Ginseng polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses ginseng polysaccharide as well as a preparation method and application thereof, and belongs to the field of polysaccharide extraction, separation and application. The invention aims to solve the problem that the ginseng polysaccharide with uniform structure and stable property is difficult to obtain by the existing ginseng polysaccharide extraction method. The ginseng polysaccharide disclosed by the invention has a clear structure, a uniform structure and stable properties, is beneficial to research on the pharmacological action mechanism of the ginseng polysaccharide, and provides technical support and theoretical basis for wide application of the ginseng polysaccharide in the fields of medicines and health products. The novel ginseng polysaccharide is successfully extracted, separated and purified, and the analysis problem caused by a complex chemical structure of the ginseng polysaccharide is remarkably solved. The ginseng polysaccharide is obtained by extracting, separating and purifying ginseng decoction pieces, the monosaccharide composition and space structure of the ginseng polysaccharide are disclosed, and basic data are provided for development and utilization of the ginseng polysaccharide. The ginseng polysaccharide disclosed by the invention is used for preparing an anti-tumor drug capable of inhibiting the biological activity of human liver cancer cells and human gastric cancer cells.

Description

Technical Field

[0001] This invention discloses a ginseng polysaccharide, its preparation method, and its application, belonging to the field of polysaccharide extraction, separation, and application. Background Technology

[0002] Ginseng (Panax ginseng CA Meyer) is a perennial herb belonging to the Araliaceae family and the Panax genus, and is abundant in the Changbai Mountains region of Jilin Province. According to the *Compendium of Materia Medica*, ginseng is sweet, slightly bitter, and slightly warm in nature, and enters the spleen, lung, heart, and kidney meridians. Ginseng polysaccharides are one of its main pharmacologically active components, possessing various biological activities such as anti-tumor and immunomodulatory effects. Ginseng polysaccharides have broad application prospects, and their potential mechanisms of action are gradually being discovered by researchers. These mechanisms are usually inextricably linked to the structure of the polysaccharides. However, as biological macromolecules, polysaccharides are characterized by complex structures and difficulties in analysis. Furthermore, obtaining polysaccharides with a uniform and stable structure during preparation is a complex and challenging process. The cellulose in the ginseng cell wall intertwines with the ginseng polysaccharides, forming a strong structural barrier that hinders sufficient contact between the solvent and the polysaccharides, making effective extraction difficult. Therefore, extraction methods need further improvement.

[0003] With advancements in analytical techniques and in-depth research, polysaccharides are poised for increasingly broad applications in disease treatment and health product development, and are expected to occupy a significant position in the global health product market. Meanwhile, due to their numerous activities and lack of toxic side effects, polysaccharides have been widely considered by researchers as potential anti-cancer candidates. The antitumor activity of polysaccharides is closely related to their structure, and molecular modification and high bioavailability can enhance their antitumor activity. However, the complex chemical structure of ginseng polysaccharides remains a key obstacle to in-depth research. The diverse combinations of monosaccharide residues, the different linkages of glycosidic bonds, and the highly branched molecular structure make it extremely difficult to resolve their spatial conformation. This is a bottleneck that urgently needs to be overcome in the research and application of ginseng polysaccharides, and will also provide crucial clues for innovative drug development. Summary of the Invention

[0004] This invention addresses the problem of existing ginseng polysaccharide extraction methods failing to yield structurally uniform and stable ginseng polysaccharides. It proposes a method for preparing ginseng polysaccharides and their applications. The ginseng polysaccharides of this invention possess a well-defined, uniform structure and stable properties, facilitating research into the pharmacological mechanisms of ginseng polysaccharides and providing technical support and theoretical basis for their widespread application in pharmaceutical and health products.

[0005] The structural formula of the ginseng polysaccharide of this invention is as follows:

[0006] In the structural formula, n and m are positive integers and n = 6m; ​​x, y and z are positive integers and x + y + z = 4.

[0007] Although arabinose is not the most abundant component in the ginseng polysaccharide composition of this invention, it is a key component for initiating antitumor activity and plays a crucial role in tumor cell recognition and anti-proliferation signal initiation during the antitumor process. At the molecular structure level, the core innovation of this ginseng polysaccharide lies in the construction of a dynamically stable three-dimensional framework based on the main chain and branches. Structurally, the three-dimensional framework of ginseng polysaccharide is constructed based on the main chain and branches. The main chain, composed of monosaccharides linked by glycosidic bonds to form a linear structure, serves as the core support of the framework. The branches originate from specific sites on the main chain and are composed of various monosaccharides, enriching the structural levels of the framework. Combined with information such as molecular weight and molecular configuration, macroscopically, ginseng polysaccharide exhibits a random coil or irregular conformation. In this conformation, the main chain and branches do not exhibit highly regular folds but interact and extend in a relatively loose and random manner, forming a three-dimensional morphology similar to a flexible network. This flexible structure facilitates its interaction with biomacromolecules such as cell surface receptors. The root-mean-square radius of 16.4 nanometers and its significant spatial extensibility reflect that the framework is not a tightly folded structure, but rather a flexible and loosely arranged spatial arrangement. Monosaccharide segments (main chain and branches) are relatively extended in space, interwoven to form a three-dimensional network with a certain span. In contrast, some existing polysaccharides may undergo significant conformational changes in their main chain and branch structures when the external environment (such as temperature and pH changes) changes slightly, leading to loss or alteration of function. They lack the stable and adjustable characteristics of the ginseng polysaccharide framework structure of this invention. The dynamically stable three-dimensional framework based on the main chain and branches of this invention also serves as a structural carrier for the antitumor effects of functional monosaccharides (arabinose). This three-dimensional framework not only provides spatial configuration support but also optimizes molecular flexibility and the exposure of functional groups. This conformation results in weaker inter-segment binding in environments such as solution, giving the molecule overall good flexibility. When interacting with tumor cells, the flexible molecular structure facilitates conformational changes, adapting to the binding requirements of cell surface targets and creating conditions for subsequent functional groups to exert their effects. Simultaneously, the flexible and loose spatial arrangement allows molecular segments to move freely within a certain range, further enhancing molecular flexibility. This enables polysaccharide molecules to more efficiently contact and interact with different targets on the tumor cell surface, creating an active environment for key units such as arabinose to bind to tumor cell surface targets and initiate anti-metastasis or pro-apoptosis signals. Without this three-dimensional framework, even the presence of functional monosaccharides such as arabinose would prevent the effective exertion of antitumor activity. This structural characteristic of framework-linked functional unit antitumor activity clarifies the decisive supporting role of the overall molecular integrity in ginseng polysaccharides in tumor suppression, further refining the relationship between structure and activity in polysaccharide molecules.

