A purified polysaccharide from pyracantha fruit with significant resistance to light damage, and its preparation and application in anti-light damage drugs

Abstract

The present invention belongs to the field of traditional Chinese medicine, and discloses a purified polysaccharide of Pyracantha fruit with significant resistance to light damage, and its preparation and application in anti-light damage drugs. Method: 1) Pyracantha fruit powder is subjected to hot water extraction, concentration, alcohol precipitation, and drying to obtain crude polysaccharide of Pyracantha fruit; 2) Pyracantha fruit crude polysaccharide is decolorized and deproteinized to obtain crude polysaccharide of Pyracantha fruit deproteinized; 3) Pyracantha fruit deproteinized crude polysaccharide is purified by DEAE-FastFlow column chromatography, and water, 0.05M NaCl solution, and 0.15M NaCl solution are used for gradient elution in sequence, and eluents are collected respectively, and the eluents obtained by eluting with 0.15M NaCl solution are concentrated, dialyzed, and dried to obtain purified polysaccharide of Pyracantha fruit. The purified polysaccharide of the present invention has significant anti-light damage activity, and the dosage is low, and the toxic and side effects at the same dosage are also low. Application of the purified polysaccharide of the present invention in the preparation of anti-light damage drugs.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and particularly relates to a pyracantha fruit polysaccharide and a preparation method thereof and an application thereof in the preparation of an anti-photodamage drug. Background Art

[0002] Human skin is the largest defense organ with many complex functions that can resist pathogenic microorganisms, radiation and harmful chemicals. Ultraviolet B (UVB) radiation is a ubiquitous environmental factor. Photodamage and aging caused mainly by UVB radiation are related to the occurrence of many diseases such as seborrheic keratosis, pigmented spots, solar keratosis and skin malignancies. Therefore, the prevention and improvement of skin photoaging has gradually become a hot topic for dermatologists.

[0003] Pyracantha fortuneana, also known as Jiujunliang and Shuichazi, is an evergreen wild shrub of the genus Pyracantha in the subfamily Apple of the Rosaceae family. It is a multifunctional flower and fruit plant that can be used as medicine, food, and ornamental. Its fruit is rich in flavonoids, saponins, pigments, polysaccharides and other active ingredients, and has multiple biological activities such as clearing away heat and detoxification, strengthening the spleen and digestion, enhancing immunity, lowering blood sugar and lipids, and anti-aging.

[0004] At present, there is little research on the development of Pyracantha fruit products. The utilization of Pyracantha fruit resources is mainly focused on the extraction of pigments. There are few studies on the preparation of Pyracantha fruit into polysaccharide extracts and then developing them into products with physiological functions. The existing Pyracantha fruit extraction process is relatively simple, the content of the main active ingredients contained is unclear, and the quality is unstable. Therefore, extracting a single main active ingredient from Pyracantha fruit and using it to prepare functional products has certain prospects.

[0005] At present, the research on the pharmacological activity of Pyracantha fruit polysaccharides at home and abroad is mainly focused on antioxidant and immune activities, while there are few studies on other activities of Pyracantha fruit polysaccharides. Different extraction methods have different composition and purity of Pyracantha fruit polysaccharides. As a new resource food, the extraction, purification and pharmacological activity of Pyracantha fruit polysaccharides need to be further explored. Comprehensively strengthening the biological utilization of Pyracantha fruit is of great significance for the development of new drugs and guiding clinical drug use. Summary of the invention

[0006] In order to overcome the deficiencies and shortcomings of the prior art and enhance the industrial application of Pyracantha fortuneana fruit, the primary purpose of the present invention is to provide a purified polysaccharide from Pyracantha fortuneana fruit with significant resistance to light damage and a preparation method thereof.

[0007] Another object of the present invention is to provide the use of the above-mentioned purified polysaccharide from Pyracantha fortuneana fruit in the preparation of anti-photodamage drugs.

