Preparation method and application of polysaccharide PJP60-Ia from north green dragon skin

Abstract

The present application relates to the preparation method and application of polysaccharide PJP60-Ia from north green dragon skin, and the polysaccharide PJP60-Ia is extracted from north green dragon skin by the flash extraction technology combined with fractional alcohol precipitation, and the polysaccharide PJP60-Ia is decolorized by AB-8 macroporous resin, and is separated and purified by DEAE-52 anion exchange chromatography column to obtain polysaccharide PJP60-I, and the polysaccharide PJP60-I is further purified by Sephadex G-50 gel chromatography column, and finally, the single component polysaccharide PJP60-Ia with a molecular weight of 11250 Da and composed of rhamnose, glucose and fructose is obtained. The polysaccharide PJP60-Ia provided by the present application has the advantages of high purity and good anti-inflammatory activity.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and in particular relates to a preparation method of PJP60-Ia polysaccharide from Psoralea corylifolia and its application. Background Art

[0002] Qinglongyi (Qinglongyi) originates from the outer green peel of the immature fruits of the walnut (Juglans regia L.) and the walnut (Juglandaceae) tree (Juglandaceae). It is mentioned in the Kaibao Materia Medica and the Jiuyi Fang (Relief Prescriptions), where it is referred to as "Hutao Qinglong" (Hutao Qinglong) or "Qinghuwalnut Skin." It was later renamed Qinglongyi (Qinglongyi) in the Shandong Handbook of Chinese Herbal Medicine, a name that has been used ever since. Qinglongyi is abundant and widely distributed, primarily in Northeast China. It was later included in the Heilongjiang Provincial Standards for Traditional Chinese Medicines, designated "Beiqinglongyi," and was included in the Heilongjiang Provincial Catalogue of Authentic Medicinal Materials in 2022. Qinglongyi has a pungent and bitter flavor, astringent properties, and a neutral nature. It is primarily used for its heat-clearing and detoxifying properties, wind-clearing and epilepsy treatment, pain relief, diarrhea relief, and swelling-reducing properties. It is a low-toxic, safe herbal remedy with a wide safety profile and abundant resources throughout history. Chemical studies have found that Qinglongyi contains a variety of chemical components, including polysaccharides, naphthoquinones, flavonoids, terpenes, and diarylheptanes. It can be used clinically to treat gastrointestinal inflammation and cancer, but current research on the chemistry and activity of Northern Qinglongyi is limited, hindering its development.

[0003] Polysaccharides, also known as polysaccharides, are natural high-molecular compounds composed of more than ten monosaccharides linked by α- or β-glycosidic bonds. They are widely used in food, medicine, and health supplements. The biological activity of polysaccharides is primarily related to their higher-order structure. A deeper understanding of their structure facilitates better study of their biological activity and holds significant implications for the research, development, and application of polysaccharides. Polysaccharides are polar macromolecules typically extracted from plants using hot or cold water. Insoluble matter is then removed from the polysaccharide solution directly or by centrifugation. Following extraction, the polysaccharides require separation, protein removal, decolorization, and purification. Currently, various methods are available for polysaccharide extraction, including ultrasonic extraction, microwave extraction, and enzyme-assisted extraction. The extraction of polysaccharides from the plant stem (Psoralea corylifolia) is often subject to interference from substances such as proteins and pigments. Existing methods suffer from low purity and poor activity of Psoralea corylifolia polysaccharides. Different preparation methods also result in varying extract compositions, impacting the activity and potential applications of the extracts. Therefore, it is necessary to select a suitable method to separate and purify the polysaccharide in order to obtain the polysaccharide with better purity and anti-inflammatory activity. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a preparation method and application of P. chinensis polysaccharide PJP60-Ia, which has the advantages of high purity, good anti-inflammatory activity, etc.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The invention provides a preparation method of PJP60-Ia polysaccharide from the capillaries of northern bluebonnet. The method comprises the following steps: extracting the supernatant of the capillaries of northern bluebonnet by adopting a graded alcohol precipitation method, and collecting the precipitate after the alcohol precipitation with a concentration of 60% ethanol.

[0007] The beneficial effects of adopting the above scheme include: the present invention tested the products after precipitation with different ethanol concentrations and found that the product after precipitation with 60% ethanol concentration had a higher polysaccharide content and anti-inflammatory activity.

[0008] Furthermore, the preparation method of the supernatant of Beiqinglongyi includes the following steps: performing flash extraction on the Beiqinglongyi liquid.

[0009] Furthermore, the parameters of the Flash extraction include: 5000 r / min, 30 s / time.

[0010] The beneficial effects of adopting the above scheme include: short extraction time, high efficiency, convenient operation, energy saving and the like.

[0011] Furthermore, the graded alcohol precipitation includes the following steps: centrifuging and filtering the supernatant of the northern blue dragon hair to remove the residue, collecting the supernatant, and concentrating the supernatant under reduced pressure to 1 / 3 of the original volume, then adding 95% ethanol to the concentrated solution while stirring, adjusting the final ethanol concentration to 20%, letting it stand overnight, and collecting precipitate I and supernatant I respectively; according to the above steps, adding ethanol to the supernatant I, adjusting the final ethanol concentration to 40%, obtaining supernatant II and precipitate II, and freeze-drying the precipitate II; then, according to the above steps, adding ethanol to the supernatant II, adjusting the final ethanol concentration to 60%, obtaining supernatant III and precipitate III, and freeze-drying the precipitate III.

[0012] The beneficial effects of adopting the above scheme include: the polysaccharide content of the Beiqinglongyi polysaccharide obtained by the above method is higher and the yield is higher.

[0013] Furthermore, the method also includes the steps of decolorization, separation and purification.

[0014] Furthermore, AB-8 macroporous resin was used for decolorization.

[0015] The method may include the following specific steps: dissolving crude polysaccharide of Northern Qinglongyi in water, adding activated AB-8 macroporous resin, stirring for 2 hours, statically adsorbing for 2 hours after stirring, separating by suction filtration, concentrating the polysaccharide solution to 1 / 3 of the original volume, and freeze-drying.

[0016] The beneficial effects of adopting the above scheme include: the above method can remove impurities such as pigments in the extract.

[0017] Furthermore, the separation and purification includes separation and purification using a DEAE-52 anion exchange chromatography column and separation and purification using a Sephadex G-50 gel chromatography column.

[0018] Furthermore, the separation and purification using a DEAE-52 anion exchange chromatography column includes the following steps: activating DEAE-52 cellulose; pretreating a DEAE-52 anion exchange chromatography column; preparing a solution of P. chinensis polysaccharide and loading the sample; preparing an eluent and collecting samples, using a 0-0.5 mol / L NaCl solution as the eluent, controlling the flow rate to 1 mL / min, collecting the eluent obtained when the NaCl solution concentration is 0, and freeze-drying it to obtain P. chinensis polysaccharide PJP60-I.

[0019] Furthermore, the separation and purification by Sephadex G-50 gel chromatography column includes the following steps: activating Sephadex G-50 gel; pretreating the Sephadex G-50 gel chromatography column; preparing PJP60-I solution and loading the sample, using deionized water as the eluent, controlling the flow rate to 0.3 mL / min, collecting, collecting 1 mL per tube, collecting samples from tubes 80 to 110, and freeze-drying them to obtain the northern blueberry polysaccharide PJP60-Ia.

[0020] The beneficial effects of adopting the above scheme include: the above method can further increase the content of polysaccharides in the extract, and the sugar content can reach 98.85%±0.27%.

[0021] The present invention provides the use of Beiqinglongyi polysaccharide PJP60-Ia in any one or more of (1) to (3), wherein Beiqinglongyi polysaccharide PJP60-Ia is prepared by the above-mentioned preparation method;

[0022] (1) Used alone or as one of the components in the preparation of anti-inflammatory preparations;

[0023] (2) As a new food ingredient alone;

[0024] (3) Used alone or as one of the components in the preparation of health foods with anti-inflammatory function.

[0025] The beneficial effects of the above scheme include: experimental detection shows that the polysaccharide PJP60-Ia prepared by the present invention has no cytotoxic effect on normal mouse macrophages, can inhibit the proliferation activity of mouse macrophages RAW264.7 induced by LPS, and can reduce the secretion of NO, the level of ROS, and Ca2+ related substances in cells. 2+The concentration of the compound can inhibit the secretion and expression of pro-inflammatory factors, promote the secretion and expression of anti-inflammatory factors, regulate the polarization state of inflammatory cells, and have a good anti-inflammatory effect. It can be used alone or in combination with other components to prepare or manufacture anti-inflammatory products, such as medicines, foods, and health foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the DEAE-52 elution curve of PJP60 polysaccharide from Psoralea corylifolia.

[0027] Figure 2 This is the infrared spectrum of the polysaccharide PJP60-Ia from the plant.

[0028] Figure 3 The molecular weight standard curve.

[0029] Figure 4 These are the Congo red test results of PJP60-Ia, a polysaccharide from Psoralea corylifolia.

[0030] Figure 5 This is the DSC-TG analysis result of the polysaccharide PJP60-Ia from Psoralea corylifolia.

[0031] Figure 6 This is the particle size distribution of PJP60-Ia, a polysaccharide from Psoralea corylifolia.

[0032] Figure 7 Zeta potential of PJP60-Ia, a polysaccharide from Psoralea corylifolia.

[0033] Figure 8 This is the X-ray diffraction curve of the polysaccharide PJP60-Ia from the plant.

[0034] Figure 9 These are scanning electron micrographs of P. chinensis polysaccharide PJP60-Ia (from left to right: 300×, 1200×, 5000×).

[0035] Figure 10 Atomic force microscopy images of PJP60-I a polysaccharide from P. chinensis (first row: 1 μm×1 μm, second row: 10 μm×10 μm).

[0036] Figure 11 The experimental results of inverted fluorescence microscopy detection of the effect of PJP60-I a on LPS-induced RAW264.7 cell morphology (10×40).

[0037] Figure 12 Inverted fluorescence microscopy was used to detect the effect of PJP60-Ia on LPS-induced intracellular Ca2+ in RAW264.7 cells. 2+ Experimental results of the influence of content (10×20). DETAILED DESCRIPTION

[0038] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0039] The present invention uses the iris as experimental material, obtains iris polysaccharide by extraction, separation and purification, studies its primary structure and higher structure, and evaluates its anti-inflammatory activity, including the following contents:

[0040] (1) The present invention uses a Flash extractor with a solid-liquid ratio of 1:30, 5000 rpm, and 30 seconds per extraction. Two extractions are performed. The extracts are combined and, in conjunction with the principle of graded alcohol precipitation, the alcohol concentrations are adjusted to 20%, 40%, and 60%. Finally, crude polysaccharides from four fractions are obtained, designated as PJP20, PJP40, and PJP60, respectively. Decolorization is performed using AB-8 macroporous resin, and the presence of protein in the crude polysaccharides is detected by full-wavelength ultraviolet spectrophotometry. The results show that no protein-related substances are found. PJP60 was selected for subsequent separation and purification, and a single symmetrical elution peak was obtained by DEAE-52 anion exchange column chromatography, named as Beiqinglongyi polysaccharide PJP60-I, with a sugar content of 80.65%±0.12% and a yield of 18.66%. Beiqinglongyi polysaccharide PJP60-I was further separated and purified by Sephadex G-50 gel column chromatography to obtain Beiqinglongyi polysaccharide PJP60-I a, with a sugar content of 98.85%±0.27% and a yield of 54.05%.

[0041] (2) The present invention studies the physicochemical properties, primary structure and advanced structure of the polysaccharide PJP60-I a of the nephrite by a series of experiments combined with instrument detection. The results show that the polysaccharide PJP60-I a of the nephrite is a homogeneous polysaccharide with good purity. It may be composed of Rha, Glc and Fru, with a molecular weight of 11250Da. The FT-IR results show that it has characteristic peaks of carbohydrates and contains α and β glycosidic bonds. The NMR results are consistent with the FT-IR results, and it is speculated that the polysaccharide PJP60-I a of the nephrite is composed of →1)-β-D-Fruf-(2→、3,4)-β-Rha-(1→、α-DG lcp. The Congo red test results show that the polysaccharide PJP60-I a of the nephrite does not have a triple helical structure. In the thermal properties experiment, the polysaccharide PJP60-I a below 140°C and has good thermal stability. Particle size and potential measurement results show that the average particle size of the polysaccharide PJP60-I a is 211.8 nm, the PDI value is 0.25, the average potential is -3.5 mV, the molecular weight distribution range is narrow, and the molecular weight distribution is well uniform, and it is easy to aggregate in the solution system. XRD results show that the polysaccharide PJP60-I a is an amorphous substance with a semi-crystalline structure. SEM observation results show that the polysaccharide PJP60-I a appears as a rolled film under low magnification, with an uneven surface, irregular protrusions, clumps and holes. Under high magnification, it is found to have a film-like structure with irregular flake structures aggregated at the edges. AFM observations show that the polysaccharide PJP60-I a contains irregular blocky particles with a high aggregation density.

