2.

[0083] Molecular weight analysis of DOP-1 and DOP-2 components was performed by high-performance gel permeation chromatography-differential detector and multi-angle laser light scattering (HPGPC-RI-MALLS) combination, and monosaccharide composition analysis was performed by PMP pre-column derivatization high-performance liquid chromatography.

[0084] The results of the molecular weight analysis of the DOP-1 and DOP-2 components are shown in Table 1, which shows that DOP-1 is a large molecular weight polysaccharide (50-300 kDa) with a retention time of about 22 minutes and good uniformity. DOP-2 is a small molecular weight polysaccharide (<5 kDa) with a retention time of about 30 minutes.

[0085] The results of the monosaccharide composition analysis of the DOP-1 and DOP-2 components are shown in Table 2, which shows that DOP-1 is mainly composed of mannose, with a content of 60% to 80%. DOP-2 is mainly composed of glucose, with a content of more than 85%.

[0086] Table 1 Molecular weight distribution of DOP-1 and DOP-2 components

[0087]

[0088] Table 2 Monosaccharide composition of DOP-1 and DOP-2 components

[0089]

[0090] Example 2 Separation and purification of DOP-1 component

[0091] Deionized water was added to the S-400 filler several times to wash away the ethanol in the original storage solution. The filler was degassed for 15 minutes, and was loaded into a chromatography column with a size of 1.6 cm x 100.0 cm, and was equilibrated with 2 column volumes of deionized water, and then was equilibrated with 2 column volumes of 0.3 M ammonium bicarbonate as the mobile phase.

[0092] 100 mg of DOP-1 was accurately weighed, and was dissolved in as little ammonium bicarbonate mobile phase as possible. The sample was loaded at a flow rate of 0.4 mL / min, and was eluted at a flow rate of 0.3 mL / min, and every 6 minutes, one tube of eluate was collected (the eluate collected in the first 6 minutes was numbered as tube 1, and so on).

[0093] The number of tubes in which the polysaccharide was present was preliminarily determined by the method of spotting the TLC plate every other tube, and developing the plate by the diphenylamine method.

[0094] The sugar substances in the eluate were detected by the phenol-sulfuric acid method: every other two tubes of eluate were taken, 100 μL of eluate was placed in a test tube, 200 μL of 6% phenol was added, 1.5 mL of concentrated sulfuric acid was quickly added, and the mixture was mixed well, and was heated in a 100°C water bath for 10 minutes. After cooling, 200 μL of the mixture was taken and was placed in a 96-well plate, and the absorbance was measured at 490 nm.

[0095] The number of tubes is taken as the horizontal coordinate and the absorbance is taken as the vertical coordinate to draw the effluent curve. According to the curve, the number of tubes receiving the eluent is selected, and specifically, the eluent components in tubes 39-71 are collected, the ammonium bicarbonate eluent is removed by rotary evaporation, and concentrated and freeze-dried to obtain polysaccharide DOP-1A.

[0096] Example 3 Separation and purification of DOP-2 components

[0097] A Q-sepharose Fast Flow strong anion exchange column is used. The Q-sepharose Fast Flow strong anion column filler is loaded into a chromatography column with a size of 5.0 cm x 20.0 cm, and 2 column volumes of deionized water are used for equilibration. 155 mg of DOP-2 is dissolved in a small amount of deionized water, and loaded at a flow rate of 3 mL / min, and allowed to stand for 30 min for adsorption. Elution is performed with deionized water at a flow rate of 3 mL / min, and eluent is collected every 3 min for two column volumes. The content of sugar substances in the eluent is detected by the phenol-sulfuric acid method, and an effluent curve is drawn.

[0098] According to the tube number corresponding to the peak of the curve, specifically, the eluent components in tubes 4-16 are collected, concentrated by evaporation, desalted by dialysis in distilled water using a 3500 Da dialysis bag, and concentrated and freeze-dried to obtain polysaccharide DOP-2A.

[0099] Example 4

[0100] The total sugar and protein content of polysaccharides DOP-1A and DOP-2A are determined, and the molecular weight and monosaccharide composition are analyzed.

