Alkali-extracted crocus sativus petal polysaccharide APPCS-1a as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an alkali-extracted saffron petal polysaccharide APPCS-1a as well as a preparation method and application thereof. The APPCS-1a provided by the invention is uniform polysaccharide, has the molecular weight of about 40KDa, better thermal stability, no triple helix, more negative charges and good antioxidant activity, and can be applied to preparation of anti-aging medicines, health care products or cosmetics. According to the extraction method of APPCS-1a, an alkali extraction and alcohol precipitation method is adopted, the extracted polysaccharide is rich in uronic acid, good in biological activity and wide in biological application value, and the extraction method is simple, low in cost and suitable for industrial mass production.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to an alkali-extracted saffron petal polysaccharide APPCS-1a and a preparation method and application thereof. Background Art

[0002] Saffron (Crocus sativus L.), also known as saffron, is a perennial herb of the genus Crocus in the family Iridaceae. It is a spice and medicinal material widely used in traditional medicine. Safflower is the dried flower of the safflower plant of the family Asteraceae. When used as medicine, the whole flower can be picked, and the yield is usually higher than that of saffron. Saffron is a perennial herb of the genus Crocus in the family Iridaceae and is a precious Chinese medicinal material.

[0003] With the advent of the 21st century, the global population structure has changed significantly, and aging research has developed rapidly, becoming a frontier field in biomedicine. The physiological mechanisms behind the human aging process mainly focus on decreased nutrient absorption capacity, mitochondrial abnormalities, and cell aging. There are currently several anti-aging drugs and natural compounds in clinical trials, but some drugs have serious side effects. For example, rapamycin can cause hyperglycemia, hyperlipidemia, nephrotoxicity, impaired wound healing, reduced platelet count, and immunosuppression, etc. It is necessary to find a natural, non-toxic, and low-side anti-aging substance.

[0004] Recent studies have shown that crocin, an extract from saffron stigma, can restore mitochondrial function by increasing oxygen diffusion, improve memory and motor coordination in elderly mice, and prolong lifespan. Genetic analysis shows that crocin increases brain energy by upregulating genes related to oxidative phosphorylation without causing oxidative stress. Saffron (pistil) has good development value and resource conditions. Currently, there are many product developments based on the clinical value of saffron, such as Tibetan medicine, pharmaceuticals, health products and cosmetics, but they are limited to the stigma part of the pistil. One saffron has only three styles, and 150 flowers can produce about 1g of dry saffron. The application value of saffron petals in anti-aging needs to be developed. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides an alkali-extracted saffron petal polysaccharide APPCS-1a and a preparation method and application thereof.

[0006] The technical scheme of the present invention is as follows: an alkali-extracted saffron petal polysaccharide APPCS-1a, which has Figure 1The structure shown in FIG. 1 is shown in FIG. 1 , wherein the main chain structure of APPCS-1a is →2)-α-Rhap-(1→4)-α-GalpA-(1→, and the side chains are mainly →4)-β-D-Galp-(1→, →3,6)-β-D-Galp-(1→, T-β-D-Galp, →5)-α-L-Araf-(, →T-α-L-Araf) linked to the C-4 position of rhamnose. The structural formula is as follows: Figure 1 shown.

[0007] APPCS-1a is a homogeneous polysaccharide with a molecular weight of about 40KDa.

[0008] APPCS-1a is composed of eight monosaccharides: galactose, arabinose, rhamnose, galacturonic acid, glucuronic acid, xylose, glucose, and mannose, with molar ratios of 64.321%, 20.419%, 6.717%, 4.313%, 1.575%, 1.560%, 0.820%, and 0.274%, respectively.

[0009] The present invention also provides a method for preparing alkali-extracted saffron petal polysaccharide APPCS-1a, comprising the following steps:

[0010] S1: heating and drying saffron petals to constant weight, crushing them, adding a solvent to the petal powder to remove pigments and fat-soluble substances, filtering, collecting the filter residue, and drying for later use;

[0011] S2 Take an appropriate amount of pretreated powder, add alkaline solution, heat in a water bath, stir and extract twice, quickly neutralize the extract to neutrality after the extraction, centrifuge and filter to obtain the supernatant, concentrate the supernatant under reduced pressure, and dialyze the polysaccharide concentrate;

[0012] After adding the aggregation promoter to the S3 polysaccharide concentrate, anhydrous ethanol is added for precipitation, the mixture is allowed to stand for filtration, and the mixture is heated and dried to obtain the crude polysaccharide;

[0013] The crude S4 polysaccharide was separated from saffron polysaccharide by a cellulose column, and deionized water, 0.1, 0.3, and 0.5 M NaCl were used for continuous elution; the 0.1 M elution fraction was dialyzed, purified by a gel column, concentrated, and freeze-dried to obtain APPCS-1a.

[0014] Preferably, the heating and drying temperature in step S1 is 50° C.; and the solvent is 95% ethanol.

[0015] Preferably, the alkaline solution in step S2 is 8% NaOH solution; the water bath heating condition is heating at 90° C. for 2 hours; the centrifugal filtration condition is 4000 r / min, 10 min; and the dialysis condition is 3500 Da dialysis for 72 hours.

[0016] Preferably, the volume ratio of the saffron polysaccharide concentrate to ethanol in step S3 is 1:4; and the aggregation promoter in step S3 is KCl.

[0017] Preferably, in step S4, the cellulose column is DEAE-52 with a specification of 5.5×50 cm; the elution rate is 1.6 mL / min; the dialysis conditions are 500 Da at 4° C. for 3 days; and the gel column is S-100.

[0018] The present invention also provides the use of saffron petal polysaccharide APPCS-1a in the preparation of anti-aging medicines, health products or cosmetics.

[0019] A medicine, health product or cosmetic for anti-aging, comprising a preventive or therapeutic effective amount of the saffron petal polysaccharide APPCS-1a and pharmaceutically acceptable pharmaceutical excipients thereof.

[0020] Beneficial effects of the present invention:

[0021] The present invention adopts high performance gel permeation chromatography, liquid chromatography, infrared spectroscopy and nuclear magnetic resonance to preliminarily identify the chemical structure of APPCS-1a extracted by alkaline solution, including determining its molecular weight, monosaccharide composition and functional group, glycosidic bond type and connection mode. According to the methylation analysis result of APPCS-1a, it can be found that GalA exists in the form of α-1,4-GalpA connection, and the connection mode of Rha is α-1,2-Rhap and α-1,2,4-Rhap. It is speculated that the disaccharide repeating unit [→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→] constitutes the backbone of APPCS-1a, and a sugar side chain is attached to the C-4 position of Rha. The sugar side chains are of different types, including arabinan, galactan and AG-II side chains. The proportion of galactan and arabinan side chains is higher in APPCS-1a. The branching degree of arabinan is 2.04%, the branching degree of galactan is 9.78%, and the branching degree of AG-II side chain is 72%. APPCS-1a has a higher branching degree, and polysaccharides with higher branching degrees usually have better activity.

[0022] Rha sugar residues exist in two forms: α-1,2- and α-1,2,4-, accounting for 7.218% and 7.564% of the total residues, respectively, and the main chain branching degree is 51.17%. The molecular weight of APPCS-1a refers to the weight average molecular weight.

[0023] APPCS-1a does not have a triple helix. The maximum absorption wavelength of the mixed solution of APPCS-1a and Congo red did not undergo an obvious red shift, and as the concentration of sodium hydroxide gradually increased, the maximum absorption wavelength of the sample also gradually decreased, without the trend of first increasing and then decreasing that is unique to the triple helix structure, indicating that APPCS-1a does not have a triple helix structure.

[0024] APPCS-1a has good thermal stability. Thermogravimetric analysis showed that the polysaccharide can maintain good stability below 229°C.

[0025] The Zeta potential of APPCS-1a is -27.5mV. The polysaccharide components with more negative charges have the ability to donate electrons, making them relatively more bioavailable.

[0026] In addition to the basic structure and molecular weight, the surface morphology of polysaccharides affects the dissolution rate and solubility of polysaccharides, thereby affecting their biological activity and application. SEM results show that when observing polysaccharides under a low-power microscope, APPCS-1a can be seen to have an irregular and rough flaky structure. Increasing the magnification, it can be seen that the surface of the polysaccharide is smooth and wavy, which may be related to the negative charge carried by the polysaccharide molecules.

[0027] The APPCS-1a of the present invention adopts an alkali extraction and alcohol precipitation method, and separates and purifies saffron polysaccharides through a cellulose column gel column. The APPCS-1a extracted in this experiment has a high uronic acid content of 22.5%, and has a wide range of biological application value. The extraction method of the present invention is simple and low in cost, and can be considered for mass production.