[0042] (3) The present invention uses LPS-induced mouse macrophage RAW264.7 as an inflammatory cell model to evaluate the anti-inflammatory activity of the polysaccharide PJP60-I a from the plant. The effect on the proliferation of inflammatory cells was detected by MTT assay. The results showed that the polysaccharide PJP60-I a from the plant inhibited the proliferation of inflammatory cells within a certain concentration range. Within the concentration range of 80-320 μg / mL, PJP60-I a could reduce the levels of inflammatory substances NO, ROS and Ca. 2+ Furthermore, PJP60-Ia modulated the M1 / M2 polarization ratio of macrophages, reversing the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. It downregulated the mRNA and protein expression of the M1 marker CD86 and upregulated the mRNA and protein expression of the M2 marker CD163. It also reduced the secretion and mRNA expression of the pro-inflammatory factors TNF-α and IL-1β, and increased the secretion and mRNA expression of the anti-inflammatory factors IL-4 and IL-10. These results indicate that PJP60-Ia, a polysaccharide from the plant stem cell lineage PJP60, has potent anti-inflammatory activity, can regulate the polarization state of inflammatory cells, and is non-cytotoxic.

[0043] A homogeneous polysaccharide PJP60-I a is obtained by extraction, separation and purification from the north green dragon skin, and shows good anti-inflammatory activity, and can be used as or for preparing an inflammation modulator.

[0044] The following will be introduced by specific examples. The experimental methods used in each example are conventional experimental methods in the art if not specifically stated. The materials, reagents and instruments used, if not specifically stated, are conventional materials, reagents and instruments in the art, which can be obtained through commercial channels or prepared by conventional methods.

[0045] Experimental materials: glucose (AR), acetonitrile (chromatographic grade), disodium hydrogen phosphate (chromatographic grade), sodium dihydrogen phosphate (chromatographic grade) were purchased from Tianjin Kermel Chemical Reagent Co., Ltd.; DEAE cellulose DE-52 (C6930), DMSO (D6370), MTT (M6180), lipopolysaccharide (L2880) were purchased from Beijing Boaotuda Technology Co., Ltd.; dextran gel G-50 (S8151), streptomycin (P1400) were purchased from Beijing Solaybao Technology Co., Ltd.; α-naphthol (AR) was purchased from China Pharmaceutical Group Chemical Reagent Co., Ltd.; 1-phenyl-3-methyl-5-pyrazolone PMP (AR), trifluoroacetic acid (chromatographic grade) were purchased from Xiamen Anyeung Technology Co., Ltd.; carbazole (AR) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; monosaccharide standard, dextran standard were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; four seasons of fetal bovine serum (11011-8611) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; DMEM medium (MA0212) was purchased from Dalian Melun Biotechnology Co., Ltd.; NO detection kit (S0021S), reactive oxygen species detection kit (S0033S), trypsin cell digestion solution (C0201) were purchased from Biyun Tian Biotechnology Co., Ltd.

[0046] The dried pericarp of north green dragon skin was identified and provided by Heilongjiang Academy of Chinese Medicine, and the mouse macrophage RAW264.7 cells were gifted by Harbin Medical University, and the public can obtain the repeated examples described in the application only for non-commercial purposes.

[0047] Instrument: Sykes extractor (SYKES-507) purchased from Ningbo Bedel Telecommunication and Electric Machinery Co., Ltd.; ordinary chromatography column (HC-0130-05, HC-0120-08) purchased from Beijing Ruida Henghui Science and Technology Development Co., Ltd.; constant flow pump (HL-2D) purchased from Shanghai Qingpu Huxi Instrument Factory; laboratory grade ultrapure water generator (EPED-E2-30TJ) purchased from Nanjing Yipuyida Science and Technology Development Co., Ltd.; Fourier infrared spectrometer (FTIR-650), ultraviolet spectrophotometer (UV-5200PC) purchased from Tianjin Gangdong Science and Technology Development Co., Ltd.; high performance liquid chromatograph (2695) purchased from Waters Technology Co., Ltd.; DSC-TG thermal gravimetric analyzer (DTG-60) purchased from Japan Shimadzu Corporation; laser particle size analyzer (Litesizer-500) purchased from Anton Paar (Shanghai) Trading Co., Ltd.; nuclear magnetic resonance spectrometer (600M), X-ray diffractometer (D8 ADVANCE) purchased from Germany Bruker Corporation; atomic force microscope (Dimens ion-XR) purchased from Agligent Technology Co., Ltd.; field emission scanning electron microscope (EVO-18) purchased from Germany Zeiss Corporation; enzyme marker (imark) purchased from Bio-Rad Corporation of the United States; fluorescence inverted microscope (IX71) purchased from Japan Olympus Corporation.

[0048] Example 1 Extraction, preparation, separation and purification of polysaccharide from north blue dragon skin

[0049] In the present application, the polysaccharide from north blue dragon skin is extracted by using flash extraction technology combined with fractional alcohol precipitation, and the AB-8 macroporous resin is used for decolorization, and the DEAE-52 anion exchange chromatography column is used for separation and purification. In order to improve the purity of the sample, the Sephadex G-50 gel chromatography column is further used for purification, and finally the polysaccharide PJP60-Ia from north blue dragon skin with single component is obtained.

[0050] 1.1 Experimental method

[0051] (1) Preparation of crude polysaccharide from north blue dragon skin

[0052] In this experiment, a Flash extractor was used for extraction, combined with the graded alcohol precipitation method to extract crude polysaccharides from the leaves of the northern blue dragonfly. The dried northern blue dragon hair was crushed, and an appropriate amount of the crushed sample was weighed. According to the material-liquid ratio of 1g:30ml, the parameters of the Flash extractor were set as follows: 5000r / min, 30s / time, and extracted twice with distilled water. The two extracts were combined, and the extracts were centrifuged (4000r / min, 10min) and filtered to remove the residue. The supernatant was collected and concentrated under reduced pressure to 1 / 3 of the original volume. Subsequently, 95% ethanol was added to the concentrate while stirring to adjust the final ethanol concentration to 20%. The concentrate was allowed to stand overnight, and the precipitate I and supernatant I were collected separately; according to the above steps, ethanol was added to the supernatant I to adjust the final ethanol concentration to 40% to obtain supernatant II and precipitate II, and the precipitate II was freeze-dried; then, according to the above steps, ethanol was added to the supernatant II to adjust the final ethanol concentration to 60% to obtain supernatant III and precipitate III, and the precipitate III was freeze-dried to obtain PJP60.

[0053] (2) Decolorization of crude polysaccharide from the iris of the Chinese hibiscus

[0054] Before decolorization, the AB-8 macroporous resin needs to be activated. First, soak it in distilled water to remove floating matter, then soak it in 95% ethanol overnight, and then wash it with distilled water until the alcohol smell is gone. Weigh 5.0g of crude polysaccharide PJP60 from the plant stem of the Northern Qinglongyi plant and add 100mL of distilled water to fully dissolve it. Then add an appropriate amount of treated AB-8 macroporous resin and stir it with a magnetic stirrer for 2 hours. After stirring, allow it to statically adsorb for 2 hours. Then filter and separate the solution. Concentrate the polysaccharide solution to 1 / 3 of its original volume and freeze-dry it.

[0055] (3) UV full wavelength scanning of crude polysaccharides from the plant

[0056] The prepared and decolorized crude polysaccharide PJP60 solution from Psoralea corylifolia was scanned at full wavelength using an ultraviolet spectrophotometer.

[0057] (4) Separation and purification using a DEAE-52 anion exchange chromatography column

[0058] ① Activation of DEAE-52 cellulose: Weigh 50.0g of DEAE-52 cellulose into a 1000mL beaker, add ultrapure water, and stir for 30 minutes to fully swell. Allow to settle and remove floating matter. Subsequently, wash with 0.5mol / L NaOH solution, then 0.5mol / L HCl solution, in that order, followed by an acid-base solution. Rinse with ultrapure water until neutral, and set aside.

[0059] ② Pretreatment of DEAE-52 anion exchange chromatography column: First, rinse the chromatography column with ultrapure water to degas the cellulose and remove bubbles. Then, slowly add the cellulose along the column wall and rinse the residual cellulose on the column wall with ultrapure water. Open the drain valve at the bottom of the column. During this process, the filler surface needs to be kept flat. When the liquid level is 4-7 cm away from the filler, close the drain valve and let it stand overnight. Connect the constant flow pump to the chromatography column, turn on the constant flow pump, open the drain valve of the chromatography column, control the flow rate to 1 mL / min, and balance the chromatography column. After balancing for 5 column volumes, use pH test paper to determine whether the outflowing liquid is neutral. Then prepare for sample loading.

[0060] ③ Preparation and loading of Beiqinglongyi polysaccharide solution: Weigh 100.0 mg of Beiqinglongyi polysaccharide sample PJP60, add 10 mL of ultrapure water to fully dissolve it, filter it with a 0.45 μm microporous filter membrane, then slowly add the polysaccharide solution along the column wall, and rinse the column wall with ultrapure water to make the sample solution evenly distributed on the filler surface, open the drain valve, let the liquid level be level with the filler surface, so that the sample solution is adsorbed on the filler surface, close the drain valve, and slowly add the eluent until the liquid level is about 5 cm higher than the filler.

[0061] ④ Preparation of eluent and sample collection: Prepare 0-0.5 mol / L NaCl solution as eluent, inject the eluent into the chromatography column with a constant flow pump, open the drain valve, control the flow rate to 1 mL / min, collect 2 mL in each tube, collect 100 tubes, determine the sample collected in each tube by the phenol-sulfuric acid method, draw an elution curve based on the measured absorbance, collect samples of each component, dialyze for 24 hours, concentrate the dialysate, and freeze-dry to obtain the northern blue dragon polysaccharide PJP60-I.

[0062] (5)Sephadex G-50 gel chromatography column separation and purification

[0063] ① Activation of Sephadex G-50 gel: Weigh 30g of Sephadex G-50 gel, soak it in ultrapure water, stir continuously to allow it to fully swell, let it settle, remove floating particles, and then prepare to load the column.

[0064] ② Pretreatment of Sephadex G-50 gel chromatography column: First, rinse the chromatography column with ultrapure water. Install the column loading device and place the swollen gel into it at once. Allow it to slowly enter the chromatography column and avoid bubbles. Wash the gel remaining on the column wall with ultrapure water. Open the drain valve and keep the packing surface flat. When the liquid level is about 3 cm away from the packing, close the drain valve and let it stand overnight. Connect a constant flow pump to the chromatography column and use ultrapure water for elution balance at a controlled flow rate of 0.3 mL / min. Balance for 3-5 column volumes. Use pH test paper to check whether the outflowing liquid is neutral, and then load the sample.

[0065] ③ Preparation and loading of Beiqinglongyi polysaccharide solution: Weigh 80.0 mg of Beiqinglongyi polysaccharide PJP60-I, add 10 mL of ultrapure water to fully dissolve it, filter it with a 0.45 μm filter membrane, then slowly add the polysaccharide solution along the column wall, and rinse the column wall with ultrapure water to make the sample solution evenly distributed on the filler surface, open the drain valve, let the liquid level be level with the filler surface, so that the sample solution is adsorbed on the filler surface, close the drain valve, and slowly add the eluent until the liquid level is about 3 cm higher than the filler.

[0066] ④ Preparation of eluent and sample collection

[0067] Ultrapure water was used as the eluent and injected into the chromatography column using a constant flow pump at a controlled flow rate of 0.3 mL / min. An automatic collector was turned on to collect 1 mL per tube. The sample collected from each tube was assayed using the phenol-sulfuric acid method. Based on the assay results, an elution curve was plotted, and the sample was collected at the optimal absorption peak. The sample was then freeze-dried to obtain the P. chinensis polysaccharide PJP60-I a.

[0068] (6) Determination of the content of polysaccharide in Beiqinglongyi

[0069] Accurately weigh 10.0 mg of glucose standard, add 10 mL of ultrapure water, and dissolve thoroughly to obtain a 1 mg / mL glucose standard solution as the mother solution. Dilute with ultrapure water to prepare glucose standard solutions of varying concentrations. Use the phenol-sulfuric acid method to determine the concentrations of glucose standard solutions: pipette 100 μL of each glucose standard solution into a test tube, add 100 μL of 6% phenol solution and 500 μL of concentrated sulfuric acid solution to each tube, mix thoroughly, heat in a boiling water bath for 15 minutes, cool, and measure the absorbance of each solution at a wavelength of 490 nm using a UV spectrophotometer. Repeat three replicate measurements and take the average value. Use ultrapure water as the blank control, plot the concentration (mg / mL) on the horizontal axis, and the absorbance (A) on the vertical axis to create a glucose standard curve.

[0070] Prepare 1 mg / mL solutions of crude PJP60 polysaccharide from the plant stem, 1 mg / mL PJP60-I polysaccharide from the plant stem, and 1 mg / mL PJP60-Ia polysaccharide from the plant stem. Perform the above-mentioned measurements three times in parallel, take the average value, and calculate the sugar content based on the resulting glucose standard curve.

[0071] (7) Physical and chemical properties of polysaccharide from Northern Qinglongyi

[0072] ① Phenol-sulfuric acid method: the method is the same as “(6) Determination of the content of polysaccharide in Beiqinglongyi”.

[0073] ②I2-KI reaction: Take 1 mL of the polysaccharide solution of the northern blue dragon hair, add 2-3 drops of the prepared I2-KI solution, shake and mix, place in a dark place, and observe the changes in the solution. If blue appears, it means that it contains starch. If there is no color change, it proves that the sample does not contain starch.

[0074] ③FeCl3 reaction: Take 1 mL of the polysaccharide solution of Northern Qinglongyi, add 2 drops of 1% FeCl3 solution, mix well, and observe the reaction phenomenon. If the color of the solution turns blue, it means that it contains phenols.

[0075] ④Molisch reaction: Take 1 mL of the polysaccharide solution of the northern blue lobelia, add 2 drops of 5% α-naphthol solution, and then carefully add 1 mL of concentrated sulfuric acid solution. Leave it for a while and observe the color change between the two liquid surfaces. If a purple ring appears, it means that the substance is a carbohydrate component.