[0101] The results of the determination of the total sugar and protein content of polysaccharides DOP-1A and DOP-2A are shown in Table 3, and the results show that the total sugar content of polysaccharides DOP-1A and DOP-2A is 96.02% and 100%, respectively, and both of them do not have impure proteins.

[0102] The results of the monosaccharide composition analysis of polysaccharides DOP-1A and DOP-2A are shown in Table 4, and the results show that DOP-1A is mainly composed of mannose (content 73.25%); and DOP-2A is mainly composed of mannose (content 66.73%) and glucose (content 33.27%).

[0103] In summary, polysaccharides DOP-1A and DOP-2A are two structurally uniform dendrobium polysaccharides.

[0104] Table 3 Determination results of total sugar content and protein content

[0105]

[0106] Table 4. Results of monosaccharide composition analysis of polysaccharides DOP-1A and DOP-2A

[0107]

[0108] Example 5

[0109] Molecular weight and monosaccharide composition analysis were performed on polysaccharides DOP-1A and DOP-2A. Infrared spectroscopy, methylation analysis, and proton nuclear magnetic resonance spectroscopy were used. 1 The structures of polysaccharides DOP-1A and DOP-2A were identified by ¹H-NMR and HSQC spectral analysis.

[0110] The molecular weight distribution of polysaccharide DOP-1A is as follows: Figure 1 As shown, the molecular weight distribution of DOP-2A is as follows: Figure 2 As shown, the results indicate that the molecular weight of DOP-1A is around 184.2 kDa, while the molecular weight of DOP-2A is around 39.36 kDa and 7.89 kDa.

[0111] The infrared spectral analysis results of polysaccharides DOP-1A and DOP-2A are as follows: Figure 3 As shown in Table 5. The results indicate that the OH stretching vibration of the hydroxyl group in the polysaccharide molecule can reach 3400 cm⁻¹. -1 A relatively broad absorption peak appears on the left and right sides, corresponding to the stretching vibration of -OH and the characteristic signal of carbohydrates, at 2927.5 cm⁻¹. -1 The strong absorption peaks in the vicinity originate from the CH stretching vibrations in the polysaccharide molecules, indicating that the Dendrobium polysaccharide samples contain characteristic functional groups of polysaccharides; the Dendrobium polysaccharide samples show a peak at 1725 cm⁻¹. -1 The presence of absorption peaks at 867.9 and 806.5 cm⁻¹ indicates the presence of a -COOH group, suggesting the stretching vibration of C=O. -1 The absorption peak indicates the presence of β-mannose residues, which corresponds to the results of the 1H NMR spectrum.

[0112] Table 5 Infrared spectral analysis results of polysaccharides DOP-1A and DOP-2A

[0113]

[0114] Polysaccharides DOP-1A and DOP-2A 1 H-NMR spectrum as shown Figure 4 As shown. The results indicate that the proton spectrum signal is mainly concentrated in the δ 3.20–5.50 ppm range. Typically, the proton signal on the antecionic carbon is in the δ 4.4–5.5 ppm range, while the proton signals of the remaining H₂–H₆ protons are concentrated in the δ 3.0–4.4 ppm range. In DOP-1A… 1The signals at δ 4.65 ppm and δ 4.41 ppm in the H NMR spectrum are attributed to β-1,4-D-Manp and β-1,4-D-Glcp, respectively. The signal at the lowest field at δ 5.41 is attributed to H-2 of (1→4)-2-O-acetyl -Manp.

[0115] The results of the glycosidic bond composition analysis of methylated DOP-1A are shown in Table 6, and the results of the glycosidic bond composition analysis of methylated DOP-2A are shown in Table 7. In combination with the methylation analysis, it is presumed that the backbone of DOP-1A is composed of β-1,4-D-Manp, β-1,4-D-Glcp and β-1-D-Arap residues, and the branches are mainly composed of β-1,3-D-Manp residues and O-acetyl groups connected to O-2 of 1,4-D-Manp.