[0028] The present invention studies the anti-aging activity of saffron polysaccharide by establishing an acute aging model of mice. It is found that saffron polysaccharide can improve the damage of intestinal villi, significantly increase the length of intestinal villi of aging mice, and improve the depth of intestinal crypts to a certain extent. Saffron polysaccharide can increase the activity of serum antioxidant enzymes SOD and CAT, reduce the level of MDA to reduce oxidative damage, thereby effectively delaying tissue damage and body aging caused by oxidative stress. Saffron polysaccharide can improve the Chao1, Shannon and Simpson index of the intestinal flora of aging mice, and increase the proportion of beneficial bacteria such as Lachnospiraceae, Lactobacillus, Bacteroides. In general, APPCS-1a can be used as a potential prebiotic for regulating the intestinal microenvironment and delaying aging. Brain metabolome analysis believes that the anti-aging effect of saffron polysaccharide is mainly through pathways such as arginine metabolism, glutathione metabolism, amino acid metabolism, pantothenic acid and coenzyme A biosynthesis, and TCA cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the structure of APPCS-1a.

[0030] Figure 2 Extraction, separation and purification of APPCS-1a: (A) Flow chart of extraction, separation and purification of APPCS-1a; (B) Sugar peak from DEAE-52 cellulose column; (C) Purification peak from 0.1M acid sugar S-100 gel.

[0031] Figure 3 Analysis of the physical and chemical properties of APPCS-1a: (A) UV full-wavelength scanning analysis; (B) infrared spectroscopy analysis; (C) monosaccharide composition determination.

[0032] Figure 4 The nuclear magnetic resonance spectra of APPCS-1a are: (A) 1H NMR spectrum; (B) 13C NMR spectrum; (C) HH COSY spectrum; (D) HSQC spectrum; (E) HMBC spectrum; (F) TOSEY spectrum.

[0033] Figure 5 SEM surface structure scans of APPCS-1a: (A) 200×; (B) 1000×; (C) 5000×; (D) 10000×.

[0034] Figure 6 Congo red analysis of APPCS-1a.

[0035] Figure 7 Stability analysis of APPCS-1a: (A) Zeta potential; (B) thermogravimetric analysis.

[0036] Figure 8 In vitro antioxidant activity analysis of APPCS-1a: (A) total antioxidant activity; (B) reducing capacity; (C) DPPH free radical scavenging activity; (D) hydroxyl free radical scavenging activity.

[0037] Fig. 9 HE sections of jejunum of aged mice: (A) jejunum pathological section; (B) intestinal villus length; (C) crypt depth; (D) intestinal villus length / crypt depth; (E) goblet cell number.

[0038] Fig.10 This is a pathological section of the liver of an aging mouse.

[0039] Fig.11 Antioxidant enzyme activities in mouse serum and tissues in vitro (A) serum SOD activity; (B) serum CAT activity; (C) serum MDA content; (D) jejunal glutathione peroxidase activity; (E) brain tissue acetylcholinesterase activity; (F) brain tissue nitric oxide synthase activity. Fig.12 Western blot analysis of brain tissue proteins (A) WB results; (B) Bcl-2; (C) Caspase3; (D) Bax.

[0040] Fig.13Analysis of mouse intestinal flora: (A) Venn diagram; (B) relative abundance of microbiota at the phylum level; (C) relative abundance of microbiota at the genus level; (D) heat map of family-level grouping and clustering; (E) LefSe map; (F) LDA score map; (G) Chao1 index; (H) Shannon index; (I) Simpson index.

[0041] Fig.14 The following are the metabolic analysis diagrams of mouse brain: (A) content of major metabolites; (B) biological roles played; (C) heat map; (D) OPLS-DA point cloud diagram; (E) OPLS-DA permutation test analysis diagram; (F) ORA enrichment analysis. DETAILED DESCRIPTION

[0042] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0043] Example 1

[0044] In order to study and utilize saffron polysaccharide APPCS-1a, the present invention makes slight modifications based on the traditional method, removes pigment, performs alkali extraction, alcohol precipitation, separation, purification and dialysis on saffron polysaccharide to obtain saffron polysaccharide APPCS-1a rich in uronic acid and good in activity.

[0045] 1.1 Experimental Materials

[0046] 1.1.1 Experimental herbs

[0047] The saffron petals used in the present invention are collected from Jiande City, Zhejiang Province, dried in an electric constant temperature drum drying oven at 50° C. to constant weight, crushed by a crusher, and the petal powder is collected for later use.

[0048] 1.1.2 Experimental reagents

[0049] Monosaccharide standards: rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, galactosamine hydrochloride monosaccharide, fucose, 1-phenyl-3-methyl-5-pyrazolone, DEAE-cellulose-52 were purchased from Shanghai Yuanye Biotechnology. Trifluoroacetic acid and potassium dihydrogen phosphate were purchased from Shanghai Yien Chemical. Acetonitrile was purchased from Hubei Futon Biochemical. Sodium hydroxide was purchased from Guangdong Guanghua Technology Co., Ltd. Shanghai Sephadex S-100 was purchased from Waterman. All chemicals used in the study were analytically pure.

[0050] 1.1.3 Experimental instruments and equipment

[0051] Electric constant temperature blast drying oven DGG-9070B Shanghai Senxin Experimental Instrument Co., Ltd., rotary evaporator RE-52A Shanghai Jinlan Instrument Manufacturing Co., Ltd., HS-S top-type electric mixer Zhengzhou Dark Horse Instrument Co., Ltd., vacuum freeze dryer ALPHA2-4Ldplus Germany Christ, vortex oscillator WH-966 Taicang Science and Education Equipment Factory, desktop high-speed refrigerated centrifuge ESJ182-4 Changsha Dongwang Experimental Instrument Co., Ltd., electronic balance ESJ182-4 Tianjin Boda Hongli Weighing Equipment Co., Ltd., UV-visible spectrophotometer UV-1150 Shanghai Meipuda Instrument Co., Ltd.

[0052] 1.2 Experimental methods

[0053] 1.2.1 Extraction method of APPCS-1a

[0054] Extraction process see Figure 2 , take the pretreated petal powder, add 95% ethanol to it, remove pigments and fat-soluble substances, collect the filter residue after filtering, and dry it for later use. Take an appropriate amount of pretreated powder, add 8% NaOH solution, heat it in a water bath at 80℃ for 2 hours, and stir it continuously with a magnetic stirrer, extract it twice, and quickly neutralize the extract to neutrality after the extraction. Centrifugal filtration (4000r / min, 10min) takes the supernatant, the supernatant is concentrated under reduced pressure, dialyzed (3500Da) for 72 hours, and after concentration, a small amount of KCl is added to the polysaccharide to promote polysaccharide aggregation, precipitated with anhydrous ethanol, filtered and dried under an infrared baking lamp to obtain crude polysaccharide. APPCS is separated by DEAE-52 cellulose column (5.5×50cm), and deionized water, 0.1, 0.3, and 0.5M NaCl are used for continuous elution at an elution rate of 1.6mL / min. It is purified by S-100 gel column, and the standard eluent is matched according to the elution curve. The eluted components were monitored by the phenol-sulfuric acid method, and the eluates of each component were combined, dialyzed using a 500Da dialysis bag at 4°C for 3 days, concentrated and dried using a vacuum freeze dryer, and the obtained APPCS-1a was used for subsequent research.

[0055] 1.2.2 Study on the physical and chemical properties of APPCS-1a

[0056] 1.2.2.1 Determination of total sugar, uronic acid and sulfate groups of polysaccharides

[0057] The total sugar content was determined by the phenol-sulfuric acid method. The dried polysaccharide sample was weighed, a polysaccharide solution of appropriate concentration was prepared, a glucose standard curve was prepared, the absorbance of the polysaccharide sample was measured, and the total sugar content was calculated by inserting it into the standard curve.

[0058] According to the carbazole sulfate method, uronic acid was determined. 50 mg of carbazole was added with 50 mL of 95% ethanol to prepare a carbazole solution. A 1 mg / mL sugar solution was prepared for testing. A glucuronic acid standard curve was prepared: 20 mg of glucuronic acid was added with distilled water to make a gradient solution. 6 mL of concentrated sulfuric acid was added and mixed in an ice water bath. The mixture was in a water bath at 85°C for 20 min. Then 0.2 mL of carbazole solution was added. After stabilization at room temperature for 2 h, the absorbance was measured at Abs 530. The measured results were plotted into a glucuronic acid standard curve. The sample solution was determined according to the above method.

[0059] According to the gelatin-barium chloride method, 100 mg of polysaccharide sample was put into an ampoule, 8 mL of 2M HCl was added, and the ampoule was sealed after the polysaccharide swelled. The ampoule was heated at 105°C for 4 h, neutralized with ammonia water, filtered with activated carbon, and the volume was fixed to 25 mL. An appropriate amount of sample solution was added with 3.8 mL of 0.2 M HCl and 1 mL of gelatin barium chloride, and the absorbance was measured at Abs 500. A potassium sulfate standard curve was prepared to calculate the sulfate content.

[0060] 1.2.2.2 Determination of protein content of polysaccharides

[0061] Take an appropriate amount of APPCS-1a solution for full-wavelength UV scanning. If APPCS-1a contains nucleic acid or protein, there will be significant absorption peaks at 260 and 280 nm.