[0076] ⑤ Ninhydrin reaction: The ninhydrin reaction is mainly used to identify whether a sample contains amino acids, peptides, or proteins. Take 1 mL of the polysaccharide solution from the northern blue dragonfly and add an appropriate amount of 1% ninhydrin solution. Heat in a water bath for 10 minutes, cool to room temperature, and observe the reaction. If a blue-purple color appears, it indicates the presence of the above components.

[0077] ⑥ Fehling's reaction: Take 1 mL of the polysaccharide solution of the northern blue lobelia in a test tube, add 1 mL of the prepared Fehling's reagent, heat it in a water bath, and observe the color change of the solution; for the blank group, use distilled water instead of the sample solution, and perform the rest of the same operation as above. If a brick-red precipitate appears, it means that reducing sugars are present.

[0078] ⑦ Sulfate-carbazole reaction: Take 1 mL of the polysaccharide solution of the northern blue dragon hair into a test tube, add the prepared sulfuric acid-carbazole solution, and observe the reaction phenomenon. If a blue-green color appears, it means that it does not contain uronic acid.

[0079] 2.1 Experimental Results and Analysis

[0080] (1) UV full wavelength scanning of crude polysaccharides from the plant

[0081] The results of full wavelength scanning of the crude polysaccharide of Beiqinglongyi using an ultraviolet spectrophotometer showed that there were no obvious absorption peaks at wavelengths of 260nm and 280nm, indicating that the crude polysaccharide of Beiqinglongyi did not contain protein or nucleic acid substances.

[0082] (2) Isolation and purification of crude polysaccharide PJP60 from the genus Psoralea corylifolia

[0083] The crude polysaccharide PJP60 from the Psoralea corylifolia was purified by DEAE-52 anion exchange chromatography column, and each tube of sample was tracked and detected by phenol-sulfuric acid method. According to the test results, the elution curve was drawn. Figure 1. Gradient elution was performed using 0-0.5 mol / L NaCl solution. When the concentration of the NaCl solution was 0, an obvious elution component was obtained. Thereafter, when eluted with 0.05-0.5 mol / L NaCl solution, almost no polysaccharide component was eluted. Therefore, only the component at the NaCl concentration of 0 needed to be collected. In order to ensure the purity of the component, half-peak collection was adopted, and the product was concentrated and freeze-dried to obtain a powdery white polysaccharide named as P. chinensis polysaccharide PJP60-I. The obtained P. chinensis polysaccharide PJP60-I was further purified using a Sephadex G-50 gel chromatography column. The phenol-sulfuric acid method was used to determine the absorbance of each tube of sample. An elution curve was drawn based on its absorbance value. According to the elution curve, a single symmetrical single peak component appeared between the 80th and 110th tubes, and the purity was good. The samples were collected, concentrated, and freeze-dried to obtain P. chinensis polysaccharide PJP60-Ia.

[0084] (3) Determination of the content of polysaccharide in Beiqinglongyi

[0085] The phenol-sulfuric acid method was used to determine the concentrations of glucose standards. A standard curve was drawn based on the measured absorbance values. The standard curve equation was obtained as follows: y = 2.5227x + 0.0844, R 2 = 0.9992, where x represents the glucose concentration (mg / mL) and y represents the absorbance (A). The polysaccharide content of PJP60, PJP60-I, and PJP60-Ia was determined to be 48.97% ± 0.16% mg / mL, 80.65% ± 0.12% mg / mL, and 98.85% ± 0.27% mg / mL, respectively. Comparison of the sugar contents of PJP60, PJP60-I, and PJP60-Ia revealed that the sugar content of PJP60 was significantly increased after purification using a DEAE-52 anion exchange chromatography column and a Sephadex G-50 gel chromatography column.

[0086] (4) Physical and chemical properties of polysaccharide from Northern Qinglongyi

[0087] The basic physicochemical properties of the polysaccharide PJP60-Ia from the genus Pseudostellariae were determined, and the results are shown in Table 1. The phenol-sulfuric acid reaction and the Molisch reaction are color reactions commonly used to identify carbohydrate components. The polysaccharide PJP60-Ia from the genus Pseudostellariae was identified by these two reactions, indicating that the polysaccharide PJP60-Ia from the genus Pseudostellariae was a carbohydrate component. The polysaccharide PJP60-Ia from the genus Pseudostellariae did not produce a color change when it reacted with I2-KI, indicating that the polysaccharide PJP60-Ia from the genus Pseudostellariae did not contain starch or glycogen. Phenolic substances will react with FeCl3 to form a colored complex, but the polysaccharide PJP60-Ia from the genus Pseudostellariae 0-Ia did not show this phenomenon, indicating that it does not contain phenolic substances; the ninhydrin reaction with most amino acids, proteins, and peptides has a special color, and the reaction with the polysaccharide PJP60-Ia of the northern blue dragon hair did not change the color of the solution, indicating that it does not contain amino acids, proteins and polypeptides; the Fehling reaction with the polysaccharide PJP60-Ia of the northern blue dragon hair did not produce a brick-red precipitate, indicating that it does not contain reducing sugars; the result of the sulfuric acid-carbazole reaction was negative, indicating that the polysaccharide PJP60-Ia of the northern blue dragon hair did not contain uronic acid.

[0088] Table 1 Physicochemical properties of PJP60-Ia polysaccharide from Psoralea corylifolia

[0089]

[0090] The present invention uses a Flash extractor combined with a graded alcohol precipitation method to extract crude polysaccharides from the iris of the Chinese iris. After alcohol precipitation, graded extraction, and decolorization, PJP60 is obtained. The iris of the Chinese iris of the Chinese iris polysaccharide PJP60 is subsequently isolated and purified using a DEAE-52 anion exchange column chromatography with a 0-0.5 mol / L NaCl solution for gradient elution. A fraction, designated as iris of the Chinese iris polysaccharide PJP60-I, is obtained. The fraction has a sugar content of 80.65% ± 0.12% and a yield of 18.66%. The iris of the Chinese iris polysaccharide PJP60-I is further isolated and purified using a Sephadex G-50 gel column chromatography using ultrapure water as the eluent. A fraction, designated as iris of the Chinese iris polysaccharide PJP60-Ia, is obtained. The fraction has a sugar content of 98.85% ± 0.27% and a yield of 54.05%.

[0091] The physical and chemical properties of the polysaccharide PJP60-Ia from the plant were determined to be a white powder with no distinctive odor and is readily soluble in water. Both the phenol-sulfuric acid method and the Molisch reaction indicated that PJP60-Ia is a carbohydrate. The I2-KI reaction indicated that it did not contain starch or glycogen. The FeCl3 reaction indicated that it did not contain phenolic compounds. The ninhydrin reaction indicated that it did not contain amino acids, proteins, or polypeptides. The Fehling reaction indicated that it did not contain reducing sugars. The sulfuric acid-carbazole reaction indicated that PJP60-Ia did not contain uronic acid.

[0092] Example 2 Primary structural analysis of PJP60-Ia polysaccharide from Psoralea corylifolia

[0093] To explore the biological activity of polysaccharides, we first need to clearly understand their chemical structure. This study used Fourier transform infrared spectroscopy, high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy to investigate the monosaccharide composition, purity, molecular weight, glycosidic bond type, connection mode, and connection site of the polysaccharide from the plant, laying the foundation for subsequent studies on the biological activity of the polysaccharide from the plant.

[0094] 2.1 Experimental methods

[0095] (1) Infrared spectrum detection:

[0096] According to the mass ratio of 1:100, the polysaccharide PJP60-Ia from Beiqinglongyi and the dried potassium bromide were accurately weighed, the two were mixed and ground in an agate mortar, and then pressed into tablets using a tablet press. The tablets were then placed in an infrared spectrometer and the spectra were analyzed at 400-4000 cm -1 Perform the test below.

[0097] (2) Purity analysis and molecular weight determination

[0098] ①Purity analysis:

[0099] Prepare a 1 mg / mL solution of PJP60-Ia polysaccharide from Northern Qinglongyi, dissolve it in ultrapure water, filter it through a 0.45 μm filter membrane, and set aside.

[0100] Assay conditions: Ultrahygrogel (7.8×300 mm) column, mobile phase: Watsons purified water, detector: differential detector (Waters 2414), column temperature: 30°C, injection volume: 10 μL, flow rate: 0.8 mL / min.

[0101] ② Molecular weight determination: Accurately weigh 1.0 mg each of the dextran standards T5, T10, T50, T70, and T110, dissolve thoroughly in 1 mL of ultrapure water, filter through a 0.45 μm filter, and set aside. The instrument parameters are the same as those for the purity determination. Plot the standard curve for the dextran standards using retention time as the horizontal axis and the logarithm of the relative molecular mass of the dextran standards as the vertical axis. Substitute the peak time of the P. chinensis polysaccharide PJP60-Ia into the standard curve to calculate its molecular weight.

[0102] (3) Monosaccharide composition

[0103] ① Acid hydrolysis treatment: Weigh 5.0 mg of P. japonica polysaccharide PJP60-Ia, add 1 mL of 2 mol / L trifluoroacetic acid solution, hydrolyze at 110 ° C for 6 h, dry under reduced pressure, wash with methanol solution several times, evaporate to remove residual trifluoroacetic acid solution, add 2 mL of ultrapure water to dissolve, and obtain the polysaccharide hydrolyzate.

[0104] ② Derivatization treatment: aspirate 0.2 mL of polysaccharide hydrolyzate, add 0.5 mol / L PMP methanol solution and 0.3 mol / L NaOH solution 0.2 mL each, place in a 70 ° C water bath and heat for 100 min to carry out the derivatization process. After the reaction is completed, add the same volume of 0.3 mol / L HCl solution and 1 mL of chloroform, extract three times, remove the organic layer, take the aqueous layer, filter through a 0.22 μm microporous membrane, and set aside.

[0105] Accurately weigh 5.0 mg each of mannose (Man), rhamnose (Rha), galacturonic acid (GalA), glucose (Glc), fructose (Fru), galactose (Gal), and xylose (Xyl), and dissolve them in ultrapure water to prepare 1 mg / mL monosaccharide standards and monosaccharide mixed standards. The derivatization process of the monosaccharide mixed standard is the same as above.

[0106] ③Liquid chromatography conditions

[0107] Chromatographic column: Waters X-BridgeC 18 Column (4.6×250 mm, 5 μm), UV detector (Waters 2487), column temperature maintained at 25°C, mobile phase: phosphate buffer solution (pH=6.8) and acetonitrile, ratio: 78:22, flow rate: 0.8 mL / min, injection volume: 10 μL, detection at a wavelength of 245 nm.

[0108] (4) Nuclear magnetic resonance analysis: 30.0 mg of PJP60-Ia was accurately weighed and dissolved in 1 mL of heavy water (D2O), freeze-dried, and then dissolved in 1 mL of D2O. The mixture was shaken to fully dissolve, and then transferred to a nuclear magnetic resonance tube and detected using a nuclear magnetic resonance spectrometer.

[0109] 2.1 Experimental Results

[0110] (1) Infrared spectrum detection results

[0111] The FT-IR results of PJP60-Ia polysaccharide from Beiqinglongyi are shown in Figure 2 ,Depend on Figure 2 It can be seen that the polysaccharide PJP60-Ia of Beiqinglongyi is at 3428cm -1 There is an obvious absorption peak at 2928cm, which is the stretching vibration absorption peak of OH. -1The absorption peak is caused by the stretching vibration of CH, at 1400-1200 cm -1 The CH angle vibration peak appeared, indicating that the polysaccharide PJP60-Ia from the northern blue dragonfly has the structural characteristics of polysaccharide. -1 The absorption peak at 1076 cm is the C=O stretching vibration peak of polysaccharide. -1 、1049cm -1 The absorption peak at 883 cm is the stretching vibration peak of CO in the pyranose ring, indicating that the polysaccharide PJP60-Ia of Beiqinglongyi is a pyranose configuration. -1 、826cm -1 There is a weak absorption peak at , which indicates that the polysaccharide PJP60-Ia may contain both α and β configurations. The specific information is shown in Table 2. According to the FT-IR results, it is speculated that the polysaccharide PJP60-Ia is a polysaccharide with α, β-pyranose as the skeleton.

[0112] Table 2 Infrared spectrum analysis of functional groups of P. chinensis polysaccharide PJP60-Ia

[0113]

[0114] (2) Purity analysis and molecular weight determination

[0115] The present invention adopts high performance liquid chromatography-differential detection to detect the purity of the polysaccharide PJP60-Ia of the plant. The experimental results show that the peak time of the polysaccharide PJP60-Ia of the plant is 13.822 min, which is a single symmetrical chromatographic peak, indicating that the polysaccharide PJP60-Ia of the plant is a uniform polysaccharide component with good purity.

[0116] The molecular weight of the polysaccharide PJP60-Ia from the Northern Qinglongyi was determined by high performance liquid chromatography. However, this method has certain limitations. The molecular weight of the polysaccharide determined within the molecular weight range of the standard curve is more accurate. Based on the chromatographic peaks of the T series dextran standards, the retention time is used as the horizontal axis and the logarithm of the relative molecular mass of the dextran standards is used as the vertical axis. Figure 3 , the standard curve equation is obtained: lgMw=-0.3161Rt+8.4204, R 2 =0.9974, indicating that the molecular weight has a good linear relationship between 5000-110000Da. According to the peak time of 13.822min of the polysaccharide PJP60-Ia from Beiqinglongyi, the molecular weight of the polysaccharide PJP60-Ia from Beiqinglongyi was calculated to be 11250Da.

