Purified fermented dendrobium polysaccharide and application thereof in skin barrier repair

Abstract

The invention belongs to the technical field of natural products, and discloses purified fermented dendrobium polysaccharides and application thereof in skin barrier repair. The invention provides a fermented dendrobium polysaccharide DOP-1A. The fermented dendrobium polysaccharide DOP-1A comprises mannose, arabinose, gluconic acid, glucose, galactose and fucose. The invention further provides the fermented dendrobium polysaccharide DOP-2A which comprises mannose and glucose. According to the method disclosed by the invention, the fermented dendrobium polysaccharides with high purity and uniform structure are obtained by performing alcohol precipitation and column chromatography purification on the dendrobium ferment powder extracting solution. Researches prove 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 invention can be used for efficiently extracting and purifying the polysaccharide from the fermented dendrobium nobile, is suitable for the fields of skin barrier repair, inflammation resistance and the like, and has good application prospect and value.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural products, and more specifically, relates to purified fermented dendrobium polysaccharide and its application in skin barrier repair. Background Art

[0002] Dendrobium is a plant of the orchid family, and Dendrobium officinale has been widely studied for its rich medicinal value. As one of the main active ingredients in Dendrobium officinale, Dendrobium polysaccharide has been proven to have multiple biological activities. With the improvement of health awareness and the increase in demand for natural products, the market demand for Dendrobium polysaccharide is growing.

[0003] Although dendrobium polysaccharide has significant pharmacological effects, its extraction and purification process still faces challenges. Traditional extraction methods are usually inefficient and difficult to remove impurities such as proteins and pigments. Studies in recent years have shown that the extraction amount of polysaccharides can be increased by fermenting dendrobium by microbial methods. 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 cells to rupture, increase intercellular spaces, accelerate the dissolution of effective ingredients of traditional Chinese medicine, and effectively improve the extraction rate of effective ingredients of traditional Chinese medicine, such as the fermented dendrobium polysaccharide prepared by Chinese patent CN114231575B, but the polysaccharide still has a large room for improvement in purity and biological activity. In addition, existing purification technologies are often costly and complex to operate, which is not conducive to large-scale production and commercial applications. Summary of the invention

[0004] The present invention aims to provide an improved method for extracting and purifying fermented dendrobium polysaccharide, by which a fermented dendrobium polysaccharide component with high purity and uniform structure is prepared, which can inhibit the inflammatory response of skin cells and can be used to repair the skin barrier.

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

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

[0007] The third object of the present invention is to provide the application of the fermented Dendrobium polysaccharide.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The invention provides a fermented dendrobium polysaccharide DOP-1A, which consists 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 consists of β-1,4-D-Manp, β-1,4-D-Glcp and β-1-D-Arap residues, and the branch mainly consists 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 100-500kDa.

[0010] As is well known in the art, due to the complex structure of polysaccharides, the content and molecular weight distribution of monosaccharide compositions measured using different batches of polysaccharide samples may be different.

[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 invention, the monosaccharide composition of the fermented Dendrobium polysaccharide DOP-1A was determined as follows: the content of mannose was 73.25%, the content of arabinose was 0.96%, the content of gluconic acid was 5.9%, the content of glucose was 10.53%, the content of galactose was 0.91%, and the content of fucose was 8.46%.

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

[0015] The present invention also provides a fermented dendrobium polysaccharide DOP-2A, which is composed of mannose and glucose; wherein 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 invention, the monosaccharide composition of the fermented dendrobium polysaccharide DOP-2A was determined as follows: the content of mannose was 66.73%, and the content of glucose was 33.27%.

[0019] In the embodiment of the present invention, the molecular weight distribution of fermented Dendrobium polysaccharide DOP-2A has peaks 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 subjecting the extract of dendrobium fermented powder to alcohol precipitation and column chromatography separation.

[0021] The present invention provides a method for separating and purifying fermented dendrobium polysaccharide, comprising the following steps:

[0022] (1) taking the supernatant of the Dendrobium fermented powder extract, adding ethanol to a volume fraction of 15-25%, ice bathing for 0.5-2h, and centrifuging to obtain supernatant A; adding ethanol to supernatant A to a volume fraction of 35-45%, ice bathing for 0.5-2h, and centrifuging to obtain supernatant B; adding ethanol to supernatant B to a volume fraction of 55-65%, ice bathing for 0.5-2h, and centrifuging to obtain supernatant C and precipitate DOP-1;

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

[0024] Specifically, the dendrobium fermented powder is a dendrobium polysaccharide powder obtained by inoculating Lactobacillus reuteri into dendrobium, fermenting, ultrasonically extracting, separating solid from liquid, removing protein, precipitating with alcohol, and freeze-drying.

