Preparation method and application of polysaccharide PJP80-Ia from north green dragon skin

Abstract

The present application relates to the preparation method and application of polysaccharide PJP80-Ia from north green dragon skin, and the polysaccharide PJP80-Ia is extracted from north green dragon skin by using the Soxhlet extraction technology combined with fractional alcohol precipitation, decolorized by AB-8 macroporous resin, separated and purified by DEAE-52 anion exchange chromatography column, further purified by Sephadex G-50 gel chromatography column, and finally obtained as a single component of north green dragon skin polysaccharide PJP80-Ia with a molecular weight of 11630 Da and composed of mannose and glucose. The north green dragon skin polysaccharide PJP80-Ia provided by the present application has the advantages of immunocompetence, antitumor activity, antioxidant activity, high purity and the like.

Description

Technical Field

[0001] This invention belongs to the fields of traditional Chinese medicine and food technology, and in particular relates to the preparation method and application of PJP80-Ia polysaccharide from the skin of *Ilex cornuta*. Background Technology

[0002] Pericarpium Juglandis is the immature exocarp of *Jugland mandshurica* Maxim. and *Juglans regia* L., mainly distributed in Northeast Asia. It was first recorded in the *Kaibao Materia Medica*, where it was called "Jugland Green Peel." The *Chinese Materia Medica* and the *Dictionary of Traditional Chinese Medicine* describe it as pungent and bitter in taste; neutral to slightly cold in nature; toxic; and entering the liver, lung, and stomach meridians. The name "Qinglongyi" first appeared in the *Shandong Handbook of Traditional Chinese Medicine*, and has been used ever since. To distinguish it from the officially designated *Qinglongyi*, the Heilongjiang Provincial Drug Administration named it "Bei Qinglongyi," also known as "Jugland Green Peel" or "Jugland Shell," which is the immature exocarp of wild mountain walnuts. As a genuine medicinal material of Heilongjiang Province, "Bei Qinglongyi" is included in the *Heilongjiang Provincial Standard for Traditional Chinese Medicine*. It is a wrinkled, hemispherical or irregularly shaped piece, with edges often curving inwards, 3-6 cm in diameter and about 0.7 cm thick. The outer surface is dark brown or dark yellow, relatively smooth, densely covered with yellow spots, and has a fruit stalk scar at one end. The inner surface is dark yellow, rough, and covered with longitudinal vascular bundles. It is brittle and easily broken. It has a faint odor, a slightly bitter and astringent taste, and a gritty feel when chewed. Immature green walnut husks are harvested from June to August. After collection, they are washed, the kernels are removed, and used immediately. The raw material is then used, impurities removed, and promptly sun-dried or dried at low temperature for preservation. It is a traditional folk medicine in my country. According to the *Jiuji Fang* (Emergency Prescriptions), the husk of *Hemiberlesia jujuba* can be used to make tea or wine for pain relief. Modern pharmacology shows that its juice, when applied externally, is effective in treating headaches caused by internal heat and neurodermatitis. my country has the world's largest walnut production and planting area. The green walnut husk is alkaline, and its natural degradation has a significant impact on water and soil. Therefore, how to effectively utilize walnut husk resources has become an urgent problem to be solved.

[0003] The main chemical components of *Cynanchum paniculatum* include quinones (such as naphthoquinones, anthraquinones, naphthoketones, and tetrahydronaphthoketones), flavonoids, phenols, diarylheptanes, coumarins, lignans, phenylpropanoids, triterpenoids, organic acids, and polysaccharides, which have good effects such as clearing heat, detoxifying, reducing swelling, relieving pain, stopping diarrhea, and improving eyesight.

[0004] Polysaccharides are widely present substances in organisms that maintain life activities. They are a class of natural high-molecular-weight polymers formed by the dehydration and polymerization of aldoses or ketoses linked by glycosidic bonds, with the molecular formula (C6H2O). 10Based on their monosaccharide composition, polysaccharides are classified into homopolysaccharides and heteropolysaccharides. The former consists of the same monosaccharide, while the latter consists of two or more monosaccharides. Based on their origin, polysaccharides can be divided into three types: plant polysaccharides, animal polysaccharides, and microbial polysaccharides. Currently isolated active polysaccharides contain a significant proportion of traditional Chinese medicine polysaccharides. Their monosaccharide molecules include glucose (Glc), mannose (Man), galacturonic acid (Gal A), glucuronic acid (Glc A), fucose (Fuc), and ribose (Rib). Traditional Chinese medicine polysaccharides exhibit different molecular structures based on the type, sequence, main chain structure, presence or absence of branches, and relative molecular mass. These different structures determine the properties of the polysaccharides, thus affecting their biological activity, and consequently, their pharmacological activities and mechanisms of action also vary.

[0005] *Ailanthus urinaria* has medicinal value, but current research is weak, hindering its further development and utilization. Traditional hot water extraction methods for polysaccharides involve large solvent consumption, long extraction times, and low extraction rates, making them inefficient and energy-saving. Impurities in polysaccharides can affect their structure and activity. Preliminary extractions of *Ailanthus urinaria* polysaccharides typically contain a large amount of impurities such as proteins and inorganic salts, necessitating their removal. Common methods include enzymatic methods, TCA methods, and the Sevag method. While enzymatic protein removal is relatively mild, it is expensive and unsuitable for large-scale applications. The Sevag method works by denaturing free proteins in the polysaccharide, forming insoluble substances without damaging the polysaccharide components, thus achieving separation. Furthermore, most natural polysaccharides contain pigments, which may affect their subsequent structure and biological activity, requiring removal. Chemical methods such as H2O2 oxidation for impurity removal may damage the polysaccharide structure. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for preparing PJP80-Ia polysaccharide from *Gynostemma pentaphyllum* and its application. The polysaccharide obtained by selecting a suitable preparation method has the advantages of high purity, and also has immune activity, anti-tumor activity, and antioxidant activity.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] This invention provides a method for preparing polysaccharide PJP80-Ia from *Gynostemma pentaphyllum*, comprising the following steps: extracting the supernatant from *Gynostemma pentaphyllum* using the Flash extraction method, preparing polysaccharide from *Gynostemma pentaphyllum* using a fractional alcohol precipitation method, collecting the precipitate after alcohol precipitation with 80% ethanol concentration, and freeze-drying it to obtain polysaccharide PJP80 from *Gynostemma pentaphyllum*.

[0009] The beneficial effects of adopting the above scheme include: the present invention tested the products after alcohol precipitation with different ethanol concentrations and found that the products obtained after alcohol precipitation with 80% ethanol concentration had higher polysaccharide content and better immune activity.

[0010] Furthermore, the preparation method of the extract of *Leymus chinensis* includes the following steps: flash extraction of the extract of *Leymus chinensis*.

[0011] Furthermore, the liquid for preparing the *Gynostemma pentaphyllum* powder consists of *Gynostemma pentaphyllum* powder and water, with a powder-to-liquid ratio of 1g:30mL and a water temperature of 90℃.

[0012] Furthermore, the parameters for the Flash extraction include: 5000 r / min, 30 s / extraction, and 2 extractions.

[0013] The advantages of adopting the above scheme include: short extraction time, high efficiency, convenient operation, and energy saving.

[0014] Further, the fractional alcohol precipitation includes the following steps: Filter the supernatant of *Agrostis chinensis* and collect the filtrate; concentrate the filtrate under reduced pressure to 1 / 3 of its original volume, add 95% ethanol, stir rapidly to achieve an ethanol concentration of 20%, and let stand overnight to obtain precipitate I and supernatant I; repeat the above steps, adding ethanol to supernatant I to achieve a final ethanol concentration of 40%, and let stand overnight to obtain precipitate II and supernatant II; repeat the above steps, adding ethanol to supernatant II to achieve a final ethanol concentration of 60%, and let stand overnight to obtain precipitate III and supernatant III; repeat the above steps, adding ethanol to supernatant III to achieve a final ethanol concentration of 80%, and let stand overnight to obtain precipitate IV and supernatant IV; wash precipitate IV with anhydrous ethanol and freeze-dry.

[0015] The beneficial effects of adopting the above scheme include: the polysaccharide content of the polysaccharide obtained by the above method is higher.

[0016] Furthermore, it also includes a decolorization step.

[0017] Further, decolorization was performed using AB-8 macroporous resin. The steps are as follows: Weigh the crude polysaccharide from *Cynanchum paniculatum* obtained after fractional alcohol precipitation, dissolve it in distilled water, add AB-8 macroporous resin, stir for 2 hours for decolorization, vacuum filter, and freeze-dry the filtrate. The ratio of crude polysaccharide from *Cynanchum paniculatum*, distilled water, and AB-8 macroporous resin is 5g:100mL:50g.

[0018] The beneficial effects of using the above scheme include: the above method can remove impurities such as pigments from the extract.

[0019] Furthermore, after decolorization, the process also includes separation and purification steps using a DEAE-52 anion exchange chromatography column and a Sephadex G-50 gel chromatography column.

[0020] Furthermore, the separation and purification using the DEAE-52 anion exchange chromatography column includes the following steps: activating DEAE-52 cellulose; packing the column and pretreating the DEAE-52 anion exchange chromatography column; preparing the *Cynanchum paniculatum* polysaccharide solution and loading the sample; preparing the eluent and collecting the sample, using 0-0.5 mol / L NaCl solution as the eluent, 200 mL for each concentration, a flow rate of 1 mL / min, and collecting the eluent at 2 mL / tube. The eluent obtained when the NaCl solution concentration is 0 is collected and freeze-dried to obtain *Cynanchum paniculatum* polysaccharide PJP80-I.

[0021] Furthermore, the Sephadex G-50 gel chromatography column separation and purification includes the following steps: activating the Sephadex G-50 gel; packing the column and pretreating the Sephadex G-50 gel chromatography column; preparing the PJP80-I solution and loading the sample, using deionized water as the eluent at a flow rate of 1 mL / 3 min, collecting the elution peak, and freeze-drying it to obtain the polysaccharide PJP80-Ia from the northern cypress bark.

[0022] The beneficial effects of adopting the above scheme include: the above method can further increase the polysaccharide content in the extract. The content of polysaccharide PJP80-Ia in *Cyclocarya paliurus* can reach 97.51±0.19%.

[0023] The present invention provides the application of PJP80-Ia polysaccharide from *Gynostemma pentaphyllum* in any one or more of (1) to (5), wherein PJP80-Ia polysaccharide from *Gynostemma pentaphyllum* is prepared by the above preparation method;

[0024] (1) Used alone or as one of the components in the preparation of immunomodulators;

[0025] (2) Used alone or as one of the components in the preparation of antitumor agents

[0026] (3) Used alone as a new food ingredient

[0027] (4) Used alone or as one of the components in the preparation of health food products with immune-modulating functions;

[0028] (5) Used alone or as one of the components in the preparation of health foods with antioxidant functions.

[0029] The beneficial effects of the above-mentioned approach include: Testing showed that PJP80-Ia, a polysaccharide from *Gynostemma pentaphyllum*, inhibited human liver cancer cells HepG2 and SMMC-7721, as well as human breast cancer cells MCF-7, and can be used for the prevention and treatment of liver cancer and breast cancer; PJP80-Ia can scavenge DPPH and ABTS free radicals and has the ability to reduce Fe... 3+ -TPTZ possesses antioxidant properties; PJP80-Ia, a polysaccharide from *Pterocarya stenoptera*, can activate mouse RAW264.7 macrophages, stimulate cell proliferation, enhance the phagocytic function of RAW264.7 macrophages, induce the secretion of NO and ROS from RAW264.7 cells, increase the expression levels of TNF-α, IL-6, IL-1β, and IFN-β mRNA, and enhance the immune activity of macrophages. It can be used to prepare or as a product for enhancing immune activity, anti-tumor activity, or anti-oxidation, including pharmaceuticals, foods, and health foods. Attached Figure Description

[0030] Figure 1 Elution curve of DEAE-52 crude polysaccharide from *Cyclocarya paliurus*.

[0031] Figure 2 The results of the antitumor activity detection of PJP80-Ia in Example 3 are as follows: A shows the detection results of the inhibitory effect of PJP80-Ia on HepG2, compared with the blank control group; *: significant P<0.05; **: highly significant P<0.01. B shows the detection results of the inhibitory effect of PJP80-Ia on SMMC-7721, compared with the blank control group; *: significant P<0.05; **: highly significant P<0.01. C shows the detection results of the inhibitory effect of PJP80-Ia on MCF-7, compared with the blank control group; *: significant P<0.05; **: highly significant P<0.01. D represents the DPPH free radical scavenging ability of Trolox, crude polysaccharide of *Cynanchum paniculatum*, and polysaccharide PJP80-Ia of *Cynanchum paniculatum*; E represents the ABTS free radical scavenging ability of Trolox, crude polysaccharide of *Cynanchum paniculatum*, and polysaccharide PJP80-Ia of *Cynanchum paniculatum*; F represents the Fe... 3+ The result of the reducing ability.

[0032] Figure 3 The results of the effect of PJP80-Ia on the morphology of RAW264.7 macrophages were obtained by inverted microscopy (magnification 400×, n=3).

[0033] Figure 4 Results of flow cytometry analysis of the effect of PJP80-Ia on the cell cycle of RAW264.7 macrophages (n=3).

[0034] Figure 5 The results show the effect of PJP80-Ia on the cell cycle of RAW264.7 macrophages (n=3).

[0035] Figure 6 The results show the effect of PJP80-Ia on ROS secretion in mouse RAW264.7 cells (n=3).

[0036] Figure 7 The results show the effect of PJP80-Ia on the expression of IL-1β, IL-6, TNF-α, and IFN-β mRNA secreted by mouse RAW264.7 cells (n=6). Among them, compared with the control group, *: significant P<0.05; **: extremely significant P<0.01.

[0037] Figure 8 The results (n=3) show the effects of TLR2 inhibitor (C29) and TLR4 inhibitor (TAK-242) on the mRNA expression levels of cytokines IL-6, IL-1β, TNF-α, and IFN-β secreted by PJP80-Ia-induced macrophages RAW264.7. In the data, ns: not significant; *: significant compared to the control group, P<0.05; **: highly significant, P<0.01; ##: highly significant compared to the data without inhibitors in each group, P<0.01. Detailed Implementation

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

[0039] This invention uses dried *Leymus chinensis* leaves as raw material and obtains high-purity *Leymus chinensis* leaf polysaccharides through a series of physical and chemical methods. Furthermore, the primary and higher-order structures of the *Leymus chinensis* leaf polysaccharides are analyzed using instruments such as infrared spectroscopy, nuclear magnetic resonance spectroscopy, and scanning electron microscopy. This provides a scientific theoretical basis for structure-activity relationship studies and new product development of *Leymus chinensis* leaf polysaccharides and further verifies their activity. The invention includes the following:

[0040] (1) In this invention, crude polysaccharide PJP80 from *Cynanchum paniculatum* was prepared by flash extraction and gradient alcohol precipitation. After decolorization with AB-8 macroporous resin, polysaccharide PJP80-I from *Cynanchum paniculatum* was obtained using DEAE-52 cellulose. Further separation and purification were carried out using dextran gel G-50 to obtain polysaccharide PJP80-Ia from *Cynanchum paniculatum*.

[0041] (2) The primary and higher structures of PJP80-Ia polysaccharide from *Gynostemma pentaphyllum* were analyzed: The purity, relative molecular weight, monosaccharide composition, and glycosidic bond type of the purified *Gynostemma pentaphyllum* polysaccharide were analyzed by high performance liquid chromatography (HPLC), Fourier transform infrared chromatography (FTIR), Raman spectroscopy (Ramam), and nuclear magnetic resonance spectroscopy (NMR); The helical structure, thermal stability, particle size, charge properties, crystal characteristics, appearance morphology, and chain conformation of the purified *Gynostemma pentaphyllum* polysaccharide were analyzed by Congo red experiment, differential scanning calorimetry, particle size and potential determination, X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), and atomic force microscopy (AFM).

[0042] (3) Tumor Activity Analysis of PJP80-Ia Polysaccharide from *Gynostemma pentaphyllum*: The effect of purified *Gynostemma pentaphyllum* polysaccharide PJP80-Ia on the proliferation of human hepatocellular carcinoma cells HepG2, SMMC-7721, and human breast cancer cells MCF-7 was determined by the MTT assay; the in vitro antioxidant activity of *Gynostemma pentaphyllum* polysaccharide PJP80-Ia was determined by three antioxidant methods: DPPH, ABTS, and FRAP. *Gynostemma pentaphyllum* polysaccharide PJP80-Ia inhibited the proliferation of hepatocellular carcinoma cells HepG2, SMMC-7721, and MCF-7, and its scavenging rates of DPPH and ABTS free radicals were 79.5% and 65%, respectively. 3+ It has a reducing power of 1.49 mg / mL and strong antioxidant capacity.

[0043] (4) Immunological activity analysis of PJP80-Ia polysaccharide from *Gynostemma pentaphyllum*: PJP80-Ia polysaccharide from *Gynostemma pentaphyllum* can activate mouse RAW264.7 macrophages, stimulate cell proliferation, enhance the phagocytic function of mouse RAW264.7 macrophages, and induce the secretion of NO and ROS in RAW264.7 cells, and increase the mRNA expression of TNF-α, IL-6, IL-1β, and IFN-β; PJP80-Ia polysaccharide from *Gynostemma pentaphyllum* can further promote the signal transduction of related pathways through Toll-like receptors (TLR), pattern recognition receptors (Dectin-1), complement receptors (CR3), and mannose receptor family (MR) on the surface of macrophages, among which the expression of Toll-like receptors (TLR2 / 4) protein is more obvious; PJP80-Ia polysaccharide from *Gynostemma pentaphyllum*... P80-Ia can synergistically participate in cellular immune responses through the MyD88-dependent MAPKs signaling pathway and the NF-κB signaling pathway, promoting the release of cytokines such as TNF-α, IL-6, and IL-1β. PJP80-Ia polysaccharide from *Gynostemma pentaphyllum* can mediate the proliferation and maturation of immune cells through the TRIF-dependent signaling pathway, prompting macrophages to secrete IFN-β to exert cellular immune functions. TLR receptor antagonists can significantly reduce the effect of PJP80-Ia in promoting the secretion of NO, TNF-α, IL-6, IL-1β, and IFN-β by RAW264.7 cells, and reduce the expression levels of NF-κB signaling pathway-related proteins, demonstrating that PJP80-Ia mainly enhances macrophage immune activity by binding to TLR2 / 4 on the cell membrane surface.

[0044] The following is a description through specific embodiments. Unless otherwise specified, the experimental methods used in each embodiment are conventional experimental methods in the art. Unless otherwise specified, the materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, which can be obtained through commercial channels or prepared by conventional methods.

