Purified polysaccharide from pyracantha fruit and its preparation method and application

Abstract

The present invention discloses a purified polysaccharide from Pyracantha fortuneana fruit, and a preparation method and application thereof. The present invention further separates and purifies the Pyracantha fortuneana fruit polysaccharide extracted by hot water extraction using a DEAE ion chromatography column, thereby obtaining a purified polysaccharide from Pyracantha fortuneana fruit having significant anti-photodamage activity. The purified polysaccharide from Pyracantha fortuneana fruit provided by the present invention can play a protective role against photodamage by significantly reducing the apoptosis level of HaCaT cells damaged by UVB, significantly improving the oxidative stress state in UVB-damaged skin cells, and inhibiting the decrease in the intracellular mitochondrial membrane potential level caused by UVB damage. The purified polysaccharide from Pyracantha fortuneana fruit provided by the present invention has a significant anti-photodamage effect, and can be used to prepare products with anti-photodamage effect, including but not limited to the preparation of drugs, functional foods or cosmetics with anti-photodamage effect.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines and relates to a purified polysaccharide of pyracantha fruit and a preparation method and application thereof. Background Art

[0002] Pyracantha fortuneana belongs to the genus Pyracantha in the family Rosaceae. Its fruit, Pyracantha fortuneana, is also known as Torch Fruit, Rescue Army Food, Chiyangzi, and Shuichazi. It has the effects of eliminating stagnation and stopping dysentery, promoting blood circulation and stopping bleeding. It is often used to treat indigestion, enteritis, dysentery, infantile malnutrition, metrorrhagia, leucorrhea, postpartum abdominal pain, etc. Pyracantha fortuneana is rich in nutrients and contains a large number of biologically active ingredients, such as phenolic acids; flavonoids; polysaccharides; fatty acids; trace metal elements; volatile components such as aldehydes, ketones, alcohols, alkanes, alkenes, esters, and terpenes. At present, more than 100 compounds have been isolated and identified from Pyracantha fortuneana.

[0003] Pyracantha fruit contains rich polysaccharide components. Wang Xiaojing et al. obtained three polysaccharides with different molecular weights mainly composed of pyranose configurations by graded alcohol precipitation (Wang Xiaojing, Chen Lihua, Xiang Mingfang. Analysis of the anti-oxidative rancidity of Pyracantha fruit polysaccharides [J]. Food and Fermentation Industries, 2016, 42(05): 175-179.); Zhang Haisheng et al. used different separation methods to separate six polysaccharide components with different compositions from Pyracantha fruit (Zhang Haisheng, Duan Yufeng, Zhou Fang, et al.). al. Extraction, separation and purification of Pyracantha fortuneana water-soluble polysaccharides [J]. Food Science, 2005, (01): 157-161.); Huang Yongchun et al. analyzed the components of Pyracantha fortuneana fruit polysaccharides PP-A2 and PP-A3 purified by DE-52 column chromatography. The results showed that PP-A3 was mainly composed of arabinose, glucose and fructose, with a molecular weight of about 210 kDa, and PP-A2 was mainly composed of arabinose, rhamnose, fructose, galactose and mannose, with a molecular weight of about 30 kDa (Huang Yongchun, Yang Feng, Duan Yufeng. Study on the physicochemical properties of Pyracantha fortuneana water-soluble polysaccharide PP-A3 [J]. Food Research and Development, 2007, (08): 75-79. / Huang Yongchun, Yang Feng, Xie Qingruo, et al. Study on the physicochemical properties of Pyracantha fortuneana water-soluble polysaccharide PP-A2. Food Science and Technology, 2007, (06): 116-119.).

[0004] At present, the research on the active effects of Pyracantha fruit polysaccharides mainly focuses on anti-oxidation, immune inflammation regulation, anti-cancer and other aspects. The research reports on the composition of polysaccharide components and their potential biological activity effects are still incomplete. Therefore, in-depth research on the polysaccharide component composition and biological activity of Pyracantha fruit polysaccharides is of great significance and value for exploring its medicinal potential and promoting the development of related products. Summary of the invention

[0005] The purpose of the present invention is to provide a purified polysaccharide from Pyracantha fortuneana fruit and a preparation method and application thereof. The present invention further separates and purifies the polysaccharide from Pyracantha fortuneana fruit extracted by hot water extraction using a DEAE ion chromatography column to obtain a purified polysaccharide from Pyracantha fortuneana fruit with significant anti-photodamage activity.

[0006] According to one aspect of the present invention, there is provided a method for preparing purified polysaccharide from Pyracantha fortuneana fruit, comprising the following steps:

[0007] The pyracantha fruit is crushed and mixed with water, and subjected to heating reflux extraction for 2 to 3 times, each time for 2 to 4 hours, at an extraction temperature of 80 to 100° C., the extracts are combined, and the extracts are concentrated and then depigmented and deproteinized to obtain pyracantha fruit polysaccharide; wherein the mass volume ratio of pyracantha fruit to water can be 1: (20 to 30) mg / mL;

[0008] The Pyracantha fruit polysaccharide is prepared into a Pyracantha fruit polysaccharide aqueous solution, and then separated by DEAE ion column chromatography, gradient eluted with a NaCl solution, and the eluate obtained by eluting with a 0.15 mol / L NaCl solution is collected, purified, and dried to obtain the product.

[0009] The purified polysaccharide of Pyracantha fortuneana fruit provided by the present invention can significantly reduce the apoptosis level of HaCaT cells damaged by UVB, significantly improve the oxidative stress state in UV-damaged skin cells, inhibit the decrease of intracellular mitochondrial membrane potential level caused by UVB damage, and exert a light damage protection effect by inhibiting the activation of NLRP3 and Caspase-1 proteins and the phosphorylation of P38, ERK and JNK proteins; in addition, it can also significantly repair the tight junction proteins Occludin and ZO-1 in UV-damaged HaCaT cells, restore their expression levels to normal levels, thereby protecting the cell barrier damage caused by UVB.

[0010] The purified polysaccharide of Pyracantha fortuneana fruit provided by the present invention has significant anti-photodamage effect and can be used to prepare products with anti-photodamage effect, including but not limited to being used to prepare drugs, functional foods or cosmetics with anti-photodamage effect.

[0011] In some embodiments, depigmentation treatment of the concentrated extract may include the following steps: adding D354F macroporous resin to the concentrated extract, stirring for 2 to 4 hours at a temperature of 50 to 60°C, filtering, collecting the filtrate, precipitating the filtrate with 3 to 5 times the volume of 95% ethanol, collecting the precipitate, and drying to obtain Pyracantha fruit crude polysaccharide.

[0012] In some embodiments, the volume ratio of the concentrated extract to the D354F macroporous resin can be 1:1.

[0013] In some embodiments, the deproteinization treatment may include the following steps: preparing a 5 mg / mL pyracantha fruit crude polysaccharide solution with water, adding 3 to 5 times the volume of Sevag solution for deproteinization, the oscillation time is 25 to 35 minutes, the deproteinization times are 4 to 6 times, and then collecting the aqueous phase polysaccharide solution, adding ethanol to a final ethanol concentration of 80% for alcohol precipitation, collecting the precipitate, and drying to obtain pyracantha fruit polysaccharide.

