Purslane homogeneous polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of cosmetics, food and medicines, and provides purslane homogeneous polysaccharide, a preparation method and application thereof. The portulaca oleracea homogeneous polysaccharide has a structural formula as shown in a figure 7. The highest peak molecular weight Mp, the weight-average molecular weight Mw and the number-average molecular weight Mn are 6.4 kDa, 7.4 kDa and 5.3 kDa respectively; the monosaccharide comprises glucose, galactose and arabinose, and the ratios of the glucose, the galactose and the arabinose are 21.54%, 62.98% and 13.21% respectively. The herba portulacae extract is extracted from herba portulacae after water extraction and alcohol precipitation, ion exchange column chromatography and gel filtration column chromatography are adopted, and it is determined through cell experiment and animal experiment research that the herba portulacae extract has the effects of promoting Hacat cell scratch healing and improving the skin barrier and can be applied to cosmetics, food and drugs.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetics, foods and medicines, and in particular to a purslane uniform polysaccharide, a preparation method and application thereof. Background Art

[0002] purslane( Portulaca oleracea Portulaca oleracea L., also known as Purslane, is an annual fleshy herb belonging to the genus Portulaca of the family Portulacaceae. Portulaca is a plant with medicinal and edible properties. It has anti-inflammatory, soothing, antibacterial, antiviral, lipid-regulating, anti-tumor, and blood sugar-lowering effects and is widely used among the people. Its active ingredients are mainly polysaccharides, organic acids, and alkaloids.

[0003] Polysaccharides are a type of complex carbohydrates that are widely distributed in nature and play an extremely important physiological function, such as participating in immune regulation, intercellular recognition, intercellular substance transport, disease diagnosis and treatment, etc. It can also regulate cell growth and aging, control cell division and differentiation, and play a wide range of roles in the fields of cosmetics, food and medicine. Summary of the invention

[0004] In view of the above technical problems, the present invention separates a uniform purslane polysaccharide with a novel structure for the first time through a specific extraction process, and finds that the polysaccharide has good skin barrier repairing effect.

[0005] In a first aspect, the present invention provides a purslane homogeneous polysaccharide having a chemical structure represented by the following general formula:

[0006] Among them, n=8, m=19.

[0007] Preferably, the highest peak molecular weight Mp, weight average molecular weight Mw and number average molecular weight Mn of the purslane homogeneous polysaccharide are 6.4 kDa, 7.4 kDa and 5.3 kDa respectively.

[0008] Preferably, the purslane homogeneous polysaccharide is mainly composed of glucose, galactose and arabinose, with molar proportions of 21.54%, 62.98% and 13.21%, respectively.

[0009] Preferably, the purslane homogeneous polysaccharide is from Portulacaceae, Portulaca purslane Portulaca oleracea The dried aerial part of L. is obtained by extraction, separation and purification. The dried aerial part refers to the other parts of the whole dried Purslane except the root.

[0010] The second aspect of the present invention provides the use of the above-mentioned Purslane uniform polysaccharide in cosmetics, food or medicine.

[0011] The third aspect of the present invention provides a cosmetic comprising the above-mentioned Purslane uniform polysaccharide.

[0012] Preferably, the cosmetic comprises phase A, phase B and phase C, wherein phase A comprises the following components by mass percentage: water, 72.05%; Glycerol, 3.0%; Pentylene glycol, 2.0%; Acrylates / C10-30 Alkyl Acrylate Crosspolymer, 0.25%; p-Hydroxyacetophenone, 0.3%; Phase B includes the following components by mass percentage: Cetearyl alcohol, cetearyl glucoside, 2.0%; Polydimethylsiloxane, 3.0%; Glyceryl tri(ethylhexanoate), 2.0%; Glyceryl stearate, 1.5%; Squalane, 2.0%; Steareth-21, 0.2%; Phase C includes the following components by mass percentage: Water, 10.0%; Bis-PEG-18 methyl ether dimethyl silane, 1.0%; Arginine, 0.2%; Purslane homogeneous polysaccharide, 0.5%.

[0013] In a fourth aspect, the present invention provides a method for preparing the above-mentioned purslane uniform polysaccharide, comprising the following steps: Step S1, crushing purslane and extracting purslane crude polysaccharide by water extraction and alcohol precipitation method, named WPOP; Step S2, subjecting the crude purslane polysaccharide to ion exchange column chromatography to obtain purslane primary polysaccharide, named WPOP-1; Step S3, subjecting the purslane polysaccharide to gel filtration column chromatography to obtain purslane homogeneous polysaccharide, which is named WPOP-1-1.

[0014] Preferably, step S1 is specifically as follows: adding 20 times the weight of deionized water to purslane, heating, stirring, refluxing and extracting at 95°C for two hours, concentrating the obtained extract and centrifuging at 11000 r / min for 10 min, taking the supernatant, and then adding 4 times the volume of the supernatant 95% ethanol to the supernatant, precipitating at 4°C for 12 hours, centrifuging at 11000 r / min for 10 min, and drying the obtained precipitate to obtain purslane crude polysaccharide.

[0015] Preferably, step S2 is specifically as follows: after loading the crude purslane polysaccharide on a DEAE-52 column, using a NaCl aqueous solution as an eluent, eluting and separating in the concentrations of 0.1 M NaCl, 0.2 M NaCl, 0.3 M NaCl, and 0.4 M NaCl, in sequence, with an elution rate of 15 mL / min, collecting the eluate, collecting 20 tubes of each eluate gradient, 10 mL in each test tube, wherein the eluate from the 6th to 18th tubes of the 0.1 M NaCl elution position is concentrated and dried under reduced pressure to obtain the purslane primary polysaccharide.

[0016] Preferably, step S3 is specifically as follows: after loading the purslane polysaccharide on a dextran G-75 column, eluting with ultrapure water, the column flow rate is 2.0 mL / min, and collecting the eluate, 5 mL per test tube, wherein the 10th to 14th tubes are dried to obtain the purslane uniform polysaccharide.