[0025] Preferably, the Lactobacillus reuteri is Lactobacillus reuteri CCFM8631 (Lactobacillus reuteri), which was deposited in the General Microbiology Center of China Culture Collection Committee CGMCC on July 7, 2017, with the deposit address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO.14394.

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

[0027] As an optional embodiment, in step (1), the Dendrobium fermented powder extract is allowed to stand for 3-4 hours and then centrifuged at 10000-14000 r / min to obtain a supernatant (preferably 12000 r / min).

[0028] As an optional embodiment, in step (1), ethanol is added to the supernatant in sequence until the ethanol volume fractions are 20%, 40%, 60%, and 80%, respectively.

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

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

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

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

[0033] The present invention also provides a method for separating and purifying fermented dendrobium polysaccharide, comprising the following steps:

[0034] S1. Take the above supernatant C and add ethanol to a volume fraction of 75-85%, place in an ice bath for 0.5-2h, and centrifuge to obtain a precipitate DOP-2;

[0035] S2. The precipitate DOP-2 is dissolved with the mobile phase, separated on a chromatography column, allowed to stand for 20 to 40 minutes of adsorption, eluted at a flow rate of 2 to 4 mL / min, and the eluate with an elution time of 12 to 48 minutes is collected. The eluate is removed, desalted, and dried to obtain the fermented Dendrobium polysaccharide DOP-2A.

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

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

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

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

[0040] As an optional embodiment, in step S2, the adsorption is allowed to stand for 30 minutes.

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

[0042] As an optional embodiment, the method for desalting in the above separation and purification method is dialysis, and the specific method is to use a 3500Da dialysis bag to dialyze and desalinate in distilled water.

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

[0044] The present invention studies have shown that the isolated and purified dendrobium polysaccharide has the effects of moisturizing the skin and repairing the skin barrier, and also has anti-inflammatory activity, so the following application scheme is claimed:

[0045] Application of the fermented dendrobium polysaccharide DOP-1A or the fermented dendrobium polysaccharide DOP-2A in the preparation of skin anti-inflammatory products.

[0046] Application of the fermented dendrobium polysaccharide DOP-1A or the fermented dendrobium polysaccharide DOP-2A in the preparation of a skin barrier repair product.

[0047] Application of the fermented dendrobium polysaccharide DOP-1A or the fermented dendrobium polysaccharide DOP-2A in the preparation of skin moisturizing products.

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

[0049] The method for preparing fermented dendrobium polysaccharide provided by the present invention uses the fermented dendrobium powder fermented by Lactobacillus reuteri as raw material, and prepares a fermented dendrobium polysaccharide component with high purity and uniform structure through graded alcohol precipitation and column chromatography purification technology. Studies have shown that the fermented dendrobium polysaccharide obtained by separation and purification has the effects of moisturizing the skin and repairing the skin barrier, and also has anti-inflammatory activity. The extraction and separation method provided by the present invention can efficiently extract and purify polysaccharides from fermented dendrobium, is suitable for the fields of skin barrier repair and anti-inflammatory, and has good application prospects and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1This is the molecular weight distribution diagram of polysaccharide DOP-1A.

[0051] Figure 2 This is the molecular weight distribution diagram of polysaccharide DOP-2A.

[0052] Figure 3 These are the infrared spectrum analysis results of polysaccharides DOP-1A and DOP-2A.

[0053] Figure 4 For polysaccharides DOP-1A and DOP-2A 1 H-NMR spectrum.

[0054] Figure 5 This is the HSQC spectrum of polysaccharide DOP-1A.

[0055] Figure 6 This is the HSQC spectrum of polysaccharide DOP-2A.

[0056] Figure 7 The chemical structures of polysaccharides DOP-1A and DOP-2A.

[0057] Figure 8 The results show the effects of different polysaccharides on the viability of HaCaT cells pretreated with LPS.

[0058] Fig. 9 These are the results of the effects of different polysaccharides on the expression of FLG, AQP3, and CAMP / LL37 proteins in HaCaT pretreated with LPS.