[0008] The purpose of the present invention is achieved through the following solutions:

[0009] A method for preparing a purified polysaccharide from Pyracantha fortuneana fruit with significant resistance to light damage comprises the following steps:

[0010] 1) extracting the Pyracantha fruit powder with hot water to obtain a Pyracantha fruit water extract; concentrating the water extract, precipitating with alcohol, and drying the precipitate to obtain a Pyracantha fruit crude polysaccharide;

[0011] 2) decolorizing and deproteinizing the crude polysaccharide from Pyracantha fortuneana fruit to obtain the deproteinized crude polysaccharide from Pyracantha fortuneana fruit;

[0012] 3) The deproteinized crude polysaccharide of Pyracantha fortuneana fruit was purified by DEAE-FastFlow column chromatography, and gradient elution was performed using water, 0.05 M NaCl solution, and 0.15 M NaCl solution in sequence. The eluates were collected respectively, and the eluate obtained by elution with 0.15 M NaCl solution was concentrated, dialyzed, and dried to obtain the purified polysaccharide of Pyracantha fortuneana fruit, which was recorded as HJGP.

[0013] The pyracantha fruit powder is obtained by drying, crushing and sieving the pyracantha fruit. The drying temperature is 40-50° C., the drying time is 12-32 hours, and the crushing and sieving is 18-60 mesh sieve.

[0014] The volume mass ratio of the pyracantha fruit powder to water in step 1) is 1 g: (15-25) mL;

[0015] The hot water extraction in step 1) refers to extraction by water immersion, heating and reflux, the number of hot water extractions is 1 to 3 times, the temperature of each extraction is 90 to 100° C., and the time of each extraction is 2 to 4 hours;

[0016] The alcohol precipitation in step 1) refers to mixing the concentrated solution with a 95% ethanol solution, and finally the final concentration of ethanol in the mixed system is 70-85%, and the concentration is a volume concentration; the alcohol precipitation is carried out at 0-4°C.

[0017] After the hot water extraction in step 1), centrifugation or filtration is performed; the rotation speed of the centrifugation is 3500-5000 r / min; and the time of the centrifugation is 5-10 min.

[0018] The concentration in step 1) is reduced pressure concentration; the temperature of the reduced pressure concentration is 60-70°C.

[0019] The drying in step 1) is heating drying and vacuum freeze drying, and the heating drying temperature is 60-70° C. That is, after heating drying, the mixture is reconstituted with water and then vacuum freeze dried.

[0020] The decolorization in step 2) refers to mixing the crude polysaccharide of Pyracantha fruit with water, then decolorizing with macroporous resin D354F, precipitating with alcohol, and drying the precipitate to obtain the decolorized crude polysaccharide of Pyracantha fruit.

[0021] During decolorization, the concentration of the Pyracantha fruit crude polysaccharide mixed with water is 4-6 mg / mL, the ratio of the Pyracantha fruit crude polysaccharide solution to the macroporous resin D354F is 1:1-1:3 (v / v), the decolorization temperature is 45-60°C, the decolorization times are 3-5 times, and the decolorization time each time is 2-4 hours. The drying is heating drying and vacuum freeze drying. That is, after heating drying, it is re-dissolved with water and then vacuum freeze dried.

[0022] The deproteinization in step 2) refers to mixing the decolorized crude polysaccharide of Pyracantha fruit with water, deproteinizing with Sevage solution, taking the aqueous phase, precipitating with alcohol, and drying to obtain the deproteinized crude polysaccharide of Pyracantha fruit. The drying is heating drying and vacuum freeze drying. That is, after heating drying, re-dissolving with water, and then vacuum freeze drying.

[0023] During the deproteinization, the concentration of the decolorized crude polysaccharide from Pyracantha fruit mixed with water is 4-6 mg / mL, the ratio of chloroform to n-butanol in the Sevag solution is 4:1-6:1 (v / v), the number of extractions is 3-5 times, and the extraction time for each time is 0.5-1.5 h.

[0024] In step 3), when the Pyracantha fruit deproteinized crude polysaccharide is purified by column chromatography, the Pyracantha fruit deproteinized crude polysaccharide is mixed with water at a concentration of 8 to 10 mg / mL; then mixed with DEAE-FastFlow column filler, and gradient elution is performed using water, 0.05 M NaCl solution, and 0.15 M NaCl solution in sequence.

[0025] The dialysis in step 3) refers to dialysis using a dialysis bag with a molecular cutoff of 8000Da, and the running water dialysis time is 3-4 days; the drying is freeze drying.