[0008] The preparation method of the ginseng polysaccharide of the present invention is carried out according to the following steps:

[0009] 1. Weigh out ginseng slices and soak them in distilled water for 8-12 hours to obtain a mixture; the content of ginseng slices in the mixture is 100g / L;

[0010] 2. The soaked ginseng slices and solution are extracted using a hot water extraction process to obtain the extract;

[0011] The hot water extraction method is as follows: under constant temperature conditions of 85~95℃, distilled water is used for extraction 2~4 times, each extraction lasting 2~4 hours, and the extracts are combined and collected.

[0012] The ratio of distilled water to ginseng slices added in each extraction is 3-8L: 400-500g;

[0013] 3. Centrifuge the collected extract to remove large particulate impurities; then remove small particulate impurities by vacuum filtration to obtain a purified extract.

[0014] The centrifugation process was carried out at a speed of 4000 rpm for 10 minutes.

[0015] 4. Concentrate the purified extract to obtain a concentrated solution, add ethanol solution to the concentrated solution and carry out alcohol precipitation at a low temperature of 2~8℃;

[0016] The concentration process is as follows: a rotary evaporation process is used, with a temperature of 55~65℃ and a time of 4~8 hours, to concentrate to 10~20% of the volume of the purified extract;

[0017] The ethanol solution has a mass fraction of 99%, the volume ratio of ethanol solution to concentrate is (5~6):1, and the alcohol precipitation time is 12~24h;

[0018] 5. Centrifuge the solution after alcohol precipitation to separate the precipitate. After freeze-drying the precipitate, the crude ginseng polysaccharide product is obtained.

[0019] The centrifugation process was carried out at a speed of 3000 rpm for 15 minutes.

[0020] 6. Ginseng crude polysaccharide was redissolved in 15-25 times its volume of water in a beaker to obtain a polysaccharide aqueous extract. The extract was then deproteinized using the Sevag method and finally freeze-dried to obtain deproteinized ginseng polysaccharide.

[0021] The volume ratio of the crude ginseng polysaccharide to distilled water is 1:15~25;

[0022] The process of deproteinizing ginseng crude polysaccharide using the Sevag method is as follows: the polysaccharide aqueous extract and Sevag reagent are mixed and extracted for 30 min, centrifuged at 4000 rpm for 10 min, the precipitate is removed, the solution is collected, and one deproteinization process is completed; the deproteinization process is repeated 5-7 times.

[0023] 7. Swell DEAE-52 cellulose in ultrapure water for 3 to 7 days, then sequentially perform alkali activation, acid activation, and alkali activation. After each activation, wash DEAE-52 cellulose with ultrapure water until the pH reaches 6-8. Pack DEAE-52 cellulose into an anion exchange chromatography column. Mix the deproteinized ginseng polysaccharide obtained in step 6 with distilled water to obtain a 10 mg / mL ginseng polysaccharide solution, then centrifuge and filter sequentially. Load the resulting solution into an anion exchange chromatography column and let it stand for 12-24 hours. After standing, inject eluent into the anion exchange chromatography column and collect the eluent discharged from the column. Dialyze the collected eluent with running water. Finally, freeze-dry to obtain purified ginseng polysaccharide. Collect 2 mL of eluent in 100 tubes.

[0024] The centrifugation process was carried out at a speed of 4000 rpm for 10 minutes.

[0025] The filtration uses a 0.45μm aqueous filter membrane;

[0026] The flow rate during sample loading is 1 mL / min;

[0027] The fluid dialysis was performed using a 3.5 kDa dialysis bag for 24 hours.

[0028] The ratio of DEAE-52 cellulose to ultrapure water is 50g: 500~800mL;

[0029] The alkaline activation uses 0.4~0.6 mol / L NaOH, and the acid activation uses 0.4~0.6 mol / L HCl;

[0030] 8. Sephacryl S-300 propylene dextran gel (preserved in anhydrous ethanol) was washed with ultrapure water to remove the anhydrous ethanol and packed into an anion exchange chromatography column. The purified ginseng polysaccharide obtained in step 7 was mixed with distilled water to obtain a 10 mg / mL ginseng polysaccharide solution. The solution was then centrifuged and filtered sequentially. The resulting solution was loaded into an anion exchange chromatography column and allowed to stand for 12–24 h. After standing, ultrapure water was injected into the anion exchange chromatography column as the eluent. The eluent discharged from the anion exchange chromatography column was collected. The collected eluent was dialyzed with running water and finally freeze-dried to obtain ginseng polysaccharide (GPS). 100 tubes of eluent were collected, with 1 mL collected from each tube.

[0031] The centrifugation process was carried out at a speed of 1500 rpm for 10 min.

[0032] The filtration uses a 0.22μm aqueous filter membrane;

[0033] The flow rate during sample loading is 1 mL / min;

[0034] The fluid dialysis uses a 3.5 kDa dialysis bag, with a dialysis time of 24 hours and distilled water changed every 4 hours;

[0035] The ginseng polysaccharide of this invention is used to prepare an antitumor drug with bioactivity that inhibits human liver cancer cells and human gastric cancer cells.

[0036] The beneficial effects of this invention are:

[0037] This invention successfully extracts, separates, and purifies novel ginseng polysaccharides, significantly improving the analytical challenges posed by the complex chemical structure of ginseng polysaccharides. Through extraction, separation, and purification of ginseng slices, ginseng polysaccharides were obtained, and their monosaccharide composition and spatial structure were revealed, providing fundamental data for the development and utilization of ginseng polysaccharides.