[0059] Fig.10 These are the results of the effects of different polysaccharides on TRPV4 protein expression in HaCaT pretreated with LPS.

[0060] Fig.11 The results show the effects of different polysaccharides on the MCP-1 protein expression of RAW264.7 cells pretreated with LPS.

[0061] Fig.12 Flow cytometry results of ROS release of RAW264.7 pretreated with LPS by different polysaccharides. DETAILED DESCRIPTION

[0062] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0063] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0064] In the following embodiments, Dendrobium fermented powder (Lactobacillus reuteri CCFM8631 is inoculated in Dendrobium, fermented, ultrasonically extracted, solid-liquid separated, protein removed, ethanol precipitated, and then freeze-dried to obtain Dendrobium polysaccharide powder), i.e., original mixed crude polysaccharide (abbreviated as MIX), is provided by Infinitus Company, and the preparation method of Dendrobium fermented powder is specifically referred to the Chinese authorized patent CN114231575B.

[0065] CCK8 test kit, brand is Bio-Tech, product number is C0038.

[0066] FLG ELISA kit, brand is ABclonal, catalog number is RK09147.

[0067] AQP3 ELISA kit, brand is ABclonal, catalog number is RK00932.

[0068] TRPV4 ELISA kit, brand is abbexa, product number is abx383968.

[0069] CAMP / LL-37 ELISA kit, brand is Feiyue Bio, product number is FY-EH3984.

[0070] DEME culture medium, brand is Seville, product number is G4511.

[0071] The references used in Example 5 for analyzing the structure of Dendrobium polysaccharides are as follows:

[0072] "Chen WH,Wu JJ,Li XF,et al.Isolation,structural properties,bioactivities of polysaccharides from Dendrobium officinale Kimura et.Migo:Areview[J].International journal of biological macromolecules,184:1000-1013[2024-11-12].";

[0073] Liu Bing. Systematic analysis of chemical structure of polysaccharides from Huoshan Dendrobium (cultivated) and study of structure-activity relationship of anti-gastric cancer activity[D]. Hefei University of Technology, 2019.

[0074] "Wang Haonan, Fan Jing, Yu Kunzi, et al. Structural analysis of polysaccharides from Dendrobium huoshanense, research progress on bioactivity and analysis of differences in polysaccharide components between different species[J]. Chinese Herbal Medicine, 2023, 54: 5044-5056."

[0075] Zhang Yi. Isolation, purification, structural characterization and protective effect of polysaccharides from Dendrobium nobile against ethanol-induced gastric mucosal injury[D]. Jiangnan University, 2019.

[0076] "Liu Xifan, Guo Shuoxuan, Lin Yingyu, et al. Research progress on the structure-activity relationship of Dendrobium polysaccharides[J]. Modern Food Science and Technology, 2021, 37(1):31."

[0077] 《Luo Zhaoyong, Jiang Yan, Yu Xiaoli, et al. Structural characteristics, biological activity, structure-activity relationship and product development progress of Dendrobium polysaccharides[J]. Food Industry Science and Technology, 2024, 45(15): 1-14.》;

[0078] Gao Yunxiao, Hu Xiaolong, Wang Yuerong, et al. Primary structural analysis of polysaccharides from Dendrobium officinale[J]. Journal of Chemistry in Universities, 2018.

[0079] "Luo Qiulian, Tang Zhuanhui, Zhang Xuefeng, et al. Isolation, purification and structural study of polysaccharides from Dendrobium officinale[J]. Journal of Guangxi University: Natural Science Edition, 2016, 41(6):7."

[0080] "Li Xueling, Bai Qinfan, Sun Tijie, et al. Structural characterization and antioxidant activity of polysaccharides from Dendrobium officinale[J]. Food Industry Science and Technology, 2024(2)."

[0081] "Zhao Xiaoliang, Lu Yun, Kang Xingxing, et al. Extraction, structural characterization and in vitro antioxidant activity of polysaccharides from Dendrobium officinale from Yandang Mountain [J]. Journal of Zhejiang Agricultural Sciences, 2024(6).".