[0026] The purified polysaccharide of Pyracantha fortuneana fruit is composed of five monosaccharides, and the molar ratio of the monosaccharides is arabinose: galactose: glucose: xylose: galacturonic acid=4.15:2.97:1:1.34:1.89.

[0027] The relative weight average molecular weight of the purified polysaccharide from Pyracantha fortuneana fruit is 70859 Da.

[0028] Application of the purified polysaccharide from Pyracantha fortuneana fruit in the preparation of drugs against light damage;

[0029] An anti-photodamage drug contains the above-mentioned Pyracantha fortuneana fruit purified polysaccharide.

[0030] The purified pyracantha fruit polysaccharide (HJGP) of the present invention is used as an inhibitor for inhibiting apoptosis of skin cells damaged by UVB.

[0031] The purified pyracantha fruit polysaccharide (HJGP) of the present invention can prolong the life span of organisms damaged by UVB.

[0032] Principle of the invention: The purified polysaccharide (HJGP) of the present invention can significantly improve the survival rate of skin cells damaged by UVB, delay the release of oxidative factors, and prolong the life span of Caenorhabditis elegans damaged by UVB, thereby playing an effect of resisting light damage.

[0033] Compared with the prior art, the present invention has the following advantages and effects:

[0034] The natural plant active ingredient, the purified polysaccharide of Pyracantha fortuneana fruit (HJGP), has significant anti-light damage activity, and has a low dosage and low toxic and side effects at the same dosage. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a molecular weight diagram of the purified polysaccharide (HJGP) from Pyracantha fortuneana fruit prepared in Example 4;

[0036] Figure 2 This is a monosaccharide composition diagram of the purified polysaccharide (HJGP) of Pyracantha fortuneana fruit prepared in Example 4;

[0037] Figure 3 This is a graph showing the cytotoxicity of the purified polysaccharide (HJGP) from Pyracantha fortuneana fruit prepared in Example 4 to HaCaT cells;

[0038] Figure 4 This is a graph showing the effect of the purified polysaccharide (HJGP) from Pyracantha fortuneana fruit prepared in Example 4 on the survival rate of HaCaT cells damaged by UVB irradiation;

[0039] Figure 5 This is a survival curve of nematodes damaged by UVB irradiation under the action of the purified polysaccharide (HJGP) of Pyracantha fortuneana fruit prepared in Example 4. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0041] Example 1 Preparation of Pyracantha fortuneana fruit crude polysaccharide from Pyracantha fortuneana fruit

[0042] (1) Dry the Pyracantha fruit in an electric hot air drying oven at 45°C for 24 hours, crush it with a grinder, and pass it through a 40-mesh sieve to obtain the Pyracantha fruit coarse powder for later use;

[0043] (2) Weigh 200 g of crude powder of Pyracantha fortuneana fruit into a container, add it to boiling pure water at a mass-to-volume ratio of 1:20 g / mL, and use hot water extraction to extract crude polysaccharides of Pyracantha fortuneana fruit at an extraction temperature of 100°C and an extraction time of 3 h; filter the extract to remove the residue, take the filtrate and evaporate and concentrate it to 1 / 20 of the original volume to obtain a concentrated solution A; mix the concentrated solution with 95% ethanol at a volume ratio of 1:4, place it in a refrigerator at 4°C for 12 h, and centrifuge to obtain a precipitate; dry it in an oven at 65°C; wait until the ethanol evaporates completely, add a small amount of pure water to re-dissolve it, and obtain crude polysaccharides of Pyracantha fortuneana fruit after vacuum freeze-drying.

[0044] Example 2 Decolorization of Pyracantha fortuneana fruit crude polysaccharide

[0045] (1) The crude polysaccharide described in Example 1 is mixed with pure water to dissolve into a 5 mg / mL crude polysaccharide solution, and an equal volume of D354F macroporous resin pre-swollen with pure water for 3 hours is added, and the solution is stirred at 55° C. for 3 hours for decolorization. The decolorized polysaccharide concentrate B is obtained by suction filtration, and the concentrate is mixed with 95% ethanol in a volume ratio of 1:4, and the solution is placed in a 4° C. refrigerator for 12 hours, and the precipitate is obtained by centrifugation; the solution is dried in an oven at 65° C.; after the ethanol evaporates completely, a small amount of pure water is added for re-dissolution, and the decolorized crude polysaccharide of Pyracantha fruit is obtained after vacuum freeze-drying.