[0014] In some embodiments, the Sevag solution is prepared by mixing chloroform and n-butanol in a volume ratio of 1:4.

[0015] In some embodiments, gradient elution with a NaCl solution may be performed by sequentially eluting with water, a NaCl solution having a concentration of 0.025 mol / L, 0.05 mol / L, and 0.15 mol / L.

[0016] In some embodiments, purifying the eluate may include the following steps: the eluate is concentrated and then dialyzed using a dialysis bag with a molecular weight cutoff of 8000 Da.

[0017] In some embodiments, the method for preparing purified polysaccharide from Pyracantha fortuneana fruit may specifically include the following steps:

[0018] The pyracantha fruit was crushed and mixed with water, and then heated and refluxed for extraction for 3 times, each time for 3 hours, at an extraction temperature of 100°C, and the extracts were combined; wherein the mass volume ratio of pyracantha fruit to water was 1:25 mg / mL;

[0019] The extract was concentrated to 1 / 20 of the original volume, and then an equal volume of D354F macroporous resin was added, stirred at 55°C for 3 hours, filtered, and the filtrate was collected. The filtrate was precipitated with 4 times the volume of 95% ethanol by mass fraction, and the precipitate was collected and dried to obtain Pyracantha fortuneana fruit crude polysaccharide;

[0020] The crude polysaccharide of Pyracantha fruit is prepared into a crude polysaccharide solution of Pyracantha fruit with water at a concentration of 5 mg / mL, and 4 times the volume of Sevag solution prepared by mixing chloroform and n-butanol in a volume ratio of 1:4 is added for deproteinization, the oscillation time is 25 to 35 minutes, and the deproteinization times are 5 times, then the polysaccharide solution in the aqueous phase is collected, ethanol is added to a final ethanol concentration of 80% for alcohol precipitation, and the precipitate is collected and dried to obtain the Pyracantha fruit polysaccharide;

[0021] The Pyracantha fruit polysaccharide is prepared into a Pyracantha fruit polysaccharide aqueous solution, and then separated by DEAE ion column chromatography, eluted with water, 0.025 mol / L, 0.05 mol / L, and 0.15 mol / L NaCl solutions in sequence, and the eluate obtained by eluting with 0.15 mol / L NaCl solution is collected, the eluate is concentrated under reduced pressure to 1 / 10 of the original volume, and dialyzed using a dialysis bag with a molecular weight cutoff of 8000 Da, and the dialyzed solution is freeze-dried to obtain the product.

[0022] Thus, the obtained Pyracantha fruit purified polysaccharide does not contain starch components, has a protein content of 0.26%, and a polysaccharide content of 75.16%. It is an acidic polysaccharide mainly composed of glucose, arabinose, galactose, xylose and galacturonic acid in a molar ratio of 1:4.15:2.97:1.34:1.89, among which arabinose, galactose and galacturonic acid are its main components. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the DEAE ion column chromatography elution curve of Pyracantha fortuneana polysaccharide;

[0024] Figures 2 to 4 The gel permeation chromatograms of PPFP-Fr.3, PPFP-Fr.4, and PPFP-Fr.5 are shown in order;

[0025] Figure 5 It is the infrared spectra of PPFP-Fr.3, PPFP-Fr.4 and PPFP-Fr.5;

[0026] Figure 6 These are SEM images of PPFP-Fr.3, PPFP-Fr.4, and PPFP-Fr.5;

[0027] Figure 7 is the total ion current chromatogram of PPFP-Fr.4;

[0028] Figure 8 Total ion current profiles were analyzed for PPFP-Fr.4 methylation results;

[0029] Fig. 9 is the 1H spectrum of PPFP-Fr.4;

[0030] Fig.10 is the 13C spectrum of PPFP-Fr.4;

[0031] Fig.11 HSQC (red) and HMBC (green) spectra of PPFP-Fr.4;

[0032] Fig.12COSY (red) and NOESY (green) spectra of PPFP-Fr.4;

[0033] Fig.13 It is the possible partial structural composition of PPFP-Fr.4;

[0034] Fig.14 The effects of different concentrations of PPFP-Fr.3, PPFP-Fr.4, and PPFP-Fr.5 on the survival rate of HaCaT cells;

[0035] Fig.15 The effect of different concentrations of PPFP-Fr.3, PPFP-Fr.4, and PPFP-Fr.5 on the survival rate of photodamaged HaCaT cells; in the figure, compared with the model group, *** P < 0.001, ** P<0.01, * P < 0.05;

[0036] Figures 16-17 The effect of PPFP-Fr.4 on apoptosis of HaCaT cells damaged by UVB. Fig.17 Compared with the model group, *** P < 0.001, ** P<0.01, * P < 0.05;

[0037] Fig.18 The effect of PPFP-Fr.4 on the expression level of antioxidant factors in HaCaT cells damaged by UVB; in the figure, compared with the model group, *** P < 0.001, ** P<0.01, * P < 0.05;

[0038] Fig.19 The effect of PPFP-Fr.4 on the mitochondrial membrane potential (MMP) level of HaCaT cells damaged by UVB; in the figure, compared with the model group, *** P < 0.001, * P < 0.05;

[0039] Fig. 20 The effect of PPFP-Fr.4 on the expression level of NLRP3 pathway proteins in HaCaT cells damaged by UVB; in the figure, compared with the model group, *** P < 0.001, ** P<0.01, * P < 0.05;

[0040] Fig.21The effect of PPFP-Fr.4 on the expression level of MAPK signaling pathway proteins in HaCaT cells damaged by UVB; in the figure, compared with the model group, *** P < 0.001, ** P<0.01, * P < 0.05;

[0041] Fig. 22 The effect of PPFP-Fr.4 on the expression level of Tjs protein in HaCaT cells damaged by UVB; in the figure, compared with the model group, *** P < 0.001, ** P<0.01, * P<0.05. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with the embodiments. The examples are only for explanation and are not intended to limit the present invention in any way. Unless otherwise specified, the raw materials and reagents used in the examples are conventional products that can be obtained commercially; the experimental methods in the examples where specific conditions are not specified are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0043] The Pyracantha fortuneana fruit used in the embodiments of the present invention was purchased from Bozhou Zhuoran Scented Tea Trading Co., Ltd.

[0044] Example 1 Preparation and structural characterization of purified polysaccharide from Pyracantha fortuneana fruit

[0045] 1. Preparation of Pyracantha fortuneana polysaccharide

[0046] Pyracantha fortuneana fruit polysaccharides were extracted using hot water extraction method.

[0047] The dried fruit of Pyracantha was crushed and passed through a 20-mesh sieve. Water was added at a mass volume ratio of 1:25 mg / mL of Pyracantha fruit to water. After mixing, the mixture was extracted by boiling water reflux for 3 hours. The extraction was repeated twice. The supernatant was combined and concentrated to about 1 / 20 of the original volume using a rotary evaporator. Then, an equal volume of D354F macroporous resin (purchased from Guangzhou Qianhui Reagent Company) was added. After stirring evenly, the mixture was placed at 55°C and stirred for 3 hours to fully remove the pigment in the polysaccharide. The decolorized polysaccharide liquid was collected and precipitated with 4 times the volume of 95% ethanol. The crude polysaccharide of Pyracantha fruit was obtained after precipitation and drying.