[0082] Example 1: Gradation and purification of polysaccharides extracted from dendrobium fermented powder

[0083] 1. Gradation and purification of polysaccharides

[0084] (1) Take 10 g of Dendrobium fermented powder, dry it at 40°C, add 100 mL of double distilled water, adjust the pH to neutral, stir at 45°C for 1 hour to obtain an extract, let the extract stand for 3-4 hours, centrifuge at 12000 r / min to obtain a supernatant, add ethanol to a volume fraction of 20%, ice bath for 1 hour, centrifuge at 12000 r / min to obtain a supernatant, add ethanol to a volume fraction of 40%, ice bath for 1 hour, centrifuge at 12000 r / min to obtain a supernatant; add ethanol to a volume fraction of 60%, ice bath for 1 hour, centrifuge at 12000 r / min to obtain a supernatant and a precipitate DOP-1; add ethanol to the supernatant to a volume fraction of 80%, ice bath for 1 hour, centrifuge at 12000 r / min to obtain a precipitate DOP-2.

[0085] The molecular weight of DOP-1 and DOP-2 components was analyzed by high performance gel permeation chromatography-differential detector coupled with eighteen-angle scattering instrument (HPGPC-RI-MALLS), and the monosaccharide composition was analyzed by PMP pre-column derivatization high performance liquid chromatography.

[0086] The molecular weight analysis results of DOP-1 and DOP-2 components are shown in Table 1. The results show 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.

[0087] The monosaccharide composition analysis results of DOP-1 and DOP-2 components are shown in Table 2. The results show that DOP-1 is mainly composed of mannose, with a content between 60% and 80%. DOP-2 is mainly composed of glucose, with a glucose content of more than 85%.

[0088] Table 1 Molecular weight distribution of DOP-1 and DOP-2 components

[0089]

[0090] Table 2 Monosaccharide composition of DOP-1 and DOP-2 components

[0091]

[0092] Example 2 Separation and purification of DOP-1 components

[0093] Add deionized water to the S-400 filler several times to wash away the ethanol in the original storage solution. Degas for 15 minutes, load the filler into a 1.6 cm × 100.0 cm chromatography column, balance it with 2 column volumes of deionized water, and then balance it with 2 column volumes of 0.3 M ammonium bicarbonate as the mobile phase for later use.

[0094] Accurately weigh 100 mg of DOP-1 and dissolve it in as little ammonium bicarbonate as possible. Load the sample at a flow rate of 0.4 mL / min and elute at a flow rate of 0.3 mL / min, collecting one tube of eluate every 6 minutes (the eluate collected in the first 6 minutes is numbered as the first tube, and so on).

[0095] By spotting the TLC plate, spot every other tube and use the diphenylamine method for color development to preliminarily determine the range of tubes where the polysaccharide is located.

[0096] The phenol-sulfuric acid method was used to detect the sugar substances in the eluate: 100 μL of the eluate was taken from every two tubes and placed in a test tube, 200 μL of 6% phenol was added, and 1.5 mL of concentrated sulfuric acid was quickly added, mixed evenly, and heated in a 100°C water bath for 10 min. After cooling, 200 μL was taken in a 96-well plate and the absorbance was measured at 490 nm.

[0097] The elution curve was drawn with the tube number as the horizontal axis and the absorbance as the vertical axis. The number of tubes to receive the eluate was selected according to the curve, specifically, 39-71 tubes of eluate components were collected respectively, the ammonium bicarbonate eluate was removed by rotary evaporation, and the polysaccharide DOP-1A was obtained by concentration and freeze drying.

[0098] Example 3 Separation and purification of DOP-2 components

[0099] Q-sepharose Fast Flow strong anion exchange column was used. The Q-sepharose Fast Flow strong anion column filler was loaded into a chromatographic column of 5.0 cm × 20.0 cm in size and balanced with 2 column volumes of deionized water. 155 mg of DOP-2 was weighed, and the polysaccharide was dissolved with a small amount of deionized water, and the sample was loaded at a flow rate of 3 mL / min, and the sample was allowed to stand for 30 minutes for adsorption, and the sample was eluted with deionized water at a flow rate of 3 mL / min for two column volumes, and one tube of eluate was collected every 3 minutes (the eluate collected in the first 3 minutes is numbered as the first tube, and so on). The phenol-sulfuric acid method was used to detect the content of sugar substances in the eluate, and the elution curve was drawn.

[0100] According to the number of tubes corresponding to the peak of the curve, 4-16 tubes of eluate components are collected, evaporated and concentrated, dialyzed and desalted in distilled water using a 3500Da dialysis bag, and the collected components are concentrated and freeze-dried to obtain polysaccharide DOP-2A.

[0101] Example 4

[0102] The total sugar and protein contents of polysaccharides DOP-1A and DOP-2A were determined, as well as molecular weight analysis and monosaccharide composition analysis.