[0046] Example 3 Deproteinization of Pyracantha fortuneana fruit crude polysaccharide

[0047] The decolorized crude polysaccharide of Pyracantha fruit was mixed with pure water to dissolve into a 5 mg / mL crude polysaccharide solution, and 4 times the volume of chloroform-n-butanol mixture (5:1, v / v) was added, stirred at room temperature for 30 minutes, extracted and separated, the aqueous phase extract was taken, and the above operation was repeated 5 times to finally obtain the decolorized polysaccharide concentrate, and the concentrate was mixed evenly with 95% ethanol in a volume ratio of 1:4, placed in a 4°C refrigerator for 12 hours, and centrifuged to obtain the precipitate; dried in a 65°C oven; after the ethanol evaporated completely, a small amount of pure water was added to re-dissolve, and vacuum freeze-dried to obtain the deproteinized crude polysaccharide of Pyracantha fruit.

[0048] Example 4 Ion column chromatography purification of Pyracantha fortuneana polysaccharide

[0049] The crude polysaccharide after deproteinization of Pyracantha fruit was mixed with pure water and dissolved into a 10 mg / mL crude polysaccharide solution, and the mass volume ratio of the crude polysaccharide solution to the DEAE-FastFlow column filler was 1 mg:1.5 mL; pure water, 0.05 and 0.15 mol / L NaCl were used for gradient elution, and the elution rate was 2.0 mL / min; the 0.15 mol / L NaCl component was evaporated and concentrated to 1 / 20 of the original volume to obtain concentrated solution 4; dialyzed with a dialysis bag with a molecular cutoff of 8000 Da for 72 hours, evaporated and concentrated to 1 / 10 of the original volume, and vacuum freeze-dried to obtain purified Pyracantha fruit polysaccharide (denoted as HJGP).

[0050] Example 5 Characteristic Detection of the Purified Polysaccharide from Pyracantha Fortuneana Fruit of the Present Invention

[0051] Determination of total sugar content in polysaccharides

[0052] An appropriate amount of the purified polysaccharide of Pyracantha fortuneana prepared in Example 4 was weighed, and the total sugar content of the purified polysaccharide of Pyracantha fortuneana was determined to be 82.02% by the phenol-sulfuric acid method with glucose as the standard reference substance; the uronic acid content in the purified polysaccharide of Pyracantha fortuneana was determined to be 11.59% by the sulfuric acid-carbazole method with galacturonic acid as the standard reference substance. The uronic acid content in the purified polysaccharide of Pyracantha fortuneana was determined to be 1.65% by the Coomassie brilliant blue method with bovine serum albumin as the standard reference substance.

[0053] An appropriate amount of the purified Pyracantha fortuneana polysaccharide prepared in Example 4 was weighed, and the molecular weight of the polysaccharide was determined by high performance gel permeation chromatography. Dextran with molecular weights of 4320, 12600, 126000, 289000, and 496000 Da was used as standard products to draw a standard curve. Chromatographic conditions: Detector: Shimadzu RID-10A differential detector; Chromatographic columns: Ultrahydrogel 1000 (7.8×300mm) and Ultrahydrogel 500 (7.8×300mm used in series; Mobile phase: 0.02M KH2PO4 buffer; Column temperature: 35°C; Flow rate: 0.8mL / min; Injection volume: 20μL. The elution volume (V) was used as the abscissa and the logarithm of the molecular weight of the standard (LogMw) was used as the ordinate. The instrument's own software was used for fitting to obtain a standard curve. The polysaccharide sample was dissolved in 0.02M KH2PO4 solution and the concentration was 2mg / mL. The sample was passed through a 0.22μm aqueous filter membrane and analyzed. The molecular weight of the polysaccharide was calculated based on the standard curve. The molecular weight distribution range of the polysaccharide was 9378Da-463846Da, and the relative weight average molecular weight was 70859Da.