[0038] The ginseng polysaccharide of this invention effectively reduces the interference caused by monosaccharide residue combinations, glycosidic bond linkages, and highly branched molecular structures, providing a clear material basis for in-depth research on the spatial conformation of ginseng polysaccharide. This helps researchers to more accurately grasp the structural characteristics of ginseng polysaccharide, thus pointing the way for subsequent research.

[0039] Meanwhile, given the close relationship between the antitumor activity of polysaccharides and their structure, as well as the enhancing effects of molecular modification and high bioavailability on their activity, the novel ginseng polysaccharides extracted, isolated, and purified in this invention, with a well-defined structure, were tested using the CCK8 assay to show that the ginseng polysaccharides inhibited tumor growth in human liver cancer cells HepG2, human gastric cancer cells SGC7901, and human breast cancer cells MCF-7. The high purity of the ginseng polysaccharides in this invention is conducive to improving bioavailability, thereby significantly enhancing antitumor activity, providing a more promising material for the research of anticancer candidate substances, and is expected to promote the development of anticancer drugs.

[0040] This invention uses a water extraction and alcohol precipitation method to extract crude ginseng polysaccharides from ginseng, with a yield of 25.2% calculated based on the weight of the ginseng material. Preliminary purification through deproteinization yielded refined ginseng polysaccharides with a yield of 30.45%, a sugar content of 74.18%, a protein content of 3.2%, and a uronic acid content of 4.2%. Polysaccharide fractions were obtained using DEAE-52 cellulose gel, with the aqueous fraction ultimately collected, yielding a yield of 52.14%, a sugar content of 86.84%, and a protein content of 1.6%. After purification using a Sephacryl S-300 propylene dextran gel, the yield was 70.09%, the sugar content was 95.32%, and the protein content was 0.5%. Using human hepatocellular carcinoma cells (HepG2) and human gastric carcinoma cells (SGC7901) as research subjects, the antitumor activity of purified ginseng polysaccharides was initially screened using the CCK8 assay. Ginseng polysaccharides showed inhibitory activity against human hepatocellular carcinoma cells (HepG2), human gastric carcinoma cells (SGC7901), and human breast cancer cells (MCF-7) in a dose-dependent manner, with maximum inhibition rates of 22.87%, 24.68%, and 39.45%, respectively.

[0041] In most purification experiments of polysaccharides and small molecule proteins, dextran gels (such as the Sephadex G series) are commonly used gel filtration chromatography media. Although propylene dextran gels (represented by Sephacryl S-300 in this experiment) belong to the same type of medium and have dextran as their core component, their performance and application scenarios differ significantly due to differences in chemical structure and preparation process. Dextran gels are formed only by cross-linking dextran with epichlorohydrin, resulting in low mechanical strength. They are easily compressed and deformed under high flow rates and high column pressures. Their chemical stability is limited to neutral to weakly acidic environments (pH 2-13), and their pore size is easily affected by solvent polarity and ionic strength, resulting in a narrow separation range (mostly <1×10⁵ Da). Furthermore, they exhibit slow swelling rates and large volume fluctuations, making them more suitable for the separation of small molecules. Acrylic dextran gels, through cross-linking of the dextran backbone with N,N'-methylenebisacrylamide, introduce acrylamide monomers to form a copolymer structure. They exhibit excellent mechanical strength and compressibility, can withstand high column pressures and flow rates, and possess extremely high chemical stability (pH 2-14). They are stable to strong acids, strong bases, oxidants, and organic solvents such as anhydrous ethanol. Their pore size is minimally affected by environmental factors, and they offer a wide separation range (e.g., Sephacryl S-300 covering 1×10⁻⁶ cells). 4 -1.5×10 6 (Da), with rapid swelling and stable volume, making it more suitable for the separation of large molecules. From the purification process perspective, the propylene dextran gel S-300 first provides crucial support for the separation of target polysaccharides through a precise molecular sieve effect. (Propylene dextran gel 1×10) 4 -1.5×10 6The separation range of the propylene dextran gel precisely matches the molecular weight range of ginseng polysaccharides, effectively separating the target polysaccharide from small molecule impurities in the system. This ensures precise enrichment of the target polysaccharide and avoids impurity aggregation affecting subsequent structure and activity. Simultaneously, the high stability of the propylene dextran gel to anhydrous ethanol prevents skeletal deformation or degradation during the pretreatment process of anhydrous ethanol storage followed by ultrapure water washing. The uniform gel bed formed after loading lays the foundation for stable subsequent loading, settling, and elution, ensuring the reproducibility of the purification process and guaranteeing a stable yield of the target polysaccharide in each purification. Crucially, the mild purification environment provided by the propylene dextran gel is the core guarantee for preserving the unique structure and activity of ginseng polysaccharides. On the one hand, the propylene dextran gel exhibits chemical stability to withstand neutral to strong acids and alkalis, ensuring that the ultrapure water elution system used in the experiment (free of corrosive reagents and high-concentration solvents) minimizes damage to the active promoter of arabinose in the polysaccharide. As a key unit for tumor cell recognition and anti-proliferation signal initiation, arabinose is sensitive to the chemical environment, and the mild elution conditions ensure its structural integrity, preserving the function of its active promoter. On the other hand, the gel's excellent mechanical strength and compressibility maintain bed stability during 12-24 hours of settling, preventing column pressure fluctuations due to gravity compression. This avoids mechanical breakage of polysaccharide molecules during elution, fully preserving the "dynamically stable three-dimensional framework of main chain-branched chains" of ginseng polysaccharide. The integrity of this three-dimensional framework, as the structural carrier for arabinose to exert its effects, directly determines whether the polysaccharide can create an active environment for arabinose to bind to tumor cell targets. If the framework is broken, even if arabinose is present, anti-tumor activity cannot be achieved. Attached Figure Description

[0042] Figure 1 The ultraviolet spectrum of ginseng polysaccharide obtained in Example 1;

[0043] Figure 2 The absolute molecular weight chromatogram of ginseng polysaccharides obtained in Example 1 is shown below.

[0044] Figure 3 This is a root mean square radius analysis diagram of the ginseng polysaccharide obtained in Example 1;

[0045] Figure 4 The molecular configuration analysis diagram of the ginseng polysaccharide obtained in Example 1 is shown. Figure 5 The standard ion chromatogram is shown in Example 1.