[0103] The results of the determination of the total sugar and protein contents of polysaccharides DOP-1A and DOP-2A are shown in Table 3. The results show that the total sugar contents of polysaccharides DOP-1A and DOP-2A are 96.02% and 100%, respectively, and both have no impurity proteins.

[0104] The results of monosaccharide composition analysis of polysaccharides DOP-1A and DOP-2A are shown in Table 4. The results show that DOP-1A is mainly composed of mannose (content 73.25%); DOP-2A is mainly composed of mannose (content 66.73%) and glucose (content 33.27%).

[0105] In summary, polysaccharides DOP-1A and DOP-2A are two Dendrobium polysaccharides with uniform structures.

[0106] Table 3 Total sugar content and protein content determination results

[0107]

[0108] Table 4 Analysis results of monosaccharide composition of polysaccharides DOP-1A and DOP-2A

[0109]

[0110] Example 5

[0111] The molecular weight and monosaccharide composition of polysaccharides DOP-1A and DOP-2A were analyzed. 1 H-NMR) and HSQC spectrum analysis were used to identify the structures of polysaccharides DOP-1A and DOP-2A.

[0112] The molecular weight distribution of polysaccharide DOP-1A is as follows Figure 1 As shown, the molecular weight distribution of DOP-2A is Figure 2 As shown, the results showed that the molecular weight of DOP-1A was distributed around 184.2 kDa, and the molecular weight of DOP-2A was distributed around 39.36 kDa and 7.89 kDa.

[0113] The infrared spectrum analysis results of polysaccharides DOP-1A and DOP-2A are as follows Figure 3 As shown in Table 5. The results show that the OH stretching vibration of the hydroxyl group in the polysaccharide molecule can -1 There are broad absorption peaks on the left and right, corresponding to the stretching vibration of -OH and the characteristic signals of sugars, 2927.5cm -1 The strong absorption peak near 1725cm comes from the CH stretching vibration in the polysaccharide molecules, indicating that the polysaccharide samples of Dendrobium have characteristic functional groups of polysaccharides; -1 There is an absorption peak at 867.9 and 806.5 cm, which is the stretching vibration of C=O, indicating the presence of -COOH groups. -1 The absorption peak indicated the presence of β-mannose residues, which corresponded to the results of H-NMR spectrum.

[0114] Table 5 Infrared spectrum analysis results of polysaccharides DOP-1A and DOP-2A

[0115]

[0116] Polysaccharides DOP-1A and DOP-2A 1 H-NMR images Figure 4As shown. The results show that the signal of the hydrogen spectrum is mainly concentrated in the range of δ3.20-5.50ppm. Usually the proton signal on the anomeric carbon is in the range of δ4.4-5.5ppm, and the proton signals of the remaining H-2 to H-6 are concentrated in the range of δ3.0-4.4ppm. 1 In the H NMR spectrum, the signals at δ4.65ppm and δ4.41ppm were assigned to β-1,4-D-Manp and β-1,4-D-Glcp, respectively, and the lowest field signal at δ5.41 was assigned to H-2 of (1→4)-2-O-acetyl-Manp.

[0117] The HSQC spectra of polysaccharides DOP-1A and DOP-2A are shown in Figure 5 and Figure 6 The analysis results of the glycosidic bond composition of methylated DOP-1A are shown in Table 6, and the analysis results of the glycosidic bond composition of methylated DOP-2A are shown in Table 7. Combined with the methylation analysis, it is inferred that the main chain 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.

[0118] In the HSQC spectrum of DOP-2A, three cross peaks were found at δ5.32 / 99.42, 4.67 / 100.15, and 4.44 / 102.751 ppm in the anomeric signal region, indicating that DOP-2A is composed of four types of residues (named A, B, and C). By combining the characterization results with relevant literature reports, the residues were confirmed to have O-acetyl groups, namely (1→4)-2-O-acetyl-Manp, residue B was β-1,4-D-Manp, and residue C was considered to be β-1,4-D-Glcp. Therefore, the main chain 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.

[0119] According to a large number of literature reports, the monosaccharide composition of Dendrobium officinale polysaccharides is mostly mannose and glucose, and the main chain is mostly 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 structure 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 main chain of DOP-1A also has β-1,4-D-Fucp residues, which are rarely mentioned in previous reports. Therefore, the structures of DOP-1A and DOP-2A are inferred. The chemical structures of DOP-1A and DOP-2A are as follows: Figure 7 shown.