[0048] The crude polysaccharide of Pyracantha fruit was re-dissolved in water to 5 mg / mL, and deproteinized by mixing thoroughly with 4 volumes of Sevag solution (chloroform: n-butanol = 1:4; V / V). Each oscillation was performed for 25 to 35 minutes. After repeated deproteinization for 5 times, the aqueous phase polysaccharide solution was collected, and the deproteinized polysaccharide was collected by alcohol precipitation method (final ethanol concentration 80%), which was Pyracantha fruit polysaccharide.

[0049] The polysaccharide content was determined by the phenol-sulfuric acid method, which specifically includes the following steps:

[0050] Dissolve 1 mg of the sample to be tested in distilled water to 1 mL to obtain the test solution. Add 1 mL of distilled water and 1 mL of 5% phenol to the test solution and mix well. Then slowly add 5 mL of concentrated sulfuric acid. After shaking well, place it in a fume hood and let it stand for 30 minutes. Measure its absorbance at 490 nm. Use glucose as the standard, with its concentration as the horizontal axis, A 490 The standard curve (Y = 11.256X-0.0106, R 2 =0.9971) is used to determine the polysaccharide content of the sample to be tested. The polysaccharide content in the sample is calculated as follows:

[0051]

[0052] The polysaccharides in Pyracantha fruit were extracted by hot water extraction method, and the content of crude polysaccharides after concentration and D354F decolorization was measured, and the content of crude polysaccharides in Pyracantha fruit was 44.59%; the content of deproteinized polysaccharides was further measured, and the content of deproteinized polysaccharides (PPFP) in Pyracantha fruit was 65.50%.

[0053] 2. Isolation and purification of Pyracantha fortuneana polysaccharides

[0054] DEAE-52 cellulose column filler (purchased from Desit Bio) was used to analyze the components with different charge properties in Pyracantha fruit polysaccharide. Before loading the column, the filler was pretreated with 0.5 mol / L hydrochloric acid solution and 0.5 mol / L sodium hydroxide solution according to the instructions. After treatment, the filler was degassed by vacuum rotary evaporation at room temperature and loaded into the column (2.6×30 cm). After the column was compacted and stabilized, the flow rate of the constant flow pump was adjusted to 5-6 s / drop. After the flow rate was balanced, the constant flow pump was turned off and a solution with 80 mg of Pyracantha fruit polysaccharide sample fully dissolved was added in small amounts and multiple times, and the sample solution was allowed to stand until the sample solution was balanced.

[0055] The Pyracantha fruit polysaccharide PPFP was eluted with distilled water, 0.025mol / L, 0.05mol / L, 0.15mol / L, 0.3mol / L, 0.5mol / L, and 0.7mol / L gradient concentrations of NaCl solution. An automatic collector was used to collect about 4-5mL per tube, and 100 tubes were collected for each component. The sugar content was determined every 3-5 tubes using the phenol-sulfuric acid method and an elution curve was drawn. The eluents obtained from each component were collected separately, concentrated under reduced pressure to about 1 / 10 of the original volume, and dialyzed with 8000Da dialysis bag running water for 48 hours, and then dialyzed with distilled water for 24 hours, and fresh distilled water was replaced every 2 hours. The dialyzed solution was freeze-dried to obtain purified polysaccharides of different components.

[0056] The elution curve is Figure 1 After elution, 7 components were obtained, namely PPFP-Fr.1 (1.68%), PPFP-Fr.2 (7.45%), PPFP-Fr.3 (23.60%), PPFP-Fr.4 (29.53%), PPFP-Fr.5 (0.89%), PPFP-Fr.6 (1.35%), and PPFP-Fr.7 (0.18%). Among them, PPFP-Fr.4 is the purified polysaccharide from Pyracantha fortuneana fruit of the present invention.

[0057] 3. Determination of polysaccharide molecular weight and homogeneity

[0058] The molecular weight distribution of PPFP-Fr.3, PPFP-Fr.4, and PPFP-Fr.5 was determined by high performance gel permeation liquid chromatography, and the molecular weight uniformity of each component polysaccharide was determined based on the peak shape. Each group of purified polysaccharides was dissolved in pure water to 2 mg / mL, filtered through a 0.22 μm aqueous phase filter, and then the molecular weight of the polysaccharide was determined at 35 °C using a Thermo Ultimate3000 high performance liquid chromatograph equipped with a RefractoMax 521 differential detector. The detection column was an Ultrahydrogel 1000 (7.8×300 mm) and Ultrahydrogel 500 (7.8×300 mm) gel column connected in series with an Ultrahydrogel (6×40 mm) guard column, the mobile phase was 0.02 mol / L potassium dihydrogen phosphate buffer solution, the injection volume was 20 μL, the flow rate was 0.8 mL / min, and the data were processed using Chromeleon software. The values ​​obtained from the sample determination were substituted into the 5200-668000Da series of dextran standard curves to calculate the molecular weight and distribution of each component polysaccharide.

[0059] The results are as follows Figures 2 to 4 As shown in the figure, the peak after 25 minutes is the impurity peak and solvent peak generated by the chromatographic column.

[0060] Among the purified polysaccharide components of Pyracantha fruit, PPFP-Fr.3 and PPFP-Fr.4 are single peaks with good symmetry and narrow peaks, while PPFP-Fr.5 is a single peak with a wider peak. The peak molecular weight Mp corresponding to each peak of PPFP-Fr.3, PPFP-Fr.4, and PPFP-Fr.5 are 22528Da, 72711Da, and 90269Da, respectively, the weight average molecular weight Mw are 28299Da, 100855Da, and 307672Da, respectively, and the dispersion coefficients PD are 1.60, 1.91, and 4.36, respectively.

[0061] 4. Determination of the physicochemical properties of polysaccharides

[0062] (1) Determination of total sugar content

[0063] The total sugar content of PPFP-Fr.3, PPFP-Fr.4 and PPFP-Fr.5 was determined by phenol-sulfuric acid method. The results are shown in Table 1.

[0064] (2) Determination of protein content

[0065] The protein content in PPFP-Fr.3, PPFP-Fr.4, and PPFP-Fr.5 was determined by referring to the instructions of the BCA protein quantification kit. The polysaccharide samples were diluted to different concentrations, mixed with the BCA working solution in the kit, and then placed at 37°C for a constant temperature reaction for 30 minutes, and the absorbance was measured at 562nm. Bovine serum albumin was used as the standard to draw a standard curve, and the protein content in each sample was calculated by substituting the sample absorbance value. The results are shown in Table 1.

[0066] (3) Determination of uronic acid content

[0067] The sulfuric acid-carbazole method was used to determine the uronic acid content in PPFP-Fr.3, PPFP-Fr.4, and PPFP-Fr.5. Using carbazole as an indicator, the uronic acid in the polysaccharide can react with sulfuric acid to generate a purple-red compound with a carboxyl or aldehyde group, and the absorbance of the product at A530 is proportional to the uronic acid content in the sample.