[0017] Preferably, in step S2, the crude purslane polysaccharide is dissolved by distillation into a 50 mg / mL aqueous solution, then centrifuged at 8000 rpm for 10 min, and then filtered with a 0.45 μm microporous filter membrane, and the filtrate is loaded onto a DEAE-52 column; in step S3, the purslane polysaccharide is dissolved by distillation into a 50 mg / mL aqueous solution, then centrifuged at 8000 rpm for 10 min, and then filtered with a 0.45 μm microporous filter membrane, and the filtrate is loaded onto a dextran G-75 column, the specification of which is φ2.6×100 cm.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects: The innovative method of the present invention is used to extract and purify the dried aerial part of purslane, and a purslane uniform polysaccharide with a novel structure is obtained. Cell experiments and animal experiments have confirmed that it has the effects of promoting Hacat cell scratch healing and repairing skin barrier damage, and has important application potential in cosmetics, food and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1The elution curve of WPOP on DEAE-52 column, the elution curve of WPOP-1 after chromatography on dextran G-75 column, the molecular weight test graph of purslane uniform polysaccharide WPOP-1-1 and the related molecular weight calibration curve: (A) elution curve of WPOP on DEAE-52 column; (B) elution curve of WPOP-1 after chromatography on dextran G-75 column; (C) molecular weight test graph of purslane uniform polysaccharide WPOP-1-1; (D1) calibration curve of weight average molecular weight (Mw) of purslane uniform polysaccharide WPOP-1-1; (D2) calibration curve of highest peak molecular weight (Mp); (D3) calibration curve of number average molecular weight (Mn); Figure 2 The monosaccharide composition analysis diagram, infrared spectrum analysis diagram, GC-MS analysis diagram and secondary mass spectrum diagram of WPOP-1-1: (A) monosaccharide composition analysis diagram; (B) infrared spectrum analysis diagram; (C1~C6) are the GC-MS analysis diagram and secondary mass spectrum diagram of WPOP-1-1; Figure 3 One-dimensional and two-dimensional NMR spectra: (A) One-dimensional NMR 1 H-NMR image; (B) one-dimensional NMR 13 C-NMR image; (C) 2D NMR HSQC image; (D) 2D NMR COSY image; (E) 2D NMR TOCSY image; (F) 2D NMR HMBC image; (G) 2D NMR NOESY image; Figure 4 The results of the cell scratch test of Purslane homogeneous polysaccharide: (A) Schematic diagram of the cell scratch test (before and after the test); (B) Cell migration rate (indicating the healing rate) after the cells were scratched; Figure 5 The results of the experiment on the repair of mouse skin barrier damage by Purslane homogeneous polysaccharide: (A) Graph of hyaluronic acid (HA) content detected by mouse serum indicators; (B) Graph of filaggrin (FLG) content detected by mouse serum indicators; (C) Schematic diagram of transepidermal horizontal loss (TEWL) detected by mouse serum indicators; Figure 6 These are the images of mouse tissue sections stained with hematoxylin-eosin (HE), Masson (Masson), and toluidine blue; Figure 7 It is a schematic diagram of the structure of the purslane uniform polysaccharide of the present invention. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0021] Unless otherwise specified, the raw materials and materials used in the examples of the present invention are purchased through general commercial channels.

[0022] Example 1: Extraction and purification of uniform polysaccharide from Purslane 1. Extraction of Purslane crude polysaccharide WPOP by water extraction and alcohol precipitation The root-free dried purslane was crushed and weighed to 400 g. It was extracted with 8 L of deionized water at 95°C for 2 h under stirring and refluxing. After the extraction, the extract was collected and repeated once. The extracts obtained from the two times were combined and concentrated to 500 mL using a rotary evaporator, and then centrifuged at 11000 r / min for 10 min to obtain the supernatant. 4 times the volume of 95% ethanol was added to the supernatant, stirred thoroughly, and then allowed to stand at 4°C for 12 h. Centrifuged at 11000 r / min for 10 min, the supernatant was discarded, and the precipitate was placed in a vacuum drying oven at 60°C. After drying, it was ground into fine powder, which was the crude purslane polysaccharide, named WPOP.

[0023] 2. Refining of Purslane Homogeneous Polysaccharide 2.1 Preparation of WPOP-1 from Portulaca oleracea by ion exchange column chromatography Pretreatment of ion exchange chromatography filler: Add 300g of DEAE sepharose FF (DEAE-52) into 600mL of water and stir to form a suspension, then pour into a Buchner funnel to extract the liquid and wash with ultrapure water about 3 times the volume of the suspension. Repeat the above operation several times until the filler has no alcohol taste, transfer to a beaker, add distilled water of one-half to one times the volume of the filler, and stir well for column loading.

[0024] Column loading and balancing: Mix the filler obtained after the above pretreatment with water and slowly add it to the XK chromatography column (φ3.0×50cm). After complete sedimentation, connect the upper column head to the peristaltic pump, start the pump at a flow rate of 250cm / h, and flush the column with ultrapure water until the gel surface is stable to complete the column loading.

[0025] Loading and elution: Dissolve the crude polysaccharide of Purslane with distilled water into a 50 mg / mL aqueous solution, then centrifuge at 8000rpm for 10 min, filter with a 0.45 μm microporous filter membrane, and load the filtrate onto the equilibrated ion exchange chromatography column. The loading volume is 30% of the column volume. After all the sample liquid enters the chromatography column, it is eluted and separated with NaCl aqueous solution according to different concentrations (0.1 M NaCl, 0.2 M NaCl, 0.3 M NaCl, 0.4 M NaCl), and the elution rate is 15mL / min. Use an automatic fraction collector to collect, and collect 20 tubes for each eluate gradient, with 10 ml collected in each tube. The phenol-sulfuric acid method is used, and the content of polysaccharides in each tube of eluate is detected by an enzyme reader at 490 nm, referred to as polysaccharide content, and then the number of tubes is used as the horizontal axis and the absorbance is used as the vertical axis to obtain the polysaccharide elution curve, see. Figure 1 A. Figure 1 As shown in A, the product after the eluate from tubes 6 to 18 was concentrated and dried under reduced pressure was taken as the purslane primary polysaccharide WPOP-1.