[0045] Experimental materials:

[0046] The dried pericarp of *Hemiberlesia lingua* (the product is a dry solid, hard and brittle, clean and odorless) was identified and provided by the Heilongjiang Academy of Traditional Chinese Medicine. RAW264.7 macrophages were donated by Harbin Medical University and are available to the public for non-commercial purposes only to replicate the embodiments described in this invention. HepG2 human liver cancer cells, SMMC-7721 human liver cancer cells, and MCF-7 human breast cancer cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0047] Experimental apparatus:

[0048] The following instruments were purchased: a flash extractor (model: SYKES-507) from Ningbo Bedell Telecommunications & Motor Co., Ltd.; a UV-Vis spectrophotometer (model: UV-5200PC) from Gangdong Technology Development Co., Ltd.; glass chromatography columns (models: 16mm and 26mm) from Jiangsu Hanbang Technology Co., Ltd.; an automatic collector (model: BS-160A) from Shanghai Kanghua Biochemical Instrument Manufacturing Plant; a high-performance liquid chromatograph (model: 2414) from Waters Technology Co., Ltd.; a Fourier transform infrared chromatograph (model: FTIR-650) from Tianjin Gangdong Technology Development Co., Ltd.; a Raman spectrometer (model: RA861) from Renishaw, UK; and a particle size analyzer (model: Litesizer). The following instruments were purchased: a -500 (model: Anton Paar (Shanghai) Trading Co., Ltd.); an X-ray diffractometer (model: D8, Advance), a 1H NMR spectrometer (model: 600M), and an atomic force microscope (model: Dimension-XR) from BRUKER GmbH, Germany; a field emission scanning electron microscope (model: G2) from ZEISS GmbH, Germany; a DSC-TG thermogravimetric analyzer (model: DTG-60) and a gas chromatograph-mass spectrometer (model: QP2010) from Shimadzu Corporation, Japan; an inverted microscope (model: CKX53) from Olympus Corporation, Japan; an ELx800 microplate reader from Bio-Tek Corporation, USA; a NanoDrop One ultra-micro spectrophotometer from Thermo Scientific Corporation, USA; and a real-time quantitative PCR instrument from Applied Biosystems Corporation, USA.

[0049] Experimental reagents:

[0050] DEAE-52 cellulose (batch number: 9013-34-7), Sephadex G-50 gel (batch number: 17-0043-01), Tris(hydroxymethyl)aminomethane (Tris, batch number: 20200803), sodium dodecyl sulfate (SDS, batch number: L-5750), DMSO (batch number: D6370), MTT (batch number: M6180), DMEM / F-12 medium (batch number: SH30023.01B), DEPC-treated water (0.1%, batch number: 0301A22), penicillin-streptomycin antibiotics (batch number: Top0016), lipopolysaccharide (LPS, batch number: L2880), and thiazolyl blue (MTT, batch number: M6180) were purchased from Beijing Bio-Top Technology Co., Ltd.; Neutral red powder (batch number: 9013-34-7) was also purchased from Beijing Bio-Top Technology Co., Ltd. DMEM high-glucose medium (batch number: N8160), DMEM high-glucose medium (batch number: 20200909), and DMSO (batch number: D8370) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; skim milk powder (batch number: 0804A20) was purchased from Beijing Regen Biotechnology Co., Ltd.; N-methylbisacrylamide (batch number: B0014) and ammonium persulfate (batch number: A6761) were purchased from Beijing Chemical Reagent Co., Ltd.; Tween-20 (batch number: ST825), Western cellular and IP lysis buffer (batch number: P0013), protein loading buffer (5X) (batch number: P0016N), trypsin EDTA digestion solution (batch number: C0201), and dimethyl sulfoxide (DMSO, batch number: N8160) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; skim milk powder (batch number: 0804A20) was purchased from Beijing Regen Biotechnology Co., Ltd.; N-methylbisacrylamide (batch number: B0014) and ammonium persulfate (batch number: A6761) were purchased from Beijing Chemical Reagent Co., Ltd.; DMEM high-glucose medium (batch number: B0014) and DMSO ... ST1276), NO kit (batch number: S0021S), ROS kit (batch number: S0033S), and BCA protein quantification kit (batch number: P0010) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; monosaccharide standard (AR) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; mouse GAPDH internal control primer (batch number: I808KA5803) was synthesized by Shanghai Sangon Biotech Co., Ltd.; glucose standard (AR) was purchased from Tianjin Gangchuan Chemical Reagent Co., Ltd.; Congo red (AR) was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.; trifluoroacetic acid (chromatographic grade) and 1-phenyl-3-methyl-5-pyrazolone (PMP, AR) were purchased from Xiamen... The following reagents were purchased from various biotechnology companies: Men'an Yongbo Technology Co., Ltd.; fetal bovine serum (batch number: 130118611) and fetal bovine serum (batch number: 20190503) were purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; DPPH, ABTS, and FRAP were purchased from Nanjing Jiancheng Bioengineering Institute; ECL chemiluminescence reagent kit (batch number: MA0186) was purchased from Dalian Meilun Biotechnology Co., Ltd.; TLR2-IN-C29 (batch number: M9063) and TAK-242 (batch number: M4838) were purchased from Aomo Biotechnology Co., Ltd.; α-naphthol (AR) was purchased from Sinopharm Chemical Reagent Co., Ltd.; and pre-stained protein marker (batch number: 26616) was purchased from Thermo Fisher Scientific.Trizol reagent (lot number: 182805) was purchased from Invitrogen, USA; TEMED (lot number: TB133) was purchased from Biosharp; PrimeScript RT reagent Kit (lot number: RR047A) and TB Green Premix Ex Taq (lot number: RR420A) were purchased from Takara, Japan; rabbit anti-TRAM1 polyclonal antibody (lot number: A15142), rabbit anti-TIRAP polyclonal antibody (lot number: A12606), rabbit anti-CD11b polyclonal antibody (lot number: A1581), and rabbit anti-GAPDH polyclonal antibody (lot number: AC001) were purchased from Wuhan Aibote Biotechnology Co., Ltd.; rabbit anti-pc-jun polyclonal antibody (lot number: AF5782) and rabbit anti-TR The following polyclonal antibodies were purchased from Shanghai Beyotime Biotechnology Co., Ltd.: IF polyclonal antibody (batch number: AF8238), rabbit anti-TBK1 polyclonal antibody (batch number: AF8103), rabbit anti-IRF3 polyclonal antibody (batch number: AF2485), rabbit anti-IRAK1 polyclonal antibody (batch number: AF7293), rabbit anti-TAK1 polyclonal antibody (batch number: AF7422), and rabbit anti-IRAK4 polyclonal antibody (batch number: P0016N). Rabbit anti-Dectin-1 polyclonal antibody was also purchased. Rabbit anti-MRC1 polyclonal antibody (batch number: bs-2455R), rabbit anti-p-TAK1 polyclonal antibody (batch number: bs-4727R), rabbit anti-p-TAK1 polyclonal antibody (batch number: bs-3438R), rabbit anti-p-IKKα polyclonal antibody (batch number: bs-3229R), rabbit anti-p-IKKβ polyclonal antibody (batch number: bs-3231R), rabbit anti-p-MEK1+MEK2 polyclonal antibody (batch number: bs-5414R), rabbit anti-p-MKK3+MKK6 polyclonal antibody (batch number: bs-2455R), rabbit anti-MRC1 polyclonal antibody (batch number: bs-4727R), rabbit anti-p-TAK1 polyclonal antibody (batch number: bs-3438R), rabbit anti-p-IKKα polyclonal antibody (batch number: bs-3229R), rabbit anti-p-IKKβ polyclonal antibody (batch number: bs-3231R), rabbit anti-p-MEK1+MEK2 polyclonal antibody (batch number: bs-5414R), rabbit anti-p-MKK3+MKK6 polyclonal antibody (batch number: bs-3231R). The following polyclonal antibodies were purchased from Beijing Bio-Sen Biotechnology Co., Ltd.: rabbit anti-pc-fos polyclonal antibody (batch number: bs-3274R), rabbit anti-ikkε polyclonal antibody (batch number: bs-3153R), rabbit anti-ikkε polyclonal antibody (batch number: bs-4114R), rabbit anti-p-ikkε polyclonal antibody (batch number: bs-8583R), rabbit anti-p-TBK1 polyclonal antibody (batch number: bs-3440R), and rabbit anti-p-IRF3 polyclonal antibody (batch number: bs-9278R).Rabbit anti-TLR4 polyclonal antibody (batch number: WL00196), rabbit anti-p38 polyclonal antibody (batch number: WLP00764), rabbit anti-β-actin polyclonal antibody (batch number: WL01372), rabbit anti-IKKα / β polyclonal antibody (batch number: WL01900), rabbit anti-p-MKK7 polyclonal antibody (batch number: WL03553), rabbit anti-IκBα polyclonal antibody (batch number: WL01936), rabbit anti-NF-κB polyclonal antibody (batch number: WL01273b), rabbit anti-ERK polyclonal antibody (batch number: WL01864), rabbit anti-TLR2 polyclonal antibody (batch number: WL01747), rabbit anti-MyD88 polyclonal antibody (batch number: WL02494), rabbit anti-TRAF6 polyclonal antibody (batch number: WL02). Rabbit anti-p-IκBα polyclonal antibody (batch number: WL02495), rabbit anti-p-NF-κB polyclonal antibody (batch number: WL02196), rabbit anti-p-ERK polyclonal antibody (batch number: WLP1512), rabbit anti-p-P38 polyclonal antibody (batch number: WLP1576), rabbit anti-JNK polyclonal antibody (batch number: WL01295), rabbit anti-p-JNK polyclonal antibody (batch number: WL01813), rabbit anti-GAPDH polyclonal antibody (batch number: WL01114), rabbit anti-c-jun polyclonal antibody (batch number: WL02863), rabbit anti-c-fos polyclonal antibody (batch number: WL03699), and rabbit anti-TRAF3 polyclonal antibody (batch number: WL04574) were purchased from Shenyang Wanlei Biotechnology Co., Ltd.

[0051] Example 1: Extraction, separation, purification and properties of polysaccharides from *Cyclocarya paliurus*.

[0052] 1.1 Extraction, Separation and Purification

[0053] This invention focuses on the dried pericarp of *Ligusticum striatum*, improving upon the traditional water extraction and alcohol precipitation method. A flash extractor is used to extract the components from *Ligusticum striatum*, followed by gradient alcohol precipitation to obtain crude polysaccharides. Pigments are removed by adsorption with macroporous resin AB-8, and the crude polysaccharides are initially separated using a DEAE-52 cellulose chromatography column. Subsequently, the polysaccharides are further purified using a Sephadex G-50 dextran gel chromatography column, and freeze-dried to obtain a single-component polysaccharide from *Ligusticum striatum*.

[0054] 1.1.1 Method

[0055] (1) The dried *Amur cork tree* leaves were pulverized into powder. 100g of the powder was weighed into a beaker, and 3000mL of 90℃ distilled water was added to the beaker at a material-to-liquid ratio of 1:30. The extract was performed twice using a flash extractor (5000r / min; 30s / time). The extract was filtered to obtain an aqueous extract. The aqueous extract was centrifuged (4500r / min) for 5 minutes, and the supernatant was collected. The filtrate was collected after filtration. The filtrate was concentrated under reduced pressure to 1 / 3 of its original volume. 95% ethanol was slowly added and stirred rapidly to make the ethanol concentration in the solution reach 20%. The solution was allowed to stand overnight to obtain precipitate I and supernatant I. The above steps were repeated. Ethanol was added to supernatant I to make the final ethanol concentration in the solution reach 40%. The solution was allowed to stand overnight to obtain precipitate II and supernatant II. The above steps were repeated. Ethanol was added to supernatant II to make the final ethanol concentration in the solution reach 60%. The solution was allowed to stand overnight to obtain precipitate III and supernatant III. Repeat the above steps, adding ethanol to supernatant III to achieve a final ethanol concentration of 80%, and let stand overnight to obtain precipitate IV and supernatant IV. Precipitates I, II, III, and IV were washed several times with anhydrous ethanol and freeze-dried to obtain crude polysaccharide powders of *Cynanchum paniculatum* PJP20, PJP40, PJP60, and PJP80, respectively. The yields were calculated.

[0056] Polysaccharide yield calculation formula: Polysaccharide yield (%) = Polysaccharide mass / Raw material mass × 100%

[0057] (2) Decolorization: Accurately weigh 5g of crude polysaccharide from the northern scabra, dissolve it in 100mL of distilled water, add 50g of AB-8 macroporous resin, stir with a magnetic stirrer for 2h to decolorize, vacuum filter, and freeze dry the filtrate.

[0058] (3) Ultraviolet spectroscopy scanning: Weigh an appropriate amount (50-100mg / mL) of decolorized crude polysaccharide, dissolve it in ultrapure water, and perform a full wavelength scan (900-190nm) using an ultraviolet-visible spectrophotometer.

[0059] (4) Determination of crude polysaccharide content in *Cyclocarya paliurus*: Accurately weigh 10 mg of constant-weight glucose standard and dilute to 10 mL with ultrapure water to obtain a concentration of 1 mg / mL as the stock solution. Using a pipette, pipette 0 μL, 100 μL, 200 μL, 400 μL, 600 μL, 800 μL, and 1000 μL of the glucose stock solution into test tubes, add ultrapure water to a volume of 1 mL, and then add phenol (5%; 1 mL) solution and 5 times the volume of concentrated sulfuric acid to each test tube. After shaking, incubate in a boiling water bath for 15 min, cool, and measure the absorbance of the solution in each test tube at a wavelength of 490 nm. Each group was measured in triplicate, and the average value was taken. Ultrapure water served as a blank control. Concentration (mg / mL) was plotted on the x-axis, and absorbance (A) was plotted on the y-axis to construct a glucose standard curve. Prepare a 0.5 mg / mL polysaccharide sample solution according to the above method, measure the absorbance, and input it into the glucose standard curve to calculate the polysaccharide content in the crude polysaccharide of *Cyclocarya paliurus*.

[0060] (5) Crude polysaccharides after separation and decolorization using a DEAE-52 cellulose chromatography column

[0061] ①DEAE-52 cellulose activation: Weigh 30g of dried DEAE-52 cellulose, add an appropriate amount of ultrapure water and stir for 30 minutes to fully swell. After standing, pour off the packing material floating on the surface, filter, add 0.5mol / L NaOH solution to the packing material and soak for 2h. Wash with ultrapure water until neutral. Repeat the above steps to soak the packing material in HCl solution and NaOH solution of the same concentration for 2h in turn. Wash the packing material repeatedly with ultrapure water until neutral, and prepare to pack the column.

[0062] ② Column packing: Add an appropriate amount of ultrapure water to the chromatography column to rinse it. Before packing, remove air bubbles from the packing material. Then, pour the cellulose along the column wall into the chromatography column. Rinse the column wall with an appropriate amount of ultrapure water to remove any remaining packing material. Open the outlet valve at the bottom of the chromatography column, ensuring the packing surface is flat. When the ultrapure water level is 3-5 cm above the packing surface, close the outlet valve and let it stand overnight.

[0063] ③ Equilibration: After connecting the constant flow pump to the chromatography column, equilibrate the chromatography column by opening the constant flow pump and the column outlet valve, controlling the speed of the constant flow pump to 1 mL / min, and equilibrate 3-5 column volumes. After equilibration, use pH test paper to check whether the eluent is neutral before loading the sample.

[0064] ④ Sample loading: Accurately weigh 80 mg of crude polysaccharide from *Cyclocarya paliurus* (after PJP80 decolorization), dissolve it in ultrapure water to prepare a 10 mg / mL solution, filter it through a 0.45 μm microporous membrane, and slowly add 8 mL of filtrate along the column wall into the chromatography column. Rinse off any remaining sample on the inner wall of the chromatography column with an appropriate amount of ultrapure water to ensure that the sample is fully distributed on the surface of the packing material. Allow the sample to stand until it has completely entered the packing material. Open the water outlet valve until the liquid level is flush with the packing material surface, then close the water outlet valve and add eluent until the liquid level is 3-5 cm above the packing material surface.

[0065] ⑤ Elution: Gradient elution with NaCl solution (0-0.5 mol / L), 200 mL for each concentration, 1 mL / min, 2 mL / tube for collecting the eluent, absorbance detected by phenol-sulfuric acid method, elution curve plotted, elution peaks of the same concentration combined, placed in dialysis bag, placed in running water for 24 h, freeze-dried after dialysis to obtain PJP80-I polysaccharide from *Gynostemma pentaphyllum*.

[0066] (6) Purification of polysaccharides using Sephadex G-50 gel chromatography column

[0067] ①Sephadex G-50 gel activation: Weigh an appropriate amount of dry gel powder, soak it in a 50-60% ethanol solution at room temperature for 24 hours, stirring constantly to allow it to swell fully, let it stand, pour off the ethanol, and repeatedly wash it with ultrapure water until there is no alcohol odor, then prepare to pack the column.

[0068] ② Column packing: Install the column packer onto the chromatography column and rinse the column with an appropriate amount of ultrapure water. Quickly pour the gel into the column packer, allowing it to slowly and evenly enter the chromatography column. Continuously tap the column to remove air bubbles from the packing material. Rinse the column wall with an appropriate amount of ultrapure water to remove any remaining packing material. Open the water outlet valve at the bottom of the chromatography column, ensuring the packing surface is flat. When the ultrapure water level is 3-5 cm above the packing surface, close the water outlet valve, remove the column packer, and let it stand overnight.

[0069] ③ Equilibration: Use ultrapure water as the eluent to equilibrate the chromatography column. Turn on the constant flow pump and the column outlet valve, and control the constant flow pump speed at 0.3 mL / min to equilibrate 5-6 column volumes.

[0070] ④ Sample loading: Accurately weigh 50 mg of PJP80-I polysaccharide from North China dragon skin, dissolve it in 5 mL of ultrapure water, filter it through a 0.45 μm microporous membrane, and slowly add 5 mL of filtrate along the column wall into the chromatography column. Rinse off any remaining sample on the inner wall of the chromatography column with an appropriate amount of ultrapure water, ensuring that the sample is fully distributed on the surface of the packing material. Allow the sample to stand until it has completely entered the packing material. Open the water outlet valve until the liquid level is flush with the packing material surface, then close the water outlet valve. Add the eluent until the liquid level is 3-5 cm above the packing material surface.

[0071] ⑤ Elution: Collect the eluent at a flow rate of 1 mL / 3 min using an automatic collector, and plot the elution curve. Freeze-dry the eluent to obtain PJP80-Ia, a polysaccharide derived from *Cynanchum paniculatum*.

[0072] 1.1.2 Experimental Results

[0073] (1) Preparation of crude polysaccharide from *Gynostemma pentaphyllum*: Based on the weight of *Gynostemma pentaphyllum* powder, the yield of crude polysaccharide from *Gynostemma pentaphyllum* was calculated to be 6.97% using the formula. Among them, the yield of PJP20 was 4.17%, the yield of PJP40 was 1.62%, the yield of PJP60 was 0.77%, and the yield of PJP80 was 0.41%. The higher yields of PJP20 and PJP40 may be due to the low alcohol concentration in the solution, which caused other impurities besides polysaccharides to be trapped in the precipitate.

[0074] (2) Ultraviolet spectral analysis of crude polysaccharide from *Cynanchum paniculatum*: The ultraviolet full-wavelength scan spectrum of crude polysaccharide from *Cynanchum paniculatum* showed that no characteristic absorption peaks appeared in the wavelength range of 260-280 nm, indicating that crude polysaccharide from *Cynanchum paniculatum* does not contain impurities such as nucleic acids and proteins.

[0075] (3) Isolation and purification of crude polysaccharides from *Cyclocarya paliurus*: The crude polysaccharides were separated using a DEAE-52 chromatography column. The elution curve is shown below. Figure 1 As shown in the figure, the elution curves revealed three distinct components, named PJP80-I, PJP80-II, and PJP80-III, with yields of 35.3%, 31.25%, and 18.75%, respectively. Since PJP80-I exhibited a single symmetrical peak during elution with ultrapure water and had a relatively high yield among the three components, this elution peak was selected for enrichment, dialysis, and lyophilization for further purification.