[0046] Figure 6 The image shows the ion chromatogram of ginseng polysaccharide obtained in Example 1.

[0047] Figure 7 The infrared spectrum of ginseng polysaccharide obtained in Example 1.

[0048] Figure 8 The image shows the GC-MS total ion chromatogram of ginseng polysaccharide obtained in Example 1 after methylation.

[0049] Figure 9 NMR analysis of the ginseng polysaccharide obtained in Example 1.

[0050] Figure 10 The results of the Congo red experiment on ginseng polysaccharides obtained in Example 1;

[0051] Figure 11 Here is a scanning electron microscope image of the ginseng polysaccharide obtained in Example 1;

[0052] Figure 12 An atomic force microscope image of ginseng polysaccharide obtained in Example 1;

[0053] Figure 13 The results of DSC-TGA for ginseng polysaccharides obtained in Example 1;

[0054] Figure 14 The inhibition rate of different concentrations of ginseng polysaccharide on human hepatocellular carcinoma cells HepG2;

[0055] Figure 15 The inhibition rate of different concentrations of ginseng polysaccharide on human gastric cancer cells SGC7901;

[0056] Figure 16 The inhibition rate of different concentrations of ginseng polysaccharide on human breast cancer cells MCF-7. Detailed Implementation

[0057] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any reasonable combination of the specific embodiments.

[0058] Specific Implementation Method 1: The structural formula of ginseng polysaccharide in this implementation method is:

[0059] In the structural formula, n and m are positive integers and n = 6m; ​​x, y and z are positive integers and x + y + z = 4.

[0060] This embodiment has the following beneficial effects:

[0061] Although arabinose is not the most abundant component in the ginseng polysaccharide composition of this embodiment, it is a key component for initiating antitumor activity and plays a crucial role in tumor cell recognition and anti-proliferation signal initiation during the antitumor process. At the molecular structure level, the core innovation of the ginseng polysaccharide in this embodiment lies in the construction of a dynamically stable three-dimensional framework based on the main chain and branches. Structurally, the three-dimensional framework of ginseng polysaccharide is constructed based on the main chain and branches. The main chain is a linear structure formed by monosaccharides linked by glycosidic bonds, serving as the core support of the framework. The branches branch from specific sites on the main chain and are composed of various monosaccharides, enriching the structural levels of the framework. Combined with information such as molecular weight and molecular configuration, macroscopically, ginseng polysaccharide exhibits a random coil or irregular conformation. In this conformation, the main chain and branches do not exhibit highly regular folds, but rather interact and extend in a relatively loose and random manner, forming a three-dimensional morphology similar to a flexible network. This flexible structure facilitates its interaction with biomolecules such as cell surface receptors. The root mean square radius of 16.4 nanometers and its significant spatial extensibility reflect that the framework is not a tightly folded structure, but rather a flexible and loosely arranged spatial arrangement. Monosaccharide segments (main chain and branches) are relatively extended in space, interwoven to form a three-dimensional network with a certain span. In contrast, some existing polysaccharides may undergo significant conformational changes in their main chain and branch structures when the external environment (such as temperature and pH changes) changes slightly, leading to loss or alteration of function. They lack the stable and adjustable characteristics of the ginseng polysaccharide framework structure of this embodiment. The dynamically stable three-dimensional framework based on the main chain and branches in this embodiment also serves as a structural carrier for the functional monosaccharide (arabinose) to exert its antitumor effect. This three-dimensional framework not only provides spatial configuration support but also allows for the regulation of molecular flexibility and optimization of functional group exposure. This conformation results in weaker inter-segment binding in environments such as solution, giving the molecule overall good flexibility. When interacting with tumor cells, the flexible molecular structure facilitates conformational changes, adapting to the binding requirements of cell surface targets and creating conditions for subsequent functional groups to exert their effects. Simultaneously, the flexible and loose spatial arrangement allows molecular segments to move freely within a certain range, further enhancing molecular flexibility. This enables polysaccharide molecules to more efficiently contact and interact with different targets on the tumor cell surface, creating an active environment for key units such as arabinose to bind to tumor cell surface targets and initiate anti-metastasis or pro-apoptosis signals. Without this three-dimensional framework, even the presence of functional monosaccharides such as arabinose would prevent the effective exertion of antitumor activity. This structural characteristic of framework-linked functional unit antitumor activity clarifies the decisive supporting role of the overall molecular integrity in ginseng polysaccharides in tumor suppression, further refining the relationship between structure and activity in polysaccharide molecules.

[0062] Specific Implementation Method Two: The preparation method of ginseng polysaccharide in this implementation method is carried out according to the following steps:

[0063] 1. Weigh out ginseng slices and soak them in distilled water for 8-12 hours to obtain a mixture; the content of ginseng slices in the mixture is 100g / L;

[0064] 2. The soaked ginseng slices and solution are extracted using a hot water extraction process to obtain the extract;

[0065] The hot water extraction method is as follows: under constant temperature conditions of 85~95℃, distilled water is used for extraction 2~4 times, each extraction lasting 2~4 hours, and the extracts are combined and collected.

[0066] The ratio of distilled water to ginseng slices added in each extraction is 3-8L: 400-500g;

[0067] 3. Centrifuge the collected extract to remove large particulate impurities; then remove small particulate impurities by vacuum filtration to obtain a purified extract.

[0068] 4. Concentrate the purified extract to obtain a concentrated solution, add ethanol solution to the concentrated solution and carry out alcohol precipitation at a low temperature of 2~8℃;

[0069] The concentration process is as follows: a rotary evaporation process is used, with a temperature of 55~65℃ and a time of 4~8 hours, to concentrate to 10~20% of the volume of the purified extract;

[0070] 5. Centrifuge the solution after alcohol precipitation to separate the precipitate. After freeze-drying the precipitate, the crude ginseng polysaccharide product is obtained.

[0071] 6. Ginseng crude polysaccharide was redissolved in 15-25 times its volume of water in a beaker to obtain a polysaccharide aqueous extract. The extract was then deproteinized using the Sevag method and finally freeze-dried to obtain deproteinized ginseng polysaccharide.