[0026] 2.2 Preparation of Purslane Homogeneous Polysaccharide WPOP-1-1 by Gel Filtration Column Chromatography Pretreatment of gel filtration chromatography filler: Add 100g of gel filtration chromatography filler Chromdex75PG (dextran G-75) into 300mL of water and stir to form a suspension. Pour into a Buchner funnel, draw out the liquid, and wash with ultrapure water about 3 times the volume of the suspension. Repeat the above operation several times until the filler has no alcohol taste. Transfer to a beaker, add distilled water of one-half to one times the volume of the filler, and stir well for column loading.

[0027] Column packing and balancing: Mix the filler obtained after the above pretreatment with water and slowly add it to the XK chromatography column (φ2.6×100 cm). After complete sedimentation, connect the upper column head to the chromatography system and rinse the column with ultrapure water at a flow rate of 30 cm / h until the gel surface is stable to complete the column packing.

[0028] Loading and elution: The purslane primary polysaccharide WPOP-1 prepared by ion exchange column chromatography was dissolved by distillation into a 50 mg / mL aqueous solution, centrifuged at 8000 rpm for 10 min, and then filtered with a 0.45 μm microporous filter membrane, and the filtrate was loaded onto the equilibrated gel filtration chromatography. The loading amount was 1% of the column volume. After all the sample liquid entered the chromatography column, it was eluted and separated with ultrapure water (φ2.6×100 cm) with a column flow rate of 2.0 mL / min. The eluate was collected using an automatic partial collector, with 5 mL collected in each tube. The phenol-sulfuric acid method was used, and the polysaccharide content in each tube of eluate was detected by an enzyme marker at 490 nm. The polysaccharide elution curve was obtained with the number of tubes as the horizontal axis and the absorbance as the vertical axis, as shown in the figure. Figure 1 Middle B.

[0029] like Figure 1 As shown in B, the eluates from tubes 10 to 14 were combined, concentrated under reduced pressure, and freeze-dried to obtain Purslane homogeneous polysaccharide WPOP-1-1, with a yield of 0.2% relative to the medicinal material (dried Purslane without roots). The properties of Purslane homogeneous polysaccharide are: white solid, tasteless, and easily soluble in water.

[0030] Example 2: Structural analysis of purslane homogeneous polysaccharide 1. Experimental Methods 1.1 Molecular weight and its distribution HPGPC-RID was used to analyze the molecular weight and distribution of the uniform polysaccharide from Purslane prepared in Example 1.

[0031] Reagent preparation: Prepare 0.05 M NaCl aqueous solution and 0.1 M NaNO3 aqueous solution respectively, then filter through 0.22 μm filter membrane respectively and ultrasonicate the filtrate for 20 min.

[0032] Preparation of samples and standards: Dissolve 5.0 mg of homogeneous polysaccharide WPOP-1-1 in 1.0 mL of 0.05 M NaCl solution to prepare a 5.0 mg / mL test solution, then centrifuge at 8000 rpm for 10 min, filter with a 0.22 μm microporous filter membrane, and place the filtrate in a 2.0 mL injection bottle for later use.

[0033] Chromatographic method: The chromatographic column was an OHpak SB-803 gel column (8 × 300 mm), the mobile phase was a 0.05 M NaCl solution, the flow rate was 0.65 mL / min, the column temperature was 40°C, the injection volume was 30 μL, and the detector was a Waters 2410 differential detector. The molecular weight and distribution results are shown in Figure 1 Middle C.

[0034] like Figure 1 As shown in C, the polysaccharide WPOP-1-1 presents a single symmetrical chromatographic peak, indicating that this polysaccharide is a uniform polysaccharide.

[0035] Figure 1 D1 is the weight average molecular weight (Mw) calibration curve of WPOP-1-1, Figure 1 D2 is the highest peak molecular weight (Mp) calibration curve. Figure 1 D3 is the calibration curve of number average molecular weight (Mn). According to the standard curve, the Mp, Mw and Mn of WPOP-1-1 were calculated to be 6.4 kDa, 7.4 kDa and 5.3 kDa, respectively.

[0036] 1.2 Monosaccharide composition analysis The monosaccharide composition of the Purslane homogeneous polysaccharide prepared in Example 1 was analyzed by high performance liquid chromatography.

[0037] Sample preparation: Accurately weigh 5.0 mg (± 0.05 mg) of the homogeneous polysaccharide WPOP-1-1, add 1.0 mL of 2 M trifluoroacetic acid (TFA) solution, heat at 121 °C for 2 h, blow dry with nitrogen, then add 3.0 mL of methanol to wash, blow dry again, wash repeatedly with methanol 2-3 times, add 5.0 mL of sterile water to dissolve, and transfer to a chromatographic bottle for testing.

[0038] Chromatographic conditions: ThermoU3000 liquid chromatography system, chromatographic column ZORBAX Eclipse XDB-C18, mobile phase acetonitrile: phosphate buffer (12 g / L potassium dihydrogen phosphate and 2 M NaOH solution, pH = 6.8) = 1:1, isocratic elution, flow rate 0.8 mL / min, column temperature 30°C, detection wavelength 250 nm, injection volume 10 μL. Results are shown in Figure 2 Middle A.

[0039] like Figure 2 As shown in A, WPOP-1-1 is mainly composed of glucose, galactose and arabinose, with molar proportions of 21.54%, 62.98% and 13.21%, respectively.

[0040] 1.3 Methylation analysis The connection mode of the purslane uniform polysaccharide prepared in Example 1 was analyzed by GC-MS.