[0116] In the HSQC spectrum of DOP-2A, three cross peaks were found in the anomeric signal region at δ 5.32 / 99.42, 4.67 / 100.15, 4.44 / 102.75 1 ppm, which indicates that DOP-2A is composed of four types of residues (named as A, B and C). By combining the characterization results with the relevant literature reports, it is confirmed that the residues have O-acetyl groups, i.e., (1→4)-2-O-acetyl -Manp, residue B is β-1,4-D-Manp, and residue C is considered to be β-1,4-D-Glcp. Therefore, the backbone of DOP-2A is composed of β-1,4-D-Manp and β-1,4-D-Glcp residues, and the branches are mainly composed of β-1,3-D-Manp residues and O-acetyl groups connected to O-2 of 1,4-D-Manp.

[0117] According to a large number of literature reports, the monosaccharide composition of Dendrobium polysaccharides is mainly mannose and glucose, and the backbone is mainly composed of β-1,4-D-Manp and β-1,4-D-Glcp residues. If O-acetylation exists, it is generally (1→4)-2-O-acetyl -Manp or (1→4)-3-O-acetyl -Manp. The structure of DOP-2A is almost consistent with the reported structures of Dendrobium polysaccharides. In addition to the common β-1,4-D-Manp, β-1,4-D-Glcp and (1→4)-2-O-acetyl -Manp residues, the backbone of DOP-1A also has β-1,4-D-Fucp residues, which are less mentioned in previous reports. Therefore, the structures of DOP-1A and DOP-2A are presumed, and the chemical structures of DOP-1A and DOP-2A are shown in Figure 5 .

[0118] Table 6 Glycosidic bond composition of methylated DOP-1A

[0119]

[0120] Table 7 Glycosidic bond composition of methylated DOP-2A

[0121]

[0122] Example 6 Effect of fermented dendrobium polysaccharides on LPS-induced HaCaT cell viability

[0123] 1) Experimental method

[0124] The experimental cells used were the human skin keratinocyte cell line HaCaT. Lipopolysaccharide (LPS) was used to induce cell inflammation, simulating the inflammatory environment after skin infection or injury.

[0125] HaCaT cells were seeded in 96-well plates at a concentration of 3 x 10 4 The cells were seeded in 96-well plates at a concentration of 3 x 10

[0126] Treatment groups were set up:

[0127] Blank control group: After HaCaT cells were cultured for 24 h, fresh DMEM medium was replaced and cultured for another 24 h.

[0128] Positive control group: LPS (5 μg / mL) was used for pretreatment for 24 h, and fresh DMEM medium was replaced and cultured for another 24 h.

[0129] Sample group: LPS (5 μg / mL) was used for pretreatment for 24 h, and fresh DMEM medium containing 100 μg / mL of samples (MIX, DOP-1A, DOP-2A) was replaced and cultured for another 24 h.

[0130] The original culture medium of each treatment group was discarded, and fresh DMEM medium containing 10% CCK8 reagent (Bi Yun Tian, C0038) was added. The cells were incubated at 37 °C, 5% CO2, and protected from light for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0131] Cell viability was calculated as follows:

[0132] Cell viability (%) = average absorbance in test wells / average absorbance in control wells x 100%.

[0133] 2) Experimental results

[0134] Damage to the skin barrier is often accompanied by an inflammatory response, and the anti-inflammatory properties of dendrobium polysaccharides may help to alleviate this response, thereby promoting the repair and regeneration of skin cells.

[0135] The results of the effect of polysaccharides DOP-1A and DOP-2A on the activity of LPS pretreated HaCaT cells are shown in Table 8 Figure 6 As shown in Table 8, the results show that the cell activity of the positive control group HaCaT after LPS treatment is 76.09%, and the cell activity is significantly inhibited by LPS. After treatment with purified polysaccharides DOP-1A, DOP-2A and crude polysaccharides MIX, the cell activity is significantly improved. The cell activity of the DOP-1A experimental group is 136.24%, which is equivalent to MIX. The cell activity of HaCaT treated with DOP-2A is 143.88%, which is significantly improved compared with the MIX group.

[0136] Therefore, the original mixed crude polysaccharide and its purified components DOP-1A and DOP-2A can improve the activity and proliferation of HaCaT cells induced by LPS. DOP-1A and DOP-2A can promote the proliferation of HaCaT cells more strongly, and have a positive effect on skin barrier repair.