[0076] PJP80-I was purified by Sephadex G-50 gel column chromatography. Elution curves showed only one symmetrical elution peak, indicating that the polysaccharide was of good purity and free of other impurities. It was named PJP80-Ia, enriched and lyophilized for further study.

[0077] (4) Determination of polysaccharide content in *Cyclocarya paliurus*: The standard curve equation for glucose concentration (mg / mL) versus absorbance (A) is Y = 2.5231X + 0.0369, R 2 =0.9993, indicating a good linear relationship between glucose concentration and absorbance value in the range of 0-1.0 mg / ml, which is statistically significant. The sugar content in crude polysaccharides PJP20, PJP40, PJP60, PJP80 and polysaccharides PJP80-I, PJP80-II, PJP80-III and PJP80-Ia of *Cephalotaxus fortunei* was calculated, as shown in Table 1.

[0078] Table 1 shows that the sugar contents of crude polysaccharides from *Ligustrum lucidum* PJP20, PJP40, PJP60, and PJP80 are 25.06% ± 0.45%, 31.47% ± 0.38%, 45.07% ± 0.51%, and 54.55% ± 0.76%, respectively. With increasing alcohol concentration, the amount of impurities such as inorganic salts and proteins in the *Ligustrum lucidum* polysaccharide precipitate decreases, and the polysaccharide content gradually increases. The sugar contents of *Ligustrum lucidum* polysaccharides PJP80-I, PJP80-II, and PJP80-III are 89.98% ± 0.52%, 64.13% ± 0.06%, and 59.30% ± 0.32%, respectively. The polysaccharide PJP80-Ia from *Cynanchum paniculatum* obtained by separation on a DEAE-52 cellulose chromatography column and purification on a G-50 gel column had a sugar content of 97.51% ± 0.19%. The sugar content of crude polysaccharide from *Cynanchum paniculatum* was significantly improved after separation and purification.

[0079] Table 1. Polysaccharide content of *Trapa natans*

[0080]

[0081] 1.2 Properties

[0082] 1.2.1 Experimental Methods

[0083] (1) I2-KI reaction: Add an appropriate amount of *Gynostemma pentaphyllum* polysaccharide solution to the prepared iodine-potassium iodide solution, mix well, react in the dark, and observe the solution color. If the solution turns blue or purplish-red, it proves that the polysaccharide contains starch; if there is no color change, it proves that the polysaccharide does not contain starch.

[0084] (2) Phenol-sulfuric acid reaction: The method is the same as 1.1.1(4) in Example 1.

[0085] (3) Molisch (α-naphthol) reaction: The Molisch reaction is a commonly used method for identifying sugars. Sugars react with furfural under strong acid to form furfural, which then reacts with α-naphthol to produce a purple-red product. Mix the prepared Molisch reagent with a solution of *Cynanchum paniculatum* polysaccharide and observe whether a purple-red ring appears.

[0086] (4) FeCl3 reaction: Mix FeCl3 solution with an appropriate amount of *Gynostemma pentaphyllum* polysaccharide solution and observe the solution color. If the solution is purple, it proves that the sugar contains phenolic substances; if it is green or bluish-black or forms a green or bluish-black precipitate, it proves that the polysaccharide contains tannins.

[0087] (5) Fehling's reagent reaction: Fehling's reagent can be used to identify reducing sugars. Pour the prepared Fehling's reagent into the polysaccharide solution of *Cynanchum paniculatum*, heat in a water bath, and observe whether a brick-red cuprous oxide precipitate is formed to identify whether there are reducing sugars in the polysaccharide.

[0088] (6) Sulfuric acid-carbazole reaction: Add the polysaccharide solution of *Cynanchum paniculatum* to the sulfuric acid-carbazole solution and observe the solution color. If the solution is purple-red, it proves that the polysaccharide contains uronic acid; if the solution is blue-green, it proves that the polysaccharide does not contain uronic acid.

[0089] (7) Determination of apparent viscosity of polysaccharide solution of *Gynostemma pentaphyllum*: Prepare a 2 g / L solution of polysaccharide of *Gynostemma pentaphyllum*, and measure the apparent viscosity of the polysaccharide at 20 °C and a shear rate of 1 r / s-100 r / s.

[0090] 1.2.2 Experimental Results and Analysis

[0091] (1) The physicochemical properties of PJP80-Ia are shown in Table 2. Table 2 shows that both the phenol-sulfuric acid and Molish reagent reactions, used to identify carbohydrates, caused color changes in the solution, further confirming that the extracted substance is a carbohydrate compound. The reaction of the *Cynanchum paniculatum* polysaccharide solution with Fehling's reagent did not produce a brick-red precipitate, proving that the polysaccharide does not contain reducing sugars. The reaction with sulfuric acid and carbazole did not change the solution color, suggesting that the polysaccharide does not contain uronic acid, consistent with the infrared results. The reaction with I2-KI did not change the solution color, proving that the polysaccharide does not contain starch. The reaction with FeCl3 did not produce a purple, green, or bluish-black color, proving that the polysaccharide does not contain phenols or tannins.

[0092] Table 2 Physicochemical Properties of PJP80-Ia

[0093]

[0094] (2) Apparent viscosity determination of PJP80-Ia: The apparent viscosity of PJP80-Ia was negatively correlated with the shear rate, exhibiting shear dilution and pseudoplastic behavior, proving that the *Cyclocarya paliurus* polysaccharide solution is a "non-Newtonian fluid". At lower shear rates, *Cyclocarya paliurus* polysaccharide PJP80-Ia has a high viscosity, possibly due to the entanglement of polysaccharide molecular chains, resulting in greater friction and electrostatic forces between molecules and higher viscous resistance. When the shear rate increases, the intermolecular forces are disrupted, and the viscous resistance decreases, leading to a decrease in the apparent viscosity of the polysaccharide. When the shear rate reaches a certain level, a more stable structure is formed between the polysaccharide molecular chains, and the apparent viscosity is no longer affected by the shear rate, exhibiting good stability.

[0095] In summary, this embodiment obtained crude polysaccharides PJP20, PJP40, PJP60, and PJP80 from *Ligusticum striatum* via flash extraction and alcohol precipitation. The calculated yields of crude polysaccharides were 4.17%, 1.62%, 0.77%, and 0.41%, respectively, with sugar contents of 25.06% ± 0.45%, 31.47% ± 0.38%, 45.07% ± 0.51%, and 54.55% ± 0.76%, respectively. PJP80, with the highest sugar content, underwent further separation and purification. AB-8 macroporous resin was used for adsorption and decolorization, followed by preliminary separation using a DEAE-52 cellulose chromatography column. NaCl solution (0-0.5 mol / L) was used as the eluent, and three fractions, PJP80-I, PJP80-II, and PJP80-III, were collected. PJP80-I showed a single, symmetrical elution peak with a yield of 35.3% and a sugar content of 89.98% ± 0.52%. PJP-I was purified using a Sephadex G-50 gel chromatography column with ultrapure water as the eluent. The elution curve showed a single symmetrical elution peak between tubes 90 and 110. The eluent with a half-peak width was collected and lyophilized to obtain PJP80-Ia, a polysaccharide from *Cyclocarya paliurus*, with a yield of 59.7% and a sugar content of 97.51% ± 0.19%, indicating good purity. PJP80-Ia is a pale yellow, odorless powder, readily soluble in water but insoluble in organic reagents. Physicochemical analysis showed that PJP80-Ia is a carbohydrate and does not contain starch, phenols, or uronic acids. Apparent viscosity measurements revealed that the PJP80-Ia solution exhibited typical characteristics of a non-Newtonian pseudoplastic fluid, demonstrating good stability.

[0096] Example 2: Primary and higher structures of PJP80-Ⅰa polysaccharide from *Cynanchum paniculatum*.

[0097] 2.1 Primary Structure

[0098] Polysaccharides are abundant biological macromolecules. Due to the different types and linkage modes of monosaccharides, polysaccharides exhibit complex and diverse structures. Influenced by their structure, polysaccharides possess various pharmacological effects. The polysaccharide structure consists of primary and higher-order structures. The primary structure includes the relative molecular weight, monosaccharide composition, glycosidic bond type, and linkage mode, forming the basis for exploring the higher-order structure of polysaccharides. This invention uses high-performance liquid chromatography, Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and nuclear magnetic resonance spectroscopy (NMR) to determine the purity, relative molecular weight, glycosidic bond type, and structural linkage sites of the polysaccharide PJP80-Ia from *Coptis chinensis*, thus analyzing its primary structure and laying the foundation for subsequent higher-order structure and bioactivity analysis.

[0099] 2.1.1 Experimental Methods

[0100] (1) Infrared spectroscopy determination: Accurately weigh 100 mg of dry potassium bromide powder and 1 mg of PJP80-Ia, grind them evenly in an agate mortar, compress them into tablets, and place the tablets into a preheated infrared spectrometer for detection. The measurement range is 400-4000 cm⁻¹. -1 between.

[0101] (2) Raman spectroscopy determination: Accurately weigh 4 mg of dried PJP80-Ia sample, distribute it evenly on a glass slide, and place it in a Raman spectrometer for measurement. Raman spectrometer operating parameters: laser power 14 mW, laser collection wavelength 785 nm, 50x long focal length objective lens for observation, measurement range 400-3000 cm⁻¹ -1 .

[0102] (3) Purity identification and molecular weight determination: The purity and relative molecular weight of the PJP80-Ia sample were determined by high performance liquid chromatography.

[0103] ① Purity determination: Weigh an appropriate amount of PJP80-Ia sample, dissolve it in ultrapure water, prepare a 2 mg / mL polysaccharide solution, filter it through a 0.45 μm microporous aqueous filter membrane, and then perform the determination. The instrument parameters are: Ultrahygrogel (7.8×300 mm) column, mobile phase: ultrapure water, flow rate: 0.8 mL / min, detector: RX, column temperature: 30℃, injection volume: 20 μL / time.

[0104] ② Molecular weight determination: Accurately weigh 2 mg of dextran standards of different molecular weights (T5, T10, T30, T50, T70, T110), dissolve in 1 mL of ultrapure water, filter through a 0.45 μm microporous aqueous filter membrane, and measure the molecular weight using the same instrument parameters as for purity determination. Plot a dextran standard curve with retention time on the x-axis and the logarithm of molecular weight on the y-axis, and calculate the molecular weight of the samples.

[0105] (4) Detection of PJP80-Ia monosaccharide composition: The composition of PJP80-Ia monosaccharide polysaccharide from North China dragon skin was determined by high performance liquid chromatography.

[0106] ① Hydrolysis of PJP80-Ia polysaccharide from *Cynanchum paniculatum*: Accurately weigh 5 mg of PJP80-Ia polysaccharide from *Cynanchum paniculatum* into a stoppered test tube, add 2 mL of 2 mol / L trifluoroacetic acid (TFA) solution, and hydrolyze at 120℃ for 6 h. Evaporate the TFA, add methanol and wash repeatedly to remove residual TFA, dry, and then add an appropriate amount of ultrapure water to dissolve and obtain the polysaccharide hydrolysate.

[0107] ②PMP derivatization: Transfer 200 μL of hydrolysate to a centrifuge tube, add an equal volume of PMP methanol solution (0.5 mol / L) and NaOH solution (0.3 mol / L), shake well, and incubate at 70℃ for 100 min. After the reaction is complete, neutralize with an equal volume of HCl solution (0.3 mol / L), extract with an appropriate amount of chloroform, repeat three times, aspirate the aqueous layer, and filter through a 0.22 μm filter membrane to obtain the derivatized polysaccharide sample. Accurately weigh 10 mg of mannose, rhamnose, galacturonic acid, glucose, galactose, and xylose, add ultrapure water to prepare 1 mg / mL monosaccharide standard and mixed standard solutions, and perform derivatization.

[0108] ③ Plotting the monosaccharide standard curve: Inject the derivatized monosaccharide standard, mixed standard and polysaccharide sample for detection to determine the monosaccharide composition in the jujube seed. Select the corresponding monosaccharide standard and inject it at concentrations of 1, 2, 4, 6, 8 and 10 μL. Plot the monosaccharide standard curve with the monosaccharide concentration on the x-axis and the peak area on the y-axis.

[0109] ④ Chromatographic conditions: Waters X-Bridge C 18 The chromatographic column was set at 30℃ and 245nm (UA). The mobile phase was acetonitrile-PBS (0.05mol / L, pH 6.8) = 20:80, with a flow rate of 0.8mL / min and an injection volume of 10μL.

[0110] (4) Nuclear magnetic resonance (NMR) determination: The polysaccharide PJP80-Ia sample from *Gynostemma pentaphyllum* was dissolved in heavy water (D2O), dried under reduced pressure, and repeated 3-4 times. 40 mg of the lyophilized sample was weighed, dissolved in 0.6 mL of D2O, and subjected to one-dimensional and two-dimensional NMR measurements using a nuclear magnetic resonance spectrometer.

[0111] 2.1.2 Analysis of Experimental Results

[0112] (1) Infrared spectroscopy analysis: Based on the infrared spectrum of PJP80-Ia polysaccharide from *Cephalotaxus fortunei*, it was inferred that polysaccharide PJP80-Ia contains the functional groups shown in Table 3. PJP80-Ia polysaccharide from *Cephalotaxus fortunei* exhibits infrared spectroscopy at a wavelength of 3402 cm⁻¹. -1 There is a strong absorption peak at 2926 cm⁻¹, which is due to the stretching vibration of OH. -1 It exhibits characteristic absorption peaks of carbohydrates, attributed to the CH stretching vibration; the CH angle-shifting vibration is located at 1423 cm⁻¹. -1 1235cm -1 Around 1635 cm⁻¹, which is a characteristic absorption peak of polysaccharides. -1 The absorption peaks at 1145 and 1051 cm⁻¹ are due to the hydration vibration of polysaccharides. -1 The absorption peak appearing at 860 cm⁻¹ may be attributed to the CO stretching vibration in the pyranose ring, suggesting that the sample may contain a pyranose ring; based on 860 cm⁻¹... -1 901cm -1The absorption peak at 1745 cm⁻¹ suggests that both α- and β-configurations of glycosidic bonds may exist simultaneously in the sample. Furthermore, the absorption peak at 1745 cm⁻¹... -1 No absorption peaks were observed on either side, indicating that the sample does not contain carboxyl groups, consistent with the results of the carbazole sulfate experiment. Therefore, it is determined that the polysaccharide PJP80-Ia from *Cypripedium pekinensis* is a polysaccharide with both α and β glycosidic bonds and a pyranose backbone.

[0113] Table 3. Infrared Absorption of PJP80-Ia Functional Groups

[0114]

[0115] (2) Raman spectroscopy analysis: According to the Raman spectrum of PJP80-Ia polysaccharide from *Cypripedium pekinensis*, it was found that at 435 cm⁻¹... -1 601cm -1 680cm -1 1080cm -1 1459cm -1 The presence of a carbohydrate absorption peak indicates that PJP80-Ia is a carbohydrate compound; based on the 851 cm⁻¹ peak... -1 and 899cm -1 The absorption peak at 926 cm⁻¹ suggests that both α- and β-configurations of glycosidic bonds may exist simultaneously in the sample; -1 The absorption peak at 1246 cm⁻¹ indicates the presence of COC stretching vibration in the polysaccharide; -1 1459cm -1 The absorption peak indicates the presence of CH angle vibration in the polysaccharide, which is consistent with the infrared spectrum analysis results of PJP80-Ia polysaccharide from *Gynostemma pentaphyllum*.

[0116] (3) Results of purity and molecular weight determination

[0117] ① Plot the standard curve for dextran: The equation for the standard curve of dextran is Y = -0.3131X + 8.3688, R0 2 =0.9982, where X is time (min) and Y is Lg (Mw). The molecular weight was measured in the range of 5000-110000 Da.

[0118] ② Purity and molecular weight of PJP80-Ia polysaccharide from *Cephalotaxus fortunei*: The chromatogram of PJP80-Ia showed an elution time of 13.744 min and only one symmetrical peak, indicating a uniform molecular weight distribution and good purity. The molecular weight of PJP80-Ia was calculated to be 11630 Da by substituting the retention time into the dextran standard curve.

[0119] (4) Monosaccharide composition: Based on the high-performance liquid chromatography (HPLC) chromatograms of mannose, rhamnose, galacturonic acid, glucose, galactose, xylose, and a mixture of monosaccharide standards, as well as the derivatized sample of *Cephalotaxus fortunei* polysaccharide, the retention times of the six monosaccharide standards and the monosaccharide mixture were obtained, as shown in Table 4. According to the retention time comparison, *Cephalotaxus fortunei* polysaccharide PJP80-Ia is composed of mannose and glucose. The mannose standard curve is Y = 31.68X - 2.5703, R0... 2 =0.9997, with monosaccharide concentration on the x-axis and peak area on the y-axis; the glucose standard curve is Y = 50.131X - 2.435, R0 2 =0.9997, with monosaccharide concentration on the x-axis and peak area on the y-axis. The calculated molar ratio of mannose to glucose in the *P. JP80-Ⅰa* polysaccharide from *P. kaempferia galanga* was 0.29:0.70.

[0120] Table 4 Retention times of components in monosaccharide and mixed monosaccharide standards

[0121]

[0122] (5) Nuclear magnetic resonance spectroscopy analysis

[0123] In polysaccharides 1 In 1H-NMR, most proton peaks on the glycocyclic carbons are concentrated between δ 3.0-4.0 ppm, and they overlap significantly, making accurate resolution impossible. Anterior hydrogen proton chemical shifts are typically concentrated between 4.3-5.5 ppm, facilitating resolution and allowing for the determination of glycosidic bond configurations in polysaccharides: α configuration δ > 4.9 ppm, β configuration δ < 4.9 ppm. According to the 1H NMR spectrum of *Cyclocarya paliurus* polysaccharide PJP80-Ia, six signal peaks exist in the anodic hydrogen region, with chemical shifts between 4.41 ppm and 5.30 ppm. These are named A, B, C, D, E, and F in descending order of shift, indicating the presence of both α- and β-configurations of glycosidic bonds in polysaccharide PJP80-Ia. Furthermore, the signal peak between 3.1-4.2 ppm corresponds to the proton signal of the pyranose ring, indicating that PJP80-Ia possesses a pyranose ring, consistent with the IR results.

[0124] polysaccharides 13In C-NMR, the chemical shifts of anodic carbons are typically distributed between 90 and 110 ppm, used to determine the type, configuration, and linkage of sugar residues. Generally, anodic carbons with chemical shifts less than 103 ppm are classified as α-configuration, while those with chemical shifts exceeding 103 ppm are classified as β-configuration. C-NMR can also distinguish the sugar ring configuration of polysaccharides. The chemical shifts of C3 and C5 of pyranoses are generally between 60 and 80 ppm, while those of furans are between δ82 and 84 ppm. According to the C-NMR spectrum of PJP80-Ia, pyranose C3 and C5 signal peaks appear between δ60.06 and 80.65 ppm, further confirming that PJP80-Ia is a polysaccharide with a pyranose ring configuration. The anodic C signal peaks of the polysaccharide *PJP80-Ia* are between δ91 and 104 ppm, indicating the presence of both α and β configurations in the polysaccharide.