[0041] Sample methylation: Accurately weigh 1.0 mg of the sample purslane homogeneous polysaccharide, add 500 μL of dimethyl sulfoxide (DMSO) to dissolve, then add 1.0 mg NaOH, incubate for 0.5 h, add 50 μL of iodomethane solution to react for 1 h, add 1 mL of water and 2 mL of dichloromethane, vortex, centrifuge, discard the aqueous phase, repeat water washing 3 times, evaporate the dichloromethane layer to dryness, add 100 μL TFA (2.0 M) solution, react at 121°C for 90 min, evaporate to dryness at 30°C, add 50 μL of ammonia (2.0 M) and 50 μL of sodium borodeuteride (NaBD4) (1.0 M) solution, mix well, react at room temperature for 2.5 h, add 20 μL of acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL of methanol, blow dry with nitrogen, add 250 μL of acetic anhydride, vortex to mix well, react at 100°C for 2.5 h, add 1 mL of water was allowed to stand for 10 min, 500 μL of dichloromethane was added, vortexed, centrifuged, the aqueous phase was discarded, and the water washing was repeated 3 times.

[0042] Chromatographic conditions: Agilent 7890A gas chromatography system (Agilent, USA), HP-5MS column 30 cm × 0.25 mm × 0.25 μm, carrier gas of high purity helium, flow rate 1.0 mL / min, injection port temperature 260 °C, injection volume 1.0 μL, split injection (split ratio 10:1), programmed temperature (50 °C for 1.0 min, 50 °C / min to 130 °C, 3 °C / min to 230 °C, hold for 2 min).

[0043] Mass spectrometry detection conditions: Agilent 5977B quadrupole mass spectrometry detection system (Agilent, USA) equipped with electron impact ion source (EI) and MassHunter workstation was used. EI inlet temperature was 230°C, quadrupole temperature was 150°C, electron energy was 70 eV, scanning mode was full scan mode (SCAN), mass scanning range (m / z): 30-600. Results are shown in Figure 2 Middle C1.

[0044] Figure 2 C1~C6 are the GC-MS analysis and secondary mass spectrum of WPOP-1-1. The main types of glycosidic bonds of WPOP-1-1 are 3-Ara(f), 5-Ara(f), t-Glc(p), t-Gal(p) and 4-Gal(p). The specific data of glycosidic bonds are shown in Table 1.

[0045] Table 1. Methylation analysis of WPOP-1-1

[0046] In the above Table 1, the Chinese name of 1,3,4-tri-O-acetyl-2,5-di-O-methyl arabinitol is 1,3,4-tri-O-acetyl-2,5-di-O-methylarabinitol; the Chinese name of 1,4,5-tri-O-acetyl-2,3-di-O-methylarabinitol is 1,4,5-tri-O-acetyl-2,3-O-methylarabinitol; the Chinese name of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol is 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol; and the Chinese name of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol is 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol.

[0047] 1.4 Infrared analysis The dried polysaccharide sample WPOP-1-1 (1-2 mg) was ground and pressed into a pellet with 200 mg KBr powder under an infrared lamp. The spectrometer was Nicolet 6700 Fourier transform infrared spectrometer (ThermoFisher, USA) was used to analyze the spectra at 4000-400 cm -1 Infrared scanning is performed within the wavelength range and infrared spectra are recorded. Figure 2 Middle B.

[0048] like Figure 2 As shown in B, infrared spectrum analysis shows that the polysaccharide sample has a characteristic absorption peak of polysaccharide: 3889.17 cm -1 There is a strong and broad absorption peak at 2929.59 cm, which is the strong absorption peak of OH stretching vibration of hydrogen bonds between or within polysaccharide molecules; -1 The medium-intensity peak nearby is the CH stretching vibration absorption peak of the methine (-CH2); 1647.22 cm -1 The absorption peak at 1450-1200 cm -1 The absorption peak is 1423.22 cm -1 , 1383.95 cm -1 ,1259.35 cm -1 , 1223.12 cm -1 It is the absorption peak of the variable angle vibration of CH. It and the stretching vibration of CH constitute the characteristic absorption of the sugar ring; 1150~1010 cm -1 The strong absorption peak between 1073.23 cm -1 、1046.88 cm -1 The presence of pyranoside was confirmed again at 890.14 cm -1 It is the characteristic region of β-pyranosidic bond, and it is speculated that the polysaccharide contains β-pyranose; 833.39 cm -1 The CH angle vibration peak of the α-anomer of pyranose; 756.44 cm -1 Showing the symmetric ring stretching vibration peaks of the pyran ring.

[0049] 1.5 Nuclear magnetic resonance analysis 50 mg of polysaccharide sample WPOP-1-1 was dissolved in 0.5 mL of heavy water and freeze-dried. After drying, the freeze-dried powder was dissolved in 0.5 mL of heavy water again. Acetone-d6 was used as an internal standard, and 1D ( 1 H. 13C) and 2D (HSQC, COSY, HMBC, NOESY, TCOSY) NMR spectra.

[0050] In order to further obtain the structural characteristics of the sample, one-dimensional NMR 1 H-NMR( Figure 3 Middle A), 13 C-NMR( Figure 3 B) and 2D NMR HSQC ( Figure 3 Middle C), COSY ( Figure 3 D), TOCSY ( Figure 3 Middle E), HMBC ( Figure 3 F), NOESY ( Figure 3 In order to obtain all H and C chemical shift information of each major sugar residue, the connection order between each sugar residue can be inferred.