[0068] Take 200 μL of polysaccharide sample of appropriate concentration into a test tube, add 5 mL of 12.5 mmol / L sodium tetraborate-sulfuric acid working solution after adding water to 1 mL, mix well and react in a boiling water bath for 20 minutes. After cooling, add 0.2 mL of 0.15% carbazole ethanol solution, mix well and continue to boil in a water bath for 5 minutes, react at room temperature for 2 hours, and measure its absorbance at 530 nm. Use galacturonic acid as the standard to draw a standard curve (Y=8.812X+0.0038), and the uronic acid content in the sample is calculated as galacturonic acid equivalent.

[0069] The results are shown in Table 1.

[0070] (4) Determination of starch content

[0071] The I-KI method was used to detect the presence of starch in PPFP-Fr.3, PPFP-Fr.4, and PPFP-Fr.5. Each group of samples with a concentration of 1 mg / mL was prepared, and I2-KI solution (1 g I2 and 10 g KI were mixed and then distilled water was added to 100 mL) was slowly added dropwise, and the mixture was allowed to stand at room temperature for 10 min while observing the color change of the mixture. The results are shown in Table 1.

[0072] Table 1 Physicochemical properties of purified polysaccharides

[0073] Polysaccharide samples Polysaccharide content% Uric acid content% Protein content% Starch content PPFP-Fr.3 70.30％ 11.51％ 0.59％ - PPFP-Fr.4 75.16％ 17.19％ 0.26％ - PPFP-Fr.5 74.17％ 18.35％ 0.68％ -

[0074] Note: “-” means it has not been detected yet.

[0075] The results showed that the polysaccharide content of each group of purified polysaccharides was greater than 70%, with high purity, the protein content of each group of samples was less than 1%, and the I-KI test results showed that each group of samples did not contain starch.

[0076] 5. FT-IT infrared analysis

[0077] Weigh 2 mg of PPFP-Fr.3, PPFP-Fr.4, and PPFP-Fr.5 polysaccharide powders respectively, mix them thoroughly with appropriate amount of KBr powder, grind them, and then evenly distribute them on the sample table by tablet pressing, making sure they fit the platform as closely as possible to avoid air layers. -1 Infrared scanning within range.

[0078] The results are as follows Figure 5 As shown, each group of samples has a wavelength of 3200-3600cm -1 The characteristic absorption peak is the common intermolecular -OH stretching vibration peak in polysaccharides, 2500-3200cm -1 The characteristic absorption peak is the -CH stretching vibration peak. In addition, the 1400-1800cm -1 The absorption peak at is usually produced by the asymmetric stretching vibration of the -COOH group, as well as the stretching vibration of COH and COC. Each group of polysaccharides has different degrees of absorption values ​​within this absorption range, which means that there are different degrees of carboxyl substitution on the monosaccharides of each group of polysaccharides.

[0079] 800-1200cm -1 This is the "fingerprint region" of polysaccharides. The absorption peaks in this range can usually reflect the information of the monosaccharide configuration in the polysaccharide structure. -1 Absorption vibration is usually caused by the stretching vibration of C-OH and COC bonds in the sugar ring. The purified polysaccharide samples in each group have more absorption peaks in this range, which may be related to the complexity of the composition of the sugar units in their structure. -1 and 890cm -1 The absorption peak may be related to the axial position configuration of C1-OH in the β-pyranose ring.

[0080] 6. Polysaccharide SEM analysis

[0081] Take appropriate amount of PPFP-Fr.3, PPFP-Fr.4, and PPFP-Fr.5 polysaccharide powders and fix them on metal plates with insulating glue, place them in vacuum for sample gold spraying, and use high-resolution scanning electron microscope to scan the surface morphology of the samples to observe the surface morphology differences of the polysaccharides. Figure 6 shown.

[0082] Depend on Figure 6 The results showed that among the three purified polysaccharides from Pyracantha fortuneana, PPFP-Fr.4 was mainly composed of large flake structures with a relatively smooth surface and few pores, while PPFP-Fr.3 showed rod-like and flake structures, some of which were curled up to present a columnar structure, and its surface roughness was greater than that of PPFP-Fr.4. The surface morphology of PPFP-Fr.5 was the most fragmented among the three purified polysaccharides, presenting a filamentous and porous flake structure, and some of which formed a spherical structure with a multi-synaptic morphology on the surface.

[0083] 7. Structural analysis of PPFP-Fr.4

[0084] (1) Monosaccharide composition analysis

[0085] The monosaccharide composition in PPFP-Fr.4 was determined using an ion chromatograph equipped with an electrochemical detector.

[0086] Sample hydrolysis: Add 4 mL of 2M TFA to 5 mg of PPFP-Fr.4, hydrolyze at 120°C for 3 h, and then use a nitrogen blower to blow dry the hydrolyzed sample. Add 5 mL of deionized water to reconstitute, dilute 20 times, centrifuge at 12000 rpm for 5 min, and take the supernatant to filter the membrane for analysis.

[0087] The chromatographic method is as follows:

[0088] Chromatographic column: Dionex CarbopacTM PA20 (3×150 mm);

[0089] The mobile phase consisted of A: H2O and B: 15 mM NaOHC, 15 mM NaOH (prepared from 100 mM NaOAC);

[0090] The sample flow rate was 0.3 mL / min;

[0091] The injection volume was set to 5 μL;

[0092] The column temperature was set at 30 °C.

[0093] Fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, and mannuronic acid were used as mixed standards to qualitatively analyze the monosaccharide composition in PPFP-Fr.4, and the molar ratio was calculated based on the molar mass of the monosaccharides.

[0094] The results are as follows Figure 7 shown.

[0095] By comparing with the standard, it can be seen that PPFP-Fr.4 is mainly composed of glucose (Glucose, Glc), arabinose (Ara), galactose (Gal), xylose (Xyl) and galacturonic acid (GalA), with a molar ratio of about 1:4.15:2.97:1.34:1.89. According to the analysis of the monosaccharide composition results, the main monosaccharides in PPFP-Fr.4 are Ara (36.52%) and Gal (26.17%), accounting for more than 50% of the total components. Other monosaccharide types GalA, Xyl and Glc account for a similar proportion in PPFP-Fr.4. The acidic sugar galacturonic acid (GalA) in PPFP-Fr.4 is relatively rich. Based on the comprehensive analysis of the monosaccharide composition, PPFP-Fr.4 can be classified as an acidic heteropolysaccharide, and a large amount of GalA in its composition may come from the pectin components present in the fruit cell wall.

[0096] (2) Methylation and GC-MS analysis

[0097] Polysaccharides are composed of a variety of basic monosaccharide units connected by a variety of glycosidic bonds. Therefore, in the process of analyzing the structure of polysaccharides, in addition to determining the proportion of monosaccharide composition, it is also necessary to understand the types of glycosidic bonds and their connection methods. Methylation analysis is currently the most commonly used method for glycosidic bond detection. Its working principle is to methylate polysaccharides, then hydrolyze and derivatize them to obtain partially methylated alditol acetate (PMAA). PMAA is analyzed by gas chromatography-mass spectrometry, and the type of sugar residues corresponding to each PMAA is analyzed using database matching based on its corresponding ion fragments.