[0125] Two-dimensional analysis of the polysaccharide PJP80-Ia from *Cephalotaxus fortunei* was performed. 1 HH COSY, HMBC, and HMQC spectra yielded the attribution information for six sugar residues. The H-1 and C-1 signals corresponding to residues A, B, C, D, E, and F were 5.30 / 92.18 ppm, 5.30 / 92.06 ppm, 5.13 / 91.94 ppm, 5.08 / 101.44 ppm, 4.55 / 95.78 ppm, and 4.41 / 102.59 ppm, respectively. Analysis showed that residue A belonged to [(1→3)-α-D-Manp], E to β-Reducing-D-Glcp, and F to [→6)-β-D-Glcp(→1]. Residues B, C, and D could not be assigned due to their weak signals. Table 5 lists the attribution information for three sugar residues. 1 H, 13 C. Chemical shift. In summary, it is inferred that the polysaccharide PJP80-Ⅰa from *Gynostemma pentaphyllum* contains α and β glycosidic bonds, composed of [(1→3)-α-D-Manp], β-Reducing-D-Glcp, and [→6)-β-D-Glcp(→1].

[0126] Table 5PJP80-Ia 1 H, 13 Chemical shift of C

[0127]

[0128] In summary, this embodiment investigated the primary structure of the polysaccharide PJP80-Ia from *Cypripedium pekinensis*. The results are as follows: characteristic absorption peaks of polysaccharide compounds appeared in both the infrared and Raman spectra of the sample, proving that PJP80-Ia is a polysaccharide compound. (The last sentence appears to be incomplete and possibly refers to a specific absorption peak at 850 cm⁻¹.) -1 901cm -1Absorption peaks were observed in all samples, indicating that the polysaccharide from *Gynostemma pentaphyllum* may contain both α- and β-configurations of glycosidic bonds. HPLC analysis was used to determine the purity, molecular weight, and monosaccharide composition of *Gynostemma pentaphyllum* polysaccharide PJP80-Ia. The results showed that polysaccharide PJP80-Ia has high purity, a molecular weight of 11630 Da, and is composed of two monosaccharides, mannose and glucose, in a molar ratio of 0.29:0.70. NMR analysis revealed that the glycan chains of PJP80-Ia are mainly linked by [(1→3)-α-D-Manp], β-Reducing-D-Glcp, and [→6)-β-D-Glcp(→1).

[0129] 2.2 Advanced Structure of PJP80-Ⅰa

[0130] This embodiment mainly uses Congo red experiment, differential scanning calorimetry, particle size and potential measurement, X-ray diffraction (XRD), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to analyze and determine the higher-order structure of PJP80-Ia polysaccharide from northern cycads, including its helical structure, thermal stability, particle size, charge properties, crystal characteristics, appearance morphology and chain conformation.

[0131] 2.2.1 Experimental Methods

[0132] (1) Congo Red Experiment: Congo red solution, NaOH solution, and PJP80-Ia polysaccharide solution were added to a test tube to form a mixed liquid (3-5 mL), so that the final concentration of PJP80-Ia polysaccharide in the mixed liquid reached 1 mg / mL, the final concentration of NaOH was gradually increased from 0.0 mol / L to 0.5 mol / L, and the final concentration of Congo red was 40 μmol / L; another test tube was used as a blank control, with the polysaccharide solution replaced by ultrapure water, and other conditions remained unchanged. The solutions in the test tubes were thoroughly mixed and allowed to stand at room temperature for a period of time. The wavelength was then scanned between 400-600 nm using a UV spectrophotometer, and the maximum absorption wavelength was recorded. The NaOH concentration was plotted on the x-axis, and the maximum absorption wavelength was plotted on the y-axis to create a curve.

[0133] (2) Thermal properties determination: Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were used to determine the thermal stability of PJP80-Ia. 13.84 mg of PJP80-Ia sample was accurately weighed and measured under nitrogen atmosphere. The measurement temperature range was set between 20-400℃, and the instrument heating rate was 10℃ / min. The TGA-DSC curve of the sample was obtained after the temperature dropped to 20℃.

[0134] (3) Particle size and potential determination: Weigh 0.2 mg of PJP80-Ia, dissolve it in ultrapure water to prepare a solution of 0.1 mg / mL, filter it through a 0.45 μm microporous membrane, and measure it. Take the average value of 3 measurements.

[0135] (4) X-ray diffraction (XRD) determination: 10 mg of *Gynostemma pentaphyllum* polysaccharide was accurately weighed, ground into a fine and uniform powder, and then the crystal properties were determined using an X-ray diffractometer. The instrument operating parameters were: Cu target, Kα as radiation source, tube voltage set to 40 kV, tube flow rate set to 50 mA, continuous scanning angle set to 10-80° (2θ), and scanning angle set to 3° / min.

[0136] (5) Field emission scanning electron microscopy (SEM) measurement: Weigh an appropriate amount of PJP80-Ia, attach conductive adhesive to the sample, place it on the sample stage, place the sample stage in the ion emission device, sputter gold and observe under the electron microscope. The accelerating voltage is 15kV, and the magnification is 200, 1K, 5K, 19.83K, 20K and 40K times respectively.

[0137] (6) Atomic Force Microscopy (AFM) Observation: A 1 mg / mL PJP80-Ia solution was prepared using ultrapure water and magnetically stirred for 2 h to ensure complete dissolution of the sample. The solution was diluted to 10 ng / mL, magnetically stirred for 4 h, and filtered through a 0.45 μm microporous membrane to obtain the filtrate. An appropriate amount of nickel chloride solution was added to a mica sheet, and after 30 s, it was rinsed with ultrapure water. 20 μL of the filtrate was then transferred to a fresh mica sheet using a pipette, rinsed with ultrapure water after 30 s, and allowed to air dry at room temperature. The morphology of the sample was then observed using an atomic force microscope.

[0138] 2.2.2 Experimental Results and Analysis

[0139] (1) Congo Red Results: Congo red is an acidic dye that can determine the triple helix structure of polysaccharides. The triple helix structure forms a complex with Congo red, and the maximum absorption wavelength decreases with increasing sodium hydroxide concentration, showing a significant red shift. According to the Congo red experimental results, the complex formed by PJP80-Ia polysaccharide from *Cyclocarya paliurus* and Congo red showed a red shift with increasing NaOH concentration, indicating that the polysaccharide has a helical structure. However, after the NaOH concentration exceeds 0.2 mol / L, the maximum absorption wavelength gradually stabilizes and does not decrease sharply. Therefore, it is inferred that PJP80-Ia polysaccharide from *Cyclocarya paliurus* does not have a triple helix structure.

[0140] (2) Results of thermal property determination: According to the thermal property experiment, the polysaccharide PJP80-Ⅰa sample of *Cyclocarya paliurus* underwent three weight loss phenomena between 20-400℃. The first weight loss occurred between the initial temperature and 140℃, with a weight loss rate of 5.7%. A small endothermic peak appeared between 105-140℃, with a peak endothermic peak of 135.48℃. This stage may be due to the evaporation of water in the polysaccharide caused by the temperature rise. The second weight loss occurred between 140-290℃, at which time the polysaccharide mass loss was relatively large, with a loss rate of 26%. A large endothermic peak also appeared between 140-224℃, with a peak endothermic peak of 173.66℃. At this time, the polysaccharide weight loss rate was the fastest, which may be due to the high temperature breaking the chemical bonds of the polysaccharide, leading to the violent decomposition of the polysaccharide. The third weight loss occurred between 290-400℃, with a relatively small polysaccharide mass loss, with a weight loss rate of 4.5%. This stage may be due to the transformation and decomposition of the remaining substances in the polysaccharide. Experiments show that qinglongyi polysaccharide has good thermal stability within 170℃.

[0141] (3) Results of particle size and potential measurement

[0142] ① Particle size distribution of PJP80-Ia polysaccharide from *Cephalotaxus fortunei*: Polysaccharides exist in solution as aggregates, and their particle size and dispersion index (PDI) reflect the degree of particle size dispersion in solution. According to the particle size distribution analysis results of PJP80-Ia, the average particle size (Z-Ave) of PJP80-Ia is 227.2 nm, and the PDI value is 0.254. It exhibits narrow and symmetrical signal peaks, indicating a relatively concentrated particle size distribution in solution. This also demonstrates the high purity of PJP80-Ia, which is consistent with the HPLC results.

[0143] ② Zeta potential determination of PJP80-Ia polysaccharide from *Cephalotaxus fortunei*: The zeta potential of a polysaccharide represents the static charge on its surface and reflects its stability in aqueous solution. A higher absolute value of the zeta potential indicates greater intermolecular repulsion, making aggregation less likely and resulting in more stable particle morphology in solution; conversely, a lower zeta potential indicates greater instability and a tendency for coagulation or aggregation. The zeta potential of PJP80-Ia polysaccharide from *Cephalotaxus fortunei* shows an average potential of -0.1 mV, indicating that the polysaccharide is almost uncharged and neutral. However, due to the low potential, it is inferred that the polysaccharide from *Cephalotaxus fortunei* is unstable in solution and prone to aggregation.

[0144] (4) X-ray diffraction (XRD) results: X-ray diffraction is commonly used to detect whether a substance has a crystalline structure. Crystalline materials generally exhibit narrow and sharp diffraction peaks, while amorphous materials exhibit broad diffraction peaks. The results (Table 6) show that the polysaccharide PJP80-Ia from *Cypripedium pekinensis* has an amorphous structure between diffraction angles of 10-80°, but two narrow and sharp strong diffraction peaks appear at 28.2° and 40.5°, indicating the presence of a partial crystalline structure in the polysaccharide sample. Analysis revealed that the interplanar spacing of the two diffraction peaks was... The grain sizes are 94.9 nm and 65.0 nm, respectively; the crystallinity is 64.04% and 73.98%, respectively. Analysis of the unit cell parameters shows that the three sides (a, b, c) of the unit cell are of equal length, with a length of... The included angles α, β, and γ are all 90°, therefore it is inferred that the crystals in the polysaccharide sample are mainly composed of cubic crystal cells. The relevant parameters of the X-ray diffraction pattern of the *PJP80-Ia* polysaccharide from *Gynostemma pentaphyllum* are shown in Table 6.

[0145] Table 6. Relevant parameters of the X-ray diffraction pattern of PJP80-Ia

[0146]

[0147] (5) Field Emission Scanning Electron Microscopy (SEM) Results: The microstructure of the polysaccharide surface can be directly observed using field emission scanning electron microscopy. The results showed that, under 200x magnification, the polysaccharide PJP80-Ia from *Scutellaria baicalensis* consisted of large, irregularly shaped sheet-like and blocky aggregates, indicating the presence of an amorphous structure, consistent with XRD analysis. High-magnification (1Kx) observation revealed granular protrusions on the aggregate surface. Magnification of these protrusions at 40Kx revealed a small number of relatively regular granular aggregates on their surface. Literature review showed that fucoidan and sea cucumber viscera polysaccharides also exhibited relatively regular granular structures under high magnification, and XRD results for both polysaccharides indicated crystalline structures. It is speculated that the small granular aggregates may be crystals in the polysaccharide sample, and their size is similar to that of the X-ray diffraction results.

[0148] (6) Atomic force microscopy (AFM) observations: Polysaccharide PJP80-Ia formed irregular chain and ring structures on mica sheets, accompanied by branching. The three-dimensional image showed peaks of varying sizes, indicating that the polysaccharide molecules had a certain degree of aggregation. It is speculated that there are branched structures in the polysaccharide molecular structure, which is consistent with the results of the planar image, and also verifies the highly branched nature of the polysaccharide chemical structure. The chain width of the ring molecular chains is between 150nm and 250nm, the chain height is between 4nm and 13nm, and the maximum ring diameter is about 1250nm. Since the height of a single polysaccharide molecular chain is usually between 0.1-1.0nm, the height of the polysaccharide molecular chain of *Cyclocarya paliurus* is much higher than that of a single polysaccharide molecular chain, further indicating that the polysaccharide may have branches that intertwine and stack to form aggregates, which corresponds to the results of scanning electron microscopy. The reasons for the aggregation of polysaccharide molecules may be as follows: the hydroxyl groups of the polysaccharide in *Gynostemma pentaphyllum* make it highly hydrophilic, which further enhances the intermolecular hydrogen bonds, van der Waals forces and other interactions, causing the polysaccharide molecules to aggregate; secondly, the Zata potential analysis of *Gynostemma pentaphyllum* polysaccharide shows that it is unstable in PJP80-Ia solution and easily aggregates, with the sugar chains intertwining to form irregular chain and ring structures of different sizes.

[0149] In summary, this embodiment analyzed and determined the higher-order structures of *P. pekinensis* polysaccharide PJP80-Ia, including its helical structure, thermal stability, particle size, charge properties, crystal characteristics, apparent morphology, and chain conformation, using methods such as the Congo red assay, differential scanning calorimetry, particle size and potential determination, XRD, SEM, and AFM observation. The Congo red assay revealed that *P. pekinensis* polysaccharide PJP80-Ia does not possess a triple-helix structure. Thermal property analysis showed that *P. pekinensis* polysaccharide underwent three weight losses between 20 and 400 °C. The first weight loss occurred between the initial temperature and 140 °C, with a loss rate of 5.7%, due to water evaporation caused by heating. The second weight loss occurred between 140 and 290 °C, with a loss rate of 26%. At a temperature of 173.66 °C, the polysaccharide underwent severe decomposition, indicating that *P. pekinensis* polysaccharide exhibits good thermal stability within 170 °C. Particle size and Zata potential measurements showed that the average particle size (Z-Ave) of *Cyclocarya paliurus* polysaccharide PJP80-Ia was 227.2 nm, and the dispersion index (PDI) was 0.254, indicating that the polysaccharide had a relatively concentrated particle size distribution and high purity in solution. Zata potential analysis indicated that *Cyclocarya paliurus* polysaccharide is a neutral polysaccharide. X-ray diffraction revealed that the polysaccharide sample contained both crystalline and amorphous structures, exhibiting a semi-crystalline structure. Scanning electron microscopy showed that *Cyclocarya paliurus* polysaccharide PJP80-Ia consisted of irregular sheet-like and blocky aggregates. High-magnification observation revealed a small number of tiny granular aggregates on the protruding surfaces, which are speculated to be crystal grains in the polysaccharide sample. Chain conformation characterization revealed that *Cyclocarya paliurus* polysaccharide PJP80-Ia molecules aggregated, with sugar chains intertwined and stacked, forming irregular chain and ring structures of varying sizes.

[0150] Example 3: Activity Study of PJP80-Ⅰa

[0151] To investigate the antitumor activity of *Gynostemma pentaphyllum* polysaccharide, this study used three cancer cells—HepG2 and SMMC-7721 hepatocellular carcinoma cells, and MCF-7 breast cancer cells—as research subjects to determine the antitumor activity of *Gynostemma pentaphyllum* polysaccharide PJP80-Ia. The in vitro antioxidant activity of *Gynostemma pentaphyllum* polysaccharide PJP80-Ia was analyzed using three antioxidant methods: DPPH, ABTS, and FRAP, to facilitate its subsequent application.

[0152] 3.1 Experimental Methods

[0153] (1) Culture of HepG2, SMMC-7721 and MCF-7 cells

[0154] ① Preparation of DMEM / F-12 medium: Inactivated fetal bovine serum and DMEM / F-12 medium were mixed evenly at a volume ratio of 1:9, filtered through a 0.22μm filter membrane, sealed with a sealing film, and stored at 4℃.

[0155] ②Preparation of PBS: Accurately weigh 8.0g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate, and 0.24g of dipotassium hydrogen phosphate. Prepare 1000mL of ultrapure water. Autoclave, seal the bottle mouth with sealing film, and refrigerate at 4℃.

[0156] ③ Preparation of MTT solution: Prepare MTT solution (5 mg / mL) with PBS buffer, protect from light, and refrigerate at 4°C.

[0157] ④ Preparation of PJP80-Ia solution of *Cyclocarya paliurus* polysaccharide: Accurately weigh an appropriate amount of PJP80-Ia, dissolve it in ultrapure water, and prepare a stock solution of 1 mg / mL. Dilute the stock solution to 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 32.25 μg / mL, and 15.625 μg / mL using the 2:1 dilution method, and set aside for later use. Filter sterilize using a 0.22 μm microporous membrane before use.

[0158] ⑤ Cell thawing: Remove the frozen cells and quickly place them in a 37°C water bath, shaking to thaw them rapidly. Transfer the cells and culture medium to centrifuge tubes, centrifuge (1000 rpm / 5 min), and discard the supernatant. Add an appropriate amount of culture medium, mix well, and then inoculate into culture flasks. Incubate in a CO2 incubator.

[0159] ⑥ Cell passage: Discard the original culture medium in the culture flask, add 3 mL of PBS buffer, shake the culture flask horizontally to rinse the cells 2-3 times, aspirate the PBS, and digest with 1 mL of trypsin. Observe under a microscope as the cells gradually become rounded, then quickly add culture medium to stop digestion. Pipette the cells into a cell suspension, add to a centrifuge tube, centrifuge at 1000 rpm for 5 min, and aspirate the supernatant. Add an appropriate amount of culture medium, mix well, and inoculate into culture flasks. Incubate in a CO2 incubator.

[0160] (2) Determination of the antitumor activity of PJP80-Ia polysaccharide from *Cyclocarya paliurus*: Cells in the logarithmic growth phase were digested according to the steps described above. The cell suspension was centrifuged, the supernatant was discarded, and culture medium was added to dilute the cell concentration to 4 × 10⁻⁶. 4Cells / mL were added to 96-well plates, 100 μL per well, with the edge of the plate filled with PBS buffer. The plates were incubated in a CO2 incubator for 24 h, ready for drug administration. Drug administration was performed 24 h later. 100 μL of PJP80-Ia solution was added sequentially at concentrations of 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 32.25 μg / mL, and 15.625 μg / mL, with six parallel experiments for each concentration. A blank control culture medium was used. After drug administration, the plates were incubated for 72 h. 10 μL of LTT solution was added to each well, and after 4 h, the solution was aspirated, and 150 μL of DMSO solution was added. The plates were shaken until the crystals were completely dissolved, and the absorbance of each well was measured at 570 nm to calculate the cell inhibition rate.

[0161] Inhibition rate (%) = (1 - mean OD value of the treated group / mean OD value of the blank group) × 100%

[0162] (3) Determination of the DPPH free radical scavenging ability of PJP80-Ia, a polysaccharide from the northern cycad, on the surface of the plant.

[0163] ① Preparation of solutions of crude polysaccharide PJP80 and polysaccharide PJP80-Ia from *Cyclocarya paliurus*: Accurately weigh 25 mg of PJP80 and PJP80-Ia samples and dilute to 10 mL with ultrapure water. Serially dilute the sample solutions to 0.5, 1.0, 1.5, 2.0, and 2.5 mg / mL solutions for later use.

[0164] ② Preparation of DPPH solution: Accurately weigh 2.5 mg of DPPH, dissolve it in anhydrous ethanol to prepare a solution with a concentration of 0.05 mg / mL, and store at room temperature away from light.

[0165] ③ Preparation of positive control solution: Prepare a positive control solution at 2.5 mg / mL, and then serially dilute it to 0.5, 1.0, 1.5, 2.0 and 2.5 mg / mL solutions. Store in the dark for later use.

[0166] ④ Mix equal volumes of PJP80 and PJP80-Ia sample solutions of various concentrations with DPPH solution, allow to stand in the dark at room temperature for 30 minutes, and then measure the absorbance of each group at a wavelength of 517 nm. Anhydrous ethanol was used as a blank control, and Trolox was used as a positive control. Each group was measured in triplicate, and the average value was taken to calculate the clearance rate. The calculation formula is as follows:

[0167] DPPH free radical scavenging rate (%) = (1-(A) 测定 -A 对照 ) / A 空白 )×100%

[0168] (3) Determination of the ABTS free radical scavenging ability of PJP80-Ia, a polysaccharide from the leaves of the Chinese scabra.