[0117] (3) Monosaccharide composition

[0118] Monosaccharide composition analysis is a crucial step in polysaccharide structural research. The monosaccharide composition of the polysaccharide PJP60-Ia from the plant was investigated using HPLC-PMP pre-column derivatization. Comparison of the HPLC chromatograms of the monosaccharide mixture standard with those of the sample reveals the specific elution times of the various monosaccharides shown in Table 3. Because Fru is reduced to Glc during the derivatization process, and the elution times of Fru and Glc are close, they overlap in the chromatogram of the monosaccharide mixture standard. Consequently, only six chromatographic peaks are present in the chromatogram of the monosaccharide mixture standard. The HPLC chromatogram of the monosaccharide composition of the polysaccharide PJP60-Ia from the plant shows two peaks with elution times of 9.953 min and 13.384 min, respectively. Comparison with the HPLC chromatogram of the monosaccharide mixture standard reveals approximately the same elution times as those of Rha, Glc, and Fru. Therefore, it is inferred that the polysaccharide PJP60-Ia from the plant is composed of Rha, Glc, and Fru.

[0119] Table 3 Comparison of peak elution time of monosaccharides and monosaccharide mixed standards

[0120]

[0121] (4) Nuclear Magnetic Resonance

[0122] Nuclear magnetic resonance spectroscopy is an essential analytical method for studying polysaccharide structure. 1 H NMR, 13 C NMR), 2D NMR ( 1 H- 1 H COSY, HMBC, HSQC) were used to analyze the structure of the polysaccharide PJP60-Ia from the plant. 1 In the H NMR spectrum, most proton signal peaks are located between δ3.0-4.0ppm. The signals overlap and are difficult to analyze. The proton hydrogen with signal peaks in the range of δ4.3-5.5ppm is anomeric hydrogen. Usually, the signal peaks in the regions of δ5.60-4.90ppm and δ4.90-4.30ppm represent α configuration and β configuration, respectively. 13 In the C-NMR spectrum, the signal peak of the anomeric carbon is mainly located between δ90-112ppm. Usually, the signal peak of the anomeric carbon of α configuration is at δ<103ppm, and the signal peak of the anomeric carbon of β configuration is at δ>103ppm. The signal peak of C3 or C5 of furanose is distributed between δ82-84ppm, while the signal peak of C3 or C5 of pyranose is δ<80ppm.

[0123] According to the polysaccharide PJP60-Ia of Beiqinglongyi 1The results of H-NMR showed that the hydrogen spectrum signals of the polysaccharide PJP60-Ia of the northern blue dragon hair were mainly concentrated between 1.00-5.22ppm, among which there were three signal peaks in the anomeric hydrogen signal region (δ5.22ppm, δ5.05ppm, δ4.47ppm). The results showed that the polysaccharide PJP60-Ia of the northern blue dragon hair had both α and β glycosidic bonds, which was consistent with the results of FT-IR analysis. The signal peaks between δ3.02-4.03ppm showed that the polysaccharide PJP60-Ia of the northern blue dragon hair had a pyranose ring. According to the results of the polysaccharide PJP60-Ia of the northern blue dragon hair 13 The C-NMR results showed that the carbon spectrum characteristic signal peaks of the polysaccharide PJP60-Ia of Beiqinglongyi were mainly concentrated in the range of δ59.49-104.35ppm. In the signal peak area of ​​the anomeric carbon, the signal peaks of the anomeric carbon of α and β configurations can be observed, indicating that both α and β configurations exist. 1 The H-NMR results were consistent.

[0124] pass 1 H- 1 Combination of H COSY, HSQC, and HMBC spectra 1 H NMR, 13 The structure of the polysaccharide was inferred from the C NMR spectrum. The sugar residue signals appearing in the HSQC spectrum were: δ3.53 / 59.00ppm, δ4.47 / 97.98ppm, δ4.31 / 101.91ppm, δ5.22 / 92.05ppm, and δ5.05 / 91.86ppm. The abundance of δ4.31 / 101.91ppm was low, making it difficult to assign. After comprehensive judgment, it was inferred that the polysaccharide PJP60-Ia from the northern blue dragonfly was probably composed of →1)-β-D-Fruf-(2→, 3,4)-β-Rha-(1→, α-D-Glcp, which also verified the results of the monosaccharide composition. The assignment information is shown in Table 4.

[0125] Table 4 Attribution information of sugar residues

[0126]

[0127] The present invention uses infrared spectroscopy analysis, purity detection, molecular weight determination, monosaccharide composition and NMR to study the primary structure of the polysaccharide PJP60-Ia from the plant. The results show that:

[0128] (1) FT-IR results showed that the polysaccharide PJP60-Ia from the plant had characteristic peaks of polysaccharides and had both α and β glycosidic bond configurations.

[0129] (2) The purity, molecular weight and monosaccharide composition of the polysaccharide were detected by HPLC. The results showed that the polysaccharide PJP60-Ia from Beiqinglongyi was a homogeneous polysaccharide with a molecular weight of 11250Da, and it was inferred that it was composed of Rha, Glc and Fru.

[0130] (3) The NMR results were consistent with the FT-IR results and corresponded to the monosaccharide composition results. The polysaccharide PJP60-Ia of P. chinensis may be composed of →1)-β-D-Fruf-(2→、3,4)-β-Rha-(1→、α-D-Glcp.

[0131] Example 3 Advanced structural analysis of the polysaccharide PJP60-Ia from the genus Beiqinglongyi

[0132] Polysaccharides are important biological macromolecules with more complex structures than proteins and nucleic acids. Currently, research on the structure of polysaccharides mainly focuses on the primary structure, while research on the higher-order structure is quite rare. Moreover, the higher-order structure directly affects the biological activity of polysaccharides. Therefore, studying the higher-order structure of polysaccharides is essential. The present invention utilizes Congo red test, DSC-TG thermogravimetric analyzer, laser particle size analyzer, X-ray diffractometer, atomic force microscope, and field emission scanning electron microscope to analyze and study the morphology, spatial structure, thermal properties, and stability in solution system of the polysaccharide PJP60-Ia from the plant of northern blue dragon hair. The Congo red test is used to determine whether the polysaccharide has a triple helical structure, SEM and AFM are used to observe the microscopic morphology and surface morphological characteristics of the polysaccharide, and XRD can determine information such as the crystal state and three-dimensional configuration of the polysaccharide.

[0133] 3.1 Experimental methods

[0134] (1) Congo red test: 10.0 mg of PJP60-Ia polysaccharide was accurately weighed and dissolved in ultrapure water to prepare a 2 mg / mL PJP60-Ia sample solution. A 0.08 mmol / L Congo red solution and a 2 mol / L NaOH solution were prepared and set aside. Five glass test tubes were taken, and equal volumes of PJP60-Ia polysaccharide sample solution and Congo red solution were drawn into each of the five glass test tubes. Then, 2 mol / L NaOH solution was added to make the final concentrations of the NaOH solution 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L, respectively. After shaking, the mixture was allowed to stand at room temperature for 30 min. The spectrum was scanned at 400-600 nm using an ultraviolet spectrophotometer, and the maximum absorption wavelength of the NaOH solution at different concentrations was recorded. Five other glass test tubes were taken, and ultrapure water was added to replace the sample solution. The same method was used for determination. Draw a curve with NaOH concentration as the horizontal axis and maximum absorption wavelength as the vertical axis.

[0135] (2) Thermal Properties: The thermal properties of the polysaccharide PJP60-Ia from the plant were investigated using thermogravimetric analysis and differential scanning calorimetry. 12.3 mg of the polysaccharide PJP60-Ia was accurately weighed and the experimental parameters were set as follows: a heating rate of 10°C / min and a test temperature of 30-600°C. The process was carried out under a nitrogen atmosphere with a gas flow rate of 30 mL / min.

[0136] (3) Particle size and potential determination: Prepare a 1 mg / mL sample solution of PJP60-Ia polysaccharide from Northern Qinglongyi, fully dissolve it, and filter it through a 0.45 μm microporous filter membrane. Use a laser particle size analyzer to measure the particle size and potential.

[0137] (4) X-ray diffraction: X-ray diffraction (XRD) is commonly used to examine the structural state and crystal structure of polysaccharides. XRD analysis of the P. chinensis polysaccharide PJP60-Ia was performed using the following diffraction conditions: CuKα radiation, 40 kV tube voltage, 40 mA tube current, continuous scanning within the 2θ range of 10°-80°, and a scan rate of 3° / min. The data were analyzed using MDI Jade 6.0 software.

[0138] (5) Field emission scanning electron microscopy observation: Weigh 2.0 mg of the dried P. chinensis polysaccharide PJP60-Ia and spread it evenly on a sample stage with conductive glue. Place the sample stage in an ion sputtering coating instrument and coat it with a layer of conductive gold powder. Then, place it under a scanning electron microscope and observe the surface morphology of the sample at an acceleration voltage of 15 kV at 100x, 1000x, and 10,000x magnifications.

[0139] (6) Atomic force microscopy observation: Prepare a 1 mg / mL solution of the polysaccharide PJP60-Ia from the plant stem, stir magnetically for 2 h to completely dissolve the sample, then dilute the polysaccharide sample solution to 10 ng / mL with ultrapure water. Stir magnetically for about 4 h to fully dissolve the sample, filter through a 0.45 μm microporous filter membrane, and use this as the working solution. Treat the mica sheet with nickel chloride solution, wait 30 seconds, then rinse it with ultrapure water. After drying, begin testing. Pipette 20 μL of the working solution and drop it onto the treated mica sheet. After 30 seconds, rinse the unadsorbed sample with ultrapure water. After drying, scan its surface morphology using an atomic force microscope.

[0140] 3.2 Experimental Results

[0141] (1) Congo red test

[0142] Congo red is an acidic dye soluble in water and anhydrous ethanol. The Congo red experiment is an effective method for studying the conformation of polysaccharide molecules. When the polysaccharide has a triple helical structure, the Congo red solution reacts with the polysaccharide solution to form a complex. The maximum absorption wavelength of the complex formed in 0-0.5 mol / L NaOH solution will shift toward the long-wave direction compared with the blank group. However, when the NaOH concentration is greater than 0.3 mol / L, the maximum absorption wavelength of the complex will decrease significantly. The results of the Congo red experiment on the polysaccharide PJP60-Ia of the Northern Qinglongyi are shown in Figure 4 When the maximum absorption wavelength of the complex formed by the polysaccharide PJP60-Ia from Beiqinglongyi and Congo red solution did not show a trend of first increasing and then decreasing as the concentration of NaOH solution increased, the maximum absorption wavelength did not increase when the concentration of NaOH solution was 0-0.2 mol / L, and the maximum absorption wavelength did not decrease significantly when the NaOH concentration was higher than 0.3 mol / L. This may be because under alkaline conditions, the generated complex was gradually degraded. It is speculated that the polysaccharide PJP60-Ia from Beiqinglongyi does not have a triple helix structure.

[0143] (2) Determination of thermal properties

[0144] DSC-TG is often used to study the thermal stability of polysaccharides. TGA analysis reflects the relationship between the thermal decomposition of polysaccharides and temperature, while DSC analysis infers the structural changes and thermal properties of polysaccharides from their endothermic and exothermic peaks. The thermal stability of polysaccharides has an important impact on their applications. Figure 5 The DSC-TG analysis results for the polysaccharide PJP60-Ia from the plant stem of the northern qinglongyi plant indicate that its thermal decomposition can be divided into three main stages. The first stage, between 30 and 140°C, saw a mass loss of 3.40%, attributed to the evaporation of crystalline and bound water during heating. The second stage, between 140 and 460°C, saw a mass loss of 39.41%, likely due to thermal decomposition of the polysaccharide and the cleavage of chemical bonds. The rate of mass loss reached its highest point at 198.87°C, indicating good thermal stability below 140°C. The third stage, between 460 and 600°C, represents the carbonization stage, with a mass loss of 3.59%. The DSC curve reveals two characteristic exothermic peaks, located at 162.19°C and 447.71°C, both occurring within the 140-460°C range and likely resulting from polysaccharide decomposition and bond cleavage.

[0145] (3) Determination of particle size and potential

[0146] ① Particle size measurement results: Polysaccharides often exist in the form of polymers in solution. The particle size and polydispersity index (PDI) of polysaccharides in solution can reflect the degree of dispersion of polysaccharides in solution. Generally speaking, the smaller the PDI value, the narrower the molecular weight distribution range of the substance. When the PDI value is less than 1, it indicates that the molecular weight distribution uniformity of the substance is good. The particle size distribution of the polysaccharide PJP60-Ia from the Northern Qinglongyi is shown in Figure 6 It can be seen that it is a single symmetrical peak, indicating that the polysaccharide PJP60-Ia from Beiqinglongyi is evenly distributed in the aqueous solution, with an average particle size of 211.8 nm and a PDI value of 0.25, which is less than 1, indicating that the molecular weight distribution range of the polysaccharide PJP60-Ia from Beiqinglongyi is narrow and the molecular weight distribution uniformity is good.

[0147] ② Potential measurement results

[0148] The Zeta potential of polysaccharides can reflect the stability of polysaccharide solution systems to a certain extent. Generally speaking, the larger the absolute value of the Zeta potential, the greater the electrostatic repulsion between molecules in the solution, the more stable the dispersion of the substance in the solution, and the less likely it is to aggregate. The potential measurement results of the polysaccharide PJP60-Ia of Beiqinglongyi are shown in Figure 7 The average potential of the polysaccharide PJP60-Ia from Northern Qinglongyi is -3.5mV, its absolute value is relatively low, and the electrostatic repulsion between molecules is small. Therefore, the polysaccharide PJP60-Ia from Northern Qinglongyi is unstable in the solution system and easy to aggregate.