[0062] 1.2.2.3 Molecular weight detection

[0063] The polysaccharide sample with a concentration of 5 mg / mL was prepared with ammonium acetate solution. HPGPC was used to analyze the molecular weight of APPCS-1a. The chromatographic conditions were HPGPC (LC-10A series), differential refractometer (RI-10A), and chromatographic column BRT105-104-102. The mobile phase was 0.05 M NaCl, the flow rate was 0.6 mL / min, and the column temperature was 40 ° C. The molecular weight was determined by a standard curve of known molecular weights (dextran 1152, 5000, 11600, 23800, 48600, 80900, 148000, 273000, 409800, and 667800).

[0064] 1.2.2.4 Determination of monosaccharide composition

[0065] First, weigh 10 mg of fucose, 5 mg each of galactose, arabinose, glucose, xylose, mannose, rhamnose, galacturonic acid, glucosamine hydrochloride, galactosamine hydrochloride, and glucuronic acid monosaccharide, and prepare 10 mL of standard stock solution, dilute to a series of gradient concentrations, filter the membrane and send it into the injection bottle. Accurately weigh 5 mg of APPCS-1a sample, add 1 mL of 2M TFA solution, heat at 120 ° C for 2 hours, blow dry with nitrogen, then wash and dry repeatedly with methanol, add 5 mL of sterile water to dissolve for testing.

[0066] Take 0.2mL of polysaccharide hydrolyzate or monosaccharide standard solution, add an equal amount of 0.5mol / L sodium hydroxide solution, 0.5mL of 0.5mol / L PMP methanol solution, vortex and mix, react in a 70℃ water bath for 1h, add 0.2mL of 0.5mol / L hydrochloric acid, add 1mL of chloroform to extract 3 times to remove excess PMP, take 0.3mL of the lower layer of chloroform layer, add water to 1mL. Use ThermoU3000 liquid chromatography system for analysis. The chromatographic column is ZORBAX EclipseXDB-C18, the mobile phase is acetonitrile and phosphate buffer (12g / L potassium dihydrogen phosphate, 2MNaOH to adjust pH to 6.8) with a volume ratio of 17:83, the flow rate is 0.8mL / min, the column temperature is 30℃, the detection wavelength is 250nm, and the injection volume is 10μL.

[0067] 1.2.2.5 Fourier infrared spectroscopy

[0068] Take 2 mg of dry APPCS-1a sample in an agate mortar, grind it into powder under an infrared lamp, add an appropriate amount of KBr and continue grinding. Put the ground and mixed powder into a tablet press for tableting. -1 Infrared spectrum scanning is performed within the range.

[0069] 1.2.2.6 Methylation detection

[0070] The previously reported technique was slightly modified. 2 mg of polysaccharide sample was dissolved in 1 mL of anhydrous DMSO, and methylation reagent A solution was quickly added and then sealed. The solution was dissolved by ultrasound, and then methylation reagent B solution was added. After 60 min in a water bath at 30 ° C, 2 mL of ultrapure water was added to terminate the methylation reaction. The methylated polysaccharide was added to 1 mL of 2M trifluoroacetic acid (TFA) for hydrolysis for 90 min and evaporated to dryness on a rotary evaporator. 2 mL of double distilled water was added to the residue, 60 mg of sodium borohydride, reduced for 8 h, neutralized with glacial acetic acid, dried in an oven, 1 mL of acetic anhydride was added for acetylation, reacted at 100 ° C for 1 h, and 3 mL of toluene was added after cooling. The excess acetic anhydride was evaporated to dryness on a rotary evaporator. The acetylation product was taken, dissolved with 3 mL of CH2Cl2 and transferred to a separatory funnel. A small amount of distilled water was added, and the upper aqueous solution was removed after thorough mixing. Repeat 3 times. CH2Cl2 was dried with anhydrous sodium sulfate, fixed to 10 mL, and placed in a liquid phase bottle.

[0071] Acetylation product samples were measured using a Shimadzu GC MS-QP 2010 gas chromatograph-mass spectrometer. GC-MS conditions: RXI-5SIL MS column 30m×0.25mm×0.25μm. Start at 120°C, increase to 250°C / min at a rate of 3°C / min, and maintain for 5min. Inlet temperature was 250°C, detector temperature was 250°C / min, carrier gas was helium, and flow rate was 1mL / min.

[0072] 1.2.2.7 Nuclear Magnetic Resonance Testing

[0073] The sample was dissolved in D2O and freeze-dried three times. Then 30 mg of the sample was dissolved in 0.5 mL D2O for NMR analysis. 1 H. 13 The C spectra and two-dimensional spectra including HSQC, HMBC and TOCSY spectra were collected on a 600 MHz NMR spectrometer (Bruker avanceAV-600, Rheinstetten, Germany).

[0074] 1.2.2.8 Congo red analysis and scanning electron microscopy (SEM)

[0075] Take 5mg APPCS-1a polysaccharide sample and add Congo red reagent, gradually add 1mol / L sodium hydroxide solution, so that the concentration of sodium hydroxide in the solution gradually increases to 0.5mol / L, and scan with UV spectrophotometer, record the maximum absorption wavelength under different sodium hydroxide concentrations, and use Congo red solution without polysaccharide as a control. Accurately weigh 2mg APPCS-1a and adhere it to the carbon conductive medium plate, and observe the surface structure of polysaccharide under scanning electron microscope at room temperature after gold spraying.

[0076] 1.2.2.9 Zeta potential and thermogravimetric analysis

[0077] Weigh 30 mg accurately, add deionized water and ultrasonicate to completely disperse the sample, measure the Zeta potential of the polysaccharide solution three times at 25°C with a capillary cuvette, and take the average value. Weigh 20 mg of polysaccharide sample accurately and place it in a crucible, nitrogen atmosphere, flow rate 50 mL / min, program heating during analysis, heating rate 10°C / min, temperature range 30-800°C.

[0078] 1.2.2.10 Total antioxidant capacity

[0079] Prepare 20 mg / mL APPCS-1a solution and ammonium molybdate reaction solution: add 2.13 g sodium phosphate and 0.99 g ammonium molybdate and dissolve them in distilled water, then add 6.67 mL concentrated sulfuric acid and make up to 200 mL with distilled water, take 6 test tubes, add 0, 25, 50, 100, 200, and 400 μL of polysaccharide solution samples respectively, make up to 1 mL with distilled water, add 4 mL of ammonium molybdate reaction solution, put on ice immediately after 90°C water bath for 90 min, and measure the absorbance at a wavelength of 690 nm. The absorbance value can represent the total antioxidant capacity of the sample.

[0080] 1.2.2.11 Reduction capability

[0081] Prepare 20 mg / mL APPCS-1a solution and potassium ferrocyanide, trichloroacetic acid, and ferric chloride solutions. Take 6 test tubes and add 0, 25, 50, 100, 200, and 400 μL of polysaccharide solution samples, respectively. Make up to 1 mL with PBS at a pH of 6.6. Add 1 mL of potassium ferrocyanide to each tube, incubate at 50°C in a water bath for 20 min, then add 2 mL of trichloroacetic acid, incubate at 50°C in a water bath for 2 min, and finally add 1.2 mL of ferric chloride. After reacting at room temperature for 30 min, measure the absorbance at Abs 700 nm. The absorbance value can represent the reducing ability of the sample.

[0082] 1.2.2.12 DPPH free radical scavenging activity

[0083] Prepare 20 mg / mL APPCS-1a solution, take 6 test tubes, add 0, 25, 50, 100, 200, 400 μL of polysaccharide solution samples, take another 5 test tubes as the sample bottom group, add 50% methanol to 1 mL, add 1 mL of methanol and 2 mL of DPPH to each tube, react in the dark for 20 minutes, measure the absorbance at Abs517 nm wavelength, and calculate the clearance rate according to the following formula

[0084] Clearance (%) = (A0-(A1-A2)) / A0×100

[0085] A0: absorbance of the control group; A1: absorbance of the polysaccharide sample group; A2: absorbance of the polysaccharide local group Hydroxyl radical scavenging activity 1.2.2.13 Hydroxyl radical scavenging activity

[0086] Prepare 20 mg / mL APPCS-1a solution, add 0, 0.25, 0.5, 1, 2, 4 μL of polysaccharide solution samples respectively, make up to 1 mL with distilled water, take another 5 test tubes as the sample bottom group, add 1 mL of FeSO4, salicylic acid, and H2O2 to each tube, bathe at 37°C for 30 min, measure the absorbance at a wavelength of 510 nm, and calculate the clearance rate according to the formula.

[0087] 1.3 Results and Analysis

[0088] 1.3.1 Purity and composition of APPCS-1a

[0089] APPCS-1a has a total sugar content of 59.28%, a uronic acid content of 22.5%, and no sulfate groups, such as Figure 3 As shown in A, APPCS-1a has no significant absorption peaks at 260 and 280 nm, proving that it does not contain nucleic acids and proteins.