[0098] When the polysaccharide uronic acid content is lower than 5%, it can be directly methylated. When the uronic acid content is higher than 5%, it needs to be uronic acid reduced before methylation. According to the previous uronic acid content determination experiment, the uronic acid content of PPFP-Fr.4 is >5%. Therefore, before the methylation operation, the uronic acid reduction instrument (Automatic reduction of carboxyl groups instrument (BR-HYY-001)) is used to reduce the uronic acid of PPFP-Fr.4. The monosaccharide composition of the sample obtained after reduction is analyzed, and the methylation experiment is performed after the acidic sugar content is determined to be <5%.

[0099] The specific steps of the methylation experiment are as follows: the polysaccharide was methylated using a methylation kit (manufacturer: Borui Saccharide), 1 mL of anhydrous DMSO was added to 20 mg of PPFP-Fr.4, followed by rapid addition of methylation reagent A solution, ultrasonic dissolution, and addition of methylation reagent B solution. The reaction was carried out in a water bath at 30°C with magnetic stirring for 60 min, and finally 2 mL of ultrapure water was added to terminate the reaction.

[0100] The methylated polysaccharide was hydrolyzed and acetylated. 1 mL of 2M trifluoroacetic acid (TFA) was added to the above product, and the product was hydrolyzed for 90 min and then evaporated to dryness using a rotary evaporator. After adding 2 mL of deionized water to dissolve, 60 mg of sodium borohydride was added to the solution for reduction for 8 hours, and finally glacial acetic acid was added to neutralize it to neutrality and evaporated to dryness. 1 mL of acetic anhydride was added to the product and acetylated at 100 ° C for 1 hour. After cooling, 3 mL of toluene was added, and the product was concentrated and evaporated to dryness under reduced pressure. This was repeated 5 times to remove excess acetic anhydride.

[0101] The acetylated product was dissolved with 3 mL CH2Cl2 and transferred to a separatory funnel. A small amount of distilled water was added to shake the mixture thoroughly. After standing for stratification, the upper aqueous solution was removed and the lower organic phase was collected. The above operation was repeated 4 times. The organic phase was dried with an appropriate amount of anhydrous sodium sulfate, fixed to 10 mL, and then filtered through a 0.22 μm filter membrane for detection.

[0102] The above products were determined using an Agilent GCMS 6890-5973 gas chromatograph-mass spectrometer equipped with a RXI-5SIL MS column (30m×0.25mm×0.25μm). The test conditions were as follows: Operating conditions: starting temperature 120°C, heating at 3°C / min to 250°C / min; maintaining for 5 minutes; injection port temperature 250°C, detector temperature 250°C / min, carrier gas helium, flow rate 1mL / min.

[0103] The results are as follows Figure 8 , as shown in Table 2.

[0104] By comparing the peak time and ion fragments and searching the spectral library, it can be seen that PPFP-Fr.4 is mainly composed of seven sugar residues: Araf-(1→, →5)-Araf-(1→, →3,5)-Araf-(1→, →2,4)-Xylp-(1→, Glcp-(1→, →4)-Galp-(1→, →3,6)-Galp-(1→). The characteristic ion fragments of each sugar residue and the normalized proportion of their peak areas are shown in Table 2.

[0105] Table 2 Analysis of methylated sugar residue fragments in PPFP-Fr.4

[0106]

[0107] Combining the monosaccharide composition and methylation results, it can be seen that PPFP-Fr.4 is an acidic polysaccharide mainly composed of arabinose and galactose. The monosaccharide composition results show that PPFP-Fr.4 contains a large amount of galacturonic acid, accounting for about 16.65%. In addition, the methylation analysis results show that the proportions of →4)-Galp-(1→ and →3,6)-Galp-(1→ in PPFP-Fr.4 are approximately 27.43% and 16.22%, respectively. Since the proportion of →3,6)-Galp-(1→ is less than the total PMAA proportion, it indicates that galacturonic acid in the form of →4)-GalpA-(1→ structure must exist in PPFP-Fr.4.

[0108] (3) NMR analysis

[0109] Weigh 60 mg of PPFP-Fr.4, add D2O water to fully dissolve, and then freeze dry it using a freeze dryer. After repeated freeze-thawing 3 times, the sample was fully dissolved in 1 mL of D2O again, centrifuged at 4000 rpm for 5 min, and the supernatant was transferred to an NMR tube (solution height>3.5 cm). The 1H, 13C, HSQC, HMBC, COSY and NOESY spectra of the polysaccharide were detected using an NMR spectrometer with a scanning frequency of 600 MHz.

[0110] The 1H, 13C, HSQC, HMBC, COSY and NOESY spectra of PPFP-Fr.4 are shown in the following order: Figures 9 to 12 The assignment of 1H / 13C chemical signal shifts in each sugar residue is shown in Table 3.

[0111] The signal peaks at 5.11, 5.02 and 5.17 ppm in the 1H spectrum can be attributed to three arabinose residues, including the terminal sugars T-α-Araf-(1→, →5)-α-Araf-(1→ and →5,3)-α-Araf-(1→. The signal peak at 4.93 ppm can be attributed to the terminal sugar t-α-Glcp-(1→, and the signal peak at 5.17 ppm can be attributed to the →2,4)-α-Xylp-(1→ sugar residue.

[0112] In addition, the signal peaks 4.56 and 4.42 ppm at 4.4-4.6 ppm can be attributed to the β-type Glap sugar residues, including →4)-β-Galp-(1→, →4)-β-GalpA-(1→ and →3,6)-β-Galp-(1→. Combined with the comprehensive analysis of 13C and HSQC spectra, it can be seen that the carbon spectrum signals of 107.32, 106.81, 99.33, 109.36, 98.36 The signal peaks at 104.08, 104.08, and 103.07 ppm can be attributed to α-Araf-(1→, →5)-α-Araf-(1→, α-Glcp-(1→, →3,5)-α-Araf-(1→, →2,4)-α-Xylp-(1→, →4)-β-Galp-(1→, →4)-β-GalpA-(1→, →3,6)-β-Galp-(1→, →3,6) respectively.

[0113] Generally, the signal peaks between 3.0-4.3ppm in the hydrogen spectrum and the signal peaks between 60-85ppm in the carbon spectrum can be attributed to the H2-H5 and C2-C5 signal peaks of each sugar residue. By analyzing the 1H-1H COSY spectrum of PPFP-Fr.4, the signal peaks at H2-H5 / H6 of each sugar residue can be inferred. For example, in the α-Araf-(1→ sugar residue, 5.11 / 4.01, 4.01 / 3.78, 3.78 / 4.06, 4.06 / 3.73ppm can be attributed to its H1 / H2, H2 / H3, H3 / H4 and H4 / H5, respectively. Combining the HSQC spectrum and the literature library and database, it can be inferred that the chemical shifts of C2-C5 are 83.87, 76.45, 81.03 and 61.12ppm, respectively. The attribution results of each C / H signal peak of other sugar residues are shown in Table 3.