[0054] Figure 1 This is a molecular weight diagram of the purified polysaccharide (HJGP) from Pyracantha fortuneana fruit prepared in Example 4.

[0055] Weigh an appropriate amount of the purified polysaccharide of Pyracantha fortuneana prepared in Example 4, and use an ion chromatograph to detect its monosaccharide composition ratio, using a ThermoFisher ICS5000 system, an electrochemical detector, a Dionex CarbopacTM PA20 (3*150mm) chromatographic column; mobile phase: A: H2O; B: 15mM NaOHC; flow rate: 0.3ml / min; injection volume: 5μL; column temperature: 30°C, and the monosaccharide composition of the purified polysaccharide of Pyracantha fortuneana is obtained according to the monosaccharide annotation curve. The purified polysaccharide of Pyracantha fortuneana fruit is composed of 5 monosaccharides, and the molar ratios are arabinose: galactose: glucose: xylose: galacturonic acid = 4.15: 2.97: 1: 1.34: 1.89.

[0056] Figure 2 This is a monosaccharide composition diagram of the purified Pyracantha fortuneana fruit polysaccharide (HJGP) prepared in Example 4.

[0057] Example 6 Determination of the protective effect of purified polysaccharide HJGP from Pyracantha fortuneana on UVB-damaged HaCaT human immortalized keratinocytes

[0058] Select HaCaT cells in the logarithmic growth phase and adjust the cell density to 6×10 4 / mL, add cell suspension to 96-well plate, 100 μL per well, place in 5% CO2, 37 ° C incubator and culture for 24h, discard supernatant, wash twice with PBS, add DMEM medium containing HJGP (prepared in Example 4) (the final concentration of HJGP is 125, 250, 500 μg / mL), 100 μL per well. After 24h of culture, pour out the sample solution quickly, wash the remaining culture medium with PBS solution, add 50 μL of diluted MTT solution to each well, and the operation needs to be carried out in the dark. After culturing the 96-well plate in an incubator for 4h, suck out the MTT solution and add 150 μL of dimethyl sulfoxide solution to terminate. After shaking in the dark for 15min, measure the absorbance at 540nm and calculate the cell survival rate. Figure 3 This is a graph showing the cytotoxicity of the purified polysaccharide (HJGP) from Pyracantha fortuneana fruit prepared in Example 4 to HaCaT cells.

[0059] like Figure 3 As shown, after culturing HaCaT cells with purified polysaccharide HJGP from Pyracantha fortuneana for 24 hours, the cell survival rates of the 125, 62.25, and 31.125 μg / mL purified polysaccharide from Pyracantha fortuneana treated groups reached 94.51%, 99.30%, and 97.42%, respectively, which were higher than 90%, showing no cytotoxicity and high safety for use in photoaging repair drugs or health products.

[0060] 100 μL per well, 6000 cells / well, 96-well ELISA plates were plated and cultured for 24 h, washed twice with PBS, and DMEM medium containing HJGP (prepared in Example 4) (the final concentrations of HJGP were 125, 62.25, and 31.125 μg / mL, respectively) was added to each well, and only DMEM medium was added to the control group. After 24 h of culture, the medium was discarded, and an equal volume of PBS was added. The cells were heated at 80 mJ / cm 2 HaCaT cells were irradiated with UVB illuminator, and the blank group was not irradiated. PBS was discarded, and DMEM medium was added to continue culturing for 24 hours. The cell suspension was discarded, the remaining medium was washed with PBS solution, and 50 μL of diluted MTT solution was added to each well. This operation should be performed in the dark. After culturing the 96-well plate in an incubator for 4 hours, the MTT solution was aspirated and 150 μL of dimethyl sulfoxide solution was added to terminate. After shaking in the dark for 15 minutes, the absorbance was measured at 540 nm and the cell survival rate was calculated.

[0061] Figure 4 This is a graph showing the effect of the purified polysaccharide from Pyracantha fortuneana fruit (HJGP) prepared in Example 4 on the survival rate of HaCaT cells damaged by UVB irradiation.

[0062] like Figure 4 As shown, the survival rates of HaCaT cells treated with 125 and 62.25 μg / mL Pyracantha fortuneana polysaccharides were 90.16% and 87.49%, respectively, which were significantly higher than 57.12% of the control group without Pyracantha fortuneana polysaccharide treatment.