[0169] ① Preparation of solutions of crude polysaccharide PJP80 and polysaccharide PJP80-Ia from *Cyclocarya paliurus*: Accurately weigh 25 mg of PJP80 and PJP80-Ia samples and dilute to 10 mL with ultrapure water. Serially dilute the sample solutions to 0.5, 1.0, 1.5, 2.0, and 2.5 mg / mL solutions for later use.

[0170] ② Preparation of positive control solution: Prepare a positive control solution at 2.5 mg / mL, and then serially dilute it to 0.5, 1.0, 1.5, 2.0 and 2.5 mg / mL solutions. Store in the dark for later use.

[0171] ③ Take 10 μL of each concentration of PJP80 and PJP80-Ia sample solution, add 190 μL of ABTS solution, mix well, react at room temperature for 6 min, and measure the absorbance of each group at a wavelength of 405 nm. Ultrapure water is used as a blank control, and Trolox is used as a positive control. Perform three parallel measurements, take the average value, and calculate the clearance rate. The calculation formula is as follows:

[0172] ABTS free radical scavenging rate (%) = (A 空白 -A 测定 ) / A 空白 ×100%

[0173] (4) Determination of the total antioxidant capacity of PJP80-Ia polysaccharide from *Scutellaria baicalensis* by FRAP method

[0174] ① Preparation of solutions of crude polysaccharide PJP80 and polysaccharide PJP80-Ia from *Cyclocarya paliurus*: Accurately weigh 25 mg of PJP80 and PJP80-Ia samples and dilute to 10 mL with ultrapure water. Serially dilute the sample solutions to 0.5, 1.0, 1.5, 2.0, and 2.5 mg / mL solutions for later use.

[0175] ② Preparation of standard solutions: Accurately weigh 25 mg of standard FeSO4-7H2O and prepare a 2.5 mg / mL solution. Dilute serially to 0.5, 1.0, 1.5, 2.0, and 2.5 mg / mL solutions. Prepare standard solutions immediately before use to avoid oxidation.

[0176] ③ Plotting the standard curve: Take each concentration of standard solution, add 180 μL of FRAP working solution, mix well, react at 37℃ for 5 min, and then measure the absorbance of each group at a wavelength of 593 nm. Ultrapure water is used as a blank control. Each group is measured in triplicate, and the average value is taken. Plot the standard curve with the OD value of the standard as the x-axis and the concentration as the y-axis.

[0177] ④ Take 5 μL of each concentrated PJP80 and PJP80-Ia sample solution, add 180 μL of FRAP working solution, mix well, react at 37℃ for 5 min, and then measure the absorbance of each group at a wavelength of 593 nm. Ultrapure water is used as a blank control. Each group is measured in triplicate, and the average value is taken. Calculate the FRAP value of each group according to the standard curve.

[0178] 3.2 Experimental Results and Analysis

[0179] (1) Effect of PJP80-Ia polysaccharide from *Cyclocarya paliurus* on the proliferation of human hepatocellular carcinoma HepG2 cells: The effect of PJP80-Ia on the proliferation of HepG2 cells was determined by the MTT assay. Figure 2 As shown in Figure A, PJP80-Ia has an inhibitory effect on HepG2 liver cancer cells, showing a dose-dependent effect between drug concentrations of 15.625 μg / mL and 500 μg / mL. At a concentration of 500 μg / mL, the polysaccharide inhibited HepG2 cells by 31.8%, which was statistically significant (P < 0.01).

[0180] (2) Effect of PJP80-Ia polysaccharide from *Cyclocarya paliurus* on the proliferation of human hepatocellular carcinoma cells SMMC-7721: The effect of PJP80-Ia polysaccharide from *Cyclocarya paliurus* on the proliferation of SMMC-7721 cells was determined by MTT assay. Figure 2 As shown in B. The polysaccharide PJP80-Ia from *Pterocarya stenoptera* exhibits an inhibitory effect on SMMC-7721 liver cancer cells in a dose-dependent manner; the higher the concentration, the higher the inhibition rate. At the high dose, the inhibition rate was 39.8%, showing a significant difference (P < 0.01).

[0181] (3) Effect of PJP80-Ia, a polysaccharide from *Cyclocarya paliurus*, on the proliferation of human breast cancer cells MCF-7: The effect of PJP80-Ia, a polysaccharide from *Cyclocarya paliurus*, on the proliferation of MCF-7 cells is as follows: Figure 2 As shown in Figure C, PJP80-Ia exhibited inhibitory effects on SMMC-7721 human breast cancer cells in a dose-dependent manner. At a concentration of 1000 μg / mL, the inhibition rate was 40.3%, which was statistically significant (P < 0.01).

[0182] (4) Results of the determination of the DPPH free radical scavenging ability of PJP80-Ia, a polysaccharide from *Cyclocarya paliurus*, on DPPH free radicals: DPPH showed strong absorption at a wavelength of 517 nm, and the alcoholic solution was purple. Upon addition of an antioxidant, the absorption gradually disappeared, and the absorbance decreased, which was used to determine the DPPH free radical scavenging ability of the sample. Figure 2 As shown in D, both PJP80 and PJP80-Ia, crude polysaccharides from *Cyclocarya paliurus*, have the ability to scavenge DPPH free radicals. Their antioxidant capacity increases with increasing sample concentration. At the same concentration, PJP80-Ia has a greater scavenging capacity for DPPH free radicals than PJP80.

[0183] (5) Results of the determination of the ABTS free radical scavenging ability of PJP80-Ia, a polysaccharide from North China scabra: ABTS was oxidized to produce green ABTS. + Antioxidants can inhibit ABTS + The antioxidant capacity of the samples was calculated by measuring the absorbance at a wavelength of 405 nm. The antioxidant capacity of Trolox, PJP80 (crude polysaccharide from *Cynanchum paniculatum*), and PJP80-Ia (coarse polysaccharide from *Cynanchum paniculatum*) against ABTS free radicals was also calculated. Figure 2 E. For example Figure 2 As shown in E, the scavenging ability of PJP-80 and PJP80-Ia is positively correlated with concentration. At the same concentration, PJP80-Ia has a greater scavenging ability for ABTS free radicals than that of crude polysaccharide from *Cyclocarya paliurus*.

[0184] (6) Results of FRAP determination of total antioxidant capacity of PJP80-Ia polysaccharide from *Cypripedium pekinensis*: Under acidic conditions, antioxidants can reduce Fe... 3+ -TPTZ reduced to Fe 2+ The reducing power of the sample was calculated by measuring the absorbance of the FeSO4 standard solution at a wavelength of 593 nm using the -TPTZ absorbance. The reducing power was expressed as the concentration of the FeSO4 standard solution. A FeSO4 standard curve was plotted, and the equation was calculated as Y = 10.806X - 0.342, R0. 2 =0.9954, where x is the OD value of the standard and y is the concentration of the standard (mg / mL). Substituting the absorbance values ​​of the sample group into the equation, the FRAP values ​​for each concentration of the sample are calculated, as follows: Figure 2 As shown in F. FRAP value is the reducing power per milligram of sample, i.e., the reducing power of a certain amount of FeSO4. The crude polysaccharides PJP80 and PJP80-Ia from *Cyclocarya paliurus* have a reducing power against FeSO4. 3+ -TPTZ all have reducing ability, which increases with increasing sample concentration. At the same concentration, the reducing ability of PJP80-Ia polysaccharide from *Cyclocarya paliurus* is greater than that of crude polysaccharide from *Cyclocarya paliurus*.

[0185] In summary, this embodiment determined the antitumor activity of *Gynostemma pentaphyllum* polysaccharide PJP80-Ia against human hepatocellular carcinoma cells HepG2, SMMC-7721, and human breast cancer cells MCF-7 using the MTT assay. The results showed that *Gynostemma pentaphyllum* polysaccharide had inhibitory effects on all three tumor cell types, exhibiting a dose-dependent effect within a certain range (15.625-1000 μg / mL). Both crude *Gynostemma pentaphyllum* polysaccharide and *Gynostemma pentaphyllum* polysaccharide PJP80-Ia can scavenge DPPH and ABTS free radicals and have the ability to reduce Fe²⁺. 3+The -TPTZ-containing polysaccharide exhibits antioxidant properties. Specifically, PJP80-Ia showed a DPPH radical scavenging rate of 79.5%, while crude polysaccharide from *Ligusticum striatum* showed a DPPH radical scavenging rate of 59.8%. Furthermore, PJP80-Ia showed a ABTS radical scavenging rate of 65.0%, while crude polysaccharide from *Ligusticum striatum* showed an ABTS radical scavenging rate of 57.0%. Experiments demonstrate that PJP80-Ia has greater antioxidant properties than crude polysaccharide from *Ligusticum striatum*.

[0186] Example 4 PJP80-Ia activity

[0187] 4.1 Effect of PJP80-Ia on RAW264.7 cell proliferation

[0188] This invention uses the MTT assay, inverted microscopy, and flow cytometry to investigate the effects of different concentrations of PJP80-Ia polysaccharide on macrophage proliferation, morphology, and cell cycle, laying the foundation for subsequent research on the immunomodulatory mechanism of PJP80-Ia polysaccharide on macrophages.

[0189] Preparation of experimental reagents

[0190] PJP80-Ia solution: Weigh 10 mg of PJP80-Ia polysaccharide from *Gynostemma pentaphyllum* and add it to 10 mL of DMEM high-glucose medium to make a 1 mg / mL PJP80-Ia stock solution. Before the experiment, take out the PJP80-Ia stock solution and dilute it to different concentrations for experimental research.

[0191] LPS solution: Prepare a 1 mg / mL LPS stock solution and store at -20℃; before use, take out the LPS stock solution and add a certain volume of DMEM culture medium to make the concentration 1 μg / mL, and store at 4℃.

[0192] PBS buffer: Take a sterile volumetric flask, add the weighed solid powder according to the formula, and make up to 500 mL with ultrapure water. Shake well. Sterilize at 121℃ for 30 min, remove and place in a clean bench to equilibrate. After cooling to room temperature, sterilize and filter, then dispense and seal. Store in a refrigerator at 4℃.

[0193] MTT solution: Weigh 20 mg MTT and add 4 mL PBS solution to make the concentration 5 mg / mL. Store in a sealed container at 4°C, protected from light.

[0194] 4.1.1 Experimental Methods

[0195] (1) RAW264.7 macrophage culture: After autoclaving, pipette tips and centrifuge tubes were sterilized on a UV-irradiated workbench for 30 minutes. The prepared complete culture medium was placed in a 37°C water bath to warm up. Cells were removed from the incubator and observed under a microscope. If the cell density was below 60% but the culture medium was bright yellow, passage was unnecessary; simply replace with fresh complete culture medium. If the cell density was between 80% and 90%, cell passage was performed. The procedure was as follows: First, aspirate the old culture medium, add 3 mL of PBS to wash the cells three times, and then aspirate the PBS. Add another 3 mL of DMEM complete culture medium and gently pipette the adherent cells, being careful not to be too vigorous to reduce foam formation. Transfer the pipette-suspended cell suspension to a new sterile culture flask according to experimental requirements and place it in a cell culture incubator. The conditions were set as follows: temperature 37°C, CO2 concentration 5%. Strict sterilization was required when removing and returning the culture flask to prevent cell contamination.

[0196] (2) MTT assay to detect the effect of PJP80-Ia on the proliferation of RAW264.7 macrophage cells: Well-grown RAW264.7 cells were prepared into a cell suspension and seeded in 96-well plates to achieve a cell density of 5 × 10⁶ cells per well. 4 Cells were inoculated at 100 μL / well (cells / mL). To prevent spontaneous sedimentation, the cells were thoroughly agitated by pipetting. After inoculation, the cells were placed in a cell culture incubator for acclimatization. After a period of time, the cells adhered completely. The experiment consisted of a blank control group and a PJP80-Ia polysaccharide treatment group, with six replicates in each group. The blank control group received 100 μL of culture medium. The PJP80-Ia polysaccharide treatment groups received 100 μL of PJP80-Ia solution to final concentrations of 6.25, 12.5, 25, 50, 100, 200, and 400 μg / mL, respectively. The cells were cultured for another 24 h. After aspirating the cell supernatant, add 20 μL of 5 mg / mL MTT solution to each well and continue culturing for 4 h. Carefully aspirate the supernatant, add 150 μL of DMSO solution, and add 150 μL of DMSO solution to each well for a new zeroing group. Shake in the dark for 10 minutes to dissolve and crystallize the cells. Measure the OD value (490 nm) of each well and calculate cell viability (cell viability = (OD value)). 药物处理组 -OD 调零组 ) / (OD 空白对照组 -OD 调零组 (×100%)

[0197] (3) Effect of PJP80-Ia on the morphology of RAW264.7 macrophages was detected by inverted microscopy: The experimental groups were a blank control group, a positive control group (LPS), and a PJP80-Ia treatment group, with three replicates in each group. RAW264.7 cells in the logarithmic growth phase were suspended in complete culture medium at a density of 1.0 × 10⁻⁶. 6RAW264.7 cells were seeded at a density of 1 mL / well in 6-well plates and thoroughly mixed by pipetting. After 24 h of culture, the cells adhered to the plate, and the supernatant of the RAW264.7 cells was aspirated. For the PJP80-Ia treatment groups, 1 mL of PJP80-Ia solution at concentrations of 50, 100, and 200 μg / mL was added. For the blank control group, 1 mL of DMEM was added per well, and for the positive control group, 1 mL of 1 μg / mL LPS was added. After 24 h of culture, the morphological changes of the RAW264.7 cells were observed under a 40x magnification inverted microscope.

[0198] (4) Flow cytometry detection of the effect of PJP80-Ia on the cell cycle of macrophage RAW264.7 cells: The experimental grouping and drug administration were the same as in method (3). After culturing for 48 h, RAW264.7 cells were collected according to the instructions for cell cycle and apoptosis detection. 1 mL of 70% pre-cooled ethanol solution was added and fixed at 4 °C. The next day, RAW264.7 cells were centrifuged for 5 min (1500 r / min). 1 mL of pre-cooled PBS solution was added to the RAW264.7 cell pellet and mixed well. The RAW264.7 cell pellet was centrifuged again and 0.5 mL of pre-prepared propidium iodide (PI) staining solution was added and stained in the dark for 30 min. The fluorescence intensity was detected by flow cytometry at an excitation wavelength of 488 nm.

[0199] (5) Data processing and analysis: Three parallel groups were set up in this experiment. The experimental data were analyzed by one-way ANOVA test using SPSS statistical software. All experimental results are expressed as mean ± standard deviation (Mean ± SD). P < 0.05 represents significant difference, P < 0.01 represents highly significant difference, and P < 0.05 is statistically significant.

[0200] 4.1.2 Experimental Results

[0201] (1) Effect of PJP80-Ia polysaccharide on the proliferation of RAW264.7 macrophage cells

[0202] This experiment used the MTT assay to determine the effect of different concentrations (6.25, 12.5, 25, 50, 100, 200, 400 μg / mL) of PJP80-Ia polysaccharide on the proliferation of RAW264.7 macrophages. As shown in Table 7, compared with the blank control group, PJP80-Ia did not have cytotoxic effects on RAW264.7 cells in the range of 6.25 μg / mL to 400 μg / mL. Moreover, the cell proliferation-promoting effect became more and more obvious with the increase of PJP80-Ia concentration, and reached a peak at 200 μg / mL, showing a trend of first increasing and then decreasing. This may be because PJP80-Ia induced excessive cell proliferation at 400 μg / mL, resulting in a decrease in survival rate. Among them, PJP80-Ia polysaccharide at 50, 100, and 200 μg / mL can significantly improve the survival rate of RAW264.7 cells in a dose-dependent manner, indicating that PJP80-Ia polysaccharide at 50, 100, and 200 μg / mL can promote macrophage proliferation. Therefore, these three concentrations were selected as the drug concentrations for subsequent experiments.

[0203] Table 7. MTT assay for the survival rate of PJP80-Ia polysaccharide in RAW264.7 macrophage cells (n=6)

[0204]

[0205]

[0206] Note: Compared with the control group, * indicates a statistically significant difference (P < 0.05); ** indicates a highly statistically significant difference (P < 0.01).

[0207] (2) Effects of PJP80-Ia on the morphology of RAW264.7 macrophages

[0208] The results are as follows Figure 3 As shown, under normal conditions, RAW264.7 cells are in an inactivated state, with small cell volume and a round shape. After treatment with 1 μg / mL LPS for 24 h, RAW264.7 cells are activated, exhibiting pseudopodia and irregular spindle shapes, showing a significant morphological difference from the blank control group. Treatment of RAW264.7 cells with different concentrations of PJP80-Ia (50, 100, 200 μg / mL) for 24 h also resulted in morphological changes similar to the LPS-treated group, with increased cell volume, elongated spindle or prismatic shapes, and elongated pseudopodia, but the proportion of deformed cells was lower than in the LPS-treated group. These experimental results indicate that PJP80-Ia can activate macrophages in RAW264.7 cells, thereby promoting macrophage proliferation and possessing potential immunomodulatory activity.

[0209] (3) Effects of PJP80-Ia on the cell cycle of RAW264.7 macrophages

[0210] The cell cycle is divided into three phases: DNA synthesis (S), mitosis (M), and interphase (G1 and G2), with the division sequence being G1-S-G2-M. After 24 hours of treatment with PJP80-Ia, a polysaccharide from *Gynostemma pentaphyllum*, mouse macrophage RAW264.7 cells underwent changes in the cell cycle (e.g., ...). Figure 4 and Figure 5 As shown in the figure, the percentage of cells in the G0 / G1 phase decreased from 63% to 53.88% in the LPS group, while the percentage of cells in the S phase increased from 19.33% to 33.62%. In contrast, the percentage of cells in the G0 / G1 phase decreased from 63% to 53.33% and the percentage of cells in the S phase increased from 19.33% to 27.65% in the 200 μg / mL *Pterocarya stenoptera* polysaccharide PJP80-Ia group. These results indicate that a certain concentration of *Pterocarya stenoptera* polysaccharide can promote the cell cycle progression of RAW264.7 macrophages, i.e., the transition from the G1 phase to the S phase, thus promoting cell proliferation. This is consistent with the MTT assay results.

[0211] In summary, the following conclusions can be drawn from the above experiments: (1) The MTT assay results showed that PJP80-Ia could promote the proliferation of RAW264.7 macrophages within the concentration range of 6.25 μg / mL to 400 μg / mL, and the concentration for subsequent experiments was determined. (2) The inverted microscope results showed that PJP80-Ia could promote the activation of macrophages, transforming them from a resting state to an activated state. This morphological change helped increase the contact area with external substances, which was beneficial for phagocytosis. (3) The cell cycle detection results by flow cytometry showed that PJP80-Ia could promote the transition of RAW264.7 cells from the G1 phase to the S phase, promoting cell proliferation. These conclusions preliminarily confirm that PJP80-Ia polysaccharide can promote the proliferation and activation of RAW264.7 cells, and that activated macrophages can initiate specific defense mechanisms and play a key role in the immune system. This indicates that PJP80-Ia polysaccharide has potential immunomodulatory functions.