[0072] The volume ratio of the crude ginseng polysaccharide to distilled water is 1:15~25;

[0073] The process of deproteinizing ginseng crude polysaccharide using the Sevag method is as follows: the polysaccharide aqueous extract and Sevag reagent are mixed and extracted for 30 min, centrifuged at 4000 rpm for 10 min, the precipitate is removed, the solution is collected, and one deproteinization process is completed; the deproteinization process is repeated 5-7 times.

[0074] 7. Swell DEAE-52 cellulose in ultrapure water for 3 to 7 days, then sequentially perform alkali activation, acid activation, and alkali activation. After each activation, wash DEAE-52 cellulose with ultrapure water until the pH reaches 6-8. Pack DEAE-52 cellulose into an anion exchange chromatography column. Mix the deproteinized ginseng polysaccharide obtained in step 6 with distilled water to obtain a 10 mg / mL ginseng polysaccharide solution, then centrifuge and filter sequentially. Load the resulting solution into an anion exchange chromatography column and let it stand for 12-24 hours. After standing, inject eluent into the anion exchange chromatography column and collect the eluent discharged from the column. Dialyze the collected eluent with running water. Finally, freeze-dry to obtain purified ginseng polysaccharide. Collect 2 mL of eluent in 100 tubes.

[0075] The ratio of DEAE-52 cellulose to ultrapure water is 50g: 500~800mL;

[0076] The alkaline activation uses 0.4~0.6 mol / L NaOH, and the acid activation uses 0.4~0.6 mol / L HCl;

[0077] 8. Sephacryl S-300 propylene dextran gel (preserved in anhydrous ethanol) was washed with ultrapure water to remove the anhydrous ethanol and packed into an anion exchange chromatography column. The purified ginseng polysaccharide obtained in step 7 was mixed with distilled water to obtain a 10 mg / mL ginseng polysaccharide solution. The solution was then centrifuged and filtered sequentially. The resulting solution was loaded into an anion exchange chromatography column and allowed to stand for 12–24 h. After standing, ultrapure water was injected into the anion exchange chromatography column as the eluent. The eluent discharged from the anion exchange chromatography column was collected. The collected eluent was dialyzed with running water and finally freeze-dried to obtain ginseng polysaccharide (GPS). 100 tubes of eluent were collected, with 1 mL collected from each tube.

[0078] The centrifugation process was carried out at a speed of 1500 rpm for 10 min.

[0079] The filtration uses a 0.22μm aqueous filter membrane;

[0080] The flow rate during sample loading is 1 mL / min;

[0081] The running water dialysis uses a 3.5kDa dialysis bag, with a dialysis time of 24 hours and distilled water changed every 4 hours.

[0082] This embodiment successfully extracted, separated, and purified a novel ginseng polysaccharide, significantly improving the analytical challenges posed by the complex chemical structure of ginseng polysaccharides. Through extraction, separation, and purification of ginseng slices, ginseng polysaccharides were obtained, and their monosaccharide composition and spatial structure were revealed, providing fundamental data for the development and utilization of ginseng polysaccharides.

[0083] This embodiment of ginseng polysaccharide effectively reduces interference from monosaccharide residue combinations, glycosidic bond linkages, and highly branched molecular structures, providing a clear material basis for in-depth research on the spatial conformation of ginseng polysaccharide. It helps researchers to more accurately grasp the structural characteristics of ginseng polysaccharide, thus pointing the way for subsequent research.

[0084] Meanwhile, given the close relationship between the antitumor activity of polysaccharides and their structure, as well as the enhancing effects of molecular modification and high bioavailability on their activity, this embodiment extracts, separates, and purifies novel ginseng polysaccharides. Based on a clear structure, the CCK8 assay was used to detect that ginseng polysaccharides inhibit tumor growth in human hepatocellular carcinoma cells HepG2, human gastric carcinoma cells SGC7901, and human breast cancer cells MCF-7. The high purity of the ginseng polysaccharides in this embodiment is conducive to improving bioavailability, thereby significantly enhancing antitumor activity. This provides a more promising material for the research of anticancer candidate substances and is expected to promote the development of anticancer drugs.

[0085] In this embodiment, crude ginseng polysaccharide was extracted from ginseng using a water extraction and alcohol precipitation method. The yield of crude polysaccharide was calculated to be 25.2% based on the weight of the ginseng raw material. Preliminary purification through deproteinization yielded refined ginseng polysaccharide with a yield of 30.45%, a sugar content of 74.18%, a protein content of 3.2%, and a uronic acid content of 4.2%. Polysaccharide fractions were obtained using DEAE-52 cellulose gel electrophoresis, with the aqueous fraction ultimately collected, yielding a yield of 52.14%, a sugar content of 86.84%, and a protein content of 1.6%. After purification using a Sephacryl S-300 propylene dextran gel electrophoresis system, the yield was 70.09%, the sugar content was 95.32%, and the protein content was 0.5%. Using human hepatocellular carcinoma cells (HepG2) and human gastric carcinoma cells (SGC7901) as research subjects, the antitumor activity of purified ginseng polysaccharides was initially screened using the CCK8 assay. Ginseng polysaccharides showed inhibitory activity against human hepatocellular carcinoma cells (HepG2), human gastric carcinoma cells (SGC7901), and human breast cancer cells (MCF-7) in a dose-dependent manner, with maximum inhibition rates of 22.87%, 24.68%, and 39.45%, respectively.