[0090] 1.3.2 Molecular weight analysis of APPCS-1a

[0091] The number average molecular weight (Mn) of APPCS-1a is 24250Da, the weight average molecular weight (Mw) is 40348Da, and the polydispersity index (Mw / Mn) is 1.66. The polydispersity index Mw / Mn is often used to evaluate the distribution of molecular weight. The higher the Mw / Mn ratio, the wider the molecular weight distribution.

[0092] 1.3.3 Analysis of monosaccharide composition of APPCS-1a

[0093] The monosaccharide composition of the polysaccharide can be determined based on the retention time of the mixed labeled 16 monosaccharide standards and the hydrolyzate of the polysaccharide in the ion chromatography. Figure 3 As shown in Figure C, APPCS-1a is mainly composed of eight monosaccharides: galactose, arabinose, rhamnose, galacturonic acid, glucuronic acid, xylose, glucose, and mannose, with molar ratios of 64.321%, 20.419%, 6.717%, 4.313%, 1.575%, 1.560%, 0.820%, and 0.274%, respectively.

[0094] 1.3.4 Infrared spectroscopy analysis of APPCS-1a

[0095] like Figure 3As shown in B, the infrared spectrum of APPCS-1a is a typical infrared spectrum of polysaccharides. The purified APPCS polysaccharide has a broad band at 3000-3700 and a peak at 3445.71 cm -1 There is a strong absorption peak at 2931.84cm, which is due to the hydrogen bonds between the OH groups of the polysaccharide, making the absorption peak wider. -1 The peak at 1640-1610 cm is attributed to the CH asymmetric stretching absorption peak in the sugar group. -1 The characteristic peak at 1618.28cm -1 The peak at 1419.09 cm is attributed to the stretching vibration of the free COO-functional group, indicating that APPCS-1a is an acidic polysaccharide, which is consistent with the results of monosaccharide composition analysis. -1 The peak at 1225.66 cm is the absorption peak caused by the stretching vibration of the methyl ester (-OCH3), indicating that the uronic acid part of APPCS-1a is esterified. -1 The peak at 800 to 1300 cm-1 may be due to the OH vibration. -1 The range between 1000-1200 cm is often referred to as the carbohydrate "fingerprint" and different types of polysaccharides can be distinguished in this region. -1 The peak at 890.78 cm is attributed to COC and COH, which are characteristic absorption peaks of pyranose. -1 The peak at 847.47 cm is the absorption peak of the C—H angle vibration of β-configuration pyranose. -1 The absorption peak at proves the existence of α-glycosidic bond configuration, and both are characteristic peaks of glycosidic bond structure.

[0096] 1.3.5 Methylation analysis of APPCS-1a

[0097] All free hydroxyl groups in various monosaccharide residues in polysaccharides are methylated, and then the glycosidic bonds in the polysaccharides are hydrolyzed. The position of the free hydroxyl group of the hydrolyzed compound is the connection site of the original sugar residue. At the same time, according to the proportion of different methylated monosaccharides, the proportion of this connection bond in the repeating structure of polysaccharides can also be deduced.

[0098] Table 1 Analysis results of bonding structure of APPCS-1a sample

[0099]

[0100]

[0101] 1.3.6 NMR analysis of APPCS-1a

[0102] In the 1H spectrum, the number of peaks and the integral of the peak area at 4.5-6.5 ppm directly correspond to the number of sugar residues. 13 In the C spectrum, the number of peaks at 90-110 ppm also corresponds to the number of sugar residues, and the two can confirm each other. Usually, δ5.0-5.4 ppm and δ90-102 ppm belong to the α configuration, and δ102-112 ppm and δ4.3-4.9 ppm belong to the β configuration, indicating that APPCS-1a contains both α and β configuration glycosidic bonds.

[0103] From the hydrogen spectrum of APPCS-1a, it can be found that there are split doublets at δ1.34 and δ1.28. There is no relevant H peak in the C spectrum interval of 170-190ppm in HSQC. It is concluded that 1.34ppm and 1.28ppm are the signals of the 6th methyl proton of rhamnose. Combined with HSQC, the H6 / C6 of α-1,2-Rhap and α-1,2,4-Rhap are 1.27 / 17.93 and 1.33 / 18.22 respectively. Generally, the strong signal generated near 3.82ppm is generated by the methoxy group (-OCH3) connected to the carboxyl end of GalA, but APPCS-1a has no obvious methoxy signal and acetyl signal. The peak near δ176ppm is the C-6 of the unesterified galacturonic acid unit.

[0104] Through 1H spectrum and HSQC spectrum, the easily distinguishable anomeric hydrogen signals of 5.29, 5.26, 5.18, 5.12, 5.10, 5.03, 4.68, 4.53, and 4.48 can be respectively labeled as AK 12 different sugar residues. Taking residue D as an example, according to 5.12 / 4.15, 4.15 / 4.03, 4.03 / 4.24, and 4.24 / 3.91 in HH-COSY, the chemical shifts of H2-H5 of residue D can be known, and according to the directly connected H\C signals provided in HSQC, the chemical shifts of C1-C5 can be obtained by 5.12 / 108.96, 4.15 / 82.52, 4.03 / 78.12, 4.24 / 83.60, and 3.91 / 68.52, and the chemical shift signals of other residues can be obtained by analogy, and the results are shown in Table 2.

[0105] Residues A and A* were assigned as →2,4-α-L-Rhap-(1→ and →2-α-L-Rhap-(1→, respectively) because their H6 / C6 ratios were 1.27 / 17.93 and 1.33 / 18.22, respectively, and the chemical shift of H4 of A* was greater than that of H4 of A. Residues BD were identified as α-Araf based on the downfield chemical shift of C-1 (110.66-108.54 ppm). The proton and carbon chemical shifts at position 3 of residue C were greater than those of residue B (Araf residue with unsubstituted end), so residue C was identified as →3,5)-α-L-Araf-(1→. Similarly, residue D was identified as →5)-α-L- Araf-(1→. Residue E can be assigned to →4)-α-D-GalpA-(1→ based on the cross peak at δ4.79 / 176.91 ppm in H5 / C6 in the HMBC spectrum, including the C-6 chemical shift at δ176.91 ppm and the downfield shifts in C-1 and C-4 of residue E. Residue GK was assigned to →4)-β-D-Galp-(1→, →3.6)-β-D-Galp-(1→, →T-β-D-Galp-(1→, →3)-β-D-Galp-(1→, →6)-β-D-Galp-(1→. This is because the H4 / C4 position of G becomes chemically enlarged after substitution. The other residues can be determined in the same way.

[0106] The HMBC spectrum was used to further characterize the linkage between sugar residues. In the HMBC spectrum, there are cross peaks between A* and A's H1 and EC4 (5.29 / 79.3), indicating that →2)-α-L-Rhap-(1 and 4)-α-D-GalAp-(1→ may be alternately connected in the main chain. Cross peaks appear at 4.58 / 79.08 (GH1 / GC4) and 4.20 / 105.78 (GH4 / GC1), indicating the existence of a →4)-α-Galp-(1→4)α-Galp-(1→ linking mode. From the cross peak of GH1 / A*C4 (4.68 / 81.51), it is not difficult to infer that the O-4 of α-D-Rhap is linked to a →4)-β-D-Galp-(1→4)-β-D-Galp-(1→ side chain. 4.53 / 71.30 (HH1 / JC3), 4.48 / 71.33 (I The cross peak at H1 / H C6) indicates the existence of →6)-β-D-Galp-(1→6)-β-D-Galp-(1→, and H-1 of T-Gal is connected to C-3 of →3.6)-β-D-Galp-(1→. There are cross peaks between C-1 / H-1 of sugar residue D and H-5 / C-5 of sugar residues C and D, indicating the existence of →5)-α-L-Araf-(1→5)-α-LAraf-(1→, and there is a branch at O-3 position. DH1 / C C3 (5.12 / 83.78) indicates that →5)-α-L-Araf-(1 is also connected to the O-3 branch, and the cross peak at 5.12 / 81.51 indicates that →5)-α-L-Araf-(1→5)-α-L-Araf-(1→ is linked to O-4 of α-D-Rhap.

[0107] Table 2 1H and 13C chemical shifts of sugar residues in APPCS-1a

[0108]

[0109] 1.3.7 Congo red analysis

[0110] Congo red is an acidic dye that forms a complex with polysaccharides with a triple helix conformation. After the complex is formed, the maximum absorption wavelength will be red-shifted. When the NaOH concentration is greater than a certain value, the maximum absorption wavelength drops sharply. Therefore, the maximum absorption wavelength of the Congo red complex of a polysaccharide with a triple helix will increase first and then decrease with the increase of NaOH concentration. Figure 6 As shown, compared with the Congo red solution, the maximum absorption wavelength of the mixed solution of APPCS-1a and Congo red did not undergo an obvious red shift, and with the gradual increase in the concentration of sodium hydroxide, the maximum absorption wavelength of the sample also gradually decreased, without the trend of first increasing and then decreasing that is unique to the triple helix structure, indicating that APPCS-1a does not have a triple helix structure.