[0212] 4.2 Effects of PJP80-Ia on the immunomodulatory activity of RAW264.7 cells

[0213] Macrophages are important cells in the immune system. Upon activation, macrophages produce NO and release various cytokines, including tumor necrosis factor and interleukins. These cytokines act as mediators of the immune response, regulating immunity and participating in pro-inflammatory and anti-inflammatory activities. Furthermore, these cytokines significantly promote macrophage function. NO enhances macrophage lysis and phagocytosis, serving as a hallmark biomarker for assessing macrophage activation. TNF-α, an early mediator of the inflammatory response, can promote macrophage activity. IL-1β plays a crucial role in macrophage activation and synergizes with TNF-α in inflammation. IFN-β stimulates macrophages to initiate antiviral responses, involving the TRIF antiviral pathway, and also possesses antitumor activity. IL-6 can regulate cellular and humoral immunity and participates in phagocytosis, antigen presentation, and inflammation regulation. Macrophages have been used as a potential cellular model for evaluating the immunomodulatory activity of bioactive compounds. As a member of the macrophage family, mouse RAW264.7 cells have been widely used in studies of immune activity. In the aforementioned studies, PJP80-Ia significantly promoted macrophage activity, inducing cells to proliferate rapidly and become activated. This invention examines the effects of PJP80-Ia, a polysaccharide from *Gynostemma pentaphyllum*, on macrophage immune function, focusing on the phagocytic and secretory functions of macrophages. Specifically, it investigates the effects of PJP80-Ia on the phagocytic capacity of RAW264.7 cells and the secretion levels and mRNA expression of NO, ROS, IL-1β, IL-6, TNF-α, and IFN-β, aiming to provide a reference for understanding the immunomodulatory activity and effects of *Gynostemma pentaphyllum* polysaccharide.

[0214] Preparation of experimental reagents

[0215] 0.1% Neutral Red Solution: Dissolve 0.01g of Neutral Red in 10mL of PBS, filter to sterilize, incubate at 37℃, and prepare fresh before use.

[0216] Cell lysis buffer: Measure equal volumes of glacial acetic acid and anhydrous ethanol, mix thoroughly, and store at 4°C for later use.

[0217] 4.2.1 Experimental Methods

[0218] (1) Neutral red assay to detect the effect of PJP80-Ia on the phagocytic capacity of RAW264.7 macrophages: RAW264.7 cells in good growth condition were prepared into cell suspension and seeded in 96-well plates at a seeding density of 1×10⁻⁶. 5The inoculation volume was 100 μL / well, with cells per mL. The cells were thoroughly mixed and cultured for 24 h to ensure cell adhesion. The supernatant was then discarded. The experiment consisted of a blank control group, a positive control group, and a PJP80-Ia polysaccharide treatment group, with six replicates per group. The PJP80-Ia polysaccharide treatment groups received 100 μL each of 50, 100, and 200 μg / mL PJP80-Ia solution. The blank control group received 100 μL of culture medium, and the positive control group received 100 μL of 1 μg / mL LLPS. Cells were cultured for 24 h. The supernatant was discarded, and the cells were gently washed twice with PBS. 100 μL of 0.1% neutral red solution was added to each well in the experimental groups, and the cells were cultured for 4 h. The cell supernatant was carefully aspirated to avoid affecting the experimental results. Wash cells twice with PBS solution, aspirate the PBS, add 100 μL of cell lysis buffer to each well, and set up a zero-adjustment group with 100 μL of cell lysis buffer per well. Incubate overnight at room temperature, measure the OD value (540 nm) using a microplate reader, and calculate the macrophage phagocytic rate (phagocytic rate = (OD value)). 药物处理组 -OD 调零组 ) / (OD 空白对照组 -OD 调零组 (×100%)

[0219] (2) Effect of PJP80-Ia on NO secretion by macrophages RAW264.7 by Griess method: The experimental grouping and drug administration were the same as in method "4.2.1(1)". After culturing for 24 h, 50 μL of supernatant of RAW264.7 was collected from each well. The operation was performed according to the Griess instructions. The absorbance value of each well (540 nm) was measured by microplate reader. NO standard curve was plotted and the NO content of macrophages at 6 h, 24 h and 48 h was calculated.

[0220] (3) Flow cytometry analysis of the effect of PJP80-Ia on ROS secretion by macrophage RAW264.7: The experimental grouping and drug administration were the same as in method “4.1.1(3)”. After culturing for 24 h, RAW264.7 cells were collected and mixed in DCFH-DA (10 μmol / L) solution and cultured for another 20 min. The RAW264.7 cell suspension was mixed every 5 min to ensure that the probe was loaded into the cells. RAW264.7 cells were washed with DMEM three times to remove DCFH-DA that failed to be loaded. The fluorescence intensity was detected by flow cytometry, with the excitation wavelength at 488 nm and the emission wavelength at 525 nm.

[0221] (4) qPCR detection of the effect of PJP80-Ia on the mRNA expression levels of cytokines IL-6, IL-1β, TNF-α, and IFN-β secreted by RAW264.7 macrophages: at 1.0 × 10⁻⁶ mRNAs. 6Cells were seeded in each well of a 6-well plate. The experimental groups and drug administration were the same as in “4.1.1(3)”. After culturing for 24 h, the mRNA expression of IL-6, IL-1β, TNF-α and IFN-β in each group of cells was detected.

[0222] (5) Extraction of total RNA from RAW264.7 cells: A suspension of well-grown RAW264.7 cells was seeded into 6-well plates at a cell density of 1.0 × 10⁻⁶ cells / well. 6 Cells per well, three replicates per group; after 24 hours, cells adhered. Add 1 mL of PBS to each well for cross-washing, aspirate the supernatant, add 1 mL of TRIzol to each well, and incubate for 5 minutes. After complete cell lysis, pipette the adherent cells completely. Transfer the TRIzol containing cells to different centrifuge tubes according to the experimental groups. Add 200 μL of CHCl3, vortex for 30 seconds, let stand for 3 minutes, and centrifuge at 15 minutes, 12,000 g, and 4°C. After centrifugation, collect 300 μL of the supernatant, add 500 μL of isopropanol, and let stand for 10 minutes. Centrifuge again for 10 minutes using the same parameters, and discard the supernatant. Add 1 mL of 75% ethanol, vortex vigorously, centrifuge for 5 min at 7500 g, centrifuge twice, discard the supernatant, and invert to air dry; add 20 μL of 0.1% DEPC-treated water to the centrifuge tube to fully dissolve the precipitate at the bottom of the tube, measure the RNA concentration and A260 / A280 absorbance, and store at -80℃ for later use.

[0223] (6) Reverse transcription to obtain total cDNA: Following the instructions of the TakaRa PrimeScript RT (RR047A) kit, the following were added sequentially: 2.0 μL of 5×gDNA Eraser, 1.0 μL of gDNA Eraser, 1.0 μg of Total RNA, and DEPC-treated water to a final volume of 10 μL. The mixture was carried out on ice for 5 min. Then, 4.0 μL of 5×PrimeScript, 1.0 μL each of PrimeScript reverse transcription complex and complex primer, and 4.0 μL of DEPC were added. After centrifugation and remixing, the reverse transcription conditions were set as follows: 37℃ for 15 min, then increased to 85℃ for 5 s, and decreased to 4℃ for a holding period to obtain cDNA. The cDNA was then frozen at -20℃ for later use. PCR reaction conditions: The temperature was increased to 95℃ and held for 30 s before cycling. Each cycle consisted of 95℃ for 5 s followed by a decrease to 60℃ for 34 s, for a total of 40 cycles. Calculation method: Using the mouse GAPDH gene as an internal control, three replicates were set up for each sample group, and measurements were performed in triplicate. Data were analyzed using a 23 -△△CT Methods for calculating IL-6, TNF-α, IL-1β and IFN-β (△CT = CT) 目的基因 -CT GAPDH , △△CT=△CT实验组 -△CT 空白组 ).

[0224] (7) Primer design and synthesis: The mRNA sequences corresponding to the mouse IL-6, TNF-α, IL-1β and IFN-β genes were searched in the Gene Bank database. Specific primers for each gene were designed using Primer5. The primer sequences are shown in Table 8.

[0225] Table 8. Target gene sequence and primer sequences

[0226]

[0227] Real-time PCR reagent composition (25μL system): SYBR Premix Ex TaqII (2×) 12.5μL, PCR Forward Primer (10M) 1.0μL, PCR Reverse Primer (10M) 1.0μL, cDNA solution 2.0μL, RNase-Free dH2O 8.5μL.

[0228] (8) Data Processing and Analysis

[0229] Data processing and analysis were performed using the same method as described in "4.1".

[0230] 4.2.2 Experimental Results

[0231] (1) Effect of PJP80-Ia on the phagocytic capacity of RAW264.7 macrophages: The previous experimental results showed that PJP80-Ia can activate RAW264.7 macrophages. Therefore, this experiment further investigated the effect of PJP80-Ia on the phagocytic capacity of RAW264.7 macrophages have pinocytosis on neutral red, and neutral red can react with acidic lysosomes to produce a red substance. The immunomodulatory activity of PJP80-Ia was evaluated by calculating the phagocytic rate. As shown in Table 9, 1 μg / mL PJP80-Ia can enhance the phagocytic capacity of RAW264.7 cells. After stimulating RAW264.7 cells with different concentrations of PJP80-Ia (50, 100, 200 μg / mL) for 24 h, PJP80-Ia promoted the enhanced phagocytic capacity of RAW264.7 cells for neutral red, which was dose-dependent and statistically significant compared with the control group (P<0.05). The results showed that PJP80-Ia could enhance the phagocytic capacity of macrophages.

[0232] Table 9. Effect of PJP80-Ia on the phagocytic capacity of RAW264.7 macrophages as determined by neutral red assay (n=6)

[0233]

[0234] (2) Effect of PJP80-Ia on NO secretion by mouse RAW264.7 cells: The effect of PJP80-Ia on the immune function of mouse RAW264.7 cells can be determined by examining its effect on NO secretion. This experiment observed the effect of different concentrations of PJP80-Ia on NO secretion by macrophages at different time points (6h, 24h, and 48h), as shown in Table 10. At the same time point, compared with the blank control group, both the PJP80-Ia treatment group and the LPS treatment group significantly promoted NO secretion in a dose-dependent manner (P<0.05). Simultaneously, the promoting effect of PJP80-Ia on NO production in macrophages was significantly lower than that in the LPS treatment group, indicating that NO secretion is not related to inflammatory stimulation but to the improvement of immune function. Therefore, PJP80-Ia can enhance the immune capacity of RAW264.7 cells by inducing NO production.

[0235] Table 10 Effects of PJP80-Ia on NO secretion from mouse RAW264.7 cells (n=3)

[0236]

[0237] (3) Effect of PJP80-Ia on ROS secretion in mouse RAW264.7 cells

[0238] The DCFH-DA probe can enter cells and be hydrolyzed into DCFH by intracellular substances. At this stage, DCFH does not show fluorescence. However, as intracellular ROS are generated, DCFH is oxidized back to DCF, displaying green fluorescence. The intensity of the fluorescence effectively reflects the ROS content of the cell; the stronger the fluorescence intensity, the higher the ROS content. Figure 6 As shown, compared with the blank control group, different concentrations of PJP80-Ia significantly promoted ROS secretion in a dose-dependent manner (P<0.05); LPS, as a positive control group, significantly stimulated macrophages to secrete ROS, and the secretion level was higher than that of the PJP80-Ia group. This indicates that *Pterocarya sibiricum* polysaccharide can participate in immune regulation by promoting ROS release from macrophages.

[0239] (4) Effects of PJP80-Ia on the expression of IL-1β, IL-6, TNF-α, and IFN-β mRNA secreted by mouse RAW264.7 cells

[0240] Upon exposure to external stimuli, macrophages secrete various cytokines such as IL-1β, IL-6, TNF-α, and IFN-β, which participate in the immune response and play important roles. After treating RAW264.7 macrophages with 50, 100, and 200 μg / mLPJP80-Ia, total RNA was extracted from the cells, and qPCR was used to quantitatively analyze the expression levels of IL-1β, IL-6, TNF-α, and IFN-β mRNA in RAW264.7 cells. Figure 7 As shown, compared with the blank group, 50, 100, and 200 μg / mL PJP80-Ia significantly promoted the expression of IL-6 and TNF-α mRNA in macrophages in a dose-dependent manner, while 100 and 200 μg / mL PJP80-Ia significantly promoted the expression of IL-1β and IFN-β mRNA (P<0.05), indicating that PJP80-Ia of *Pterocarya stenoptera* has a strong immunomodulatory effect.

[0241] In summary, this embodiment draws the following conclusions from the above experiments: (1) Neutral red assay results show that PJP80-Ia polysaccharide enhances the phagocytic function of mouse macrophages in a dose-dependent manner. (2) Griess assay results show that PJP80-Ia polysaccharide promotes NO secretion by cells. (3) Flow cytometry results show that PJP80-Ia polysaccharide promotes ROS secretion by cells. (4) qPCR results show that PJP80-Ia polysaccharide promotes the secretion of IL-1β, IL-6, TNF-α and IFN-β mRNA expression by macrophages. Therefore, PJP80-Ia from *Eriocaulon buergerianum* has immune activity that promotes macrophage phagocytosis and cytokine secretion, and shows great potential to become a natural immune enhancer.

[0242] 4.3 Effects of PJP80-Ia on RAW264.7 cell surface receptors

[0243] Preparation of experimental reagents:

[0244] 30% Acrylamide (AB): Weigh 14.5g of Acrylamide and 0.5g of Bis-acrylamide into a volumetric flask, and dilute to 50mL with ultrapure water. Store at 4℃ protected from light.

[0245] 1.5M Tris-HCl (8.8): Weigh 4.54g of Tris into a volumetric flask, dilute to 50mL with ultrapure water, slowly add concentrated hydrochloric acid until the pH value is 8.8, filter, and store at 4℃.

[0246] 0.5M Tris-HCl (6.8): Weigh 3.028g of Tris into a volumetric flask, dilute to 50mL with ultrapure water, slowly add concentrated hydrochloric acid until the pH value is 6.8, filter, and store at 4℃.

[0247] 1M Tris-HCl (7.5): Weigh 11.365g of Tris into a volumetric flask, dilute to 50mL with ultrapure water, slowly add concentrated hydrochloric acid until the pH value is 7.5, filter, and store at 4℃.

[0248] 10% SDS: Weigh 5g of SDS into a volumetric flask, dilute to 50mL with ultrapure water, dissolve by heating at 50℃, filter, and store at room temperature.

[0249] 10% Ammonium Persulfate (AP): Add 0.1g of AP to an EP tube and dissolve it in 1mL of ultrapure water before use. The solution must be prepared fresh each time.

[0250] 20% Tween: Measure 10 mL of Tween into a volumetric flask, dilute to 50 mL with ultrapure water, and store at 4°C.

[0251] TBS buffer: Weigh 4.4g of NaCl, add 5mL of Tris-HCl (7.5), and bring the volume up to 500mL with ultrapure water. Store at 4℃.

[0252] TBST: Take 100mL of TBST into a beaker, add 240μL of Tween20 to the beaker containing 100mL of TBST and mix well. Prepare fresh before use.

[0253] Blocking solution: Weigh 5g of skim milk powder and dissolve it in 100mL of TBST.

[0254] Electrophoresis buffer: Weigh 1.818g Tris, 11.262g glycine and 0.6g SDS, dissolve in 600mL ultrapure water and store at 4℃.

[0255] Electroporation solution: Weigh 1.5g Tris, 7.2g g glycine and 7.2g Tris into a volumetric flask, add 100mL methanol, and dilute to 500mL with ultrapure water to completely dissolve it. Store at 4℃.

[0256] 12% separating gel (15 mL): Take 4.9 mL of ultrapure water, 3.8 mL of 1.5 mol / L Tris·HCl (8.8), 6.0 mL of 30% AB, 150 μL of 10% SDS, 150 μL of 10% AP, and 6 μL of TEMED into a beaker and mix thoroughly.

[0257] Stacking gel (6 mL): Pipette 4.2 mL of ultrapure water, 0.9 mL of 30% AB, 0.8 mL of 0.5 mol / L Tris·HCl (pH 6.8), 0.06 mL of 10% SDS, 0.06 mL of 10% AP, and 6 μL of TEMED into a beaker and mix thoroughly.

[0258] 4.3.1 Experimental Methods

[0259] (1) Macrophage RAW264.7 cells were cultured using the same method as in “4.1”.

[0260] (2) Western blot analysis was used to detect the expression levels of TLR4, TLR2, Dectin-1, CD11b, and MRC1 proteins in macrophages.

[0261] ① Drug administration: The experimental grouping and drug administration were the same as in “4.1.1(3)”.

[0262] ② Extraction of total cell protein: Aspirate the cell culture medium, wash each well with PBS 3 times, scrape off adherent cells with a cell scraper, put the suspension into a centrifuge tube, centrifuge at 12000 rpm for 15 min, aspirate the supernatant, add 0.5 mL of cell lysis buffer to each tube, repeatedly pipette for 30 min to allow the cells to fully lyse. The entire experiment was performed on ice and stored at -80℃.

[0263] ③ Protein quantification: Follow the BCA instructions to perform the experimental procedures to obtain a standard curve of protein, calculate the protein concentration of the experimental sample group, and use PBS to adjust the protein samples of different concentrations to the same concentration.

[0264] ④ Protein denaturation: Take out the prepared protein sample, add 1 / 4 of the total volume of loading buffer, mix well by pipetting, heat in a water bath for 10 min (100℃), and freeze at -80℃.

[0265] ⑤ Electrophoresis: Neatly install the cleaned glass plates on the gel casting rack, fill the grooves with water, and check if the liquid seal on the glass plates is good. If the liquid level does not drop, the experiment can proceed. Prepare the appropriate separating gel according to the molecular weight of the target protein to achieve better separation. Quickly inject the separating gel into the glass plates, with the distance between the injected liquid level and the edge based on the length of the comb teeth. Use anhydrous ethanol to remove air bubbles from the plates. When obvious creases appear in the glass plates, it indicates that the separating gel has completely solidified. Remove the anhydrous ethanol and immediately add the stacking gel. Insert the comb (1.5 mm) when the stacking gel level is 0.5 cm from the edge of the glass plates to prevent the stacking gel from overflowing and affecting electrophoresis. Take care to avoid generating air bubbles. Once the stacking gel has completely solidified, add freshly prepared electrophoresis buffer, taking into account the liquid level difference between the inside and outside of the glass plate. Slowly remove the comb and align the sample wells. Add 5× loading buffer, pre-stained protein ladder, blank control group, LPS group, and PJP80-I polysaccharide (50, 100, 200 μg / mL) groups in sequence, followed by 5× loading buffer. Connect the electrophoresis apparatus and start electrophoresis (constant voltage 80V). Observe for bubble formation in the electrophoresis tank; bubble formation indicates the start of electrophoresis. After the samples reach the separating gel, increase the voltage to continue electrophoresis (constant voltage 120V) until the bottom of the separating gel is reached, then stop electrophoresis.

[0266] ⑥ Transfer: Determine the location of the target protein based on the molecular weight of the pre-stained protein marker and cut the gel. Soak the sponge pad, filter paper, and NC membrane in transfer buffer. Place them in the transfer tank in the following order: positive electrode - soaked sponge - three layers of filter paper - NC membrane - gel - three layers of filter paper - sponge - negative electrode, and add transfer buffer. Be careful to remove air bubbles between the NC membrane and the gel. Transfer mode settings: constant current 200mA, 90min ice bath.

[0267] ⑦ Sealing: Place the transferred NC film in the sealing solution and seal for 1-2 hours, then place it on a shaker and shake.