[0086] In most purification experiments of polysaccharides and small molecule proteins, dextran gels (such as the Sephadex G series) are commonly used gel filtration chromatography media. Although propylene dextran gels (represented by Sephacryl S-300 in this experiment) belong to the same type of medium and have dextran as their core component, their performance and application scenarios differ significantly due to differences in chemical structure and preparation process. Dextran gels are formed only by cross-linking dextran with epichlorohydrin, resulting in low mechanical strength. They are easily compressed and deformed under high flow rates and high column pressures. Their chemical stability is limited to neutral to weakly acidic environments (pH 2-13), and their pore size is easily affected by solvent polarity and ionic strength, resulting in a narrow separation range (mostly <1×10⁵ Da). Furthermore, they exhibit slow swelling rates and large volume fluctuations, making them more suitable for the separation of small molecules. Acrylic dextran gels, through cross-linking of the dextran backbone with N,N'-methylenebisacrylamide, introduce acrylamide monomers to form a copolymer structure. They exhibit excellent mechanical strength and compressibility, can withstand high column pressures and flow rates, and possess extremely high chemical stability (pH 2-14). They are stable to strong acids, strong bases, oxidants, and organic solvents such as anhydrous ethanol. Their pore size is minimally affected by environmental factors, and they offer a wide separation range (e.g., Sephacryl S-300 covering 1×10⁻⁶ cells). 4 -1.5×10 6 (Da), with rapid swelling and stable volume, making it more suitable for the separation of large molecules. From the purification process perspective, the propylene dextran gel S-300 first provides crucial support for the separation of target polysaccharides through a precise molecular sieve effect. (Propylene dextran gel 1×10) 4 -1.5×10 6The separation range of the propylene dextran gel precisely matches the molecular weight range of ginseng polysaccharides, effectively separating the target polysaccharide from small molecule impurities in the system. This ensures precise enrichment of the target polysaccharide and avoids impurity aggregation affecting subsequent structure and activity. Simultaneously, the high stability of the propylene dextran gel to anhydrous ethanol prevents skeletal deformation or degradation during the pretreatment process of anhydrous ethanol storage followed by ultrapure water washing. The uniform gel bed formed after loading lays the foundation for stable subsequent loading, settling, and elution, ensuring the reproducibility of the purification process and guaranteeing a stable yield of the target polysaccharide in each purification. Crucially, the mild purification environment provided by the propylene dextran gel is the core guarantee for preserving the unique structure and activity of ginseng polysaccharides. On the one hand, the propylene dextran gel exhibits chemical stability to withstand neutral to strong acids and alkalis, ensuring that the ultrapure water elution system used in the experiment (free of corrosive reagents and high-concentration solvents) minimizes damage to the active promoter of arabinose in the polysaccharide. As a key unit for tumor cell recognition and anti-proliferation signal initiation, arabinose is sensitive to the chemical environment, and the mild elution conditions ensure its structural integrity, preserving the function of its active promoter. On the other hand, the gel's excellent mechanical strength and compressibility maintain bed stability during 12-24 hours of settling, preventing column pressure fluctuations due to gravity compression. This avoids mechanical breakage of polysaccharide molecules during elution, fully preserving the "dynamically stable three-dimensional framework of main chain-branched chains" of ginseng polysaccharide. The integrity of this three-dimensional framework, as the structural carrier for arabinose to exert its effects, directly determines whether the polysaccharide can create an active environment for arabinose to bind to tumor cell targets. If the framework is broken, even if arabinose is present, anti-tumor activity cannot be achieved.

[0087] Specific Implementation Method 3: This implementation method differs from Specific Implementation Method 2 in that the centrifugation speed in step 3 is 4000 rpm and the centrifugation time is 10 min.

[0088] Specific Implementation Method Four: This implementation method differs from Specific Implementation Method Two in that: the mass fraction of the ethanol solution in step four is 99%, the volume ratio of the ethanol solution to the concentrate is (5~6):1, and the alcohol precipitation time is 12~24h.

[0089] Specific Implementation Method 5: This implementation method differs from Specific Implementation Method 2 in that the centrifugation speed in step 5 is 3000 rpm and the centrifugation time is 15 min.

[0090] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method Two in that the centrifugation speed in step seven is 4000 rpm and the centrifugation time is 10 min.

[0091] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Method Two in that the filtration described in step seven uses a 0.45μm aqueous filter membrane.

[0092] Specific Implementation Method 8: This implementation method differs from Specific Implementation Method 2 in that the flow rate during sample loading in step 7 is 1 mL / min.

[0093] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Method Two in that the flushing dialysis described in step seven uses a 3.5kDa dialysis bag, and the dialysis time is 24 hours.

[0094] Specific Implementation Method 10: In this implementation method, ginseng polysaccharide is used to prepare an anti-tumor drug with biological activity that inhibits human liver cancer cells and human gastric cancer cells.

[0095] This embodiment extracts, separates, and purifies a novel ginseng polysaccharide. Based on a well-defined structure, the CCK8 assay was used to detect that the ginseng polysaccharide inhibits tumor growth in human hepatocellular carcinoma cells HepG2, human gastric carcinoma cells SGC7901, and human breast cancer cells MCF-7. The high purity of the ginseng polysaccharide in this embodiment is beneficial to improving bioavailability, thereby significantly enhancing antitumor activity. It provides a more promising material for the research of anticancer candidate substances and is expected to promote the development of anticancer drugs.

[0096] Using human hepatocellular carcinoma cells (HepG2) and human gastric carcinoma cells (SGC7901) as research subjects, the antitumor activity of purified ginseng polysaccharides was initially screened using the CCK8 assay. Ginseng polysaccharides showed inhibitory activity against human hepatocellular carcinoma cells (HepG2), human gastric carcinoma cells (SGC7901), and human breast cancer cells (MCF-7) in a dose-dependent manner, with maximum inhibition rates of 22.87%, 24.68%, and 39.45%, respectively.

[0097] Example 1:

[0098] The preparation method of ginseng polysaccharide in this embodiment is carried out according to the following steps:

[0099] 1. Weigh 500g of ginseng slices and soak them in distilled water for 10 hours to obtain a mixture; the content of ginseng slices in the mixture is 100g / L.

[0100] 2. The soaked ginseng slices and solution are extracted using a hot water extraction process to obtain the extract;

[0101] The hot water extraction method is as follows: extracting three times with distilled water under constant temperature of 90℃, and combining and collecting the extracts;

[0102] The ratio of distilled water to ginseng slices added for the first extraction was 8L:500g, and the extraction time was 4 hours.

[0103] The ratio of distilled water to ginseng slices added during the second extraction was 7L:500g, and the extraction time was 3 hours.

[0104] The ratio of distilled water to ginseng slices added in the third extraction was 6L:500g, and the extraction time was 2 hours.

[0105] 3. Centrifuge the collected extract to remove large particulate impurities; then remove small particulate impurities by vacuum filtration to obtain a purified extract.