[0137] Table 8 Effect of different polysaccharides on the activity of LPS induced HaCaT cells

[0138]

[0139] Note: In the table, different lowercase letters indicate significant differences (P<0.05), and the same letters indicate no significant difference (P>0.05).

[0140] Example 7 Study on the skin barrier repair activity of fermented dendrobium polysaccharides

[0141] 1) Experimental method

[0142] DOP-1A, DOP-2A, and the original mixed crude polysaccharide MIX were weighed and dissolved in water to form a 1 mg / mL solution. The solution was filtered through a 0.22 μm filter to obtain DOP-1A solution, DOP-2A solution and MIX solution, respectively.

[0143] The experimental cell model was human skin keratinocyte cell line HaCaT, and the cell density was adjusted to 1×10 6 / well. After the cells were cultured in a 6-well plate until the cells adhered, the original culture medium was discarded, and different treatments were performed.

[0144] Treatment groups were set up:

[0145] Sample group: DOP-1A solution, DOP-2A solution and MIX polysaccharide solution were used as different sample solutions, and 200 μL of different sample solutions were mixed with 1600 μL of culture medium and added to the 6-well plate.

[0146] Control group (LPS): 200 μL of PBS was mixed with 1600 μL of culture medium and added to the 6-well plate.

[0147] After incubation of each group at 37℃, 5% CO2 for 24 h, 200 μL 50 μg / mL LPS solution was added to each well of the sample group and the control group, and after incubation at 37℃, 5% CO2 for 24 h, the cells in each treatment group were collected into a centrifuge tube, and the cell protein was extracted.

[0148] The protein expression content of the target cells was detected by an ELISA kit (for specific detection method, refer to the instructions thereof).

[0149] The percentage calculation formula is as follows:

[0150] Promotion percentage (%) = (sample group - control group) / control group x 100%;

[0151] 2) Experimental results

[0152] The results of the influence of different polysaccharides on the FLG, AQP3, CAMP / LL37, and TRPV4 protein expression of LPS pretreated HaCaT are shown in Tables 8, Figure 7 , Figure 8 and 9, the promotion percentage of different polysaccharides on LPS-induced related skin barrier proteins is shown in Table 10, and the expression of different target points is as follows:

[0153] (1) In the epidermis of human skin, FLG produces natural moisturizing factor (NMF) and plays a key role in the epidermal barrier function. The results show that MIX, DOP-1A, and DOP-2A can effectively promote the expression of FLG and maintain the integrity of the epidermal barrier function compared with the control group; and compared with MIX, DOP-1A and DOP-2A can significantly enhance the expression level of FLG.

[0154] (2) AQP3 is a key water channel protein that regulates skin hydration and helps regulate skin hydration and barrier function. AQP3 can transport water molecules, glycerol, and small molecules, and is essential for skin hydration and moisturizing function. The experimental results show that DOP-1A and DOP-2A can significantly increase the expression level of AQP3 in an inflammatory environment and enhance the regulation ability of skin hydration, and the effect is better than that of MIX.

[0155] (3) TRPV4 is a calcium ion channel related to temperature perception and skin barrier function, which participates in the regulation of skin water and electrolyte balance and affects the skin barrier function. MIX has no significant effect on TRPV4, while DOP-1A and DOP-2A can promote TRPV4, which helps to enhance the maintenance of skin water and electrolyte balance, and DOP-2A is more effective.

[0156] (4) CAMP / LL-37 itself has anti-inflammatory effect, in patients with atopic dermatitis (AD), the expression of CAMP / LL-37 is reduced, leading to impaired skin barrier function, more prone to bacterial infection. The results show that DOP-1A and DOP-2A can improve the activity of CAMP / LL-37 in the inflammatory environment, and can regulate the occurrence and development of inflammation in the body by regulating the expression or activity of CAMP / LL-37, directly killing bacteria or enhancing natural immunity. Among them, DOP-2A promotes the release activity of CAMP / LL37 is better than MIX.

[0157] Table 9 Average expression of LPS-induced related skin barrier proteins treated with different polysaccharides

[0158]

[0159] Note: In the table, different lowercase letters indicate significant differences (P <0.05), and the same letters indicate no significant difference (P >0.05).