[0268] ⑧ Primary antibody incubation: After blocking, wash once with TBST for 10 min. Prepare the primary antibody dilution buffer according to the target protein manufacturer's instructions, distinguish the target band, and place it in the primary antibody incubation chamber. Incubate at 4°C until the next day. Secondary antibody incubation: Remove the NC membrane incubated with the primary antibody, wash three times with TBST for 10 min each time. Prepare the secondary antibody dilution buffer according to the manufacturer's instructions, and place it together with the NC membrane in the secondary antibody incubation chamber. Incubate on a shaker at room temperature for a total of 2 hours.

[0269] ⑨ ECL color development: Wash the membrane with TBST washing solution for 10 minutes each time, for a total of 3 times. Mix equal volumes of chemiluminescence reagent A and B, add the color development solution to the NC membrane, adjust the equipment parameters, and develop the color in the dark for 2 minutes.

[0270] (3) Data processing and analysis: Data processing and analysis are performed in the same way as in method “4.1”.

[0271] 4.3.2 Experimental Results

[0272] TLR2 / 4, Dectin-1, CD11b, and MRC1 surface receptors are the main recognition receptors for polysaccharides to exert their immune effects. Therefore, this example first investigated the effect of PJP80-Ia on the expression levels of macrophage surface receptor proteins. The experimental results are shown in Tables 11 and 12. Compared with the control group, PJP80-Ia can increase the expression of TLR4, TLR2, Dectin-1, CD11b, and MRC1 proteins. 50 μg / mL of PJP80-Ia significantly upregulated the expression level of CD11b in RAW264.7 cells (P<0.05) and extremely significantly upregulated the expression level of MRC1 (P<0.01); 100 and 200 μg / mL... PJP80-Ia significantly upregulated the expression levels of TLR4, TLR2, Dectin-1, CD11b, and MRC1 in RAW264.7 cells (P<0.01). Within the experimental concentration range, the expression of TLR4, TLR2, and CD11b in RAW264.7 cells showed a dose-dependent increasing trend with increasing PJP80-Ia concentration. The protein expression levels of TLR4, TLR2, Dectin-1, CD11b, and MRC1 in RAW264.7 cells were significantly increased in the LPS group. The increase in TLR4 expression in RAW264.7 cells by 200 μg / mL PJP80-Ia was significantly higher than that of other receptors. These results indicate that PJP80-Ia can facilitate signal transduction through macrophage surface receptors TLR4, TLR2, Dectin-1, CD11b, and MRC1.

[0273] Table 11 Effects of PJP80-Ia on the expression of TLR2 and TLR4 receptor proteins in macrophages (n=3)

[0274]

[0275] Table 12 Effects of PJP80-Ia on the expression of Dectin-1, CD11b, and MRC1 receptor proteins in macrophages (n=3)

[0276]

[0277] In summary, the above experiments lead to the following conclusions: Western blot results show that different concentrations of PJP80-Ia can promote the protein expression of TLR2 / 4, Dectin-1, CD11b, and MRC1 in RAW264.7 cells, indicating that the immune effect of PJP80-Ia is related to the cell surface receptors TLR2 / 4, Dectin-1, CD11b, and MRC1 in RAW264.7 cells. Toll-like receptors are highly expressed receptors on the surface of macrophages. PJP80-Ia's promoting effect on TLR4 expression in RAW264.7 cells is relatively stronger than its effect on the expression of other receptors in RAW264.7 cells. Therefore, further investigation will be conducted using Toll-like receptors (TLR2 / 4) to explore their mediated signal transduction pathways.

[0278] 4.4 Effects of PJP80-Ia on the MyD88-dependent pathway in RAW264.7 cells

[0279] The TLR family (TLR1, TLR2, TLR4, TLR5, etc.) can be divided into myeloid differentiation protein 88 (MyD88)-dependent and β-interferon TIR domain adaptor protein (TRIF)-dependent pathways based on different adaptor proteins. Activation of TRAF6 in the MyD88 signaling pathway can activate two different downstream signal transduction pathways: mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB). The above results indicate that PJP80-Ia can promote macrophage phagocytosis and increase the release of macrophage-secreted cytokines through TLR2 / 4, thereby exerting an immune effect. However, the specific mechanism by which *Gynostemma pentaphyllum* polysaccharide exerts its immunomodulatory effect remains unclear. To further elucidate the mechanism of action of *Gynostemma pentaphyllum* polysaccharide in exercising its immunomodulatory function, this invention uses Western blot technology to study the effect of *Gynostemma pentaphyllum* polysaccharide on the MyD88-dependent signaling pathway, thereby revealing the immunomodulatory mechanism of *Gynostemma pentaphyllum* polysaccharide.

[0280] Preparation of experimental reagents: The method is the same as in "4.3".

[0281] 4.4.1 Experimental Methods

[0282] (1) Macrophage RAW264.7 cell culture: The method is the same as "4.1".

[0283] (2) Western blot was used to detect the expression level of MyD88-dependent pathway-related proteins in macrophages: the method was the same as in “4.3”.

[0284] (3) Data processing and analysis: The same method as in “4.1” is used for data processing and analysis.

[0285] 4.4.2 Experimental Results

[0286] (1) Effects of PJP80-Ia on the expression of TIRAP and MyD88 proteins in RAW264.7 cells

[0287] MyD88 and TIRAP are important members of the TLR2 / 4 pathway. After the polysaccharide recognizes the receptor, it forms a complex, initiating the signaling pathway. The adaptor protein TIRAP, located in the TIR domain, serves as a substrate, enabling MyD88 to bind to TLR2 / 4 and transmit signals downstream. To investigate the effect of PJP80-Ia, a polysaccharide from *Cyclocarya paliurus*, on the activation of the MyD88 signaling pathway in RAW264.7 cells, this experiment examined the effects of three different concentrations of PJP80-Ia on macrophages after 24 hours of treatment, and detected the influence of PJP80-Ia on the expression of TIRAP and MyD88 proteins in RAW264.7 cells. The experimental results showed that, compared with the blank control group, PJP80-Ia could increase the expression of TIRAP and MyD88 proteins; 50, 100 and 200 μg / mL PJP80-Ia significantly upregulated the expression of TIRAP and MyD88 in RAW264.7 cells (P<0.01), with MyD88 showing a dose-dependent effect; the LPS group significantly upregulated the expression of TIRAP and MyD88 proteins; the experimental results indicated that PJP80-Ia could activate the TLR2 / 4 / TIRAP / MyD88 signaling pathway for signal transduction.

[0288] (2) Effects of PJP80-Ia on the expression of IRAK4, IRAK1 and their phosphorylated proteins in RAW264.7 cells

[0289] The experimental results showed that, compared with the control group, PJP80-Ia increased the relative expression of p-IRAK4 / IRAK4 and p-IRAK1 / IRAK1 proteins. PJP80-Ia at concentrations of 50, 100, and 200 μg / mL significantly increased the expression of p-IRAK4 and p-IRAK1 proteins in RAW264.7 cells (P<0.01), and this effect was dose-dependent. It did not significantly change the expression levels of IRAK1 and IRAK4 proteins. The LPS group showed a significant increase in the relative expression of p-IRAK4 / IRAK4 and p-IRAK1 / IRAK1 proteins (P<0.01). These results indicate that PJP80-Ia can promote the phosphorylation of IRAK4 and IRAK1 in RAW264.7 cells, suggesting that *Gynostemma pentaphyllum* polysaccharide can induce macrophages to exert an immune effect through the (TLR2 / 4 / TIRAP / MyD88 / IRAK4 / IRAK1) signaling pathway.

[0290] (3) Effect of PJP80-Ia on TRAF6 protein expression in RAW264.7 cells

[0291] The experimental results showed that, compared with the control group, PJP80-Ia increased the expression of TRAF6 protein in RAW264.7 cells. 100 and 200 μg / mL PJP80-Ia significantly affected TRAF6 expression in RAW264.7 cells (P<0.01) in a dose-dependent manner. The LPS-positive control group significantly promoted TRAF6 protein expression. These results indicate that PJP80-Ia can promote TRAF6 expression in RAW264.7 cells, further clarifying the signaling pathway of PJP80-Ia (TLR2 / 4 / TIRAP / MyD88 / IRAK4 / IRAK1 / TRAF6).

[0292] (4) Effects of PJP80-Ia on the expression of TAK1 and p-TAK1 proteins in RAW264.7 cells

[0293] The experimental results showed that, compared with the control group, PJP80-Ia increased the relative expression of p-TAK1 / TAK1 protein. 100 μg / mL PJP80-Ia significantly increased the expression of p-TAK1 in RAW264.7 cells (P<0.05), and 200 μg / mL PJP80-Ia significantly increased the expression of p-TAK1 in RAW264.7 cells (P<0.01). There was no significant change in TAK1 protein expression, but 100 and 200 μg / mL PJP80-Ia significantly upregulated the relative expression of p-TAK1 / TAK1 in RAW264.7 cells. In the LPS group, p-TAK1 and p-TAK1 / TAK1 were significantly increased (P<0.01), while there was no significant difference in TAK1 expression. Experimental results show that PJP80-Ia can promote the expression of p-TAK1 / TAK1 in RAW264.7 cells and promote TAK1 phosphorylation, indicating that PJP80-Ia can exert immunomodulatory effects through the signaling pathway (TLR2 / 4 / TIRAP / MyD88 / IRAK4 / IRAK1 / TRAF6 / TAK1 / p-TAK1).

[0294] (5) Effects of PJP80-Ia on the expression of NF-κB signaling pathway-related proteins in RAW264.7 cells

[0295] The experimental results showed that, compared with the control group, PJP80-Ia could increase the relative expression of p-IKKα / IKKα / β, p-IKKβ / IKKα / β, p-IкBα / IкBα, and p-NF-кB / NF-кB proteins. The three concentrations of PJP80-Ia did not significantly change the expression of IKKα / β and NF-кB in RAW264.7 cells, but showed a dose-dependent inhibition of IкBα protein expression in RAW264.7 cells. All three concentrations of PJP80-Ia promoted the phosphorylation of IKKα / β, IкBα, and NF-кB, and their ability to promote the phosphorylation levels of these three proteins was enhanced. The LPS-positive control group significantly promoted the relative expression of p-IKKα / IKKα / β, p-IKKβ / IKKα / β, p-IкBα / IкBα, and p-NF-кB / NF-кB. Experimental results show that PJP80-Ia can activate IKKα / β in RAW264.7 cells, promote IκBα activation, and enable activated NF-κB to enter the cell nucleus to promote the secretion of cytokines by RAW264.7 cells. This also indicates that PJP80-Ia polysaccharide from *Gynostemma pentaphyllum* can transmit signals through the MyD88-dependent NF-κB signaling pathway (IKKα / β / p-IKKα / p-IKKβ / IκBα / p-IκBα / NF-κB / p-NF-κB).

[0296] (6) Effects of PJP80-Ia on the expression of MAPK signaling pathway-related proteins in RAW264.7 cells

[0297] Phosphorylation of TAK1 can also activate downstream MAPK signaling pathways. The main members of the MAPK family include the ERK, P38, and JNK signaling pathways. ERK1 / 2 is activated by MEK1 / 2; P38 is activated by MKK3 / 6; and JNK is activated by MKK4 / 7. AP-1 is an intracellular transcription activator, a heterodimer composed of c-Fos and c-Jun. Phosphorylation of JNK, ERK1 / 2, and P38 activates c-Fos and c-Jun, thereby activating AP-1. These three signaling pathways participate in important immune processes such as immune regulation and signal transduction. Therefore, this embodiment mainly determines whether the polysaccharide PJP80-I from *Cyclocarya paliurus* transmits signals through the MAPK signaling pathway. The experimental results showed that, compared with the control group, PJP80-Ia could increase the relative expression of p-ERK / ERK protein in macrophages. 200 μg / mL PJP80-Ia had a highly significant effect on the expression of p-MEK1 / 2 protein in RAW264.7 cells (P<0.01). There was no significant difference in the effect of three different concentrations of PJP80-Ia on the ERK1 / 2 protein level in RAW264.7 cells. PJP80-Ia had a highly significant effect on p-ERK protein expression, which was dose-dependent (P<0.01). The LPS positive control group significantly upregulated the expression levels of p-MEK1 / 2, p-ERK, and p-ERK / ERK proteins, indicating that PJP80-Ia polysaccharide from *Gynostemma pentaphyllum* can be used for signal transduction through the ERK signaling pathway (P-MEK1 / 2 / ERK / PERK). Compared with the control group, PJP80-Ia increased the relative expression of p-P38 / P38 protein in macrophages in a dose-dependent manner. 200 μg / mL PJP80-I significantly promoted the expression of p-MKK3 / 6 in RAW264.7 cells (P<0.01). Three different concentrations of PJP80-Ia had no significant effect on the P38 protein level in RAW264.7 cells. 50, 100, and 200 μg / mL PJP80-Ia significantly affected the expression of p-P38 in RAW264.7 cells (P<0.01). The LPS-positive control group significantly upregulated the expression levels of p-MKK3 / 6, p-P38, and p-P38 / P38 proteins. The results indicate that PJP80-Ia can transmit signals through the P38 signaling pathway (p-MKK3 / 6 / P38 / p-P38).Compared with the control group, PJP80-Ia increased the relative expression of p-JNK / JNK proteins in macrophages. 50 μg / mL PJP80-Ia significantly promoted p-MKK7 expression in RAW264.7 cells (P<0.05), while 100 and 200 μg / mL PJP80-Ia significantly promoted p-MKK7 expression in RAW264.7 cells (P<0.01). The three different concentrations of PJP80-Ia polysaccharide had no significant effect on the JNK protein level in RAW264.7 cells, but significantly promoted p-JNK expression in RAW264.7 cells (P<0.01). The LPS-positive control group significantly upregulated the expression levels of p-MKK7, p-JNK, and p-JNK / JNK proteins. The results indicate that PJP80-Ia polysaccharide can transmit signals through the JNK signaling pathway (p-MKK7 / JNK / p-JNK). Compared with the control group, PJP80-Ia increased the relative expression of pc-fos / c-fos and pc-jun / c-jun proteins in macrophages. Three different concentrations of *Pterocarya stenoptera* polysaccharide had no significant effect on the levels of c-fos and c-jun proteins in RAW264.7 cells. 50 μg / mL PJP80-Ia significantly promoted pc-fos expression in RAW264.7 cells (P<0.05). 100 and 200 μg / mL PJP80-Ia had a highly significant effect on the expression of pc-fos and pc-jun in RAW264.7 cells (P<0.01). 50 μg / mL PJP80-Ia had a highly significant effect on the expression of pc-jun in RAW264.7 cells (P<0.01). The LPS-positive control group significantly upregulated the expression levels of pc-fos, pc-jun, pc-fos / c-fos, and pc-jun / c-jun proteins. Experimental results show that PJP80-Ia can promote the secretion of cytokines by macrophages RAW264.7 through signal transduction via the MyD88-dependent signaling pathway MAPKs (p-MEK1 / 2 / ERK / p-ERK, p-MEK3 / 6 / P38 / p-P38, p-MKK7 / JNK / p-JNK, AP-1).

[0298] In summary, this embodiment draws the following conclusions from the above experiments: PJP80-Ia *Gynostemma pentaphyllum* polysaccharide can activate the TIRAP / MyD88 / IRAK4 / p-IRAK4 / IRAK1 / p-IRAK1 / TRAF-6 signaling pathway, subsequently promoting TAK phosphorylation, leading to phosphorylation of NF-κB (IKK complex, IκBα, NF-κB) and MAPKs (MEK1 / 2, MKK3 / 6, MKK7, ERK1 / 2, p38, and JNK1 / 2), promoting intracellular AP-1 (c-fos, c-jun) expression and phosphorylation, and promoting macrophage secretion of cytokines (such as NO, IL-6, IL-1β, and...). The presence of TNF-α and other enzymes in PJP80-I enhances immunity, indicating that the specific mechanism by which PJP80-I regulates immunity can be achieved through the TLR2 / 4MyD88-dependent NF-κB (IKKα / β / p-IKKα / p-IKKβ / IκBα / p-IκBα / NF-κB / p-NF-κB) and MAPKs (p-MEK1 / 2 / ERK / p-ERK, p-MKK3 / 6 / P38 / p-P38, p-MKK7 / JNK / p-JNK, c-fos / pc-fos / c-jun / pc-jun) signaling pathways, thereby promoting the secretion of cytokines IL-1β, IL-6, and TNF-α by macrophages. To more comprehensively elucidate the specific mechanism by which PJP80-I participates in the immune response, investigating the TRIF-dependent signaling pathway is of great significance.

[0299] 4.5 Effects of PJP80-I on the TRIF-dependent pathway in RAW264.7 cells

[0300] The MyD88-independent pathway is activated by binding to TRAM via the TLR3 / 4 domains, recruiting and activating TRIFs. Activated TRIFs interact with and promote the activation of TRAF3. TRAF3 recruits and phosphorylates TBK1 and IKKε kinases of the IKK family. Both can act as IRF3 / 7 kinases, binding to them to form a complex, which in turn phosphorylates and activates IRF3, inducing the transcription of a series of genes. Therefore, this study uses Western blot technology to investigate the effects of PJP80-Ia polysaccharide from *Cyclocarya paliurus* on the MyD88-independent signaling pathway, thereby revealing the specific mechanism of immunomodulation by *Cyclocarya paliurus* polysaccharide.

[0301] Preparation of experimental reagents: The method is the same as "4.3".

[0302] 4.5.1 Experimental Methods

[0303] (1) Macrophage RAW264.7 cell culture: The method is the same as "4.1".

[0304] (2) Western blot analysis of TRIF-dependent pathway protein expression levels in macrophages: the method is the same as in "4.3".

[0305] (3) Data processing and analysis: The same method as in “4.1” is used for data processing and analysis.

[0306] 4.5.2 Experimental Results

[0307] (1) Effects of PJP80-Ia on the expression of TRAM and TRIF proteins in macrophages

[0308] TRAM is a adaptor protein in the TIR domain that can activate the downstream signal TRIF for signal transduction. Therefore, this experiment used three different concentrations of PJP80-Ia to treat macrophages for 24 hours to detect the effects of PJP80-Ia on the expression of TRAM and TRIF proteins in RAW264.7 cells. The results showed that compared with the control group, PJP80-Ia increased the expression of both TRAM and TRIF proteins. 100 μg / mL PJP80-Ia had a significant effect on TRAM expression in RAW264.7 cells (P<0.05), while 200 μg / mL PJP80-Ia had a highly significant effect (P<0.01). The three different concentrations of PJP80-Ia had a highly significant dose-dependent effect on TRIF expression in RAW264.7 cells (P<0.01). The LPS-positive control group significantly promoted the expression levels of both TRAM and TRIF proteins. These results indicate that PJP80-Ia participates in the immune response by transmitting signals through a TRIF-dependent pathway.