[0106] The centrifugation process was carried out at a speed of 4000 rpm for 10 minutes.

[0107] 4. Concentrate the purified extract to obtain a concentrated solution, add ethanol solution to the concentrated solution and perform alcohol precipitation at a low temperature of 5°C.

[0108] The concentration process is as follows: a rotary evaporation process is used at a temperature of 60°C for 6 hours to concentrate the extract to 20% of its volume.

[0109] The ethanol solution has a mass fraction of 99%, the volume ratio of ethanol solution to concentrate is 5.5:1, and the alcohol precipitation time is 12h.

[0110] 5. Centrifuge the solution after alcohol precipitation to separate the precipitate. After freeze-drying the precipitate, the crude ginseng polysaccharide product is obtained.

[0111] The centrifugation process was carried out at a speed of 3000 rpm for 15 minutes.

[0112] 6. Ginseng crude polysaccharide was redissolved in 20 times its volume of water in a beaker to obtain a polysaccharide aqueous extract. The extract was then subjected to deproteinization using the Sevag method and finally freeze-dried to obtain deproteinized ginseng polysaccharide.

[0113] The volume ratio of the ginseng crude polysaccharide to distilled water is 1:20;

[0114] The process of deproteinizing ginseng crude polysaccharide using the Sevag method is as follows: the polysaccharide aqueous extract and Sevag reagent are mixed and extracted for 30 min, centrifuged at 4000 rpm for 10 min, the precipitate is removed, the solution is collected, and one deproteinization process is completed; the deproteinization process is repeated 7 times.

[0115] 7. DEAE-52 cellulose was swollen in ultrapure water for 5 days, then successively activated by alkali, acid, and alkali. After each activation, DEAE-52 cellulose was washed with ultrapure water until the pH reached 6-8. DEAE-52 cellulose was then packed into an anion exchange chromatography column. The deproteinized ginseng polysaccharide obtained in step 6 was mixed with distilled water to obtain a 10 mg / mL ginseng polysaccharide solution. This solution was then centrifuged and filtered. The resulting solution was loaded into an anion exchange chromatography column and allowed to stand overnight (12 h). After standing, eluent was injected into the anion exchange chromatography column, and the eluent discharged from the column was collected. The collected eluent was dialyzed with running water. Finally, the eluent was freeze-dried to obtain purified ginseng polysaccharide. 100 tubes of eluent were collected, with 2 mL collected from each tube.

[0116] The centrifugation process was carried out at a speed of 4000 rpm for 10 minutes.

[0117] The filtration uses a 0.45μm aqueous filter membrane;

[0118] The flow rate during sample loading is 1 mL / min;

[0119] The fluid dialysis was performed using a 3.5 kDa dialysis bag for 24 hours.

[0120] The ratio of DEAE-52 cellulose to ultrapure water is 50g:700mL;

[0121] The alkali activation uses 0.4 mol / L NaOH, and the acid activation uses 0.4 mol / L HCl;

[0122] 8. Sephacryl S-300 propylene dextran gel (preserved in anhydrous ethanol) was washed with ultrapure water to remove the anhydrous ethanol and packed into an anion exchange chromatography column. The purified ginseng polysaccharide obtained in step 7 was mixed with distilled water to obtain a 10 mg / mL ginseng polysaccharide solution. The solution was then centrifuged and filtered sequentially. The resulting solution was loaded into an anion exchange chromatography column and allowed to stand overnight (12 h). After standing, ultrapure water was injected into the anion exchange chromatography column as the eluent. The eluent discharged from the anion exchange chromatography column was collected. The collected eluent was dialyzed with running water and finally freeze-dried to obtain ginseng polysaccharide (GPS). 100 tubes of eluent were collected, with 1 mL collected from each tube.

[0123] The centrifugation process was carried out at a speed of 1500 rpm for 10 min.

[0124] The filtration uses a 0.22μm aqueous filter membrane;

[0125] The flow rate during sample loading is 1 mL / min;

[0126] The fluid dialysis uses a 3.5 kDa dialysis bag, with a dialysis time of 24 hours and distilled water changed every 4 hours;

[0127] The crude ginseng polysaccharides extracted in this embodiment were tested, and the yield was calculated to be 25.2% based on the weight of the ginseng slices. Preliminary purification through deproteinization yielded refined ginseng polysaccharides with a yield of 30.45%, a sugar content of 74.18%, a protein content of 3.2%, and a uronic acid content of 4.2%. Polysaccharide components were obtained using DEAE-52 cellulose gel electrophoresis, with the aqueous fraction ultimately collected, yielding a yield of 52.14%, a sugar content of 86.84%, and a protein content of 1.6%. After purification using a Sephacryl S-300 propylene dextran gel electrophoresis system, the ginseng polysaccharide fraction was obtained with a yield of 70.09%, a sugar content of 95.32%, and a protein content of 0.5%.

[0128] Physicochemical analysis showed that the ginseng polysaccharide extracted in this embodiment was a white flocculent substance, easily soluble in water, and odorless. The phenol-sulfuric acid method experiment showed that the ginseng polysaccharide was a sugar substance; the sulfuric acid-carbazole experiment showed that the ginseng polysaccharide did not contain uronic acid; the ninhydrin experiment showed that the ginseng polysaccharide did not contain polypeptides, amino acids, or proteins; the iodine-potassium iodide experiment showed that the ginseng polysaccharide did not contain starch; and the ferric chloride experiment showed that the ginseng polysaccharide did not contain polyphenolic substances.

[0129] Figure 1 The image shows the ultraviolet spectrum of the ginseng polysaccharide obtained in Example 1. Ultraviolet spectroscopy analysis showed that no significant absorption peaks were detected at 260 nm and 280 nm, indicating that the ginseng polysaccharide obtained in Example 1 does not contain protein or nucleic acid substances.

[0130] Figure 2 The chromatogram shows the absolute molecular weight of the ginseng polysaccharide obtained in Example 1; the molecular weight of the ginseng polysaccharide was determined to be 1.121 × 10⁻⁶. 4 (±2.395%) g / mol, and its polydispersity index (Mw / Mn) is 1.149 (±3.931%). This low polydispersity index indicates that the molecular weight distribution is relatively concentrated, exhibiting uniform and well-distributed characteristics.