[0051] One-dimensional H NMR spectroscopy ( 1 H-NMR) was used to further identify the glycosidic bond configuration of the polysaccharide sample. Most of the hydrogen spectrum signals of the polysaccharide were in the range of δ 3.0 to 5.5 ppm, and usually the resonance region of the anomeric proton (H-1) was between δ 4.5 and 5.5 ppm. 1 H- 1 H COSY, HSQC, and TOCSY can assign signals to the main sugar residues in the methylation results. In the HSQC spectrum, 11 anomeric signals of various sugar residues can be found, namely δ 5.31 ppm / δ 99.46 ppm, δ 5.28 ppm / δ 99.87 ppm, δ 4.88 ppm / δ 99.54 ppm, δ 4.95 ppm / δ 99.38 ppm, δ 4.55 ppm / δ 104.32 ppm, δ 5.06 ppm / δ 106.87 ppm, δ 5.10 ppm / δ 106.85 ppm, δ 5.06 ppm / δ106.25 ppm, δ 5.13 ppm / δ 106.87 ppm, δ 5.03 ppm / δ 107.17 ppm, δ 5.01 ppm / δ 107.40 ppm 1 H- 1H COSY, HSQC, and TOCSY can be used to assign signals at other positions corresponding to the anomeric signals. Combined with the H and C signal characteristics of each position of the sugar residue and the methylation results, the sugar residues corresponding to these anomeric signals are assigned as →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, α-D-Glcp-(1→, α-D-Galp-(1→, →4)-β-D-Galp-(1→, α-L-Araf-(1→, →2,3,5)-α-L-Araf-(1→, →3)-α-L-Araf-(1→, →3,5)-α-L-Araf-(1→, →5)-α-L-Araf-(1→, marked as Glc 1,4 , Glc 1,4,6 , Glc t , α Gal t , β Gal 1,4 , A t 1 , A t 2 , A 1,2,3,5 , A 1,3 , A 1,3,5 , A 1,5 .

[0052] Glc 1,4 For example, the anomeric signals δ 5.31 ppm (H-1) and δ 99.46 ppm (C-1) indicate that the sugar residue is in α configuration. The H-2, H-3, H-4, and H-5 signals were identified by COSY spectrum and TOCSY cross peaks. The chemical shifts of the H-2, H-3, H-4, and H-5 of the sugar residue were assigned to δ 3.49 ppm, δ 3.87 ppm, δ 3.56 ppm, and δ 3.74 ppm, respectively, and the H6a and H6b signals were determined to be δ 3.75 ppm and δ 3.67 ppm by COSY spectrum and HSQC spectrum. After assigning the chemical shift of hydrogen on the sugar ring, the chemical shift of each carbon on the sugar ring was assigned by HSQC correlation spectrum, which were δ 99.46 ppm, δ 71.29 ppm, δ 73.04 ppm, δ 76.73 ppm, δ 71.29 ppm and δ 60.61 ppm respectively. The chemical shifts of C-1 and C-4 shifted to the low field, indicating that the residue was substituted at the C-1 and C-4 positions of the sugar ring. Combined with the methylation results, the sugar residue was inferred to be →4)-α-D-Glcp-(1→. The hydrogen and carbon signals of other residues were derived by similar methods. The assignment of H and C chemical shifts of the main sugar residues in the sample is summarized in Table 2.

[0053] Table 2. Sugar residues of WPOP-1-1 1 H and 13 Chemical shift assignment of C

[0054] In the HMBC spectrum, sugar residue Glc 1,4 H-1 (δ 5.31 ppm) and sugar residue Glc 1,4 There is a coupling signal (Glc 1,4 H-1 / Glc 1,4 C-4), indicating the existence of a connection between the →4)-α-D-Glcp-(1→ and →4)-α-D-Glcp-(1→ sugar residues, with the connection site located at the O-4 position; sugar residue Glc 1,4 H-1 (δ 5.31 ppm) and sugar residue Glc 1,4,6 There is a coupling signal (Glc 1,4 H-1 / Glc 1,4,6 C-4), indicating the existence of a connection between the →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→ sugar residues, with the connection site located at the O-4 position; sugar residue Glc t H-1 (δ 4.88 ppm) and sugar residue Glc 1,4,6 There is a coupling signal (Glc t H-1 / Glc 1,4,6 C-6), indicating that there is a connection between the sugar residues α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→, and the connection site is located at the O-6 position; the sugar residue Glc 1,4,6 H-1 (δ 5.28 ppm) and sugar residues β Gal 1,4 There is a coupling signal (Glc 1,4,6 H-1 / β Gal 1,4 C-4), indicating the existence of a connection between the →4,6)-α-D-Glcp-(1→ and →4)-β-D-Galp-(1→ sugar residues, with the connection site located at the O-4 position; sugar residue β Gal 1,4 H-1 (δ 4.55 ppm) and sugar residues β Gal 1,4 There is a coupling signal at C-4 (δ 77.56ppm) ( β Gal 1,4 H-1 / βGal 1,4 C-4), indicating the existence of a connection between the →4)-β-D-Galp-(1→ and →4)-β-D-Galp-(1→ sugar residues, with the connection site located at the O-4 position; sugar residue A 1,5 H-1 (δ 5.00 ppm) and sugar residue Glc 1,4,6 There is a coupling signal (A 1,5 H-1 / Glc 1,4,6 C-6), indicating the existence of a connection between the →5)-α-L-Araf-(1→ and →4,6)-α-D-Glcp-(1→ sugar residues, with the connection site located at the O-6 position; sugar residue A 1,5 H-1 (δ 5.00 ppm) and sugar residue A 1,3,5 There is a coupling signal (A 1,5 H-1 / A 1,3,5 C-5), indicating the existence of a connection between the →5)-α-L-Araf-(1→ and →3,5)-α-L-Araf-(1→ sugar residues, with the connection site located at the O-5 position; sugar residue A 1,3,5 H-1 (δ 5.03 ppm) and A 1,5 There is a coupling signal (A) at C-5 (δ 66.23 ppm) of the sugar residue 1,3,5 H-1 / A 1, 5 C-5), indicating that there is a connection between the →3,5)-α-L-Araf-(1→ and →5)-α-L-Araf-(1→ sugar residues, and the connection site is located at the O-5 position; sugar residue A 1,3 H-1 (δ 5.13 ppm) and A 1,2,3,5 There is a coupling signal (A) at C-3 (δ79.92 ppm) of the sugar residue 1,3 H-1 / A 1,2,3,5 C-3), indicating the existence of a connection between the →3)-α-L-Araf-(1→ and →2,3,5)-α-L-Araf-(1→ sugar residues, with the connection site located at the O-3 position; sugar residue A t 1 H-1 (δ 5.06 ppm) and sugar residue A 1,3,5 There is a coupling signal (A t 1 H-1 / A 1,3,5 C-3), indicating that there is a connection between the α-L-Araf-(1→ and →3,5)-α-L-Araf-(1→ sugar residues, and the connection site is located at the O-3 position; sugar residue α Gal tH-1 (δ 4.95 ppm) and A 1,2,3,5 There is a coupling signal at C-2 (δ 84.67 ppm) of the sugar residue ( α Gal t H-1 / A 1,2,3,5 C-2), indicating the existence of a connection between the α-D-Galp-(1→ and →2,3,5)-α-L-Araf-(1→ sugar residues, with the connection site located at the O-2 position; sugar residues α Gal t H-1 (δ 4.95 ppm) and A 1, 3 There is a coupling signal at C-3 (δ 84.14 ppm) of the sugar residue ( α Gal t H-1 / A 1, 3 C-3), indicating the existence of a connection between α-D-Galp-(1→ and →3)-α-L-Araf-(1→ sugar residues, with the connection site located at the O-3 position.