[0063] Example 7: The purified polysaccharide HJGP from Pyracantha fortuneana fruit prolongs the lifespan of nematodes damaged by UVB in HaCaT.

[0064] The Caenorhabditis elegans strains and Escherichia coli OP50 used in the experiment were purchased from Caenorhabditis Genetics Center (CGC); Caenorhabditis elegans strains: wild-type nematode N2;

[0065] M9 buffer: weigh 0.3g KH2PO4, 0.5g NaCl, and 0.6g Na2HPO4 into a conical flask, add 100mL distilled water for sterilization, and after sterilization, add 1mL sterile 1mol / L MgSO4 solution for later use.

[0066] NGM solid culture medium: weigh 1.25g Tryptone, 8.5g agar, and 1.5g NaCl into a 1000mL conical flask, add 12.5mL 1M phosphate buffer and 488mL distilled water for sterilization, wait for the culture medium to cool to 60℃, add 0.5mL each of 1M MgSO4, 1M CaCl2, and 5mg / mL cholesterol sterile solution, shake well, pour into a plate while hot, and store inverted at 4℃ after the culture medium solidifies.

[0067] Cultivation of C.elegans

[0068] (1) Nematode resuscitation: Take out the frozen Caenorhabditis elegans from a -80°C refrigerator, dissolve them in 20°C running water, let them stand until the nematodes settle, then aspirate the supernatant, wash the precipitate twice with M9 solution, inoculate it on an NGM plate with Escherichia coli, and culture it in a nematode incubator at 20°C.

[0069] (2) Transfer of nematodes: When the E. coli in the plate is about to be consumed, the nematodes need to be transferred to a new plate. Use M9 solution to wash off the nematodes in the old plate, place them in a 1.5 mL EP tube to settle, discard the supernatant, transfer the precipitate to a new plate with E. coli, and continue culturing.

[0070] (3) Cryopreservation of nematodes: Place the nematodes on an NGM plate without E. coli and culture them for 2 days. Rinse the nematodes off the plate with M9, discard the supernatant after sedimentation, wash the precipitate twice with M9 solution, add an equal volume of nematode freezing solution, place in a programmed cooling box and freeze at -80°C.

[0071] Synchronized culture of C.elegans

[0072] Pick a plate containing a large number of female worms, rinse the female worms with M9 solution, and place them in a 1.5mL EP tube. After the female worms settle, discard the supernatant by suction, repeat several times to wash the worms, leaving 0.5mL of nematode M9 mixed solution, add 1mL of nematode lysis solution (prepared and used immediately), vortex for 3.5min, and immediately centrifuge at 2000r / min for 2min. After centrifugation, discard the supernatant by suction, add M9 solution and centrifuge repeatedly three times to remove the residual lysis solution. The white precipitate left is the nematode eggs.

[0073] Transfer the eggs to an empty NGM plate and culture at 20°C for 23 hours to obtain L1 stage nematodes. Transfer the L1 stage nematodes to a plate containing E. coli OP50 and continue to culture for 43 hours to obtain L4 stage nematodes.

[0074] Selection of safe sample concentration: Pick synchronized L4 nematodes into each group of standard nematode NGM culture medium, 30 in each group, 30 in each plate. Mix the purified polysaccharide solution of Pyracantha fruit with the inactivated bacterial suspension in equal volumes to obtain a mixture of purified polysaccharide of Pyracantha fruit and Escherichia coli with a concentration of 250μg / mL and 500μg / mL, and take 100μL of the mixture and spread it on an NGM plate containing 25mg / L 55FU. Place the same number of L4 nematodes on the plates coated with samples of different concentrations, culture at 20℃ for 48h, record the number of nematodes that died, and the nematode survival rate>90% is a safe concentration.