[0149] (4) X-ray diffraction analysis

[0150] XRD is often used for high-level structural analysis of polysaccharides to determine their crystal structure. Generally speaking, crystals show sharp and narrow characteristic peaks, while amorphous substances show broad and diffuse diffraction peaks. Polysaccharides generally exist in non-crystalline amorphous or semi-crystalline forms. Different extraction methods, processing methods and other factors will affect the crystal structure. The XRD results of the polysaccharide PJP60-Ia from the Northern Qinglongyi are shown in Figure 8 As shown in the data, between 2θ=10°-80°, the polysaccharide PJP60-Ia from Beiqinglongyi has an amorphous structure, and sharp and narrow diffraction peaks appear above 20°, at 2θ=28.4°, 40.7°, 66.4°, and 73.8°, respectively, indicating that the polysaccharide PJP60-Ia from Beiqinglongyi has a locally ordered structure and tends to be semi-crystalline. This feature is similar to that of soybean polysaccharides in aqueous solution. The results show that the polysaccharide PJP60-Ia from Beiqinglongyi is an amorphous substance with a semi-crystalline structure.

[0151] (5) Field emission scanning electron microscopy observation

[0152] Field emission scanning electron microscopy (SEM) is often used to observe the surface morphology of polysaccharide samples. It can effectively analyze the structure and morphology of polysaccharides, such as molecular morphology, porosity, etc. The surface morphology of polysaccharides varies greatly and is related to the source and extraction method of polysaccharides. Figure 9 , observed under 300×, 1200×, and 5000× magnifications, respectively. Under 300× magnification, the polysaccharide was in the form of a rolled film with an uneven surface and irregular protrusions, clumps, or holes; under 1200× magnification, the polysaccharide surface was relatively smooth with granular protrusions or depressions; under 5000× magnification, the polysaccharide was a continuous membranous structure with wrinkles and irregular flaky structures gathered at the edges, but not completely stacked together, which may be due to the repulsive force between polysaccharide molecules and the weak molecular attraction.

[0153] (6) Atomic force microscopy observation

[0154] Atomic force microscopy (AFM) helps in studying the structure of polysaccharides in terms of morphological and molecular characteristics such as height (diameter), width, and dispersion, and can provide two-dimensional and three-dimensional images of polysaccharides. Figure 10 The two-dimensional and three-dimensional morphologies of the polysaccharide PJP60-Ia from the plant stem of the northern blue dragonfly are shown in different fields of view. The two-dimensional image shows a large number of bright, irregular, and densely aggregated particles. This is likely due to the hydroxyl groups on the polysaccharide chains, which strengthen intermolecular and intramolecular interactions, leading to molecular aggregation. Van der Waals forces, hydrogen bonds, and other factors may also contribute to the aggregation. The three-dimensional image shows a large number of peak-like protrusions of varying heights and a relatively dense distribution, allowing for a more intuitive observation of the degree of polysaccharide aggregation. The AFM results correspond to those of the zeta potential measurement, indicating that the polysaccharide PJP60-Ia from the northern blue dragonfly is unstable in solution and prone to aggregation.

[0155] The present invention measured the spatial conformation and stability of the polysaccharide PJP60-Ia of the genus P. japonica by Congo red test, thermal property measurement, particle size potential measurement, XRD, AFM and SEM. The results showed that:

[0156] (1) The results of the Congo red experiment showed that the polysaccharide PJP60-Ia from the genus Beiqinglongyi did not have a triple helical structure.

[0157] (2) The thermal properties experiment showed that the polysaccharide PJP60-Ia of Northern Qinglongyi experienced three weight loss events. The first one occurred between 30-140℃, with a mass loss of 3.40%, which was caused by the evaporation of crystalline water and bound water; the second one was between 140-460℃, with a mass loss of 39.41%, which may be caused by the thermal decomposition of the polysaccharide and the breaking of chemical bonds; the third one was between 460-600℃, with a mass loss of 3.59%, which was the carbonization stage.

[0158] (3) According to the results of particle size potential, the average particle size of the polysaccharide PJP60-Ia from Beiqinglongyi is 211.8 nm, the PDI value is 0.25, and the average potential is -3.5 mV. The molecular weight distribution uniformity of the polysaccharide PJP60-Ia from Beiqinglongyi is good, but the sample is unstable in the solution system and is easy to aggregate.

[0159] (4) XRD results showed that the polysaccharide PJP60-Ia from the plant is an amorphous substance with a semi-crystalline structure.

[0160] (5) The morphology of the polysaccharide PJP60-Ia from Psoralea corylifolia was observed by SEM, and it was found that the polysaccharide PJP60-Ia from Psoralea corylifolia was in the form of a rolled film with an uneven surface, irregular protrusions or holes, and a film-like and sheet-like structure.

[0161] (6) Through AFM observation, it was found that the polysaccharide PJP60-Ia of the northern blueberry hair was irregular and blocky, and the aggregation density was relatively large, which was consistent with the SEM observation results and Zeta potential measurement results. The surface of the polysaccharide PJP60-Ia of the northern blueberry hair was irregular and blocky, and it was unstable in the solution and easily aggregated.

[0162] Example 4 Study on the anti-inflammatory activity of PJP60-Ia polysaccharide from Northern Qinglongyi on LPS-induced mouse macrophages RAW264.7

[0163] Inflammation is the immune system's first protective response to a variety of harmful stimuli. Its process is highly dynamic and can be regulated by complex molecular cascades. Appropriate inflammatory responses are generally beneficial to the human body, while excessive and uncontrolled inflammation is pathological and fatal. As an important endogenous messenger, NO is involved in many physiological activities and pathological processes of the system, including immune and inflammatory responses. ROS is a family of chemical substances derived from oxygen, including superoxide anions, hydrogen peroxide, and hydroxyl free radicals. High levels of ROS are harmful to the body, interfere with normal cell metabolism, cause oxidative stress, produce oxidative decomposition products in the body, induce inflammation, activate inflammatory cells to secrete a large number of pro-inflammatory factors, and lead to oxidative damage to cells. [Ca 2+ ]i changes can directly participate in regulating the signal transduction in cells. After being stimulated by external signals such as LPS, the [Ca 2+]i will rise rapidly, thereby regulating the basic processes of inflammatory cells such as secretion, metabolism and differentiation. Cytokines are a class of soluble small molecule proteins produced by various cells after stimulation. They have a wide range of biological functions such as regulating immunity, participating in inflammation, regulating cell differentiation and development, and tissue repair. They are often released with the occurrence of inflammation. Cytokines mainly include tumor necrosis factor (TNF), interferon (IFN), interleukin (IL), etc. According to their different functions, they can be divided into two types: pro-inflammatory and anti-inflammatory. The balance between the two is extremely important for maintaining the homeostasis of the tissue environment. The occurrence and development of inflammation is regulated by various cytokines under different external stimuli by changing the balance between the pro-inflammatory and anti-inflammatory systems. Current studies have confirmed that the production of cytokines is closely related to the M1 / M2 polarization of macrophages in the body. Under normal circumstances, M1 and M2 macrophages are in a dynamic equilibrium, or exist in a non-polarized M0 state. However, when the body is stimulated by endogenous or exogenous inflammatory substances, M0 macrophages are activated in large numbers to become M1 macrophages. These macrophages then respond to inflammatory signals by releasing pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β, and IFN-β, promoting the inflammatory cascade and leading to irreversible damage and necrosis of cells and tissues. These large amounts of pro-inflammatory cytokines, in turn, activate macrophages and recruit more inflammatory mononuclear phagocytes from the circulation, contributing to various inflammatory diseases. Targeting macrophage polarization during inflammatory responses is an effective treatment approach, reversing the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, thereby increasing the secretion of the anti-inflammatory cytokines IL-4 and IL-10 and reducing the expression of inflammatory stimuli.

[0164] In this example, LPS was used to induce mouse RAW264.7 macrophages to establish an in vitro inflammatory model. The effects of PJP60-Ia on the morphology and proliferation of inflammatory cells were observed under an inverted microscope. The levels of cellular ROS were detected by flow cytometry. The content of NO was detected by the Griess method. The intracellular Ca2+ expression was observed under an inverted fluorescence microscope. 2+ The expression of M1 / M2 polarization markers CD86 and CD163 mRNA and protein was detected by qPCR and Western blot. The mRNA expression of TNF-α, IL-1β, IL-4, and IL-10 was detected by qPCR. Finally, the expression of proteins related to the TLR2 / 4 signaling pathway and cGAS-STING pathway in RAW264.7 cells was detected by Western blot.

[0165] Reagent preparation

[0166] Prepare PBS buffer: Accurately weigh 4.0 g NaCl, 0.1 g KCl, 0.78 g Na2HPO4, and 0.1 g KH2PO4, fully dissolve them in ultrapure water, and make up to volume in a 500 mL sterilized saline bottle. Sterilize by high temperature and high pressure, filter through a 0.22 μm microporous membrane, and store at 4°C until use.

[0167] Prepare culture medium: Use a syringe to draw 90 mL of DMEM medium and 10 mL of inactivated fetal bovine serum, then add 1 mL of penicillin and streptomycin, mix well, seal the tube, and store at 4°C.

[0168] Preparation of MTT solution: Accurately weigh 10.0 mg of MTT powder and add 2 mL of PBS to prepare 5 mg / mL MTT solution. Seal the tube with sealing film, protect from light, and store at 4°C.

[0169] Prepare neutral red solution: weigh an appropriate amount of neutral red, dissolve it in PBS, filter it through a 0.22 μm microporous filter membrane to obtain a 0.1% neutral red solution, and store it at 4°C until use.

[0170] Prepare LPS solution: Weigh an appropriate amount of lipopolysaccharide powder, dissolve it in culture medium, filter it through a 0.22 μm microporous filter, protect it from light, and store it at 4°C. Dilute it to the desired concentration before use.

[0171] Prepare the solution of PJP60-Ia polysaccharide from Beiqinglongyi: accurately weigh 20.0 mg of PJP60-Ia polysaccharide from Beiqinglongyi, add 10 mL of culture medium to dissolve, protect from light, seal the container, and store at 4°C until use. Dilute with culture medium to the desired concentration when using.

[0172] Prepare DCFH-DA stock solution: dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L. Prepare and use immediately.

[0173] 4.1 Experimental methods

[0174] (1) Cell culture

[0175] ① Cell recovery: Remove mouse macrophage RAW264.7 cells from the liquid nitrogen tank and quickly thaw them in a 37°C water bath. Remove the cells as soon as possible after thawing. Working in a clean bench, transfer the cells in the cryopreserved tube to a sterile centrifuge tube containing 5 mL of culture medium. Pipette the cells to mix them evenly. Centrifuge (1500 rpm) for 5 minutes, discard the supernatant in the centrifuge tube, aspirate 1 mL of culture medium, gently pipette the cells to mix them evenly, and transfer them to a culture flask containing fresh culture medium. Finally, place the cells in an incubator (37°C, 5% CO2) for culture and label them.

[0176] ②Cell passaging: Take the cells out of the incubator and observe the cell morphology under a microscope. When the cell growth density reaches more than 80%, the cell can be passaged. The passage work should be carried out in a clean bench. Operation steps: Pour out the old culture medium, then aspirate 3mL PBS to rinse the wall cells. This process should be repeated 2-3 times. Pour out the PBS, add 1mL trypsin cell digestion solution to digest the cells, and observe under a microscope. When the cells become round, aspirate the trypsin, add culture medium to stop digestion, and then pipette the cells up and down to make a cell suspension. Do not generate bubbles. Add 3mL culture medium and centrifuge (1500r / min) for 5min. Discard the supernatant and add 3mL culture medium. Pipet the cells to mix well to make a cell suspension. Pipette an appropriate amount of cell suspension into a new culture bottle, add an appropriate amount of culture medium, and then culture in the incubator.

[0177] ③ Cell freezing: Take cells in the logarithmic growth phase, digest them with trypsin, then add culture medium to terminate the digestion, transfer them to a 10mL centrifuge tube, centrifuge (1500r / min) for 5min, discard the supernatant, add freezing solution to the centrifuge tube, gently blow the cells to mix, then transfer them to a cryopreservation tube, seal it, mark the frozen cell name and date, and then store in the following order: 4℃ for 30min, place at -20℃ for 2h, and then store at -80℃.

[0178] ④ Cell counting: In a clean ultra-clean operating table, digest the cells with trypsin, then add culture medium to terminate the digestion. Wipe the cell counting plate clean with an alcohol cotton ball. After the counting plate is dry, place the cover glass in the appropriate position of the counting plate. Take 20μL of cell suspension and slowly add it to one side of the counting plate. Let the cell suspension slowly spread. Be careful not to generate bubbles. Place the counting plate under a microscope and observe the four large squares of the counting plate. Record the number of cells in the four large squares and calculate the average value and multiply by 10. 4 That is, the number of cells per milliliter.