[0114] Based on the above results, it can be seen that the main chain structure of PPFP-Fr.4 is mainly composed of →6)-β-Galp-(1→, →6,3)-β-Galp-(1→, →4)-β-Galp-(1→4)-β-Galp-(1→, →4)-β-Galp-(1→4)-β-GalpA-(1→), among which H-3 in →6,3)-β-Galp-(1→ may be replaced by t-α-Araf-(1→, →5,3)-α-Araf-(1→, →4,2)-α-Xylp-(1→ and t-α-G lcp-(1→ is replaced. As a component of the branch chain, the H-5 position of →5)-α-Araf-(1→ and →5,3)-α-Araf-(1→ may be replaced by t-α-Araf-(1→. In addition, the H-2 and H-4 positions of →4,2)-α-Xylp-(1→ are also replaced by terminal sugar residues. According to the NMR signal, its main substituted sugar residues may include t-α-Glcp-(1→ and t-α-Araf-(1→. A comprehensive analysis of the PPFP-Fr.4 structure suggests that its partial sugar chain structure may be as follows Fig.13 shown.

[0115] Table 3 Assignment of NMR signals in sugar residues of PPFP-Fr.4

[0116]

[0117] Experimental Example 1 Study on the Anti-Photodamage Activity of Polysaccharides

[0118] In the experimental examples of the present invention, the HaCaT human immortalized keratinocytes used were cultured in DMEM high-glucose complete medium, and the DMEM complete medium was a DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody (penicillin + streptomycin).

[0119] (1) MTT method to determine cell survival rate

[0120] HaCaT cells in the logarithmic growth phase were inoculated in a 96-well plate at a seeding density of 5 × 10 3 Each well was then placed in an incubator and incubated for 24 hours until the cells adhered to the wall. The supernatant was then discarded, and sample solutions of different concentrations were added to the cells and incubated for another 24 hours. After the incubation, the supernatant was discarded, and 100 μL of complete medium containing 0.5 mg / mL MTT was added and incubated for another 4 hours. The supernatant was then discarded, and 150 μL of DMSO solution was added to each well. After shaking for 1 minute, the absorbance was measured at 570 nm. The blank control group was replaced with complete medium instead of the medium solution containing the sample. The cell viability calculation method is as follows:

[0121]

[0122] (2) Determination of anti-photodamage activity

[0123] A blank group, a sample group, and a model group were set up. HaCaT cells in the logarithmic growth phase were inoculated in a 96-well plate at a seeding concentration of 5×10 3 Each well was placed in an incubator and incubated for 24 hours until adhered. The supernatant was then discarded, and DMEM complete medium was added to the blank group and model group, and DMEM complete medium containing different concentrations of drugs was added to the sample group. After culturing for 24 hours, the supernatant was discarded from the blank group and replaced with fresh DMEM complete medium for another 24 hours; after the supernatant was discarded from the sample group and model group, 100 μL PBS solution was added and placed in a dark box with 80 mJ / cm 2 After the irradiation, the PBS solution was discarded and 100 μL DMEM complete medium was added to continue culturing for 24 hours. The cell survival rate of each group was determined by referring to the above MTT method. The protection rate of each group of samples against photodamaged HaCaT cells (Protection rate) was calculated as follows:

[0124]

[0125] The results are as follows Fig.14 , 15 shown.

[0126] like Fig.14 As shown, PPFP-Fr.3, PPFP-Fr.4, and PPFP-Fr.5 had no significant effect on cell growth at concentrations below 125 μg / mL.

[0127] like Fig.15 As shown, compared with the model group, among the PPFP-Fr.5 treatment groups, only the high-concentration treatment group could significantly improve the survival rate of HaCaT cells damaged by UVB (P<0.05). 62.5-125μg / mL PPFP-Fr.4 treatments can improve the survival rate of HaCaT cells damaged by UVB, and the medium concentration (62.5μg / mL) treatment group has a very significant effect on improving the survival rate of HaCaT cells damaged by UVB (P<0.001). PPFP-Fr.3 and PPFP-Fr.4 have good protective effects on HaCaT cells damaged by UVB, and the survival rates of the treatment groups treated with PPFP-Fr.4 at various concentrations are significantly different from those of the model group. It can be seen that the purified polysaccharide PPFP-Fr.4 from Pyracantha fortuneana fruit provided by the present invention has significant anti-photodamage activity.

[0128] Experimental Example 2 Effect of PPFP-Fr.4 on apoptosis of HaCaT cells damaged by UVB

[0129] HaCaT cells in the logarithmic growth phase were taken at 3×10 5The cells were inoculated into 6-well plates at a density of 100 / well, placed in an incubator for incubation for 24 hours, and the supernatant was discarded. The cells were divided into a blank group, a model group, a sample group, and a positive control group. Among them, DMEM complete medium was added to the blank group and the model group, DMEM complete medium containing different concentrations of PPFP-Fr.4 was added to the sample group, and DMEM complete medium containing 100μg / mL ascorbic acid (AA) was added to the positive control group. After culturing for 24 hours, the supernatant of the blank group was discarded, and fresh DMEM complete medium was added to continue culturing for 24 hours; after discarding the supernatant of the model group, the sample group and the positive control group, 100μL PBS solution was added, and they were placed in a dark box and 80mJ / cm 2 After irradiation, the PBS solution was discarded and 100 μL DMEM complete medium was added to continue culturing for 24 h.

[0130] Annexin V-FITC apoptosis detection kit (manufacturer: Bio-Tech Biotechnology Co., Ltd.) was used to stain the cells according to the instructions of the kit, and then the stained cells were grouped using flow cytometry to determine the level of cell apoptosis.

[0131] The results are as follows Figures 16-17 shown.

[0132] Figures 16-17 The results showed that compared with the blank group, UVB damage significantly increased the apoptosis level of HaCaT cells, and the apoptosis rate after damage was as high as 42.91%. The positive control AA (100μM) and PPFP-Fr.4 (62.5-125μg / mL) treatment significantly reduced the apoptosis rate of cells exposed to UVB (P<0.05). After treatment with high-concentration PPFP-Fr.4, the apoptosis rate of HaCaT cells damaged by UVB could be reduced to 27.58%.

[0133] Experimental Example 3 Effect of PPFP-Fr.4 on the expression of antioxidant factors in UVB-damaged HaCaT cells

[0134] HaCaT cells in the logarithmic growth phase were taken at 3×10 5The cells were inoculated into 6-well plates at a density of 100 / well, placed in an incubator for incubation for 24 hours, and the supernatant was discarded. The cells were divided into a blank group, a model group, a sample group, and a positive control group. Among them, DMEM complete medium was added to the blank group and the model group, DMEM complete medium containing different concentrations of PPFP-Fr.4 was added to the sample group, and DMEM complete medium containing 100μg / mL ascorbic acid (AA) was added to the positive control group. After culturing for 24 hours, the supernatant of the blank group was discarded, and fresh DMEM complete medium was added to continue culturing for 24 hours; after discarding the supernatant of the model group, the sample group and the positive control group, 100μL PBS solution was added, and they were placed in a dark box and 80mJ / cm 2 After irradiation, the PBS solution was discarded and 100 μL DMEM complete medium was added to continue culturing for 24 h.