[0120] Table 6 Glycosidic bond composition of methylated DOP-1A

[0121]

[0122] Table 7 Glycosidic bond composition of methylated DOP-2A

[0123]

[0124]

[0125] Example 6 Effect of fermented Dendrobium polysaccharide on LPS-induced HaCaT cell viability

[0126] 1) Experimental methods

[0127] The experimental cells used were human skin keratinocyte cell line HaCaT. Lipopolysaccharide (LPS) was used to induce cellular inflammatory response to simulate the inflammatory environment after skin infection or injury.

[0128] Press 3×10 4 HaCaT cells were inoculated in a 96-well plate at a concentration of cells / well until the cells adhered to the wall, the original culture medium was discarded, and different treatments were performed.

[0129] Set up the processing group:

[0130] Blank control group: After HaCaT cells were cultured for 24 h, fresh DMEM medium was replaced and cultured for another 24 h.

[0131] Positive control group: pre-treated with LPS (5 μg / mL) for 24 h, and then replaced with fresh DMEM medium and cultured for another 24 h.

[0132] Sample group: pretreated with LPS (5 μg / mL) for 24 h, and then replaced with fresh DMEM medium containing 100 μg / mL sample (MIX, DOP-1A, DOP-2A) and cultured for another 24 h.

[0133] The original culture medium of each treatment group was discarded, and fresh DMEM culture medium containing 10% CCK8 reagent (Biyuntian, C0038) was added, and the cells were incubated in the dark at 37°C and 5% CO2 for 2 h, and the absorbance was measured at 450 nm using an enzyme reader.

[0134] Cell viability was calculated as follows:

[0135] Cell viability (%) = average absorbance in test wells / average absorbance in control wells × 100%.

[0136] 2) Experimental results

[0137] Damage to the skin barrier is often accompanied by an inflammatory response, and the anti-inflammatory properties of Dendrobium polysaccharides may help alleviate this response, thereby promoting the repair and regeneration of skin cells.

[0138] The results of the effects of polysaccharides DOP-1A and DOP-2A on the viability of HaCaT cells pretreated with LPS are shown in Figure 8 As shown in Table 8, the results showed that the cell viability of HaCaT in the positive control group after LPS treatment was 76.09%, and the cell viability was significantly inhibited by LPS. After treatment with purified polysaccharides DOP-1A, DOP-2A and crude polysaccharide MIX, the cell viability was significantly improved. The cell viability of the DOP-1A experimental group was 136.24%, which was equivalent to MIX; the cell viability of HaCaT after DOP-2A treatment was 143.88%, which was significantly improved compared with the MIX group.

[0139] This shows that the original mixed crude polysaccharide and its purified components DOP-1A and DOP-2A can improve the viability and proliferation activity of HaCaT cells induced by LPS. DOP-1A and DOP-2A promote the proliferation activity of HaCaT cells more strongly and have a positive effect on skin barrier repair.

[0140] Table 8 Effects of different polysaccharides on LPS-induced HaCaT cell viability

[0141]

[0142] Note: Different lowercase letters in the table indicate significant differences (P<0.05), and the same letters indicate insignificant differences (P>0.05).

[0143] Example 7 Study on the activity of fermented Dendrobium polysaccharide in repairing skin barrier

[0144] 1) Experimental methods

[0145] DOP-1A, DOP-2A and original mixed crude polysaccharide MIX were weighed, reconstituted with water to form a 1 mg / mL solution, and filtered through a 0.22 μm filter membrane to obtain DOP-1A solution, DOP-2A solution and MIX solution, respectively.

[0146] The experimental cell model was the human skin keratinocyte cell line HaCaT, and the cell density was adjusted to 1×10 6 / well, culture the cells in 6-well plates until the cells adhere to the wall, then discard the original culture medium and perform different treatments.

[0147] Set up the processing group:

[0148] Sample group: DOP-1A solution, DOP-2A solution and MIX polysaccharide solution were used as different sample solutions. 200 μL of different sample solutions were taken and mixed with 1600 μL of culture medium and added into 6-well plates.

[0149] Control group (LPS): 200 μL PBS and 1600 μL culture medium were mixed and added to a 6-well plate.