[0111] 1.3.8 Scanning electron microscopy analysis

[0112] The morphology of polysaccharides not only affects their structure in solution, but also affects the dissolution rate and solubility of polysaccharides, thereby affecting their biological activity and application. Using SEM to observe the microstructure of polysaccharides can provide detailed information about their morphology and organization. Figure 5 The results showed that when observing the polysaccharide under a low-power microscope, APPCS-1a could be seen to have an irregular and rough flaky structure. When the magnification was increased, the surface of the polysaccharide could be seen to have a smooth wavy shape, which may be related to the negative charge carried by the polysaccharide molecules.

[0113] 1.3.9 Zeta potential

[0114] The value of Zeta potential is related to the stability of colloidal dispersion. It can affect the stability of particles in the dispersed solution through the electrostatic repulsion between particles and has a significant effect on the biological activity of the particles. The larger the absolute value of Zeta potential, the more stable the system. Figure 7 As shown in A, the Zeta potential of APPCS-1a is -27.5mV. Studies have shown that polysaccharide components with more negative charges have electron-donating ability, making them relatively more bioavailable. Therefore, APPCS-1a is more easily absorbed and utilized in medicines and foods, and has a wider application value in production and manufacturing.

[0115] 1.3.10 Thermogravimetric analysis

[0116] Thermogravimetric analysis helps to understand the thermal behavior of polysaccharides. Figure 7 B shows that APPCS-1a experienced two mass losses during the heating process of 25-800℃. The first mass loss was around 30-170℃, and the mass loss of polysaccharide was about 11.5%, which may be due to the incomplete water loss of polysaccharide freeze-drying. Sugar contains abundant hydrophilic groups, which can absorb water. As the temperature rises, the water evaporates, causing the mass of the polysaccharide sample to decrease. Around 170-350℃, the polysaccharide experienced a second mass loss, with a weight loss of about 46.5%, which may be caused by the rapid depolymerization and thermal decomposition of polysaccharides due to high temperature. A large number of chemical bonds of polysaccharides were destroyed, and they were decomposed into oligosaccharides and monosaccharides, and then further decomposed into CO2 and water vapor. The thermal weight loss reached the maximum at 255℃, and the inflection point was at 229℃, indicating that the polysaccharide can maintain good stability below 229℃. After 350℃, the weight loss tended to be slow, and at the end of the reaction, the sample had a residual of about 25.7%, which may be due to the fact that most of the polysaccharides became stable carbonized structures after high temperature treatment.

[0117] 1.3.11 Total antioxidant activity

[0118] Within the experimental concentration range, APPCS-1a has a certain antioxidant capacity, and the total antioxidant activity of APPCS-1a shows a dose-dependent effect. When APPCS-1a is 5 mg / mL, its antioxidant capacity is relatively strong, and the absorbance reaches 1.29.

[0119] 1.3.12 Restore Capability

[0120] Reducing ability can be used as a method to evaluate the antioxidant activity of natural products. Within the experimental concentration range, APPCS-1a has a certain reducing ability, and its reducing ability increases with the increase of polysaccharide concentration, showing a dose-dependent effect. When APPCS-1a is 4 mg / mL, its reducing ability is strong, and the absorbance reaches 1.69. The quality of reducing ability is usually related to the content of reducing ketones, and it is speculated that APPCS-1a may contain a small amount of reducing ketones.

[0121] 1.3.13 Scavenging effect of APPCS-1a on DPPH free radicals

[0122] The DPPH free radical scavenging test is a quick and easy method to evaluate the antioxidant activity of natural substances. DPPH is a synthetic free radical. The principle is that DPPH free radicals can act as hydrogen acceptors, and polysaccharides can react with them as electron donors to reduce the effective concentration of hydroxyl free radicals, peroxy free radicals, and alkyl free radicals, or terminate lipid peroxidation chain reactions. Figure 8 C It can be seen that the scavenging rate of APPCS-1a increases with the increase of concentration. When APPCS-1a is 4 mg / mL, its free radical scavenging activity reaches 81.52%, which has a strong ability to scavenge DPPH free radicals. Modern pharmacological studies have found that saffron can effectively scavenge oxygen free radicals. The uronic acid content of alkali-extracted saffron polysaccharides is higher than that of water-extracted saffron polysaccharides. Uric acid can activate the hydrogen atoms of the anomeric carbon in the polysaccharide, which may allow APPCS-1a to provide more hydrogen to react with free radicals, thereby making APPCS-1a have stronger antioxidant activity.

[0123] 1.3.14 Scavenging effect of APPCS-1a on hydroxyl radicals

[0124] Hydroxyl radicals are a type of reactive oxygen species (ROS). They are the most toxic and active free radicals that cause the most serious oxidative damage. They can react quickly with any biological macromolecule in the cell. 2 +) to generate hydroxyl radicals under the catalysis of alkali-extracted polysaccharides. Hydrogen peroxide can also be decomposed to generate hydroxyl radicals under ultraviolet irradiation. The hydroxyl radical scavenging activity of APPCS-1a is dose-dependent. When the concentration of APPCS-1a is 10 mg / mL, its hydroxyl radical scavenging activity reaches 88.46%, indicating that APPCS-1a has a certain ability to provide proton hydrogen to reduce free radicals. Studies have found that polysaccharides with lower molecular weight usually have more reducing groups and free hydroxyl groups. Alkaline-extracted polysaccharides have lower molecular weights, which enhances the ability of APPCS-1a to remove free radicals.

[0125] Example 2

[0126] This example uses oral administration to study the mitigation effect of APPCS-1a on the acute aging model of mice induced by D-gal, including the effects on antioxidant enzymes, tissue pathological changes, changes in apoptotic protein expression, intestinal flora, and brain metabolism, aiming to explore the effects of APPCS-1a on delaying aging and benefiting health based on the "gut-brain axis" theory. The results of the present invention help to better understand the mitigation and therapeutic mechanism of APPCS-1a on aging, as well as its effects on intestinal microorganisms, intestinal health, and brain metabolism, and provide a theoretical basis for the application and development of APPCS-1a.

[0127] 2.1 Experimental Materials

[0128] 2.1.1 Experimental herbs and animals

[0129] Medicinal materials: The crocus petal polysaccharide APPCS-1a obtained by extraction, separation and purification in Example 1 was used.

[0130] Experimental animals: 60 KM male mice, SPF grade, purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd., weighing about 30g, 6 weeks old, experimental animal certificate number: 20240314Abzz0100999357, experimental animal production license: SCXK (Zhejiang) 2022-0005. The experimental animal use license number is: SYXK (Zhejiang) 2022-0024. The quality inspection of mice was completed in Hangzhou Hospital. They were raised in an SPF environment (temperature: 25±0.5℃, relative humidity: 60%~70%, light 12h / dark 12h), fed with standard feed (Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.), and had free access to water. All procedures involving animals in the entire experimental process were approved by the Experimental Animal Ethics Committee of Huzhou Normal University.

[0131] 2.1.2 Experimental reagents

[0132] Crocus polysaccharide; TP kit, Nanjing Jiancheng Bioengineering Institute; T-SOD, MDA, CAT, GSH kits, Nanjing Jiancheng Bioengineering Institute; T-NOS, AchE kits, Nanjing Jiancheng Bioengineering Institute; RIPA tissue / cell lysate, Jiangsu Biyuntian; BCA protein concentration determination kit, Jiangsu Biyuntian; SDA-PAGE protein loading buffer (4×), Jiangsu Biyuntian; Western blot preparation kit, Yazyme; β-Actin Mouse Monoclonal Antibody, Biyuntian; p21 antibody (mouse monoclonal antibody), Biyuntian; p53 antibody (mouse monoclonal antibody), Biyuntian; Bax antibody (mouse monoclonal antibody), Biyuntian; horseradish peroxidase-labeled goat anti-mouse IgG (H+L), Biyuntian.

[0133] 2.1.3 Experimental instruments and equipment

[0134] Vortex oscillator WH-966 Taicang Science and Education Equipment Factory; desktop high-speed refrigerated centrifuge ESJ182-4 Changsha Dongwang Experimental Instrument Co., Ltd.; electronic balance ESJ182-4 Tianjin Boda Hongli Weighing Equipment Co., Ltd.; microplate reader SpectraMax 190

[0135] MD Company, USA; UV-visible spectrophotometer UV-1150 Shanghai Mepta Instrument Co., Ltd.; portable pressure steam sterilizer DSX-280B Shanghai Shen'an Medical Equipment Factory.