[0055] In the NOESY spectrum, sugar residue A 1,3,5 H-1 (δ 5.03 ppm) and A 1,5 There is a coupling signal (A) at H-5 (δ 3.80 ppm) of the sugar residue 1,3,5 H-1 / A 1, 5 H-5), indicating that there is a connection between the →3,5)-α-L-Araf-(1→ and →5)-α-L-Araf-(1→ sugar residues, and the connection site is located at the O-5 position; sugar residue A 1,2,3,5 H-1 (δ 5.06 ppm) and A 1,5 There is a coupling signal (A) at H-5 (δ 3.71 ppm) of the sugar residue 1,2,3,5 H-1 / A 1, 5 H-5), indicating that there is a connection between the →2,3,5)-α-L-Araf-(1→ and →5)-α-L-Araf-(1→ sugar residues, and the connection site is located at the O-5 position; sugar residue A 1,3 H-1 (δ5.13 ppm) and A 1,2,3,5 The H-3 δ 79.92 ppm of the sugar residue has a coupling signal (A 1,3 H-1 / A 1,2,3,5 H-3), indicating the existence of a connection between the →3)-α-L-Araf-(1→ and →2,3,5)-α-L-Araf-(1→ sugar residues, with the connection site located at the O-3 position; sugar residue A 1,5 H-1 (δ 5.00 ppm) and sugar residue A 1,3,5 The H-5 (δ 3.75 ppm) has a coupling signal (A1,5 H-1 / A 1,3,5 H-5), indicating the existence of a connection between the →5)-α-L-Araf-(1→ and →3,5)-α-L-Araf-(1→ sugar residues, with the connection site located at the O-5 position; sugar residue α Gal t H-1 (δ 4.95 ppm) and A 1, 3 The H-3 (δ 3.86 ppm) of the sugar residue has a coupling signal ( α Gal t H-1 / A 1, 3 H-3), indicating that there is a connection between the α-D-Galp-(1→ and →3)-α-L-Araf-(1→ sugar residues, and the connection site is located at the O-3 position; sugar residue β Gal 1,4 H-1 (δ 4.55 ppm) and sugar residues β Gal 1,4 The H-4 (δ 4.08 ppm) has a coupling signal ( β Gal 1,4 H-1 / β Gal 1,4 H-4), indicating the existence of a connection between the →4)-β-D-Galp-(1→ and →4)-β-D-Galp-(1→ sugar residues, with the connection site located at the O-4 position.

[0056] Based on the above monosaccharide composition and methylation information, combined with the nuclear magnetic resonance HMBC and NOESY related signal information, a possible structural model is proposed: the main chain of the polysaccharide structure is composed of →4)-α-D-Glcp-(1→ sugar residues and →4)-β-D-Galp-(1→ sugar residues, and there is a branching site at the C-6 position of some →4)-α-D-Glcp-(1→. There may be two types of polysaccharide branched structures, one is α-D-Glcp-(1→, and the other is a polysaccharide composed of galactose and arabinose.

[0057] Example 3: Cell scratch test of uniform polysaccharide from Portulaca oleracea 1. Source of experimental materials: Human immortalized keratinocytes (Hacat cells) were obtained from Wuhan Pronocell Biotechnology Co., Ltd. The culture medium was: 90% DMEM high glucose + 10% FBS (fetal bovine serum).

[0058] 2. Experimental groups: Hacat cells were inoculated in six-well plates and divided into 5 groups: blank control group, model group, low-, medium-, and high-dose (1.25 μg / mL, 2.5 μg / mL, 5 μg / mL) Purslane homogeneous polysaccharide groups.

[0059] 3. Experimental Methods Experiment on the effect of Purslane homogeneous polysaccharide on the healing of Hacat cells after scratching: When the density of immortalized human keratinocytes reached 95% or more, the cells were cultured at 1×10 5 The cells were inoculated at a density of 100 μg / mL in a sterile, marked 6-well plate, and the 6-well plate was placed in a 37 °C, 5% CO2 incubator for 24 h. After the human immortalized keratinocytes were completely attached to the wall, each well was scratched with a pipette tip and the cells were washed with PBS. Then, they were intervened with 0, 1.25, 2.5, and 5 μg / mL of purslane homogeneous polysaccharide for 24 h. After the intervention, the old culture medium was discarded and gently rinsed with PBS 3 times, 3 min each time. The position changes of the cells were observed under a microscope and photographed.