[0150] After incubation for 24 h at 37°C and 5% CO2, 200 μL of 50 μg / mL LPS solution was added to each well of the sample group and the control group. After incubation for 24 h at 37°C and 5% CO2, the cells of each treatment group were collected into a centrifuge tube and the cell protein was extracted.

[0151] The protein expression level of target cells was detected using an ELISA kit (refer to its instructions for specific detection methods).

[0152] The percentage calculation formula is as follows:

[0153] Promotion percentage (%) = (sample group - control group) / control group × 100%;

[0154] 2) Experimental results

[0155] The effects of different polysaccharides on the expression of FLG, AQP3, CAMP / LL37, and TRPV4 proteins in HaCaT pretreated with LPS are shown in the following table. Fig. 9 , Fig.10 As shown in Table 9, the percentage of promoting effect of different polysaccharide treatments on LPS-induced related skin barrier proteins is shown in Table 10, and the expression of different targets is as follows:

[0156] (1) In the epidermis of human skin, FLG produces natural moisturizing factor (NMF), which plays a key role in the epidermal barrier function. The results showed that compared with the control group, MIX, DOP-1A, and DOP-2A can effectively promote the expression of FLG and maintain the integrity of the epidermal barrier function; and compared with MIX, DOP-1A and DOP-2A can enhance the expression level of FLG more significantly.

[0157] (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 under inflammatory conditions and enhance the ability to regulate skin hydration, and the effect is better than MIX.

[0158] (3) TRPV4 is a calcium ion channel related to temperature perception and skin barrier function. It is involved in regulating the moisture and electrolyte balance of the skin and affects the skin barrier function. MIX has no significant effect on TRPV4, while DOP-1A and DOP-2A can promote TRPV4 and help maintain the moisture and electrolyte balance of the skin, among which DOP-2A has a more significant effect.

[0159] (4) CAMP / LL-37 itself has anti-inflammatory effects. In patients with atopic dermatitis (AD), the expression of CAMP / LL-37 is reduced, resulting in impaired skin barrier function and greater susceptibility to bacterial infection. The results showed that DOP-1A and DOP-2A can increase the activity of CAMP / LL-37 under inflammatory conditions, and can directly kill bacteria or enhance natural immunity by regulating the occurrence and development of inflammation in the body by regulating the expression or activity of CAMP / LL-37. Among them, DOP-2A is better than MIX in promoting the release of CAMP / LL37.

[0160] Table 9 Effects of different polysaccharide treatments on the average expression of LPS-induced skin barrier proteins

[0161]

[0162] Note: Different lowercase letters in the table indicate significant differences (P<0.05), and the same letters indicate insignificant differences (P>0.05).

[0163] Table 10 The percentage of promoting effect of different polysaccharide treatments on LPS-induced skin barrier proteins

[0164]

[0165] Example 8 Study on the anti-inflammatory activity of purified polysaccharide components from Dendrobium officinale

[0166] 1) Experimental methods

[0167] The experimental cells were mouse macrophage RAW264.7.

[0168] DOP-1A solution, DOP-2A solution and MIX solution were prepared according to the method of Example 7, with a concentration of 100 μg / mL. A 50 μM indomethacin solution was prepared, where indomethacin is a nonsteroidal anti-inflammatory drug.

[0169] Adjust the cell density to 1×10 6 / well, culture the cells in 6-well plates until the cells adhere to the wall, discard the original culture medium, and then perform different treatments.

[0170] Set up the processing group:

[0171] Sample group: Indomethacin solution, DOP-1A solution, DOP-2A solution and MIX solution were used as different sample solutions. 200 μL of different sample solutions were taken and mixed with 1600 μL of culture medium and added into 6-well plates.

[0172] Positive control group (LPS): 200 μL PBS and 1600 μL culture medium were mixed and added to a 6-well plate.

[0173] Blank control group (CON): 200 μL PBS and 1600 μL culture medium were mixed and added to a 6-well plate.

[0174] After each group was incubated at 37°C and 5% CO2 for 24 h, 200 μL of 10 μg / mL LPS solution was added to each well of the sample group and the positive control group, and 200 μL of PBS was added to the blank control group. After each group was incubated at 37°C and 5% CO2 for 24 h, the cell supernatant and cells were collected into centrifuge tubes.

[0175] The MCP-1 content in the cell supernatant was detected by ELISA kit (ABclonal, RK00381) according to the kit instructions.

[0176] The cells were tested for reactive oxygen species using a reactive oxygen species detection kit (Biyuntian, S00033S) according to the instructions, and the fluorescence intensity of each group, i.e., the ROS level, was detected by flow cytometry.