[0179] (2) MTT assay of the effect of PJP60-Ia on LPS-induced RAW264.7 cell proliferation

[0180] The experiment was divided into blank group, control group, LPS inflammation model group and LPS+PJP60-Ia administration group. Blank group: no cells; control group: cells, only 200μL DMEM was added; LPS inflammation model group: only 200μL 1μg / mL LPS solution was added; LPS+PJP60-Ia administration group: first 100μL 1μg / mL LPS solution was added for induction for 1h, and then 100μL of different concentrations of PJP60-Ia solution were added to the final mass concentrations of 10, 20, 40, 80, 160, 320, and 640μg / mL respectively. RAW264.7 cells with good growth status were made into cell suspension and seeded in 96-well plates so that the cell density per well was 5×10 4 / mL, add 100μL to each well except the blank group. To prevent the cells from naturally settling, blow evenly. After inoculation, place them in a cell culture incubator for adaptive culture for 24h. Discard the supernatant and treat the cells according to the above grouping, with 6 replicates per group. After continuing to culture for 24h, add 20μL of 5mg / mL MTT solution to each well and incubate at 37℃ for 4h. Finally, discard the supernatant, add 150μL DMSO to each well, shake in the dark for 10min, measure the absorbance of each well at 490nm with a microplate reader, and calculate the cell activity according to the formula: Cell activity = (OD 给药组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%.

[0181] (3) Fluorescence inverted microscopy was used to detect the effect of PJP60-Ia on the morphology of LPS-induced RAW264.7 cells

[0182] RAW264.7 cells with good growth status were prepared into a cell suspension and inoculated into a 6-well plate at 1 mL / well to make the cell density of each well 5×10 5 The supernatant was discarded, and the control group was treated with 2 mL of DMEM, while the LPS inflammation model group was treated with 2 mL of 1 μg / mL LPS solution. The low-, medium-, and high-dose LPS+PJP60-Ia groups were first induced with 1 mL of LPS solution (final concentration of 1 μg / mL) for 1 hour, followed by the addition of 1 mL of PJP60-Ia solution (final concentrations of 80, 160, and 320 μg / mL), respectively. After a further 24 hours of culture, the morphology of RAW264.7 cells was observed using an inverted fluorescence microscope.

[0183] (4) GRIESS assay to detect the effect of PJP60-Ia on LPS-induced NO production in RAW264.7 cells

[0184] RAW264.7 cells in good growth condition were prepared into a cell suspension and inoculated into a 96-well plate at 100 μL / well to make the cell density per well 1×10 5 Cells were cultured for 24 hours. The supernatant was discarded, and the control group was treated with 200 μL of DMEM, while the LPS inflammation model group was treated with 200 μL of 1 μg / mL LPS solution. The low-, medium-, and high-dose LPS+PJP60-Ia groups were first induced with 100 μL of LPS solution (final concentration of 1 μg / mL) for 1 hour, followed by the addition of 100 μL of PJP60-Ia solution (final concentrations of 80, 160, and 320 μg / mL), respectively, with three replicates per group. Cell supernatants were collected after 1.5, 6, 24, 48, and 72 hours of culture and subjected to Griess reaction using a NO assay kit. The absorbance was measured at 540 nm using a microplate reader, and the NO concentration in the supernatant was calculated based on a standard curve.

[0185] (5) Flow cytometry was used to detect the effect of PJP60-Ia on ROS production induced by LPS in RAW264.7 cells

[0186] The experimental groups and treatments were the same as above, except that a reactive oxygen species (ROS)-positive control group was added and treated with 2 mL of DMEM. After 24 hours of culture, RAW264.7 cells from each group were collected using the ROS detection kit and placed in 1.5 mL centrifuge tubes. The cells were centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. 1 mL of 10 μM DCFH-DA fluorescent dye was added to each tube. The cells were incubated at 37°C in the dark for 40 minutes, mixed by inversion every 5 minutes, and washed three times with DMEM by centrifugation. The cells were then resuspended in 1 mL of PBS. 1 μL of Rosup was added to the ROS-positive control group and incubated for 20 minutes. Finally, fluorescence intensity was measured by flow cytometry at 488 nm.

[0187] (6) Observation of the effect of PJP60-Ia on LPS-induced Ca2+ in RAW264.7 cells using an inverted fluorescence microscope 2+ Effect of content

[0188] RAW264.7 cells with good growth status were prepared into a cell suspension and inoculated into a 6-well plate at 1 mL / well to make the cell density of each well 1×10 5 / mL, cultured for 24h. The supernatant was discarded, and the control group was treated with 2mL DMEM, and the LPS inflammation model group was treated with 2mL 1μg / mL LPS solution. The low, medium, and high dose groups of LPS+PJP60-Ia were first induced with 1mL LPS solution (final mass concentration of 1μg / mL) for 1h, and then 1mL PJP60-Ia solution (final mass concentration of 80, 160, 320μg / mL) was added respectively, with 3 replicates in each group. After continuing to culture for 24h, the cells were washed 3 times with PBS, and then 1mL of 2.5μM Fluo-3AM calcium ion fluorescent dye was added to each well. After Fluo-3AM enters the cells, it will be cleaved into Fluo-3 by the esterase in the cells, thereby reacting with Ca 2+ The binding produces strong fluorescence. Incubate at 37°C in the dark for 45 minutes to ensure complete conversion of fluo-3AM to fluo-3 in the cells. Finally, wash away excess dye with PBS. Observe fluorescence intensity using an inverted fluorescence microscope.

[0189] (7) qPCR detection of the effect of PJP60-Ia on LPS-induced RAW264.7 cytokine mRNA expression

[0190] ① Extract total RNA and pipette 1 μL of RNA into the NanoDrop ultra-micro nucleic acid analyzer. Detect the RNA concentration and A260 / A280 ratio of each group. If the ratio is between 1.8-2.2, the RNA purity is high.

[0191] ② Check RNA integrity using 1% agarose gel electrophoresis at a constant voltage of 120 V for 30 min. Run the sample from the negative electrode to the positive electrode. After electrophoresis, remove the gel and visualize and analyze it using a gel imaging system. Adjust the concentration of high-quality RNA to 1000 ng / μL per tube and store at -80°C until ready for use.

[0192] ③ Reverse transcription to obtain cDNA

[0193] (a) Remove genomic DNA using the following reaction system: 2 μL of 5× gDNA Eraser Buffer, 1 μL of gDNA Eraser, 1 μL of Total RNA, and 10 μL of RNase-free dH2O. Refer to the reverse transcription kit instructions and add the sample to each reaction tube according to the reaction system listed in the table. Incubate at room temperature for 20 minutes and place on ice until ready to use.

[0194] (b) Reverse transcription reaction. The RNA reverse transcription reaction system includes: 10 μL of the reaction solution obtained in step (a), 1 μL of PrimeScript RT Enzyme Mix 1, 1 μL of RT Primer Mix, 4 μL of 5× PrimeScript Buffer 2 (for Real Time), and 4 μL of RNase-free dH2O. The reaction solution was prepared on ice. After adding the sample to each reaction tube, the reaction solution was centrifuged briefly to ensure that the reaction solution accumulated at the bottom of the tube. The reaction was then performed using a reverse transcription PCR instrument at 37°C for 15 min, 85°C for 20 s, and terminated at 4°C to obtain cDNA. The cDNA was then stored at -20°C until further use.

[0195] ④ Primer design and synthesis

[0196] The mRNA sequences and related information corresponding to mouse TNF-α, IL-1β, IL-4, and IL-10 genes were searched in the NCBI and Gene Bank databases. Primers were screened according to the design principles. Finally, the specific primers determined by blast were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The primer sequences are shown in the table.

[0197] Table 5 Target gene primer sequences

[0198]

[0199] ⑤ Real-time fluorescence quantitative PCR

[0200] (a) According to the experimental design, PCR reaction solution was prepared in batches on ice according to the table. The reaction system was 10 μL, including: TBGreen Premix Ex Taq (2×) 5 μL, upstream primer (10 μM) 0.2 μL, downstream primer (10 μM) 0.2 μL, ROX Reference Dye (50×) 0.2 μL, template cDNA 1 μL, and sterile water 3.4 μL.

[0201] (b) Perform real-time PCR: After adding the sample and centrifuging, the sample was tested using a real-time PCR instrument. The reaction conditions were as follows: pre-denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. After completion of the reaction, the amplification curve, melting curve, and Ct value were obtained.

[0202] (c) Mouse GAPDH gene was used as internal reference, and three replicate wells were set up in each group. -△△CT The relative expression levels of each cytokine mRNA were calculated by the method.

[0203] (8) Western blot analysis of the effect of PJP60-Ia on LPS-induced protein expression in RAW264.7 cells

[0204] ① Preparation of protein samples

[0205] (a) Total protein extraction: After 24 h of drug treatment, cells from each group were collected into 1.5 mL EP tubes and centrifuged at 1500 rpm for 5 min. The supernatant was discarded and the tubes were washed once with 1 mL of PBS and centrifuged. The supernatant was discarded and 100 μL of PBS was added again. After pipetting and mixing, an equal amount of lysis buffer was added and the tubes were boiled at 100°C for 10 min.

[0206] (b) Protein quantification: Use the Bradford protein assay kit to measure the absorbance of the standard and protein samples. Calculate the total protein concentration in each sample using the standard curve. Dilute each sample to the same concentration in 5× protein loading buffer and store at -20°C until ready for use.

[0207] ② Detect protein expression

[0208] (a) Gel Laying. (b) Electrophoresis: Remove the spread gel from the refrigerator and place it in the electrophoresis tank. Add the electrophoresis buffer, ensuring a slight difference in the liquid level between the inside and outside of the glass plate. Then, slowly remove the comb and use a fine needle to align the sample well. Sequentially add 6 μL of 5× protein loading buffer, 3 μL of prestained protein marker, 10 μL each of the control group, the LPS inflammation model group, and the LPS+PS-1 treatment group at three concentrations (40, 80, and 160 μg / mL), and 6 μL of 5× protein loading buffer. Connect the power supply to a constant voltage of 120 V. Stop electrophoresis when the gel reaches the bottom of the separation gel. (c) Transfer: Locate the target protein based on the marker position and determine the order before cutting the gel. Arrange the NC membrane, filter paper, and cotton pad in the electrotransfer tank, which have been pre-soaked in electrotransfer solution, in order, following the principle of "gel on the negative side, membrane on the positive side." Ensure that the membrane completely covers the gel and there are no bubbles between the membrane and gel. Then, place the membrane in the electrotransfer tank and add electrotransfer solution. Connect the power supply to a constant current of 200mA and electrotransfer on ice for 90 minutes. (d) Blocking: Place the transferred NC membrane in blocking solution, ensuring that the membrane is completely immersed in the blocking solution. Block on a shaker at room temperature for 2 hours. (e) Primary Antibody Incubation: Wash the blocked membrane three times with TBST for 15 minutes each time. Prepare primary antibodies for different target proteins according to the dilution ratio in the manufacturer's instructions. Place the washed membrane and antibodies in the corresponding incubation box and incubate overnight at 4°C. (f) Secondary antibody incubation: After the primary antibody incubation is completed, the membrane is washed with TBST 3 times, each time for 15 minutes, and the secondary antibody is prepared in the incubation box according to the dilution ratio in the instructions, and the washed membrane is placed in it and incubated on a shaker at room temperature for 90 minutes. (g) ECL color development: After the secondary antibody incubation is completed, the membrane is washed with TBST 3 times, each time for 15 minutes, and then the prepared color development solution is used in the dark for 2 minutes, and the imaging system is used for scanning and analysis. (h) Statistical analysis: The experimental data were analyzed using SPSS Statistics 21.0 software. The experimental results of each group are shown in Figure 2. The results were shown in Figure 3. One-way analysis of variance was used to compare the means of multiple samples. P < 0.05 and P < 0.01 were considered statistically significant. Origin software was used to plot the experimental results.

[0209] 4.2 Experimental Results

[0210] (1) Effects of PJP60-Ia polysaccharide on LPS-induced RAW264.7 cell proliferation and cell morphology

[0211] ①Effect of PJP60-Ia on LPS-induced RAW264.7 cell proliferation

[0212] The effect of PJP60-Ia on LPS-induced RAW264.7 cell viability was analyzed. MTT assays were used to assess the effect of PJP60-Ia on LPS-induced RAW264.7 cell viability. As shown in Table 6, increasing PJP60-Ia concentrations decreased RAW264.7 cell viability. When PJP60-Ia concentrations ranged from 80 to 320 μg / mL, cell viability was significantly different from that in the LPS-induced group (P < 0.01). However, no significant difference was observed between the 320 μg / mL treatment group and the control group (P > 0.05). Based on these results, 80, 160, and 320 μg / mL were selected as the dosing concentrations for subsequent experiments.

[0213] Table 6 Cell viability of RAW264.7 cells in each group (n=6)

[0214]

[0215] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0216] ②Effects of PJP60-Ia on LPS-induced RAW264.7 cell morphology

[0217] like Figure 11 As shown, RAW264.7 cells in the blank group, in a resting state without antigen stimulation, exhibited a translucent, quasi-round morphology, clustered in sheets, and firmly adhered to the wall. In the model group, RAW264.7 cells induced with LPS for 24 hours developed pseudopodia, increased in size, and transformed into elongated, spindle-shaped, and polygonal shapes, with cytoplasmic vacuoles. Three concentrations of PJP60-Ia (80, 160, and 320 μg / mL) reversed the polarization of RAW264.7 cells and significantly reduced the number of pseudopodia in a dose-dependent manner. At a concentration of 320 μg / mL, the proportion of pseudopodia was lowest, with the majority of cells returning to their original quasi-round shape. These results demonstrate that PJP60-Ia can regulate differentiated RAW264.7 cells to a resting state and inhibit the onset of inflammatory responses.