[0136] 2.2 Experimental methods

[0137] 2.2.1 Animal experiments and grouping

[0138] Sixty KM male mice were randomly divided into 6 groups according to their body weight, with 10 mice in each group, namely normal control group (CK group), model group (M group), positive control group (P group) and alkali-extracted saffron polysaccharide low, medium and high dose groups (XL, XM, XH). They were allowed to adapt for one week before the experiment. Except for the CK group, which was subcutaneously injected with 0.2 mL of saline in the back of the neck every afternoon, the other groups of mice were subcutaneously injected with D-galactose (150 mg / kg / D, 0.2 mL) in the back of the neck for 8 consecutive weeks. At the same time, the XL group was gavaged with 0.3 mL APPCS-1a (100 g / kg / D), the XM group was gavaged with 0.3 mL APPCS-1a (200 g / kg / D), the XH group was gavaged with 0.3 mL APPCS-1a (400 g / kg / D) (the dosage of APPCS-1a was determined by referring to the results of the preliminary experiment), the P group was gavaged with 0.3 mL Vc (200 g / kg / D), and the CK group was gavaged with the same amount of saline for 8 consecutive weeks. During the modeling experiment, the weight of the mice was weighed weekly using an electronic balance, and the behavioral activities, appearance (hair volume, hair color, glossiness, whether hair loss), appetite, mental state, skin elasticity, etc. of the mice in each group were observed and recorded.

[0139] 2.2.2 Sample collection and processing

[0140] At the end of the 8th week of the experiment, the mouse feces were taken, placed in a sterile centrifuge tube, and stored at -80℃ for later use. After taking the feces, the mice were fasted for 12h. Weigh before sampling and calculate the amount of anesthetic. The mice were anesthetized with ether, the eyeballs were removed to take blood, the blood samples were placed in a centrifuge tube, and left at 4℃ for 30min to allow the serum to precipitate naturally, and centrifuged at 3000r / min and 4℃ for 10min to take the serum for testing. After the eyeballs were bled, the mice were quickly dissected, the mouse jejunal tissue was taken out, washed with pre-cooled saline, dried with filter paper, weighed, and placed in a cryopreservation tube at -80℃ for later use. Weigh 50mg of mouse tissue sample, put it in a 1.5mL centrifuge tube, add pre-cooled saline at a ratio of 1:9, and add magnetic beads, homogenize with a tissue homogenizer to make a 10% tissue homogenate, centrifuge at 2500 rpm for 10min, and take the supernatant for testing.

[0141] 2.2.3 Observation of mouse jejunal tissue morphology

[0142] Fresh jejunal tissue blocks were obtained, fixed with 4% paraformaldehyde, dehydrated in gradients, embedded in paraffin, and cut longitudinally. After HE staining of jejunal and liver tissues, the morphological changes of small intestinal tissues were observed under a x100 light microscope, the number of goblet cells on jejunal villi was observed and counted, and imageJ was used to analyze the length of jejunal villi and the depth of crypts, and their ratios were calculated.

[0143] 2.2.4 Determination of serum biochemical indicators

[0144] According to the operating instructions of the kit, the thiobarbituric acid method was used to detect the MDA content in serum, the xanthine oxidase method was used to detect the T-SOD activity in serum, and the ammonium molybdate method was used to detect the CAT activity in serum.

[0145] 2.2.5 Western Blot Analysis

[0146] Brain homogenate was prepared and the expressions of Bax, Bcl-2 and Caspase3 were detected.

[0147] 2.2.6 16S rRNA gene sequencing and intestinal microbial analysis

[0148] Six samples were randomly selected from each group, fecal DNA was extracted, and the intestinal flora was compared using a high-throughput 16SrRNA detection method. The original sequencing data were filtered to obtain valid data, and OTU clustering / denoising and species classification analysis were performed based on the valid data to form an OTU species abundance spectrum. Based on sequence reads and OTUs, α diversity, β diversity, and linear discriminant analysis effect size (LEfSe) were analyzed.

[0149] 2.2.7 UPLC-MS detection of brain tissue supernatant

[0150] The metabolites of mouse brain tissue were separated and detected using a Waters 2777C UPLC and a Q Exactive HF high-resolution mass spectrometer tandem system. First, the brain tissue samples were placed in a mortar, and liquid nitrogen was added to quickly grind them. 100 mg of the sample was accurately weighed and placed in a centrifuge tube. 500 μL of 80% methanol aqueous solution was added, mixed, and placed in an ice-water bath for 5 minutes. The supernatant was taken for dilution before liquid chromatography-mass spectrometry analysis and centrifuged again at 4°C for 20 minutes. Finally, the treated supernatant was injected into the LC-MS / MS system for subsequent analysis. The raw signal data generated by the mass spectrometer was imported into the metabolomics analysis software Progenesis QI. After processing, a data matrix containing information such as retention time, mass-to-charge ratio, and peak area was generated. In order to predict the functions of differential metabolites, the obtained sequencing data was compared with the KEGG reference gene database containing known metabolic information.

[0151] 2.2.8 Data Analysis

[0152] Statistical analysis was performed using IBM SPSS Statistics 26. One-way analysis of variance (ANOVA) was used to assess differences among groups. A value of P < 0.05 was considered statistically significant.

[0153] 2.3 Results and Analysis

[0154] 2.3.1 Tissue section observation

[0155] 2.3.1.1 Changes in jejunal mucosal morphology

[0156] like Fig. 9 As shown in the figure, the villi of the jejunum in the normal group were neatly arranged, with clear and complete structures, without hypertrophy or loss, and the glands were arranged tightly and neatly. The villi of the jejunum in the model group became shorter and sparse, and there were phenomena such as shedding, breaking, and dissolution. After intervention with saffron polysaccharide, the damage to the intestinal villi was improved. The effects of the medium and high dose groups of polysaccharide were similar to those of the positive control group, and the jejunal tissue structure basically returned to normal.

[0157] 2.3.1.2 Changes in jejunal villus length, crypt depth, villus length / crypt depth (V / C value), and goblet cells

[0158] Aging affects the structure and function of the intestine. With age, the digestion and absorption capacity of the intestine decreases, the intestinal villi shorten, the intestinal mucosa becomes thinner, the crypt depth becomes deeper, and the number of goblet cells decreases. Compared with the normal group, the length of the intestinal villi in the model group was significantly shortened (P < 0.01), the crypt depth became significantly deeper (P < 0.05), and the V / C ratio was significantly reduced (P < 0.01). The length of the intestinal villi in the polysaccharide administration group gradually increased with the increase of polysaccharide dose, which was the same as the trend of V / C ratio. Although the crypt depth became shallower, it was not statistically significant, indicating that saffron polysaccharide can significantly increase the length of the intestinal villi of aging mice and alleviate the crypt depth to a certain extent. The normal group had abundant goblet cells, and the number of goblet cells in the model group was significantly reduced compared with the normal group (P < 0.01). The medium and high dose groups of polysaccharides can significantly improve this condition.

[0159] 2.3.1.3 Liver pathological sections

[0160] Liver tissue is sensitive to oxidative damage and can be used as an important indicator for evaluating mouse aging. Fig.10 As shown, the CK group was normal liver tissue, with no obvious pathological changes in HE sections, and the liver tissue structure was clear and complete. With the central vein as the center, the hepatocytes were arranged in a single layer radially, and blood sinusoids were visible between the hepatocyte cords. The hepatocytes were plump and round, with pink cytoplasm and uniform cytoplasm; the nuclear boundaries were obvious, there was no obvious shrinkage, and there were many binuclear hepatocytes. In the M group, the hepatic cords were disordered, the hepatocytes were edematous and degenerated, the cytoplasm was loose and lightly stained, the nuclear boundaries were blurred, and there were few binuclear hepatocytes. The liver damage in the other four groups of liver tissue staining sections was alleviated, and the improvement effect of the P group and the high-dose polysaccharide group was particularly obvious.

[0161] 2.3.2 Antioxidant enzyme activity

[0162] 2.3.2.1 Effects of APPCS-1a on serum SOD, CAT and MDA in mice

[0163] SOD enzyme can catalyze the dismutation of superoxide anion free radicals to generate oxygen and hydrogen peroxide, and decompose them into harmless water through other pathways. SOD enzyme plays a vital role in the balance of oxidation and anti-oxidation in the body. Fig.11 The SOD activity in the serum of M shown in A was significantly lower than that of the other four groups (P<0.05), indicating that the D-gal model was successfully established. As the dose of saffron polysaccharide increased, the SOD activity also gradually increased, showing a dose-dependent effect, and the SOD activity of the saffron dose group was better than that of the normal group, indicating that saffron polysaccharide can effectively improve the SOD activity of serum in D-gal-induced aging damage and increase the body's antioxidant capacity.

[0164] CAT enzyme, full name Catalase, its main function is to catalyze the decomposition of hydrogen peroxide into water and oxygen, remove hydrogen peroxide in the body, thereby protecting cells from H2O2 toxicity. It is one of the key enzymes in the biological defense system. Fig.11 The CAT activity in the serum of M group (B) was significantly lower than that of the other four groups (P<0.05), indicating that the D-gal model was successfully established. As the dose of saffron polysaccharide increased, the CAT activity also gradually increased, showing a dose-dependent effect.