[0177] The inhibition rate calculation formula is as follows:

[0178] Inhibition rate (%) = (positive control group - sample group) / positive control group × 100%.

[0179] 2) Experimental results

[0180] MCP-1 is a chemokine that can induce a variety of inflammatory mediators, which can lead to chronic inflammation and hinder the repair of the skin barrier. The effects of different polysaccharides on the expression of MCP-1 protein in RAW264.7 cells pretreated with LPS are shown in the following table. Fig.11 As shown in Table 11, the results showed that in the LPS-induced inflammatory environment, MIX could not inhibit the release of MCP-1, while DOP-1A and DOP-2A could effectively inhibit the activity of MCP-1 and control the inflammatory response, and the effect of DOP-1A in inhibiting MCP-1 was comparable to that of indomethacin.

[0181] Inflammatory response will produce a large amount of ROS, further aggravating local tissue damage and even leading to chronic inflammation. The flow cytometry results of different polysaccharides on ROS release of LPS-pretreated RAW264.7 are shown in Figure 2. Fig.12 As shown in Table 12, the results showed that DOP-1A and DOP-2A could reduce oxidative stress, regulate inflammatory response, and protect tissues from further damage by inhibiting the production of ROS and clearing excess ROS, and the effect of DOP-1A was more significant.

[0182] The above results show that fermented Dendrobium polysaccharides, especially DOP-1A, have significant inhibitory activity on ROS and MCP-1 and have good anti-inflammatory activity.

[0183] Table 11 Average expression of MCP-1 protein in different groups and inhibition rate of MCP-1

[0184]

[0185] Note: Different lowercase letters in the table indicate significant differences (P<0.05), and the same letters indicate insignificant differences (P>0.05).

[0186] Table 12 Average fluorescence intensity of ROS in different groups and inhibition rate of ROS

[0187]

[0188]

[0189] Note: Different lowercase letters in the table indicate significant differences (P<0.05), and the same letters indicate insignificant differences (P>0.05).

[0190] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

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 0-5%, 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 100-500kDa.

2. A fermented dendrobium polysaccharide DOP-2A, characterized in that: It 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 3-100kDa.

3. A method for separating and purifying fermented dendrobium polysaccharide, characterized in that: The steps include: (1) taking the supernatant of the Dendrobium fermented powder extract, adding ethanol to a volume fraction of 15-25%, ice bathing for 0.5-2h, and centrifuging to obtain supernatant A; adding ethanol to supernatant A to a volume fraction of 35-45%, ice bathing for 0.5-2h, and centrifuging to obtain supernatant B; adding ethanol to supernatant B to a volume fraction of 55-65%, ice bathing for 0.5-2h, and centrifuging to obtain supernatant C and precipitate DOP-1; the Dendrobium fermented powder is a polysaccharide powder obtained by fermenting Dendrobium by Lactobacillus reuteri. (2) After the precipitate DOP-1 is dissolved with the mobile phase, it is separated on a chromatography column and eluted at a flow rate of 0.2-0.3 mL / min. The eluate with an elution time of 234-426 min is collected, the eluate is removed and dried to obtain the fermented Dendrobium polysaccharide DOP-1A.

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 filler is Sepharose S-400HR.

5. A method for separating and purifying fermented dendrobium polysaccharide, characterized in that: The steps include: S1. Take the supernatant C described in claim 3, add ethanol to a volume fraction of 75-85%, ice bath for 0.5-2h, and centrifuge to obtain a precipitate DOP-2; S2. The precipitate DOP-2 is dissolved with the mobile phase, separated on a chromatography column, allowed to stand for 20 to 40 minutes of adsorption, eluted at a flow rate of 2 to 4 mL / min, and the eluate with an elution time of 12 to 48 minutes is collected. The eluate is removed, desalted, and dried to obtain the fermented Dendrobium polysaccharide DOP-2A.

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

7. A product for repairing skin barrier, characterized in that: Contains the fermented Dendrobium polysaccharide DOP-1A according to claim 1 and / or the fermented Dendrobium polysaccharide DOP-2A according to claim 2.

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

9. Use of the fermented Dendrobium polysaccharide DOP-1A according to claim 1 or the fermented Dendrobium polysaccharide DOP-2A according to claim 2 in the preparation of a product for repairing skin barrier.

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

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