[0160] Table 10 Percentage of promotion of LPS-induced related skin barrier proteins treated with different polysaccharides

[0161]

[0162] Example 8 Anti-inflammatory activity of purified dendrobium polysaccharide components

[0163] 1) Experimental method

[0164] The experimental cells were mouse macrophage RAW264.7.

[0165] DOP-1A solution, DOP-2A solution and MIX solution were prepared according to the method of Example 7, and the concentration was 100 μg / mL. 50 μM indomethacin solution was prepared, and indomethacin was a non-steroidal anti-inflammatory drug.

[0166] Adjust the cell density to 1×10 6 / well, and culture the cells in a 6-well plate until the cells adhere, then discard the original culture medium, and then perform different treatments.

[0167] Set the treatment groups:

[0168] Sample group: indomethacin solution, DOP-1A solution, DOP-2A solution and MIX solution were used as different sample solutions, and 200 μL of different sample solutions were mixed with 1600 μL of culture medium and added to the 6-well plate.

[0169] Positive control group (LPS): 200 μL of PBS was mixed with 1600 μL of culture medium and added to the 6-well plate.

[0170] CON: 200 μL PBS mixed with 1600 μL medium was added to the 6-well plate.

[0171] After incubation at 37°C, 5% CO2 for 24 h, 200 μL 10 μg / mL LPS solution was added to each well of the sample group and the positive control group, and 200 μL PBS was added to the blank control group. Then, after incubation at 37°C, 5% CO2 for 24 h, the cell supernatant and cells were collected into a centrifuge tube.

[0172] The MCP-1 content of the cell supernatant was detected by an ELISA kit (ABclonal, RK00381) according to the kit instructions.

[0173] The reactive oxygen species of the cells were detected using a reactive oxygen species detection kit (Bi Yun Tian, S00033S), and the specific scheme was performed according to the instructions. The fluorescence intensity of each group, i.e., the ROS level, was detected by flow cytometry.

[0174] The inhibition rate calculation formula is as follows:

[0175] Inhibition rate (%) = (positive control group - sample group) / positive control group x 100%.

[0176] 2) Experimental results

[0177] MCP-1 is a chemokine that can induce various inflammatory mediators and can cause chronic inflammation, hindering the repair of the skin barrier. The results of the effect of different polysaccharides on the MCP-1 protein expression of LPS pretreated RAW264.7 are shown in Table 10 and Table 11. Figure 9 As shown in Table 10 and Table 11, the results show that MIX cannot inhibit the release of MCP-1 in the LPS-induced inflammatory environment, while DOP-1A and DOP-2A can effectively inhibit the activity of MCP-1, control the inflammatory response, and the effect of DOP-1A on inhibiting MCP-1 is comparable to that of indomethacin.

[0178] Inflammatory reactions can produce a large amount of ROS, further aggravating local tissue damage, and even leading to chronic inflammation. The flow cytometry detection results of the ROS release of LPS pretreated RAW264.7 by different polysaccharides are shown in Table 11 and Table 12. Figure 10 As shown in Table 11 and Table 12, the results show that DOP-1A and DOP-2A can reduce oxidative stress, regulate inflammatory response, and protect tissues from further damage by inhibiting the production of ROS and removing excess ROS, and DOP-1A is more effective.

[0179] In summary, the results show that the fermented dendrobium polysaccharides, especially DOP-1A, have a more significant inhibitory activity on ROS and MCP-1, and have good anti-inflammatory activity.

[0180] Table 11 Average expression of MCP-1 protein and inhibition rate of MCP-1 in different groups

[0181]

[0182] Note: Different lower case letters in the table represent significant difference (P <0.05), and the same letter represents no significant difference (P >0.05).

[0183] Table 12 Average fluorescence intensity of ROS and inhibition rate of ROS in different groups

[0184]

[0185] Note: Different lower case letters in the table represent significant difference (P <0.05), and the same letter represents no significant difference (P >0.05).