[0135] The expression levels of CAT, SOD, MDA and GSH in HaCaT cells were determined using a catalase detection kit, a total SOD activity detection kit, a lipid oxidation detection kit and a GSH detection kit (all purchased from Bio-Tech Biotechnology Co., Ltd.).

[0136] The results are as follows Fig.18 shown.

[0137] UVB damage can lead to increased levels of intracellular oxidative stress, which further induces rapid progression of the cascade reaction in the intracellular damage mechanism, resulting in a series of harmful effects on the body. By measuring the level of intracellular oxidative stress, the protective effect of PPFP-Fr.4 on UVB-damaged HaCaT cells can be further evaluated.

[0138] Fig.18 The results showed that compared with the model group, 125 μg / mL PPFP-Fr.4 could significantly improve the oxidative stress level of UVB-damaged HaCaT cells, and its effects included significantly increasing the concentration of antioxidant GSH, the activities of antioxidant enzymes CAT and SOD, and significantly reducing the concentration of lipid peroxide product MDA in cells; while 62.5 μg / mL PPFP-Fr.4 only had a significant effect on the expression of SOD enzyme activity in UVB-damaged HaCaT cells, and had no obvious effect on the expression levels of GSH, CAT, and MDA in the cells.

[0139] Experimental Example 4 Effect of PPFP-Fr.4 on mitochondrial membrane potential (MMP) level of UVB-damaged HaCaT cells

[0140] HaCaT cells in the logarithmic growth phase were taken at 3×10 5The cells were inoculated into 6-well plates at a density of 100 / well, placed in an incubator for incubation for 24 hours, and the supernatant was discarded. The cells were divided into a blank group, a model group, a sample group, and a positive control group. Among them, DMEM complete medium was added to the blank group and the model group, DMEM complete medium containing different concentrations of PPFP-Fr.4 was added to the sample group, and DMEM complete medium containing 100μg / mL ascorbic acid (AA) was added to the positive control group. After culturing for 24 hours, the supernatant of the blank group was discarded, and fresh DMEM complete medium was added to continue culturing for 24 hours; after discarding the supernatant of the model group, the sample group and the positive control group, 100μL PBS solution was added, and they were placed in a dark box and 80mJ / cm 2 After the irradiation, the PBS solution was discarded and 100 μL DMEM complete medium was added to continue culturing for 24 hours. The supernatant was aspirated and rinsed with PBS for 3 times. After 300 μL trypsin was added to each well for 1 minute, 600 μL complete medium was added to terminate the digestion, and the cells were collected into a 1.5 mL centrifuge tube by pipetting. Centrifuge at 1000 rpm for 5 minutes. After centrifugation, the supernatant was removed, and 1 mL JC-1 staining working solution (5 μL JC- added to 1 mL JC-1 staining buffer) was added to each centrifuge tube, incubated at 37 ° C for 20 minutes, and then centrifuged at 1000 rpm for 3 minutes, the supernatant was aspirated, PBS was added to resuspend, and centrifuged again. Repeat 3 times to remove the influence of excess dye. Finally, the cells of each group were resuspended in the basal medium, transferred to the flow tube and stored on ice in the dark, and tested on the machine within 30 minutes.

[0141] The results are as follows Fig.19 shown.

[0142] JC-1 is a cationic dye that can assess mitochondrial status by evaluating MMP levels through the quantitative ratio of its red / green fluorescent cell population.

[0143] Fig.19 The results showed that compared with the blank control group, the MMP level in the model group decreased significantly, indicating that mitochondrial damage caused by UVB may lead to mitochondrial depolarization, thereby inducing mitochondrial dysfunction. Compared with the model group, both AA and PPFP-Fr.4 treatment groups significantly increased the MMP level of UVB-damaged HaCaT cells, among which the PPFP-Fr.4 high concentration group (125μg / mL) had the best effect. Compared with the model group, although the mitochondrial membrane potential of the cells in the PPFP-Fr.4 low concentration group (62.5μg / mL) was improved to a certain extent, the MMP value of the cells in this treatment group was only 1 / 2 of that in the high concentration treatment group. In general, PPFP-Fr.4 treatment had a significant recovery effect on the mitochondrial membrane potential of UVB-damaged keratinocyte HaCaT cells.

[0144] Experimental Example 5 Effect of PPFP-Fr.4 on the expression levels of NLRP3 / MAPK pathway proteins and Tjs proteins in UVB-damaged HaCaT cells

[0145] HaCaT cells in the logarithmic growth phase were taken at 3×10 5 The cells were inoculated into 6-well plates at a density of 100 μg / well, placed in an incubator for incubation for 24 hours, and the supernatant was discarded. The cells were divided into a blank group, a model group, a sample group, and a positive control group. DMEM complete medium was added to the blank group and the model group, 125 μg / mL PPFP-Fr.4 DMEM complete medium was added to the sample group, and 100 μg / mL ascorbic acid (AA) was added to the positive control group. After culturing for 24 hours, the supernatant of the blank group was discarded, and fresh DMEM complete medium was added to continue culturing for 24 hours; after discarding the supernatant of the model group, the sample group and the positive control group, 100 μL PBS solution was added, and they were placed in a dark box and exposed to 80 mJ / cm 2 After the irradiation, the PBS solution was discarded and 100 μL DMEM complete medium was added to continue culturing for 24 h. After the culture was completed, the culture medium was discarded, PBS solution was slowly added to wash twice, and then the total cell protein was extracted using RIPA.

[0146] The BCA protein kit was used to detect the cell protein concentration, and then 1 / 4 volume of loading buffer was added to the cell protein solution, which was evenly blown with a pipette and placed in a metal bath at 100°C for 10 minutes to complete protein denaturation. The denatured protein sample was then used to detect the expression of MAPK / NLRP3 pathway proteins and tight junction (Tjs)-related proteins in the protein sample.

[0147] The results are as follows Figures 20-22 shown.

[0148] (1) Effect of PPFP-Fr.4 on the expression level of NLRP3 pathway proteins in UVB-damaged HaCaT cells

[0149] The mechanism of cell damage induced by light damage is relatively complex, usually involving a series of intracellular signaling pathways and physiological and biochemical reactions. NLRP3 inflammasome is a protein complex composed of multiple units, usually present in the cytoplasm. NLRP3 inflammasome in cells is usually activated by conditions that cause cell stress response, such as light damage and inflammation. NLRP3 inflammasome can activate caspase-1. On the one hand, activated Caspase-1 can cleave pro-IL-1β and pro-IL-18 to convert them into mature cytokines that can function. On the other hand, activated Caspase-1 can induce cell pyroptosis or apoptosis. Cleaved caspase-1 fragment can be used as an important indicator to judge whether Caspase-1 is activated or not.

[0150] Effects of PPFP-Fr.4 on the expression levels of NLRP3 and Caspase-1 in UVB-damaged HaCaT cells Fig. 20 As shown. Compared with the blank group, the expression levels of NLRP3 and Cleaved Caspase-1 in HaCaT cells treated with UVB damage were significantly increased. Keratinocytes will induce intracellular NLRP3 activation and Caspase-1-mediated cell pyroptosis under UVB radiation, thereby inducing inflammatory skin diseases.