[0060] 4. Model establishment and drug administration method Blank control group (control): The blank wells were scratched with a pipette tip and then no intervention was given.

[0061] Experimental group: (low, medium and high doses of Purslane homogeneous polysaccharide): Each well was scratched with a pipette tip and then Purslane homogeneous polysaccharide was administered at low doses (1.25 μg / mL), medium doses (2.5 μg / mL) and high doses (5 μg / mL). The administration was terminated after 24 hours.

[0062] 5. Experimental Results The comparison results before and after the scratch test are shown in Figure 4 Middle A. Figure 4 In figure A, the area between the two vertical lines indicates the scratch.

[0063] from Figure 4 As shown in Figure A, compared with the blank group, the healing area of ​​the purslane uniform polysaccharide treatment groups (1.25, 2.5, 5 μg / mL) increased significantly, with statistically significant differences (P<0.05), and was time- and concentration-dependent. This indicates that the Hacat cells intervened by purslane uniform polysaccharide can enhance the migration ability of cells.

[0064] Depend on Figure 4 A is calculated Figure 4 The healing rate after scratching shown in B is calculated as follows: healing rate % = 100% - (scratch area at the end / scratch area at the beginning of the experiment) %.

[0065] from Figure 4 As shown in Figure B, compared with the blank control group, the healing rates of the purslane uniform polysaccharide treatment groups (1.25, 2.5, 5 μg / mL) were all higher than those of the blank control group, and there were significant differences between the medium and high dose groups of purslane ( * P<0.05, **P<0.01), and the results showed a good dose dependence.

[0066] Example 4: Experiment on repairing skin barrier damage in mice by homogeneous purslane polysaccharide 1. Source of experimental materials: 36 ICR mice (female, 6-8 weeks old) were provided by Liaoning Changsheng Biotechnology Co., Ltd.; Portulaca oleracea plants were provided by Yunnan Qiancaoyuan Pharmaceutical Co., Ltd.

[0067] 2. Experimental groups: ICR mice were randomly divided into 6 groups, 6 mice in each group, namely blank control group, model group, positive control group and low, medium and high dose (50 mg / kg, 100 mg / kg, 200 mg / kg) groups.

[0068] 3. Experimental Methods Mouse damaged skin model: 30 healthy female ICR mice weighing 20-30 g were selected and intraperitoneally injected with 1.0 mL of sodium pentobarbital aqueous solution. After the mice entered anesthesia, they were fixed and the hair in a circular area with a diameter of about 3 cm in the center of their backs was cut. After disinfection with povidone iodine, a circular skin tissue with a diameter of 1.5 cm was cut from the depilated area and exposed to the air without any interference to establish a mouse skin damage model.

[0069] Blank control group (control): no modeling, free drinking water, and oral administration of sterile water.

[0070] Model group (model): A skin damage model was established according to the modeling method.

[0071] Positive group (mometasone furoate cream, MFC): The skin damage model was established according to the method of the model group, and mometasone furoate cream was applied from the day of model establishment, 200 μL per mouse.

[0072] Experimental group (low-, medium- and high-dose groups of Purslane homogeneous polysaccharide): The skin damage model was established according to the method of the model group. Starting from the day the model was established, low-dose (50 mg / kg body weight), medium-dose (100 mg / kg body weight) and high-dose (200 mg / kg body weight) of Purslane homogeneous polysaccharide (Purslane homogeneous polysaccharide was prepared by adding sterile water), 200 μL per mouse.

[0073] 4. Specific sampling operations On the 20th day, the transepidermal water loss of each mouse was measured using a TE007 Swiss SKT skin water loss instrument. On the 21st day, which was the end of the experiment, the mouse serum was collected and the expression levels of FLG and HA in the mouse serum were determined by ELISA; the damaged skin was cut off and the HE, Masson, and toluidine blue staining of the mouse tissue sections in each group were compared.

[0074] 5. Experimental Results Figure 5 A and B are the contents of HA and FLG detected by serum indicators. Figure 5 As shown in A and B, the contents of both in the model group decreased significantly compared with the blank group ( # P<0.05, ### P<0.001). Compared with the model group, the contents of HA and FLG in the high, medium and low dose groups of Portulaca oleracea uniform polysaccharide were increased, and the difference was statistically significant ( * P<0.05, ** P<0.01, *** P<0.001). This indicates that the administration of purslane homogeneous polysaccharide can repair the production of hyaluronic acid and filaggrin in mice.

[0075] Figure 5 C is the TEWL value of mouse skin. Transepidermal Water Loss (TEWL), also known as transdermal water loss, is a commonly used indicator reflecting the barrier function of the skin stratum corneum and is also the most intuitive value reflecting the skin barrier repair ability. Figure 5 As can be seen from C, the model group values ​​were significantly higher than those of the blank control group ( ### P<0.001). After low, medium and high doses of Purslane homogeneous polysaccharide were given, as well as in the positive group, the values ​​gradually decreased, and the difference was statistically significant ( * P<0.05, ** P<0.01, *** P<0.001), and showed a good dose-dependency.

[0076] Figure 6 The following are the pictures of the skin tissue sections of mice in each group stained with hematoxylin-eosin (HE), Masson (Masson), and toluidine blue. Figure 6HE staining showed that after modeling, the model group had inflammatory cell infiltration, swelling and thickening of the epidermis and dermis, fewer collagen fibers in the dermis, twisting, breaking and disordered arrangement, and damaged skin barrier, while the positive group and low, medium and high dose groups of purslane homogeneous polysaccharide were significantly improved compared with the model group. Masson staining showed that after modeling, the collagen fibers in the dermis of the model group were loose and unevenly distributed, while the collagen fibers in the positive group and low, medium and high dose groups of purslane homogeneous polysaccharide and the control group were more regular and basically returned to normal, the collagen fibers were significantly thickened and widened, and the collagen fibers in the high dose group and the positive group were arranged regularly and orderly, and the morphology was similar to that of the blank control group. Toluidine blue staining showed that after modeling, the model group had a large number of mast cells, and the number of mast cells in the positive group and low, medium and high dose groups of purslane homogeneous polysaccharide was reduced to a certain extent compared with the model group. Among them, the number of mast cells in the high dose group was significantly reduced and basically returned to normal.