[0165] MDA is a toxic product produced by free radical-induced lipid peroxidation. Excessive accumulation of MDA can cause cross-linking and polymerization of macromolecules such as proteins and nucleic acids, leading to changes in the structure and function of cell membranes. It is usually used to evaluate the degree of tissue peroxidation damage. Fig.11 As shown in Figure C, after intervention with saffron polysaccharide, the MDA content in the serum of the high-dose polysaccharide group decreased significantly with the increase in dose (P<0.05), indicating that saffron polysaccharide has a certain ability to reduce the body's MDA and significantly restore the body's antioxidant level.

[0166] 2.3.2.2 Effect of APPCS-1a on GPx in mouse intestinal tissue

[0167] GPx (glutathione peroxidase) is an important antioxidant enzyme that is widely present in the body and can catalyze reduced glutathione (GSH) to generate oxidized glutathione (GSSG), thereby accelerating the reduction of toxic hydrogen peroxide to non-toxic hydroxyl compounds. GPx plays an important role in maintaining the redox balance in cells, especially in protecting cells from oxidative stress damage. Fig.11 As shown in D, the GPx activity of intestinal tissue in group M was significantly lower than that in the normal group, indicating that the D-gal model was successfully established. After intervention with saffron polysaccharide, the GPx activity in the low-dose group increased, but not significantly, while the GPx activity in the medium- and high-dose groups increased significantly (P<0.05).

[0168] 2.3.2.3 Effects of APPCS-1a on AchE and T-NOS in the Mouse Brain

[0169] Acetylcholinesterase (AChE) is a hydrolase of acetylcholine (ACh). ACh is a neurotransmitter that transmits signals between neurons. A decrease in the Ach content in the brain can cause a decline in memory and learning abilities. In brain tissue homogenate, the activity of AChE can reflect changes in the Ach content in the brain. In addition, an increase in AchE promotes the aggregation of Aβ and aggravates neuronal damage in AD patients. The AchE activity in the brain tissue of mice in the aging model group increased significantly, accelerating the decomposition of Ach, resulting in insufficient Ach content, causing central nervous system disorders, decreased learning and cognitive functions, and even loss of intelligence. Fig.11 As shown in E, the activity of AchE in the polysaccharide dosage group was significantly lower than that in the model group, indicating that saffron polysaccharide can reduce the decomposition of Ach by reducing the activity of AchE and improve the higher neural function of the brain in D-gal-induced aging mice.

[0170] T-NOS is a nitric oxide synthase (neuronal nitric oxide synthase), which is widely expressed in the nervous system. T-NOS can catalyze the conversion of L-arginine into nitric oxide. NO is an important neurotransmitter that plays a variety of physiological functions in the nervous system, including regulating vascular tension, participating in learning and memory processes, and playing a role in neurodegenerative diseases. NO is involved in a variety of functional changes during the aging process. Excessive NO may cause tissue damage through cytotoxic and cytostatic effects, induce neuronal apoptosis, and is one of the causes of neurodegenerative diseases such as Parkinson's and Alzheimer's. Fig.11 As shown in Figure F, the activity of T-NOS in the model group was significantly higher than that in the blank control group. After polysaccharide intervention, the activity of T-NOS decreased, which was consistent with the dose-dependent relationship.

[0171] 2.3.3 Effect of APPCS-1a on apoptotic proteins in brain tissue

[0172] Bcl-2 is mostly located on the outer membrane of mitochondria. Its increased expression will cause the dissociation of Bax / Bcl-2 dimers, thereby preventing the release of apoptosis-related factors and preventing cell apoptosis. When cells are damaged or stressed, Bax will increase mitochondrial permeability, activate the Caspase family, promote the release of apoptosis signals, and lead to cell apoptosis. Caspase-3 is cysteine ​​protease-3, also known as death protein execution enzyme. It usually exists as an inactive enzyme element. After activation, it becomes active Caspase-3, which can cause cell apoptosis to enter an irreversible stage. Fig.12As shown in the figure, the expression of Bcl-2 was down-regulated, while the expression of Caspase3 and Bax was up-regulated in the model group, which was reversed in the polysaccharide administration group.

[0173] 2.3.4 Effect of APPCS-1a on the intestinal flora of aged mice

[0174] Fig.13 The distribution of intestinal microbiota shown in Figure 1 indicates that saffron polysaccharides can delay aging by improving the imbalance of intestinal flora in mice, especially the ratio of Bacteroidetes to Firmicutes. Fig.14 As shown in the data, there were no significant differences in the Chao, Shannon and Simpson indices between the normal group and the polysaccharide intervention group, indicating that the diversity, richness and uniformity of the microbiota in the two groups were similar. However, compared with the model group, saffron polysaccharide intervention could improve the richness and diversity of the intestinal microbiota in aging mice, and make the functions of the dominant microbiota more prominent.

[0175] In conclusion, saffron polysaccharides can improve the Chao1, Shannon and Simpson indexes of the intestinal flora of aged mice, and increase the proportion of beneficial bacteria such as Lachnospiraceae, Lactobacillus and Bacteroides.

[0176] 2.3.5 Effect of APPCS-1a on brain metabolism in aged mice

[0177] like Fig.14 As shown in the metabolome study, it is believed that the anti-aging effect of saffron polysaccharides is mainly through arginine metabolism, glutathione metabolism, amino acid metabolism, pantothenic acid and coenzyme A biosynthesis, and TCA cycle. Changes in the levels of some important amino acids and changes in metabolic genes are related to aging.

[0178] Comparative Example 1 CN114751995A A saffron petal polysaccharide CSP1 with anti-inflammatory effect, preparation method and application thereof

[0179] Although both APPCS-1a and CSP1 are polysaccharides from saffron petals, they have many differences in extraction methods and polysaccharide structures.

[0180] (1) The monosaccharide composition is different.

[0181] APPCS-1a is a heteropolysaccharide composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose, with molar ratios of 0.274:6.717:1.575:4.313:0.820:64.321:1.560:20.419. CSP1 is mainly composed of mannose, glucuronic acid, rhamnose, glucose, galactose, and arabinose, with a content ratio of 8.31:1.73:43.97:3.77:31.56:10.65, and the remaining molar content is other components. CSP1 rhamnose and galactose account for the vast majority, with a ratio of 43.97:31.56, and APPCS-1a galactose accounts for the vast majority, accounting for about 64.32%.

[0182] Secondly, APPCS-1a contains about 1.56% xylose and very little about 0.27% mannose, while CSP1 contains no xylose and has a higher content of mannose.

[0183] Finally, APPCS-1a is extracted by alkali extraction instead of distilled water, so the obtained APPCS-1a is rich in uronic acid, especially galacturonic acid. CSP1 contains only a small amount of glucuronic acid and no galacturonic acid.

[0184] (2) Different extraction methods

[0185] APPCS-1a was extracted with dried petals at a solid-liquid ratio of 1:30, while CSP1 was extracted with fresh petals at a solid-liquid ratio of 1:60. APPCS-1a was extracted twice with 8% alkali solution (NaOH) in a 90°C water bath for 2 h, while CSP1 was extracted three times in a 100°C oil bath.

[0186] Previous studies in our laboratory have found that when APPCS-1a is extracted with alkaline solution, the sugar content of the extract is still high when extracted a second time under the same conditions. When extracted a third time, the sugar content of the extract is extremely low, almost non-existent. From the perspective of economic saving, APPCS-1a is only extracted with alkaline twice.

[0187] Alkaline solution extraction has many advantages over aqueous solution extraction. Alkaline solution can fully destroy the plant cell wall, making polysaccharides more easily dissolved, and the final polysaccharide yield is higher. In this study, the single extraction yield of saffron petal polysaccharides was about 14%, and the double extraction yield was about 24%. Polysaccharides rich in uronic acid are also more soluble in alkaline solutions. Polysaccharides with a higher uronic acid content will have better antioxidant activity and a wider range of application value. The molecular weight of polysaccharides extracted by alkali solution is also smaller, APPCS-1a is about 40.3KDa, and polysaccharides with smaller molecular weights are easier to absorb and utilize.

[0188] (3) Different alcohol precipitation conditions

[0189] When APPCS-1a is precipitated with alcohol, 95% ethanol is used, and the ratio of ethanol to polysaccharide concentrate is 4:1. Potassium chloride is added to ethanol to promote better coagulation of polysaccharides and reduce losses during the alcohol precipitation process. When CSP1 is precipitated with alcohol, its final concentration is 70 vol%.

[0190] Different concentrations of ethanol precipitation of polysaccharides will affect their purity, yield and structure. The precipitation selectivity is relatively poor at lower ethanol concentrations. As the ethanol concentration increases, the purity of the precipitated polysaccharides may increase, but other impurities may also co-precipitate. When the ethanol concentration is too low, the polysaccharide precipitation is incomplete and the yield will be low. If the ethanol concentration is too high, the polysaccharide yield may be artificially high due to factors such as co-precipitation of impurities. 4 times 95% ethanol precipitation is a relatively good alcohol precipitation condition obtained by our laboratory based on many years of experience.