[0309] (2) Effects of PJP80-Ia on the expression of TRAF3, TBK1 and p-TBK1 proteins in macrophages

[0310] Once activated, TRIF can activate the downstream signal TRAF3, which in turn activates TBK1 and phosphorylates it for signal transduction. Therefore, in this part of the experiment, macrophages were treated with three different concentrations of PJP80-Ia for 24 hours to detect the effect of PJP80-Ia on the levels of TRAF3, TBK and p-TBK1 proteins in RAW264.7 cells. The experimental results showed that, compared with the control group, PJP80-Ia could increase the expression of TRAF3 and p-TBK1 / TBK1 proteins in a dose-dependent manner (P<0.01). 50 and 100 μg / mL PJP80-Ia had no significant effect on TBK1 expression in RAW264.7 cells, but it significantly promoted the expression of TRAF3 and p-TBK1 in RAW264.7 cells in a dose-dependent manner (P<0.01). The LPS-positive control group significantly promoted the expression levels of TRAF3, p-TBK1, TBK1, and p-TBK1 / TBK1 proteins. These results indicate that PJP80-Ia can activate TRAF3 protein in RAW264.7 cells and promote TBK1 phosphorylation in RAW264.7 cells.

[0311] (3) Effects of PJP80-Ia on the expression of ikkε and p-ikkε, IRF3 and p-IRF3 proteins in macrophages

[0312] Phosphorylation of TBK1 activates downstream signal transduction pathways such as ikkε and IRF3, leading to their phosphorylation and signal transduction. Therefore, this experiment used three different concentrations of PJP80-I to treat macrophages for 24 hours, and detected the effects of PJP80-Ia on the expression levels of IRF3 / p-IRF3 and ikkε / p-ikkε in RAW264.7 cells. The results showed that PJP80-Ia increased the relative expression of p-IRF3 / IRF3 and p-ikkε / ikkε proteins. 50 μg / mL PJP80-Ia showed no significant difference in IRF3 protein expression levels, while all three concentrations significantly promoted p-IRF3 expression in RAW264.7 cells (P<0.01). The three concentrations of PJP80-Ia showed no significant difference in ikkε expression levels in RAW264.7 cells, but 100 and 200 μg / mL PJP80-Ia showed significant differences. PJP80-Ia showed a highly significant difference in p-ikkε protein expression in a dose-dependent manner. The LPS-positive control group significantly promoted the expression levels of p-IRF3, p-ikkε, p-IRF3 / IRF3, and p-ikkε / ikkε proteins. The experimental results indicate that PJP80-Ia can activate and phosphorylate ikkε and IRF3 proteins in RAW264.7 cells, which also demonstrates that PJP80-I polysaccharide from *Gynostemma pentaphyllum* can promote macrophage secretion of cytokines through the TLR2 / 4TRIF-dependent pathway (TLR2 / 4 / TRAM / TRIF / TRAF3 / TBK1 / p-TBK1 / IRF3 / p-IRF3).

[0313] In summary, this embodiment draws the following conclusions from the above experiments: PJP80-Ia can activate TRAM, TRIF, and TRAF3 in RAW264.7 macrophages and promote the phosphorylation of TBK1, ikkε, and IRF3. This indicates that PJP80-Ia can also activate macrophages through the TLR2 / 4TRIF-dependent signaling pathway (TLR2 / 4 / TRAM / TRIF / TRAF3 / TBK1 / p-TBK1 / IRF3 / p-IRF3 / ikkε / p-ikkε). The preliminary study on the immunomodulatory activity of PJP80-Ia on RAW264.7 macrophages demonstrates that PJP80-Ia can promote the secretion of the cytokine IFN-β by macrophages. The main reason for this immunomodulatory activity is that PJP80-Ia activates macrophages and promotes their secretory function through the TLR2 / 4TRIF-dependent signaling pathway.

[0314] 4.6 Effects of PJP80-Ia on the immune activity of RAW264.7 cells under TLR2 / 4 inhibitor intervention

[0315] Polysaccharides bind to PRRs on the surface of macrophages, triggering immune signaling pathways, including the TLRs-MyD88-MAPK / NF-κB dependent signaling pathway and the TLRs-TRIF dependent signaling pathway, enhancing cell viability, promoting the release of cytokines and NO, and improving macrophage immune activity. In the above experiments, our results confirmed that PJP80-Ia can activate RAW264.7 macrophages in vitro and enhance their immune activity. Whether this mechanism is the basis for the immunomodulatory effect of PJP80-Ia requires further investigation. Therefore, to investigate whether TLRs are upstream signals for the in vitro immune-enhancing effect of PJP80-Ia, this embodiment conducted a specific receptor antagonism experiment. By comparing the effects of PJP80-Ia on macrophage immunity before and after the treatment of TLR2 and TLR4 receptor antagonists (measuring NO release, mRNA expression levels of IL-6, IL-1β, TNF-α, and IFN-β), the binding of PJP80-Ia to macrophage receptors was studied (Western blot was used to detect the expression levels of key proteins and their corresponding phosphorylation in the macrophage NF-κB signaling pathway after treatment with TLR2 and TLR4 receptor antagonists), thus verifying the interaction between PJP80-Ia and TLRs on the macrophage membrane surface.

[0316] Preparation of experimental reagents: Same as in "4.3"

[0317] 4.6.1 Experimental Methods

[0318] (1) Macrophage RAW264.7 cells were cultured using the same method as in “4.1”.

[0319] (2) Effect of TLR2 / 4 inhibitors on the ability of PJP80-Ia to regulate NO secretion in RAW264.7 macrophages: Logarithmic growth phase cells were taken and subjected to 1×10 5 Cells were seeded at a density of 100 μL / mL in 96-well plates. After culturing for 24 h, cells were treated with TLR2 inhibitor C29 and TLR4 inhibitor TAK-242 for 4 h each. After removing the supernatant, the cells were washed three times with PBS and cultured in PJP80-I medium for 24 h. The supernatant was collected, and the procedure was performed according to the Griess instructions, as in section 4.2.1. The specific experimental groups are shown in Table 13.

[0320] Table 13 Effects of TLR2 and TLR4 inhibitors on the NO secretion capacity of PJP80-Ia-regulated macrophage RAW264.7 cells Experimental grouping and cell treatment

[0321]

[0322] (3) Effects of TLR2 / 4 inhibitors on the mRNA expression levels of macrophage RAW264.7 cytokines IL-6, IL-1β, TNF-α and IFN-β regulated by PJP80-Ia: The experimental grouping and administration methods were the same as in “4.6.1(2)”.

[0323] (4) Effect of TLR2 / 4 inhibitors on the expression of NF-κB signaling pathway-related proteins regulated by PJP80-Ia in RAW264.7 macrophages. The administration method is as described in “4.6.1(2)”, the experimental groups are as shown in Table 14, and the experimental procedures are the same as “4.3.1(2)”.

[0324] Table 14. Experimental grouping and cell treatment of TLR2 and TLR4 inhibitors on the expression of NF-κB signaling pathway-related proteins in PJP80-Ia-regulated macrophage RAW264.7 cells.

[0325]

[0326] (5) Data processing and analysis: Data processing and analysis shall be performed in the same manner as in method “4.1.1”.

[0327] 4.6.2 Experimental Results

[0328] (1) Effects of TLR2 / 4 inhibitors on PJP80-Ia regulation of NO secretion in RAW264.7 macrophages

[0329] To further investigate whether TLR2 / 4 is the receptor for PJP80-Ia activation of RAW264.7 macrophages, RAW264.7 cells were pretreated with C29 and TAK-242. The effect of the inhibitor on PJP80-Ia-stimulated NO secretion by macrophages was analyzed to verify the main polysaccharide receptor. The experimental results are shown in Table 15. Compared with the control group, the NO secretion of the single C29 and TAK-242 inhibitor treatment groups was significantly reduced (P<0.01). The LPS positive control group and the PJP80-Ia polysaccharide treatment group significantly promoted the secretion of NO by macrophages (P<0.01). After cell pretreatment with the TLR2 inhibitor (C29), the NO secretion of LPS positive drug and PJP80-Ia polysaccharide was significantly inhibited (P<0.01). After pretreatment with the TLR4 inhibitor (TAK-242), the NO secretion of LPS positive drug and PJP80-Ia polysaccharide was significantly reduced (P<0.01), and the inhibitory effect was better than that of the TLR2 inhibitor. The experimental results show that the effect of PJP80-Ia in promoting NO secretion in RAW264.7 cells is related to the TLR2 and TLR4 signaling pathways.

[0330] Table 15 Effects of TLR2 and TLR4 pathway inhibitors on PJP80-Ia on NO secretion in mouse RAW264.7 cells (n=3)

[0331]

[0332] (2) Effects of TLR2 / 4 inhibitors on the expression levels of IL-6, IL-1β, TNF-α, and IFN-β of macrophage RAW264.7 cells regulated by PJP80-Ia

[0333] IL-6, IL-1β, TNF-α, and IFN-β are important pro-inflammatory cytokines related to the host's innate immune response and play a role in clearing pathogenic microorganisms. Previously, we used qPCR to detect the transcriptional levels of IL-6, IL-1β, TNF-α, and IFN-β mRNA in macrophages induced by PJP80-Ia. The results showed that PJP80-Ia enhanced the expression of these four cytokines in a dose-dependent manner. To further investigate the role of Toll-like receptors in inducing the expression of inflammatory cytokines, macrophages were pretreated with TLR2 and TLR4 receptor inhibitors to explore the expression levels of IL-6, IL-1β, TNF-α, and IFN-β mRNA. The experimental results are as follows: Figure 8 As shown, compared with the control group, the expression level of TNF-α mRNA was significantly reduced in the single C29 and TAK-242 inhibitor treatment groups (P<0.01), and the expression level of IFN-β mRNA was significantly reduced in the single TAK-242 inhibitor treatment group; while the LPS positive control group and the PJP80-I polysaccharide treatment group promoted the expression levels of IL-6, IL-1β, TNF-α and IFN-β mRNA in macrophages (P<0.01). After cell pretreatment with the TLR2 inhibitor (C29), LPS... There was no significant difference in TNF-α mRNA expression levels between the LPS-positive drug and PJP80-I polysaccharide, but the expression levels of IL-6, IL-1β, and IFN-β mRNAs decreased significantly (P<0.01). Pretreatment with the TLR4 inhibitor (TAK-242) significantly inhibited the expression levels of IL-6, IL-1β, TNF-α, and IFN-β mRNAs in both the LPS-positive drug and PJP80-I polysaccharide (P<0.01), with TAK-242 showing a more significant inhibitory effect than C29. These results indicate that Toll-like receptor activation is a key receptor pathway for PJP80-I to promote the upregulation of pro-inflammatory cytokine expression in RAW264.7 macrophages.

[0334] (3) Effects of TLR2 / 4 inhibitors on the regulation of NF-κB signaling pathway-related protein expression in RAW264.7 macrophages by PJP80-I

[0335] This part of the experiment used C29 and TAK-242 inhibitors to pretreat RAW264.7 cells and then further investigated the effect of PJP80-Ia on the NF-κB pathway in cells. The experimental results are shown in Tables 16 to 19. Compared with the control group, the TAK-242 inhibitor treatment group significantly reduced the expression of IKKα / β, p-IKKα, p-IкBα, and p-NF-кB (P<0.01) and significantly enhanced the expression of IкBα (P<0.01); the C29 inhibitor treatment group significantly reduced the expression of p-IKKα, p-IкBα, and p-NF-кB (P<0.01), significantly reduced the expression of p-IKKβ (P<0.05), and significantly enhanced the expression of IкBα (P<0.01); the PJP80-Ia group significantly enhanced the expression of p-IKKα, p-IKKβ, p-IкBα, and p-NF-кB in RAW264.7 cells and significantly inhibited the expression of IкBα (P<0.01), which is consistent with the previous experimental results. Pretreatment of cells with the TLR2 inhibitor (C29) resulted in a significant decrease in the expression of p-IKKβ, p-IκBα, and p-NF-κB proteins regulated by PJP80-Ia, and an increase in the expression of IκBα protein (P<0.01). Treatment of cells with the TLR4 inhibitor (TAK-242) resulted in a highly significant decrease in the expression of p-IKKα, p-IKKβ, and p-NF-κB proteins regulated by PJP80-I (P<0.01), and a significant increase in the expression of IκBα protein (P<0.05). These results indicate that PJP80-Ia treatment of RAW264.7 cells promotes the expression of NF-κB-related proteins in the downstream key signaling pathway of the TLR2 / 4 receptor, but this effect is weakened by the inhibition of the TLR2 / 4 receptor on the surface of RAW264.7 cells. Furthermore, PJP80-I induces IκBα protein degradation, but this effect is weakened by the inhibition of TLR2 / 4 receptors. Experiments using specific inhibitors confirmed that the immunogenicity of RAW264.7 macrophages activated by *Gynostemma pentaphyllum* polysaccharide is closely related to the MyD88-dependent NF-κB signaling pathway mediated by TLR2 / 4 receptors on the cell surface.

[0336] Table 16 Effects of TLR2 inhibitor (C29) and TLR4 inhibitor (TAK-242) on the expression of IKKα / β and p-IKKα proteins in PJP80-Ia-induced macrophages (n=3)

[0337]

[0338] Table 17 Effects of TLR2 inhibitor (C29) and TLR4 inhibitor (TAK-242) on the expression of IKKα / β and p-IKKβ proteins in PJP80-I induced macrophages (n=3)

[0339]

[0340] Table 18 Effects of TLR2 inhibitor (C29) and TLR4 inhibitor (TAK-242) on the expression of IκBα and p-IκBα proteins in PJP80-I induced macrophages (n=3)

[0341]

[0342] Table 19 Effects of TLR2 inhibitor (C29) and TLR4 inhibitor (TAK-242) on the expression of NF-κB and p-NF-κB proteins in PJP80-I-induced macrophages (n=3)

[0343]

[0344] In summary, the experiments yielded the following conclusions: when the TLR2 / 4 receptors on the surface of RAW264.7 cells were inhibited, the NO secretion-promoting effect of PJP80-Ia was weakened, and the mRNA expression levels of cytokines IL-6, IL-1β, TNF-α, and IFN-β were downregulated. Furthermore, the TLR2 / 4 inhibitor weakened the regulatory effect of PJP80-I on the expression of NF-κB (IKK complex, IκBα, NF-κB) and its phosphorylated proteins. These results indicate that PJP80-Ia's regulation of RAW264.7 cell immune activity is related to the downstream NF-κB signaling pathway mediated by the cell surface receptor TLR2 / 4.

[0345] The immunomodulatory mechanism of PJP80-Ia, a polysaccharide from *Cyclocarya paliurus*, on mouse macrophage RAW264.7 cells may involve its binding to TLR2 / 4 receptors on the cell surface of RAW264.7 cells. This binding occurs through the TLR2 / 4MyD88-dependent NF-κB (IKKα / β / p-IKKα / p-IKKβ / IκBα / p-IκBα / NF-κB / p-NF-κB) signaling pathway and the MAPK signaling pathway (p-MEK1 / 2 / ERK / p-ERK, p-MK). K3 / 6 / P38 / p-P38, p-MKK7 / JNK / p-JNK, c-fos / pc-fos, c-jun / pc-jun synergistically promote the release of cytokines such as TNF-α, IL-6, and IL-1β from mouse macrophages RAW264.7. Through the TRIF-dependent signaling pathway (TRAM / TRIF / TRAF3 / p-TBK1 / IRF3 / p-IRF3 / ikkε / p-ikkε), RAW264.7 is stimulated to secrete IFN-β, thereby enhancing its phagocytic activity and immune function.

[0346] 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 within the protection scope of the present invention.

Claims

1. A method for preparing PJP80-Ia polysaccharide from the North Green Dragon Skin, characterized in that, The method comprises the following steps: (1) extracting the supernatant of the north green dragon skin by fractional alcohol precipitation, and collecting the precipitate obtained by alcohol precipitation at an ethanol concentration of 80%; The fractional alcohol precipitation comprises the following steps: after the supernatant of the north green dragon skin is filtered, the filtrate is collected; the filtrate is concentrated under reduced pressure to 1 / 3 of the original volume, 95% ethanol is added, and the solution is quickly stirred to make the final ethanol concentration in the solution reach 20%, and then the solution is left overnight to obtain precipitate I and supernatant I; the above steps are repeated, 40% ethanol is added to the supernatant I to make the final ethanol concentration in the solution reach 40%, and then the solution is left overnight to obtain precipitate II and supernatant II; the above steps are repeated, 60% ethanol is added to the supernatant II to make the final ethanol concentration in the solution reach 60%, and then the solution is left overnight to obtain precipitate III and supernatant III; the above steps are repeated, 80% ethanol is added to the supernatant III to make the final ethanol concentration in the solution reach 80%, and then the solution is left overnight to obtain precipitate IV and supernatant IV; the precipitate IV is washed with anhydrous ethanol and freeze-dried to obtain the north green dragon skin polysaccharide PJP80; (2) decolorizing the north green dragon skin polysaccharide PJP80 obtained after the fractional alcohol precipitation by AB-8 macroporous resin, comprising the following steps: the north green dragon skin polysaccharide PJP80 obtained after the fractional alcohol precipitation is weighed, dissolved in distilled water, and then AB-8 macroporous resin is added and stirred to decolorize, and then the filtrate is obtained by vacuum filtration and freeze-dried; the ratio of the north green dragon skin crude polysaccharide, distilled water and AB-8 macroporous resin is 5 g:100 mL:50 g; (3) after the decolorization, further comprising the steps of DEAE-52 anion exchange chromatography column separation and purification and Sephadex G-50 gel chromatography column separation and purification; The DEAE-52 anion exchange chromatography column separation and purification comprises the following steps: activating DEAE-52 cellulose; column loading, DEAE-52 anion exchange chromatography column pretreatment; preparing a north green dragon skin polysaccharide solution and loading; preparing an eluent and collecting a sample, collecting the eluent obtained by washing with water, and freeze-drying to obtain the north green dragon skin polysaccharide PJP80-I; The Sephadex G-50 gel chromatography column separation and purification comprises the following steps: activating Sephadex G-50 gel; column loading, Sephadex G-50 gel chromatography column pretreatment; preparing a PJP80-I solution and loading, using deionized water as the eluent, collecting the elution peak, and freeze-drying to obtain the north green dragon skin polysaccharide PJP80-Ia.

2. The method of claim 1, wherein, The preparation method of the supernatant of the north green dragon skin comprises the following steps: performing a Soxhlet extraction on the feed liquid of the north green dragon skin.

3. The preparation method according to claim 2, characterized in that, The feed liquid of the north green dragon skin comprises north green dragon skin powder and water, and the ratio of the north green dragon skin powder to water is 1 g:30 mL, and the temperature of the water is 90°C.

4. The preparation method according to claim 2, characterized in that, The parameters of the Soxhlet extraction include: 5000 r / min, 30 s / time, and 2 times of extraction.

5. The process according to any one of claims 1 to 4, characterized in that, In step (2), the stirring time is 2 h.

6. The process according to any one of claims 1 to 4, characterized in that, The DEAE-52 anion exchange column separation and purification in step (3) is carried out by using 0-0.5 mol / L NaCl solution as eluent, 200 mL for each concentration, flow rate 1 mL / min, 2 mL / tube for collecting eluent, and the eluent obtained when the concentration of NaCl solution is 0 is collected, and then freeze-dried to obtain the polysaccharide PJP80-I from north blue dragon skin.

7. The process according to any one of claims 1 to 4, characterized in that, The flow rate of Sephadex G-50 gel chromatography column separation and purification in step (3) is 1 mL / 3 min.

8. The polysaccharide PJP80-Ia prepared by the preparation method of any one of claims 1-7 is used in any one or more of (1) to (5): (1) used alone or as one component to prepare an immunomodulator; (2) used alone or as one component to prepare an antitumor preparation; (3) used alone as a food raw material; (4) used alone or as one component to prepare a health food with immunomodulatory function; (5) used alone or as one component to prepare a health food with antioxidant function.

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