[0077] Figure 5 and Figure 6 The results showed that the model group was successfully established and the skin barrier of the mice in the experimental group was significantly repaired, indicating that Purslane homogeneous polysaccharide can repair the skin barrier damage of model mice.

[0078] Example 5: Application of Purslane Homogeneous Polysaccharide The purslane homogeneous polysaccharide obtained in Example 1 was applied to a cosmetic facial cream, and the proportions of the components in the cream formula were shown in Table 3 below: Table 3. Facial cream ingredients

[0079] Preparation method of cosmetic cream: heat phase A raw material and phase B raw material to 80℃~85℃ respectively, after the two phases are completely dissolved and stirred evenly, transfer phase B raw material to phase A, place in a homogenizer at 5000 rpm for homogenization for 5 min, stir and cool to 40℃~45℃ after mixing evenly, then add phase C raw material, continue stirring until uniform, and prepare cosmetic cream containing purslane uniform polysaccharide.

[0080] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A purslane homogeneous polysaccharide, characterized in that: The purslane homogeneous polysaccharide has a structure represented by the following general formula: ， Among them, n=8, m=19.

2. The purslane homogeneous polysaccharide according to claim 1, characterized in that: The highest peak molecular weight Mp, weight average molecular weight Mw and number average molecular weight Mn of the purslane homogeneous polysaccharide are 6.4 kDa, 7.4 kDa and 5.3 kDa respectively.

3. The purslane homogeneous polysaccharide according to claim 1, characterized in that: The purslane uniform polysaccharide is mainly composed of glucose, galactose and arabinose, accounting for 21.54%, 62.98% and 13.21% respectively.

4. The purslane homogeneous polysaccharide according to claim 1, characterized in that: The purslane homogeneous polysaccharide is obtained from the genus Portulaca of the family Portulacaceae. Portulaca oleracea The dried aerial parts of L. were extracted, separated and purified.

5. Use of the Portulaca oleracea homogeneous polysaccharide according to any one of claims 1 to 4 in cosmetics, food or medicine.

6. A cosmetic, characterized in that: The cosmetic comprises the Portulaca oleracea homogeneous polysaccharide according to any one of claims 1 to 4.

7. The cosmetic according to claim 6, characterized in that The cosmetic comprises phase A, phase B and phase C, wherein phase A comprises the following components by mass percentage: water, 72.05%; Glycerol, 3.0%; Pentylene glycol, 2.0%; Acrylates / C10-30 Alkyl Acrylate Crosspolymer, 0.25%; p-Hydroxyacetophenone, 0.3%; The phase B comprises the following components by mass percentage: Cetearyl alcohol, cetearyl glucoside, 2.0%; Polydimethylsiloxane, 3.0%; Glyceryl tri(ethylhexanoate), 2.0%; Glyceryl stearate, 1.5%; Squalane, 2.0%; Steareth-21, 0.2%; The C phase includes the following components by mass percentage: Water, 10.0%; Bis-PEG-18 methyl ether dimethyl silane, 1.0%; Arginine, 0.2%; Purslane homogeneous polysaccharide, 0.5%.

8. The method for preparing the uniform purslane polysaccharide according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step S1, crushing purslane and extracting crude purslane polysaccharide by water extraction and alcohol precipitation method; Step S2, subjecting the crude purslane polysaccharide to ion exchange column chromatography to obtain primary purslane polysaccharide; Step S3, subjecting the purslane polysaccharide to gel filtration column chromatography to obtain purslane homogeneous polysaccharide.

9. The method for preparing uniform purslane polysaccharide according to claim 8, characterized in that: The step S1 is specifically as follows: adding 20 times the weight of deionized water to the purslane, heating and stirring under reflux extraction at 95°C for two hours, concentrating the obtained extract and centrifuging at a speed of 11000 r / min for 10 minutes, taking the supernatant, and then adding 4 times the volume of 95% ethanol to the supernatant, precipitating at 4°C for 12 hours, centrifuging at a speed of 11000 r / min for 10 minutes, and drying the obtained precipitate to obtain the crude purslane polysaccharide; The step S2 specifically comprises: loading the crude purslane polysaccharide onto a DEAE-52 column, using a NaCl aqueous solution as an eluent, eluting and separating in the concentrations of 0.1 M NaCl, 0.2 M NaCl, 0.3 M NaCl, and 0.4 M NaCl in sequence, with an elution rate of 15 mL / min, collecting the eluate, collecting 20 tubes of each eluate gradient, and 10 mL per test tube, wherein the eluate from the 6th to 18th tubes of the 0.1 M NaCl elution position is concentrated and dried under reduced pressure to obtain the crude purslane polysaccharide; The step S3 is specifically as follows: after loading the purslane polysaccharide onto a dextran G-75 column, eluting with ultrapure water, the column flow rate is 2.0 mL / min, and collecting the eluate, 5 mL per test tube, wherein the 10th to 14th tubes are dried to obtain purslane uniform polysaccharide.

10. The method for preparing uniform purslane polysaccharide according to claim 8, characterized in that: In step S2, the crude purslane polysaccharide is dissolved into a 50 mg / mL aqueous solution by distillation, and then centrifuged at 8000 rpm for 10 min, and then filtered with a 0.45 μm microporous filter membrane, and the filtrate is loaded onto a DEAE-52 column; In step S3, the purslane polysaccharide is dissolved into a 50 mg / mL aqueous solution by distillation, and then centrifuged at 8000 rpm for 10 min, and then filtered with a 0.45 μm microporous filter membrane, and the filtrate is loaded onto a dextran G-75 column with a specification of φ2.6×100 cm.

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