[0218] (2) Effect of PJP60-Ia on LPS-induced NO production in RAW264.7 cells

[0219] NO is a signaling mediator with a bidirectional regulatory effect in the immune system. Excessive NO will activate inflammatory factors and promote the occurrence of inflammatory reactions. In this experiment, the Griess method was used to measure the release of NO in RAW264.7 cells at five time points: 1.5h, 6h, 24h, 48h, and 72h, to investigate whether PJP60-Ia can inhibit the production of NO by inflammatory cells. The NaNO2 standard curve is Y=0.0276X+0.1675(R 2 =0.998). As shown in Table 7, the LPS model group showed a highly significant increase in NO secretion in the RAW264.7 cell supernatant compared to the control group (P < 0.01), and this increase was time-dependent, indicating that the inflammatory model was successfully established. At 1.5 hours of administration, NO secretion levels remained unchanged. At 6 hours of administration, PJP60-Ia at three concentrations (80, 160, and 320 μg / mL) downregulated NO secretion, demonstrating significant differences compared to the LPS model group (P < 0.05). At 24, 48, and 72 hours of administration, PJP60-Ia at three concentrations (80, 160, and 320 μg / mL) showed highly significant differences compared to the LPS model group (P < 0.01). These results suggest that PJP60-Ia can alleviate inflammatory responses by inhibiting excessive NO release.

[0220] Table 7 NO secretion levels in RAW264.7 cells in each group (n=3)

[0221]

[0222]

[0223] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0224] (3) Effect of PJP60-Ia on LPS-induced ROS production in RAW264.7 cells

[0225] After macrophages phagocytose pathogens, they produce large amounts of endogenous ROS, which, coupled to signal transduction pathways, regulate inflammatory gene expression. In this study, the fluorescent probe DCFH-DA was used to monitor ROS levels. This probe penetrates the cell membrane and enters the cell interior, where it is hydrolyzed by intracellular esterases to produce DCFH. DCFH, under the action of intracellular ROS, then generates fluorescent DCF. Flow cytometry was used to measure intracellular fluorescence intensity to assess ROS levels. The results showed that, compared with the control group, the mean ROS fluorescence intensity in RAW264.7 cells increased from 46.9% to 85.3% after LPS treatment. Treatment with PJP60-Ia at three concentrations (80, 160, and 320 μg / mL) decreased the fluorescence intensity to 77.0%, 65.4%, and 53.6%, respectively, in a dose-dependent manner. These results suggest that PJP60-Ia can alleviate inflammatory responses by inhibiting excessive ROS production.

[0226] (4) Effect of PJP60-Ia on LPS-induced Ca2+ in RAW264.7 cells 2+ Effect of content

[0227] LPS induces Ca2+ on the surface of macrophage membrane 2+ The channel opens, activates inflammation-related signaling pathways, and stimulates the release of pro-inflammatory factors. 2+ ]i can be used as an indicator to measure the degree of cellular inflammatory response. After entering the cell, Fluo-3 / AM is hydrolyzed by endogenous esterases into Fluo-3, which reacts with free Ca in the cytoplasm. 2+ After binding, fluorescence is generated, and the intensity of the fluorescence can indicate [Ca 2+ In this study, the effect of PJP60-Ia on LPS-induced macrophage [Ca] was studied by using Fluo-3 / AM fluorescent probe to label cytoplasmic calcium ions and imaging with an inverted fluorescence microscope. 2+ ]i's regulatory effect. The experimental results are as follows Figure 12 As shown in the figure, compared with the control group, the fluorescence intensity of the LPS group was significantly increased (P<0.01), while the three concentrations of PJP60-Ia (80, 160, 320 μg / mL) reduced the fluorescence intensity in a dose-dependent manner, and the inhibitory effect was strongest when the administration concentration reached 320 μg / mL (P<0.01). The above results indicate that PJP60-Ia may reduce intracellular Ca 2+ Content to suppress inflammation.

[0228] (5) Effect of PJP60-Ia on LPS-induced TNF-α, IL-10, IL-1β, and IL-4 mRNA expression in RAW264.7 cells

[0229] Under normal physiological conditions, the levels of anti-inflammatory factors and pro-inflammatory factors secreted by macrophages tend to be in dynamic balance, maintaining the homeostasis of the immune system. When macrophages are stimulated by the external environment, pro-inflammatory factors are expressed in large quantities to mediate the development of inflammatory reactions. Therefore, changes in the release of pro-inflammatory and anti-inflammatory factors can be used to test the anti-inflammatory activity of drugs. In order to determine whether PJP60-Ia can regulate the balance of inflammatory cytokines, this experiment detected the levels of pro-inflammatory factors and two anti-inflammatory factors. The melting curve and amplification curve of the target gene have good specificity and meet the experimental requirements. The RT-qPCR results are shown in Table 8. In the detection of pro-inflammatory factor genes, the expression of TNF-α and IL-1β mRNA in the LPS-induced group was significantly increased compared with the control group (P<0.01). Compared with the LPS-induced group, 80 μg / mL of PJP60-Ia significantly downregulated the expression of TNF-α and IL-1β mRNA (P < 0.05), while 160 μg / mL and 320 μg / mL of PJP60-Ia extremely significantly downregulated the expression of TNF-α and IL-1β mRNA (P < 0.01). In the detection of anti-inflammatory gene genes, the LPS-treated group extremely significantly downregulated the expression of IL-4 and IL-10 mRNA compared with the control group (P < 0.01). Compared with the LPS-induced group, 80 μg / mL of PJP60-Ia significantly upregulated the expression of IL-4 and IL-10 mRNA (P < 0.05). PJP60-Ia at 160 μg / mL and 320 μg / mL extremely significantly upregulated the expression of IL-4 and IL-10 mRNA (P < 0.01).

[0230] Table 8 Expression levels of TNF-α, IL-1β, IL-4, and IL-10 mRNA in RAW264.7 cells in each group (n=3)

[0231]

[0232] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0233] (6) Effect of PJP60-Ia on the expression of polarization markers CD86 and CD163

[0234] Macrophages are divided into two phenotypes, M1 and M2, based on their functional differences after polarization. Regulating the ratio of M1 and M2 cells can control the progression of the inflammatory response. CD86 and CD163 are membrane surface proteins specific to M1 and M2 macrophages, respectively. Therefore, in this study, we measured the levels of these two polarization markers to assess whether PJP60-Ia could exert anti-inflammatory effects by regulating macrophage differentiation. Western blot results are shown in Table 9. Compared with the control group, the expression of CD86, a marker for M1 macrophages, was significantly increased in the LPS-induced group (P < 0.01), while the expression of CD163, a marker for M2 macrophages, was significantly decreased (P < 0.01). Compared with the LPS-induced group, 80 μg / mL PJP60-Ia had no significant effect on CD86 protein expression. However, 160 μg / mL and 320 μg / mL PJP60-Ia significantly downregulated CD86 protein expression in a dose-dependent manner (P < 0.01). Compared with the LPS-induced group, 80 μg / mL, 160 μg / mL, and 320 μg / mL all significantly upregulated CD163 protein expression (P<0.01). This suggests that PJP60-Ia can promote the conversion of M1 macrophages to M2 macrophages and inhibit LPS-induced inflammation.

[0235] Table 9 Expression levels of CD86 and CD163 proteins in RAW264.7 cells in each group (n=3)

[0236]

[0237] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0238] (7) Effect of PJP60-Ia polysaccharide on the TLR2 / 4-NF-κB pathway in RAW264.7 macrophages induced by LPS

[0239] ①Effect of PJP60-Ia on LPS-induced TLR2 and TLR4 protein expression in RAW264.7 cells

[0240] LPS binds to the pattern recognition receptors TLR2 / 4 on the surface of macrophages, activating inflammatory responses. To investigate whether PJP60-Ia could modulate TLR2 / 4-mediated inflammatory signaling pathways, we first measured the protein expression levels of these two receptors. Western blot results, shown in Table 10, show that LPS stimulation increased the expression levels of TLR2 and TLR4 proteins, with extremely significant differences compared to the control group (P < 0.01). Compared with the LPS-induced group, 80 μg / mL of PJP60-Ia had no effect on TLR2 protein expression but significantly downregulated TLR4 protein expression (P < 0.05). Both 160 μg / mL and 320 μg / mL of PJP60-Ia significantly downregulated TLR2 and TLR4 protein expression (P < 0.01). This suggests that PJP60-Ia can compete with LPS for TLR2 and TLR4 receptors, regulating inflammatory responses.

[0241] Table 10 Expression levels of TLR2 and TLR4 proteins in RAW264.7 cells in each group (n=3)

[0242]

[0243] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0244] ②Effect of PJP60-Ia on the expression of key upstream proteins of the MyD88-dependent pathway in LPS-induced RAW264.7 cells

[0245] (a) Effect of PJP60-Ia on MyD88 protein expression

[0246] After TLR2 / 4 specifically binds to its ligand, it connects to the myeloid differentiation factor MYD88 through the TIR region, initiating a MYD88-dependent pathway, activating the relevant inflammatory factor receptor kinase in the cytoplasm, and inducing an inflammatory signaling cascade. This experiment investigated whether PJP60-Ia regulates the adaptor protein MYD88 in this pathway and tested its protein expression level. The Western blot results are shown in Table 11. The expression level of MyD88 protein increased after LPS stimulation, with a significant difference compared to the control group (P < 0.01). Compared with the LPS-induced group, all PJP60-Ia-treated groups significantly reduced MyD88 protein expression (P < 0.01), and the decrease was dose-dependent.

[0247] Table 11 MyD88 protein expression levels in RAW264.7 cells in each group (n=3)

[0248]

[0249] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0250] (b) Effects of PJP60-Ia on the expression of IRAK4, IRAK1 and their phosphorylated proteins

[0251] IRAK4 and IRAK1 are members of the interleukin-1 (IL-1) receptor-associated kinase IRAK family. They are activated by phosphorylation of MyD88 and positively regulate TLR-mediated signaling pathways. Western blot results, as shown in Table 12, showed that LPS stimulation increased the expression levels of IRAK4 and IRAK1, as well as their phosphorylated proteins, significantly different from those in the control group (P < 0.01). Compared with the LPS-induced group, 80 μg / mL of PJP60-Ia extremely significantly downregulated the expression of p-IRAK1 protein, and had no significant effect on the expression of IRAK4, p-IRAK4, and IRAK1 proteins (P>0.05); 160 μg / mL of PJP60-Ia significantly downregulated the expression of IRAK4 protein (P<0.05), and extremely significantly downregulated the expression of p-IRAK4, IRAK1, and p-IRAK1 proteins (P<0.01); 320 μg / mL of PJP60-Ia extremely significantly downregulated the expression of IRAK4 and IRAK1 and their phosphorylated proteins (P<0.01). Except for 80 μg / mL, 160 μg / mL and 320 μg / mL of PJP60-Ia had an inhibitory effect on the expression of p-IRAK4 / IRAK4, while all three concentrations of PJP60-Ia exerted an inhibitory effect on p-IRAK1 / IRAK1.

[0252] Table 12 Expression levels of IRAK4, IRAK1 and their phosphorylated proteins in RAW264.7 cells of each group (n=3)

[0253]

[0254] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0255] (c) Effect of PJP60-Ia on TRAF6 protein expression

[0256] Phosphorylated IRAK1 and IRAK4 dissociate from MYD88, activating the downstream protein TRAF6. TRAF6 is a ubiquitin-protein ligase that promotes the activation of downstream signaling pathways. Western blot results, as shown in Table 13, show that TRAF6 protein expression levels increased after LPS stimulation, with a highly significant difference compared to the control group (P < 0.01). Compared with the LPS-induced group, all PJP60-Ia treatment groups (80, 160, and 320 μg / mL) significantly downregulated TRAF6 protein expression (P < 0.01), with a dose-dependent decrease.

[0257] Table 13 Expression levels of TRAF6 protein in RAW264.7 cells in each group (n=3)

[0258]

[0259] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0260] (d) Effects of PJP60-Ia on the expression of TAK1 and its phosphorylated proteins

[0261] Ubiquitinated TRAF6 recruits and activates transforming growth factor-β-activated kinase TAK1, further activating the downstream inhibitor of NF-κB kinase, promoting NF-κB pathway signaling. Western blot results, as shown in Table 14, show that LPS stimulation increased the expression levels of TAK1 and its phosphorylated protein, with a highly significant difference compared with the control group (P < 0.01). Compared with the LPS-induced group, 80 μg / mL of PJP60-Ia had no significant effect on the expression of TAK1 and p-TAK1 proteins (P > 0.05). However, both 160 μg / mL and 320 μg / mL of PJP60-Ia significantly downregulated the expression of TAK1 and p-TAK1 proteins (P < 0.01). Except for the 80 μg / mL dose, 160 μg / mL and 320 μg / mL of PJP60-Ia had a highly significant inhibitory effect on the expression of p-TAK1 / TAK1 (P < 0.01). Judging from the above experimental results, PJP60-Ia can inhibit the transduction of TLR2 / 4-MyD88-IRAK4 / IRAK1-TRAF6-TAK1 signaling pathway in RAW264.7 cells induced by LPS.