[0191] (4) No need to remove protein

[0192] The alkaline solution extraction of APPCS-1a does not require a protein removal step, as the alkaline solution will cause excessive denaturation and degradation of the protein, thereby reducing the protein content. The results of ultraviolet full-wavelength scanning analysis also show that the alkaline-extracted saffron petal polysaccharide does not contain protein, so there is no need to remove protein from the extracted polysaccharide, thereby avoiding the loss of polysaccharide during the protein removal process.

[0193] (5) Different purification columns

[0194] CSP1 uses Sephadex G-200 gel chromatography column, and APPCS-1a uses Sephadex S-100. The advantage is that Sephadex S-100 has good mechanical stability, can withstand high flow rates, and can achieve relatively fast separation. The separation range of Sephadex S-100 is 1000-100000, which is more suitable for separating polysaccharides with relatively small molecular weight. SephadexG-200 has strong hydrophilicity and is generally suitable for separation and purification in aqueous solution systems. Sephadex S-100: has good stability in various solvents and a wider range of applicable solvents.

[0195] Comparative Example 2 WO2019137349A1 A safflower polysaccharide, a preparation method thereof and its application in anti-tumor drugs

[0196] HH1-1 was extracted five times with boiling water at a ratio of 1:50 and dried in a vacuum dryer at 50°C, while APPCS-1a was extracted with alkaline solution at a ratio of 1:30 and the extract concentrate was dried in a vacuum freeze dryer.

[0197] HH1-1 uses a Cl-type DEAE-cellulose anion column, and APPCS-1a uses a DEAE-52 cellulose anion column and Sephadex S-100. DEAE-52 cellulose anion column usually refers to a column with a diethylaminoethyl ion exchange group. It can be transformed with different ion solutions as needed when used, and it is not necessarily a Cl-type. In general, after good column packing and balancing operations, DEAE-52 cellulose anion column can provide good column efficiency and resolution, and can effectively separate biomacromolecules with similar molecular weights. DEAE-52 cellulose anion column has good chemical stability and can tolerate a variety of buffers.

[0198] Safflower polysaccharide HH1-1 mainly contains galactose (59.2wt%) and arabinose (40.8wt%), while APPCS-1a mainly contains galactose (64.32%) and arabinose (20.42%). Compared with HH1-1, it contains more galactose and relatively less arabinose. The difference in the polysaccharide structure may lie in the length, degree of branching and number of arabinose and galactose side chains, which leads to differences in their activity.

[0199] Comparative Example 3 CN109678981B A method for preparing safflower polysaccharide, product and application

[0200] Safflower polysaccharide was extracted twice with water at 60°C, with the solid-liquid ratios being 1:10 and 1:8 respectively. APPCS-1a was extracted twice with a solid-liquid ratio of 1:30 and was bathed in alkaline solution at 90°C for 2 hours.

[0201] Safflower polysaccharide used polar macroporous adsorption resin and Sephadex LH-20 dextran gel to collect two fractions, and APPCS-1a used DEAE-52 cellulose anion column and Sephadex S-100 to collect one fraction.

[0202] Safflower polysaccharide A is composed of six monosaccharides, including rhamnose, arabinose, glucose, galactose, xylose, and mannose, connected by β-chains; safflower polysaccharide B is composed of four monosaccharides, including arabinose, glucose, galactose, and rhamnose, connected by β-chains. APPCS-1a is composed of eight monosaccharides, including mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose, connected by β- and α-chains.

[0203] Comparative Example 4 CN109422820 A Preparation method of safflower polysaccharide

[0204] Safflower is mostly extracted by boiling sugar in boiling water for 3 times, each time for 0.5h, stirring while extracting, adding 4 times the volume of 85% ethanol during alcohol precipitation, and repeating the ethanol precipitation for 2 times. APPCS-1a is extracted twice by bathing in 90℃ alkali solution for 2h, stirring at a constant speed with a hanging magnetic stirrer, adding 4 times the volume of 95% ethanol during alcohol precipitation, and alcohol precipitation once.

[0205] Comparative Example 5 CN104288201A A safflower soft capsule and its preparation method

[0206] The safflower extract was extracted by decoction in water for 1.5 hours, with a solid-liquid ratio of 1:10, and extracted again for 1 hour. Graded alcohol precipitation was used. APPCS-1a was bathed in 90℃ alkaline solution for 2 hours and extracted twice. During alcohol precipitation, 4 times the volume of 95% ethanol was added and the precipitation was performed once.

[0207] The safflower extract was purified by AB-8 macroporous adsorption resin column chromatography, and APPCS-1a was separated and purified using DEAE-52 cellulose anion column and Sephadex S-100.

[0208] The safflower extract was microwave dried for 120 minutes, and the APPCS-1a extract concentrate was dried in a vacuum freeze dryer. The safflower extract contained polysaccharides and flavonoids, with a total flavonoid weight content of 17%, while APPCS-1a was only a single polysaccharide component and did not contain flavonoids.

[0209] APPCS-1a of the present invention is extracted from saffron petals, while comparative examples 2-5 are safflower extracts. Safflower: It is the dried flower of safflower of the Asteraceae family. Compared with saffron, the whole flower can be picked, and the yield is usually higher than that of saffron. Saffron: It is a perennial herb of the genus Crocus of the Iridaceae family and is a precious Chinese medicinal material.

Claims

1. An alkali-extracted saffron petal polysaccharide APPCS-1a, which has the structure shown in Figure 1, wherein the main chain structure of APPCS-1a is →2)-α-Rhap-(1→4)-α-GalpA-(1→, and the side chains are mainly →4)-β-D-Galp-(1→, →3,6)-β-D-Galp-(1→, T-β-D-Galp, →5)-α-L-Araf-(, →T-α-L-Araf) linked to the C-4 position of rhamnose, and its structural formula is shown in Figure 1.

2. The alkali-extracted saffron petal polysaccharide APPCS-1a according to claim 1, characterized in that: APPCS-1a is a homogeneous polysaccharide with a molecular weight of about 40KDa.

3. The alkali-extracted saffron petal polysaccharide APPCS-1a according to claim 1, characterized in that: APPCS-1a is composed of eight monosaccharides: galactose, arabinose, rhamnose, galacturonic acid, glucuronic acid, xylose, glucose, and mannose, with molar ratios of 64.321%, 20.419%, 6.717%, 4.313%, 1.575%, 1.560%, 0.820%, and 0.274%, respectively.

4. The preparation method of alkali-extracted saffron petal polysaccharide APPCS-1a according to claim 1, comprising the following steps: S1: heating and drying saffron petals to constant weight, crushing them, adding a solvent to the petal powder to remove pigments and fat-soluble substances, filtering, collecting the filter residue, and drying for later use; S2 Take an appropriate amount of pretreated powder, add alkaline solution, heat in a water bath, stir and extract twice, quickly neutralize the extract to neutrality after the extraction, centrifuge and filter to obtain the supernatant, concentrate the supernatant under reduced pressure, and dialyze the polysaccharide concentrate; After adding the aggregation promoter to the S3 polysaccharide concentrate, anhydrous ethanol is added for precipitation, the mixture is allowed to stand for filtration, and the mixture is heated and dried to obtain the crude polysaccharide; The crude S4 polysaccharide was separated from saffron polysaccharide by a cellulose column, and deionized water, 0.1, 0.3, and 0.5 M NaCl were used for continuous elution; the 0.1 M elution fraction was dialyzed, purified by a gel column, concentrated, and freeze-dried to obtain APPCS-1a.

5. The preparation method according to claim 4, characterized in that: In step S1, the heating and drying temperature is 50° C.; the solvent is 95% ethanol.

6. The preparation method according to claim 4, characterized in that: In step S2, the alkaline solution is 8% NaOH solution; the water bath heating condition is heating at 90° C. for 2 hours; the centrifugal filtration condition is 4000 r / min, 10 min; and the dialysis condition is 3500 Da dialysis for 72 hours.

7. The preparation method according to claim 4, characterized in that: The volume ratio of the saffron polysaccharide concentrate to ethanol in step S3 is 1:4; and the aggregation promoter in step S3 is KCl.

8. The preparation method according to claim 4, characterized in that: In step S4, the cellulose column is DEAE-52 with a specification of 5.5×50 cm; the elution rate is 1.6 mL / min; the dialysis condition is 500 Da at 4° C. for 3 days; and the gel column is S-100.

9. Use of the saffron petal polysaccharide APPCS-1a according to claim 1 in the preparation of anti-aging drugs, health products or cosmetics.

10. An anti-aging medicine, health product or cosmetic, comprising a preventive or therapeutic effective amount of the saffron petal polysaccharide APPCS-1a according to claim 1 and pharmaceutically acceptable pharmaceutical excipients thereof.

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