[0186] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A fermented Dendrobium polysaccharide DOP-1A, characterized in that, It is composed of mannose, arabinose, gluconic acid, glucose, galactose, and fucose; the content of mannose is 60-80%, the content of arabinose is 0.96-5%, the content of gluconic acid is 1-10%, the content of glucose is 5-15%, the content of galactose is 0.91-5%, and the content of fucose is 5-15%; the main chain of the fermented Dendrobium polysaccharide DOP-1A is composed of β-1,4-D-Manp, β-1,4-D-Glcp, and β-1-D-Arap residues, and the branches are mainly composed of β-1,3-D-Manp residues and O-acetyl groups attached to the O-2 of 1,4-D-Manp; the molecular weight distribution of the fermented Dendrobium polysaccharide DOP-1A is 100-500 kDa.

2. A fermented Dendrobium polysaccharide DOP-2A, characterized in that, It is composed of mannose and glucose; wherein the mannose content is 55-75% and the glucose content is 25-45%; the main chain of the fermented Dendrobium polysaccharide DOP-2A is composed of β-1,4-D-Manp and β-1,4-D-Glcp residues, and the branches are mainly composed of β-1,3-D-Manp residues and O-acetyl groups attached to the O-2 of 1,4-D-Manp; the molecular weight distribution of the fermented Dendrobium polysaccharide DOP-2A is 3-100 kDa.

3. A method for isolating and purifying fermented Dendrobium polysaccharides, characterized in that, Includes the following steps: (1) Take the supernatant of the Dendrobium fermentation powder extract, add ethanol to a volume fraction of 15-25%, incubate on ice for 0.5-2 h, centrifuge to obtain supernatant A, add ethanol to supernatant A to a volume fraction of 35-45%, incubate on ice for 0.5-2 h, centrifuge to obtain supernatant B; add ethanol to supernatant B to a volume fraction of 55-65%, incubate on ice for 0.5-2 h, centrifuge to obtain supernatant C and precipitate DOP-1; the Dendrobium fermentation powder is a polysaccharide powder obtained by fermenting Dendrobium with Lactobacillus reuteri; (2) After the precipitate DOP-1 is dissolved in the mobile phase, it is separated by the upper chromatography column and eluted at a flow rate of 0.2-0.3 mL / min. The eluent with an elution time of 234-426 min is collected, the eluent is removed and dried to obtain the fermented Dendrobium polysaccharide DOP-1A as described in claim 1.

4. The separation and purification method according to claim 3, characterized in that, The mobile phase in step (2) is ammonium bicarbonate, and the chromatography column packing is Sepharose S-400HR.

5. A method for isolating and purifying fermented Dendrobium polysaccharides, characterized in that, Includes the following steps: S1. Take the supernatant C described in claim 3, add ethanol to the volume fraction of ethanol to 75-85%, incubate on ice for 0.5-2 h, and centrifuge to obtain precipitate DOP-2; S2. After dissolving the precipitate DOP-2 with the mobile phase, it is separated on a chromatography column, allowed to stand for 20-40 min for adsorption, eluted at a flow rate of 2-4 mL / min, and the eluent is collected after elution for 12-48 min. After removing the eluent, desalting, and drying, the fermented Dendrobium polysaccharide DOP-2A of claim 2 is obtained.

6. The separation and purification method according to claim 5, characterized in that, The mobile phase in step S2 is water, and the chromatography column packing material is Q-sepharose Fast Flow.

7. A product for repairing the skin barrier, characterized in that, It contains the fermented Dendrobium polysaccharide DOP-1A of claim 1 and / or the fermented Dendrobium polysaccharide DOP-2A of claim 2.

8. The use of fermented Dendrobium polysaccharide DOP-1A according to claim 1 or fermented Dendrobium polysaccharide DOP-2A according to claim 2 in the preparation of anti-inflammatory skin products.

9. The use of fermented Dendrobium polysaccharide DOP-1A according to claim 1 or fermented Dendrobium polysaccharide DOP-2A according to claim 2 in the preparation of products for repairing the skin barrier.

10. The use of fermented Dendrobium polysaccharide DOP-1A according to claim 1 or fermented Dendrobium polysaccharide DOP-2A according to claim 2 in the preparation of skin moisturizing products.

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