[0151] Compared with the model group, 125 μg / mL PPFP-Fr.4 treatment significantly downregulated the expression of related proteins in the inflammasome. This downregulation indicates that PPFP-Fr.4 can attenuate the inflammatory response in UVB-damaged skin cells. Compared with the positive control group of 100 μM ascorbic acid (AA), the level of inhibition of Caspase-1 activation by PPFP-Fr.4 was slightly lower than that of the positive control, but its level of inhibition of NLRP3 expression was comparable to that of the positive control.

[0152] (2) Effect of PPFP-Fr.4 on the expression level of MAPK signaling pathway proteins in UVB-damaged HaCaT cells

[0153] After exposure to ultraviolet rays, the MAPK signaling pathway in skin cells is activated and the phosphorylation level of its key proteins increases, thereby triggering a series of physiological responses to cell damage such as inflammation and aging, causing cell growth cycle stagnation or leading to cell apoptosis.

[0154] like Fig.21The results showed that compared with the blank group, the phosphorylation levels of P38, JNK and ERK proteins in the MAPK signaling pathway of UVB-damaged HaCaT cells increased significantly, while PPFP-Fr.4 and AA treatments could significantly reduce the phosphorylation levels of proteins in these three pathways, making the phosphorylation levels of P38, JNK and ERK proteins in UVB-damaged cells tend to be normal. The MAPK signaling pathway is closely related to the physiological reactions of cells such as mitochondrial function, oxidative stress, and immune response. The activation of P38, JNK and ERK proteins in the pathway usually means the generation of cell stress response. The overactivation of these key proteins can induce cell DNA damage, growth cycle arrest, increased inflammation levels, and thus cause cell apoptosis. Therefore, it can be inferred that PPFP-Fr.4 regulates the mitochondrial state and improves the level of cellular oxidative stress, thereby regulating the phosphorylation level of key proteins in the MAPK signaling pathway, thereby playing a protective role against UVB-damaged HaCaT cells.

[0155] (3) Effect of PPFP-Fr.4 on Tjs protein expression in UVB-damaged HaCaT cells

[0156] Tight junctions (Tjs) are important components of skin cells. They are widely located at the junctions between epithelial cells and cells. They are complexes composed of a series of protein families such as transmembrane proteins and cytoplasmic proteins. Tjs can connect epithelial cells to each other, forming a protective barrier in the outer layer of the skin. They play a key role in maintaining tissue integrity and regulating the passage of substances. They are also an important indicator for judging the health of the skin. Occludin and ZO-1 play a key role in maintaining the integrity of the cell junction complex. Impaired expression of Occludin and ZO-1 will lead to a disruption of the balance of proliferation and differentiation of skin cells, which will eventually lead to the destruction of the skin barrier function.

[0157] like Fig. 22 The results showed that compared with the blank control group, UVB damage significantly reduced the expression of ZO-1 and Occludin proteins in keratinocyte HaCaT cells, indicating that ultraviolet irradiation damaged the tight junction protein structure in skin cells. Both AA and PPFP-Fr.4 treatments significantly increased their expression levels, of which PPFP-Fr.4 could effectively restore the expression levels of these two tight junction proteins to normal cell levels, and the effect of PPFP-Fr.4 on tight junction proteins was better than that of the positive control AA.

[0158] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing purified polysaccharide from Pyracantha fortuneana fruit, characterized in that: The steps include: The pyracantha fruit is crushed and mixed with water, and subjected to heating reflux extraction for 2 to 3 times, each time for 2 to 4 hours, at an extraction temperature of 80 to 100°C, and the extracts are combined, and the extracts are concentrated and then depigmented and deproteinized to obtain pyracantha fruit polysaccharide; the mass volume ratio of the pyracantha fruit to water is 1: (20 to 30) mg / mL; The depigmentation treatment comprises the following steps: adding D354F macroporous resin to the concentrated extract, stirring for 2-4 hours at a temperature of 50-60°C, filtering, collecting the filtrate, using 3-5 times the volume of 95% ethanol to precipitate the filtrate, collecting the precipitate, and drying to obtain Pyracantha fortuneana fruit crude polysaccharide; the volume ratio of the concentrated extract to the D354F macroporous resin is 1:1; The deproteinization treatment comprises the following steps: preparing a 5 mg / mL pyracantha fruit crude polysaccharide solution with water, adding 3 to 5 times the volume of Sevag solution for deproteinization, oscillating for 25 to 35 min, deproteinizing for 4 to 6 times, then collecting the aqueous phase polysaccharide solution, adding ethanol to a final ethanol concentration of 80% for alcohol precipitation, collecting the precipitate, and drying to obtain pyracantha fruit polysaccharide; the Sevag solution is prepared by mixing chloroform and n-butanol in a volume ratio of 1:4; The pyracantha fruit polysaccharide is prepared into a pyracantha fruit polysaccharide aqueous solution, and then separated by DEAE ion column chromatography, eluted with water, 0.025 mol / L, 0.05 mol / L, and 0.15 mol / L NaCl solutions in sequence, and the eluate obtained by eluting with 0.15 mol / L NaCl solution is collected, purified, and dried to obtain; Purifying the eluate includes the following steps: the eluate is concentrated and then dialyzed using a dialysis bag with a molecular weight cutoff of 8000 Da.

2. The preparation method according to claim 1, characterized in that: The steps include: The pyracantha fruit was crushed and mixed with water, and the mixture was heated and refluxed for extraction for 3 times, each time for 3 hours, and the extraction temperature was 100°C, and the extracts were combined; the mass volume ratio of the pyracantha fruit to water was 1:25 mg / mL; The extract was concentrated to 1 / 20 of the original volume, and then an equal volume of D354F macroporous resin was added, stirred at 55°C for 3 hours, filtered, and the filtrate was collected. The filtrate was precipitated with 4 times the volume of 95% ethanol by mass fraction, and the precipitate was collected and dried to obtain Pyracantha fruit crude polysaccharide. The crude polysaccharide of Pyracantha fruit was prepared into a crude polysaccharide solution of Pyracantha fruit with water at a concentration of 5 mg / mL, and 4 times the volume of Sevag solution prepared by mixing chloroform and n-butanol in a volume ratio of 1:4 was added for deproteinization, the oscillation time was 25-35 min, and the deproteinization times were 5 times, then the polysaccharide solution in the aqueous phase was collected, ethanol was added to a final ethanol concentration of 80% for alcohol precipitation, and the precipitate was collected and dried to obtain Pyracantha fruit polysaccharide; The Pyracantha fruit polysaccharide is prepared into a Pyracantha fruit polysaccharide aqueous solution, and then separated by DEAE ion column chromatography, eluted with water, 0.025 mol / L, 0.05 mol / L, and 0.15 mol / L NaCl solutions in sequence, and the eluate obtained by eluting with 0.15 mol / L NaCl solution is collected, the eluate is concentrated under reduced pressure to 1 / 10 of the original volume, and dialyzed using a dialysis bag with a molecular weight cutoff of 8000 Da, and the dialyzed solution is freeze-dried to obtain the method.

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