[0262] Table 14 Expression levels of TAK1 and its phosphorylated proteins in RAW264.7 cells in each group (n=3)

[0263]

[0264] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0265] ③Effect of PJP60-Ia on the expression of NF-κB signaling pathway-related proteins in LPS-induced RAW264.7 cells

[0266] (a) Effects of PJP60-Ia on the expression of IKKα / β and its phosphorylated proteins

[0267] TLR2 / 4 can regulate immune responses by activating NF-κB through a MYD88-dependent pathway. p50 and p52, members of the NF-κB family, induce the expression of multiple cytokines, including TNF-α, IL-6, and IL-1β. Therefore, this study examined key NF-κB pathway proteins and phosphorylated proteins to assess whether PJP60-Ia could regulate NF-κB pathway conduction and inhibit inflammatory responses. The IKK protein kinase complex is central to the NF-κB pathway, and activated IKKs are crucial for NF-κB activation. Western blot results, as shown in Table 15, showed no statistically significant differences in IKKα / β expression among the groups (P>0.05). However, LPS treatment promoted the phosphorylation of IKKα / β proteins in RAW264.7 cells, with a highly significant difference compared to the control group (P<0.01). Compared with the LPS-induced group, 80 μg / mL PJP60-Ia had no significant effect on p-IKKβ (P>0.05), but significantly reduced the expression level of p-IKKα (P<0.01). Both 160 μg / mL and 320 μg / mL PJP60-Ia significantly downregulated the expression of p-IKKα and p-IKKβ proteins (P<0.01). In addition, all three concentrations of PJP60-Ia inhibited the expression of p-IKKα / IKKα / β and p-IKKβ / IKKα / β.

[0268] Table 15 Expression levels of IKKα / β and its phosphorylated proteins in RAW264.7 cells of each group (n=3)

[0269]

[0270] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0271] (b) Effects of PJP60-Ia on the expression of IκBα, NF-κB and their phosphorylated proteins

[0272] In unstimulated cells, the RHD domain of NF-κB binds to the inhibitory protein IκBα and remains in a quiescent state. The IKKs complex phosphorylates IκBα, which then undergoes ubiquitination catalyzed by the E3 ubiquitin ligase. Ultimately, NF-κB is recognized by the 26S proteasome and rapidly degraded, releasing NF-κB. Activated NF-κB enters the cell nucleus and initiates the transcription of inflammation-related target genes. Western blot results are shown in Table 16. In the IκBα protein assay, LPS treatment significantly downregulated IκBα protein expression and upregulated its phosphorylated protein expression compared to the control group (P < 0.01). Compared with the LPS-induced group, 80 μg / mL PJP60-Ia had no significant effect on IкBα protein expression (P>0.05), but significantly downregulated p-IкBα protein expression (P<0.05). PJP60-Ia at 160 μg / mL and 320 μg / mL significantly upregulated IкBα protein expression and very significantly downregulated p-IкBα protein expression (P<0.01). All three concentrations of PJP60-Ia inhibited p-IкBα / IκBα expression. In the NF-кB protein assay, NF-кB expression did not differ significantly among the groups (P>0.05). However, LPS treatment promoted NF-кB phosphorylation, with a highly significant difference compared with the control group (P<0.01). Compared with the LPS-induced group, PJP60-Ia at 80, 160, and 320 μg / mL significantly downregulated p-NF-кB protein expression, and all three concentrations of PJP60-Ia inhibited p-NF-кB / NF-κB expression. These experimental results indicate that PJP60-Ia can inhibit LPS-induced phosphorylation of the IKKα / β-IкBα-NF-кB pathway in RAW264.7 cells, reduce NF-кB nuclear translocation, and inhibit inflammatory factor DNA transcription.

[0273] Table 16 Expression levels of IκBα, NF-κB and their phosphorylated proteins in RAW264.7 cells of each group (n=3)

[0274]

[0275] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0276] (8) Effect of PJP60-Ia polysaccharide on the cGAS-STING pathway in RAW264.7 macrophages induced by LPS

[0277] ①Effect of PJP60-Ia on cGAS and STING protein expression

[0278] To elucidate whether PJP60-Ia regulates LPS-induced inflammation by inhibiting cGAS-STING activation, this study investigated key proteins in this pathway. Studies have shown that LPS activates the NF-κB pathway through TLR2 / 4 receptors, inducing the release of inflammatory factors and damaging mitochondrial DNA (mtDNA). The cytoplasmic DNA sensor cGAS is a cyclic GMP-AMP synthase that recognizes abnormal cytoplasmic DNA and catalyzes the chemical reaction between ATP and GTP to synthesize the cyclic dinucleotide cGAMP. cGAMP is a messenger of the innate immune system, carrying signals to the endoplasmic reticulum where it binds to the transmembrane adaptor protein STING, forming an active STING dimer and activating the type I interferon signaling pathway. Western blot results, shown in Table 17, show that LPS stimulation increases the expression levels of cGAS and STING proteins, with a highly significant difference compared to the control group (P < 0.01). Compared with the LPS-induced group, 80 μg / mL of PJP60-Ia had no significant effect on the expression of cGAS protein (P>0.05), but could significantly downregulate the expression of STING protein (P<0.01); 160 μg / mL and 320 μg / mL of PJP60-Ia could both significantly downregulate the expression of cGAS and STING proteins (P<0.01).

[0279] Table 17 Expression levels of cGAS and STING proteins in RAW264.7 cells in each group (n=3)

[0280]

[0281]

[0282] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0283] ②Effects of PJP60-Ia on the expression of TBK1, IRF3 and their phosphorylated proteins

[0284] The carboxyl terminus of the activated STING dimer can bind and phosphorylate TBK1, activating it and translocating it from the endoplasmic reticulum to the Golgi apparatus. Activated TBK1 prompts the STING C-terminus to dock with the type I interferon regulatory factor IRF3 to form an active dimer, which then enters the cell nucleus and initiates transcription of target genes, including type I interferons, mediating inflammation and autoimmune diseases. Western blot results, as shown in Table 18, show no statistically significant differences in the expression of TBK1 and IRF3 among the groups (P>0.05). However, LPS stimulation increased the expression levels of p-TBK1 and p-IRF3 proteins, with highly significant differences compared to the control group (P<0.01). Compared with the LPS-induced group, 80 μg / mL of PJP60-Ia had no significant effect on the expression of p-TBK1 and p-IRF3 proteins (P>0.05). However, both 160 μg / mL and 320 μg / mL of PJP60-Ia significantly downregulated the expression of p-TBK1 and p-IRF3 proteins (P<0.01). Furthermore, all three concentrations of PJP60-Ia exhibited inhibitory effects on the p-TBK1 / TBK1 ratio and p-IRF3 / IRF3 ratio. These results suggest that PJP60-Ia can inhibit LPS-induced cGAS-STING signaling in RAW264.7 cells and suppress the release of inflammatory factors mediated by the TBK1-IRF3 pathway.

[0285] Table 18 Expression levels of TBK1, IRF3 and their phosphorylated proteins in RAW264.7 cells in each group (n=3)

[0286]

[0287] Note: *P<0.05, **P<0.01 vs. control group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0288] The above results show that the polysaccharide PJP60-Ia from Psoralea corylifolia has good anti-inflammatory effect.

[0289] In summary, the present invention clarifies the structural characteristics of Beiqinglongyi polysaccharide from the extraction, separation and purification to the use of analytical methods, and performs anti-inflammatory activity detection on the prepared samples, laying the foundation for subsequent research on the structure-activity relationship of Beiqinglongyi polysaccharide.

[0290] The present invention adopts a Flash extraction method combined with ethanol graded precipitation to extract and separate the polysaccharide of the iris of the Chinese iris, adopts ethanol (20%, 40%, 60%) to carry out graded precipitation, selects 60% of the precipitate for further research, selects DEAE-52 anion exchange column chromatography, uses 0-0.5 mol / L NaCl solution as eluent for preliminary separation and purification, and then selects Sephadex G-50 gel chromatography column for further purification, finally obtaining the polysaccharide PJP60-Ia of the iris of the Chinese iris, with a sugar content of 98.85%±0.27%. The results of physical and chemical property determination further show that the polysaccharide PJP60-Ia of the iris of the Chinese iris of the Chinese iris is a carbohydrate substance and does not contain components such as amino acids, proteins, starch, and reducing sugars.

[0291] The present invention studies the polysaccharide PJP60-Ia from the capillaries of northern blue dragonfly through FT-IR, HPLC and NMR. The results show that the polysaccharide PJP60-Ia from the capillaries of northern blue dragonfly is a homogeneous polysaccharide with high purity and a molecular weight of 11250 Da. The polysaccharide is mainly composed of Rha, Glc and Fru, and contains α and β glycosidic bonds. According to relevant literature, it is inferred that the polysaccharide PJP60-Ia from the capillaries of northern blue dragonfly may be composed of α-D-Fruf-(2→, 3,4)-β-Rha-(1→, α-D-Glcp).

[0292] Polysaccharides are important bioactive macromolecules. Their biological activity is not only related to their primary structure, but also directly affected by their higher-order structure. However, little research has been done on the higher-order structure of polysaccharides.

[0293] The present invention uses Congo red test, XRD, SEM, AFM and other means to detect the high-level structure of the polysaccharide PJP60-Ia of the plant. It is found that the polysaccharide PJP60-Ia of the plant does not have a triple helical structure, is an amorphous substance with a semi-crystalline structure, has a rolled film-like surface, is uneven, has holes, has a membrane structure and a sheet structure, and has irregular protrusions and clumps. The average particle size is 211.8 nm, the PDI value is 0.25, the average potential is -3.5 mV, the molecular weight distribution is well uniform, but the plant is unstable in a solution system and is easily aggregated.

[0294] Beiqinglongyi polysaccharide PJP60-Ia has anti-inflammatory effects. LPS was used to induce mouse RAW264.7 macrophages to establish an in vitro inflammation model. A certain concentration of PJP60-Ia can inhibit the proliferation of inflammatory cells and reduce NO, ROS and Ca 2+In addition, PJP60-Ia modulates the M1 / M2 polarization ratio of macrophages, downregulating the mRNA and protein expression of the M1 marker CD86 and upregulating the mRNA and protein expression of the M2 marker CD163. It also reduces the secretion and mRNA expression of proinflammatory cytokines TNF-α and IL-1β, while increasing the secretion and mRNA expression of anti-inflammatory cytokines IL-4 and IL-10. PJP60-Ia exerts anti-inflammatory effects through TLR2- and TLR4-mediated pathways, reducing TLR2 / 4 receptor protein expression in LPS-induced RAW264.7 cells. It also inhibits the expression of key upstream proteins of the MyD88-dependent signaling pathway, including MyD88, IRAK4, p-IRAK4, IRAK1, p-IRAK1, TRAF6, TAK1, and p-TAK1. It also suppresses phosphorylation and activation of the IKKα / β-IκBα-NF-κB pathway, reduces NF-κB nuclear translocation, and suppresses the transcription of inflammatory factors. PJP60-Ia inhibits LPS-induced cGAS-STING signaling in RAW264.7 cells and suppresses the release of inflammatory factors mediated by the TBK1-IRF3 pathway. PJP60-Ia is a promising polysaccharide with anti-inflammatory activity and could be used in the development of anti-inflammatory preparations.

[0295] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing PJP60-Ia polysaccharide from Psoralea corylifolia, characterized in that: The following steps are involved: The supernatant of the Northern Qinglongyi was extracted by graded alcohol precipitation, and the precipitate after precipitation with 60% ethanol was collected. The stepwise alcohol precipitation comprises the following steps: centrifuging and filtering the supernatant of the iris var. chinensis to remove the residue, collecting the supernatant, and concentrating the supernatant under reduced pressure to 1 / 3 of the original volume; then adding 95% ethanol to the concentrate while stirring, adjusting the final ethanol concentration to 20%, standing overnight, and collecting precipitate I and supernatant I respectively; according to the above steps, adding ethanol to the supernatant I to adjust the final ethanol concentration to 40% to obtain supernatant II and precipitate II, and freeze-drying the precipitate II; then, according to the above steps, adding ethanol to the supernatant II to adjust the final ethanol concentration to 60% to obtain supernatant III and precipitate III, and freeze-drying the precipitate III to obtain the iris var. chinensis polysaccharide PJP60; The method also includes the steps of decolorization and separation and purification; AB-8 macroporous resin is used for decolorization; separation and purification include separation and purification using a DEAE-52 anion exchange chromatography column and a Sephadex G-50 gel chromatography column; The separation and purification using a DEAE-52 anion exchange chromatography column comprises the following steps: activating DEAE-52 cellulose; pre-treating the DEAE-52 anion exchange chromatography column; preparing a solution of P. chinensis polysaccharide and loading the sample; preparing an eluent and collecting the sample, using a 0-0.5 mol / L NaCl solution as the eluent at a flow rate of 1 mL / min, collecting the eluent obtained when the NaCl solution concentration is 0, and freeze-drying the eluent to obtain P. chinensis polysaccharide PJP60-I; Sephadex G-50 gel chromatography column separation and purification includes the following steps: activating Sephadex G-50 gel; pretreating the Sephadex G-50 gel chromatography column; preparing PJP60-I solution and loading the sample, using deionized water as the eluent, controlling the flow rate to 0.3 mL / min, collecting the eluent, collecting 1 mL in each tube, collecting samples from tubes 80 to 110, and freeze-drying them to obtain the northern blueberry polysaccharide PJP60-Ia.

2. The preparation method according to claim 1, characterized in that The preparation method of the supernatant of the northern blue dragon cap comprises the following steps: performing flash extraction on the liquid of the northern blue dragon cap.

3. The preparation method according to claim 2, characterized in that: The parameters of the Flash extraction included: 5000 r / min, 30 s / time.

4. Use of the polysaccharide PJP60-Ia from the plant stem of the Chinese yarrow genus, alone or as one of its components, in the preparation of an anti-inflammatory preparation, characterized in that: The Psoralea corylifolia polysaccharide PJP60-Ia is prepared by the preparation method according to any one of claims 1 to 3.

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