[0075] Pick synchronized L4 nematodes to each group of standard nematode NGM culture medium, 30 in each group, and 30 in each plate. Calculate the number of nematode survival days from the time of transfer, and the day of transfer is recorded as day 0 of the life span experiment (day 0). Mix equal volumes of purified polysaccharide solutions of Pyracantha fortuneana fruit with inactivated bacterial suspensions at different concentrations to obtain a mixture of purified polysaccharide of Pyracantha fortuneana fruit and Escherichia coli at concentrations of 250 μg / mL and 500 μg / mL, and take 100 μL of the mixture and spread it on the NGM plate. Place the same number of L4 nematodes on the plates coated with samples of different concentrations and culture at 20°C for 48 hours. Use a UVB illuminator to apply 1.8 J / cm 2 After irradiation, the nematodes were transferred to new culture medium for cultivation. The nematodes were observed every day and the number of deaths was recorded.

[0076] Nematode death criteria: loss of movement ability and no response to repeated touch.

[0077] Blank control group: no irradiation treatment; model group: no drug was added, that is, the drug-free Escherichia coli mixed solution was added in the above steps.

[0078] The survival rates of nematodes treated with 250μg / mL and 500μg / mL of purified polysaccharides from Pyracantha fortuneana fruit were 96.67% and 93.33% respectively. These two concentrations can be selected as safe drug concentrations for lifespan experiments. Figure 5 The results showed that the lifespan of nematodes exposed to UVB was drastically shortened to only 12 days. Treatment with 250 and 500 μg / mL of purified polysaccharides from Pyracantha fortuneana fruit extended the lifespan of nematodes exposed to UVB to 17 and 18 days, respectively.

[0079] Figure 5 This is a survival curve of nematodes damaged by UVB irradiation under the action of the purified polysaccharide (HJGP) of Pyracantha fortuneana fruit prepared in Example 4.

[0080] Purified polysaccharides from Pyracantha fortuneana fruit can protect HaCaT cells damaged by UVB and reduce the apoptosis rate of HaCaT cells damaged by UVB.

[0081] Purified polysaccharides from Pyracantha fortuneana fruit can protect Caenorhabditis elegans damaged by UVB and extend the lifespan of Caenorhabditis elegans damaged by UVB.

[0082] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a purified polysaccharide from Pyracantha fortuneana fruit having significant resistance to light damage, characterized in that: The following steps are included: 1) extracting the Pyracantha fruit powder with hot water to obtain a Pyracantha fruit water extract; concentrating the water extract, precipitating with alcohol, and drying the precipitate to obtain a Pyracantha fruit crude polysaccharide; 2) decolorizing and deproteinizing the crude polysaccharide from Pyracantha fortuneana fruit to obtain the deproteinized crude polysaccharide from Pyracantha fortuneana fruit; 3) The crude polysaccharide from the deproteinized Pyracantha fruit was purified by DEAE-FastFlow column chromatography, and water, 0.05 M NaCl solution, and 0.15 M NaCl solution were used for gradient elution in sequence, and the eluates were collected respectively, and the eluate obtained by eluting with 0.15 M NaCl solution was concentrated, dialyzed, and dried to obtain the purified polysaccharide from Pyracantha fruit, which was recorded as HJGP; The purified polysaccharide from Pyracantha fortuneana fruit is composed of five monosaccharides: arabinose, galactose, glucose, xylose, and galacturonic acid. The molar ratio of arabinose: galactose: glucose: xylose: galacturonic acid is 4.15:2.97:1:1.34:1.

89.

2. The method for preparing the purified polysaccharide from Pyracantha fortuneana fruit having significant resistance to light damage according to claim 1, characterized in that: The hot water extraction in step 1) refers to extraction by water immersion, heating and reflux, the number of hot water extractions is 1 to 3 times, the temperature of each extraction is 90 to 100° C., and the time of each extraction is 2 to 4 hours; The alcohol precipitation in step 1) refers to mixing the concentrated solution with a 95% ethanol solution, and finally the final concentration of ethanol in the mixed system is 70-85%, and the concentration is the volume concentration; the alcohol precipitation is carried out at 0-4°C; In step 3), when the Pyracantha fruit deproteinized crude polysaccharide is purified by column chromatography, the Pyracantha fruit deproteinized crude polysaccharide is mixed with water, and the concentration of the mixed solution is 8-10 mg / mL; then mixed with DEAE-FastFlow column filler, and gradient elution is performed using water, 0.05M NaCl solution, and 0.15M NaCl solution in sequence; The dialysis in step 3) refers to dialysis using a dialysis bag with a molecular cutoff of 8000Da; the drying is freeze-drying.

3. The method for preparing the purified polysaccharide from Pyracantha fortuneana fruit having significant resistance to light damage according to claim 1, characterized in that: The decolorization in step 2) refers to mixing the crude polysaccharide of Pyracantha fruit with water, then decolorizing with macroporous resin D354F, precipitating with alcohol, and drying the precipitate to obtain the decolorized crude polysaccharide of Pyracantha fruit.

4. The method for preparing the purified polysaccharide from Pyracantha fortuneana fruit having significant resistance to light damage according to claim 3, characterized in that: During decolorization, the concentration of the solution after the Pyracantha fruit crude polysaccharide is mixed with water is 4-6 mg / mL, and the volume ratio of the Pyracantha fruit crude polysaccharide solution to the macroporous resin D354F is 1:1-1:3; the decolorization temperature is 45-60°C, the decolorization times are 3-5 times, and the decolorization time each time is 2-4 hours; The alcohol precipitation refers to mixing the decolorized solution with a 95% ethanol solution, and finally the final concentration of ethanol in the mixed system is 70-85%, and the concentration is the volume concentration; the alcohol precipitation is carried out at 0-4°C; The drying is performed by heating and drying, re-dissolving with water, and then vacuum freeze-drying; the temperature of the heating and drying is 60-70°C.

5. The method for preparing the purified polysaccharide from Pyracantha fortuneana fruit having significant resistance to light damage according to claim 1, characterized in that: The deproteinization in step 2) refers to mixing the decolorized crude polysaccharide of Pyracantha fruit with water, deproteinizing with Sevage solution, taking the aqueous phase, precipitating with alcohol, and drying to obtain the deproteinized crude polysaccharide of Pyracantha fruit.

6. The method for preparing the purified polysaccharide from Pyracantha fortuneana fruit having significant resistance to light damage according to claim 5, characterized in that: In the deproteinization, the concentration of the solution after the decolorized crude polysaccharide of Pyracantha fruit is mixed with water is 4-6 mg / mL, the volume ratio of chloroform and n-butanol in the Sevag solution is 4:1-6:1, the number of extractions is 3-5 times, and the extraction time for each time is 0.5-1.5 h; The alcohol precipitation refers to mixing the aqueous phase with a 95% ethanol solution, and finally the final concentration of ethanol in the mixed system is 70-85%, and the concentration is a volume concentration; the alcohol precipitation is carried out at 0-4°C; The drying refers to heating and drying, re-dissolving with water, and then vacuum freeze-drying; the temperature of the heating and drying is 60-70°C.

7. The method for preparing the purified polysaccharide from Pyracantha fortuneana fruit having significant resistance to light damage according to claim 1, characterized in that: The pyracantha fruit powder is obtained by drying, crushing and sieving pyracantha fruit; The volume mass ratio of the pyracantha fruit powder to water in step 1) is 1 g: (15-25) mL; After the hot water extraction in step 1), centrifugation or filtration is performed; the centrifugal speed is 3500-5000 r / min; the centrifugal time is 5-10 min; The concentration in step 1) is concentration under reduced pressure; The drying in step 1) is heating drying and vacuum freeze drying, and the heating drying temperature is 60-70°C.

8. A purified polysaccharide from Pyracantha fortuneana fruit with significant resistance to light damage prepared by the method according to any one of claims 1 to 7, characterized in that: The purified polysaccharide of Pyracantha fortuneana fruit is composed of five monosaccharides: arabinose, galactose, glucose, xylose, and galacturonic acid. The molar ratio of arabinose: galactose: glucose: xylose: galacturonic acid is 4.15:2.97:1:1.34:1.89; The molecular weight distribution range is 9378Da-463846Da, and the relative weight average molecular weight is 70859Da.

9. The use of the purified polysaccharide from Pyracantha fortuneana fruit having significant resistance to light damage according to claim 8, characterized in that: The purified polysaccharide of pyracantha fruit with significant resistance to light damage is used for preparing drugs for resisting light damage and / or improving skin photoaging.

10. The use according to claim 9, characterized in that: The photodamage and photoaging refer to the photodamage and photoaging caused by UVB.

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