[0131] Figure 3 The image shows the root mean square radius (RMS) analysis of the ginseng polysaccharide obtained in Example 1. The RMS radius is 16.4 (±39.2%) nanometers, indicating that the ginseng polysaccharide has a large spatial expansion capacity and exhibits a flexible or loose structure.

[0132] Figure 2 and Figure 3In this context, LS (Light Scattering) determines the absolute molecular weight and other information of polymers by detecting scattered light, directly obtaining molecular mass-related data. The graph reflects the change in molar mass over time based on the principle of light scattering. dRI (Differential Refractive Index) determines the concentration of a sample by measuring the difference in refractive index between the sample and the mobile phase.

[0133] Figure 4 The image shows the molecular configuration analysis of the ginseng polysaccharide obtained in Example 1; the slope of the molecular weight distribution is 0.65±0.06, indicating that it is a random coil or irregular conformation.

[0134] Figure 5 The image shows the ion chromatogram of the standard in Example 1. In the monosaccharide standard chromatogram, from left to right, according to the elution time order, the peaks are fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), guluronic acid (Gul-UA), glucuronic acid (Glc-UA), and mannuronic acid (Man-UA).

[0135] Figure 6 The image shows the ion chromatogram of ginseng polysaccharide obtained in Example 1. Analysis of the monosaccharide composition by ion chromatography revealed that the ginseng polysaccharide contains five monosaccharides: glucose, arabinose, galactose, rhamnose, and fucose, with a molar ratio of 91.60:5.91:1.62:0.58:0.30.

[0136] Figure 7 The infrared spectrum of the ginseng polysaccharide obtained in Example 1 is shown; the infrared absorption peak is at 3417.69 cm⁻¹. -1 The peak at 2927.83 cm⁻¹ corresponds to the stretching vibration of the hydroxyl group (-OH), a characteristic peak of carbohydrates. -1 The absorption peak at 1423.99 cm⁻¹ corresponds to the stretching vibration of CH₄. -1 and 1367.16 cm -1 The absorption peak at 1151.49 cm⁻¹ corresponds to the bending vibration of CH₄. -1 1080.6 cm -1 1028.57 cm -1 and 928.36 cm -1 The absorption peak at that point corresponds to the vibration of the pyranose ring.

[0137] Figure 8The image shows the GC-MS total ion chromatogram of ginseng polysaccharide obtained in Example 1 after methylation. Seven sugar alcohol acetyl ester products (PMAA) were detected in the methylated products of ginseng polysaccharide. By comparing each peak with the CCRC standard mass spectrometry library and considering the peak time, monosaccharide composition and ratio, the connection mode corresponding to each sugar peak can be deduced. The assignment is shown in Table 1. The monosaccharide composition is mainly composed of glucose (Glc, accounting for 88.790%), especially the molar ratio of →4)-Glcp-(1→ (64.762%) to →4,6)-Glcp-(1→ (11.413%), which is approximately 6:1. This indicates that the polysaccharide sample is mainly composed of a backbone of →4)-α-D-Glcp-(1→. In addition, ginseng polysaccharides also contain 1,3,5-arabinose residues (3.466%) and 1,5-arabinose residues (3.374%), with a molar ratio of approximately 1:1. The molar ratio of →3,5)-Araf-(1→ (3.466%), →5)-Araf-(1→ (3.374%), and →4,6)-Glcp-(1→ (11.413%) is approximately 1:1:3.

[0138] Table 1. Methylation analysis of ginseng polysaccharides

[0139]

[0140] Figure 9 NMR analysis of ginseng polysaccharides obtained in Example 1, wherein A: 1H NMR, B: 13C NMR, C: DEPT-135, D: 1H-1H COSY, E: HSQC, F: NOESY, G: HMBC, H: TOCSY; ¹H NMR of ginseng polysaccharides ( Figure 9 A) and ¹³ CNMR spectra ( Figure 9 In B), eleven anomeric hydrogen signals were found at δH 5.31, 4.88, 5.26, 5.30, 5.14, 4.56, 4.55, 5.06, 5.03, 5.00, and 5.15 ppm, and eleven anomeric carbon signals were found at δC 99.63, 98.45, 99.77, 99.63, 91.98, 95.76, 104.33, 107.03, 107.31, 107.42, and 109.23 ppm, corresponding to the H1 / C1 ratios of different residues. In HSQC spectroscopy (… Figure 9 In C), the crossover signal of 5.31 / 99.63 ppm is assigned to the H1 / C1 of residue (A), the H1 / C1 of residue (A') is at 4.88 / 98.45 ppm, residue (B) is at 5.26 / 99.77 ppm, residue (C) is at 5.30 / 99.63 ppm, and residue (R) is at 5.30 / 99.63 ppm. α The concentrations were 5.14 / 91.98 ppm, and the residues (R) were...β Residue (A) is located at 4.56 / 95.76 ppm, residue (D) is located at 4.55 / 104.33 ppm, and residue (A) is located at 4.55 / 104.33 ppm. t The concentration of residue (A) was 5.06 / 107.3 ppm. 1,3,5 The concentrations were 5.03 / 107.03 ppm, and residues (A) 1,5 The concentrations were 5.00 / 107.42 ppm, with residue (A) t' The concentrations were located at 5.15 / 109.23 ppm. The remaining signals were assigned according to HSQC spectra. Signals H-2, H-3, H-4, and H-5 were generated by COSY (…). Figure 9 D) and TOCSY Figure 9 E) Based on the cross-peak inference in the spectrum, residue (A) is →4)-α-D-Glcp-(1→, (A') is →4)-α-D-Glcp-(1→, (B) is →4,6)-α-D-Glcp-(1→, (C) is α-D-Glcp-(1→, (R) α ) is →4)-α-D-Glcp, (R β (A) is →4)-β-D-Glcp, (D) is →4)-β-D-Galp-(1→, (A) t ) is α-L-Araf-(1→,(A 1,3,5 ) is→3,5)-α-L-Araf-(1→, (A 1,5 ) is→5)-α-L-Araf-(1→, (A t' ) is α-L-Araf-(1→. The proton and carbon signal assignments of ginseng polysaccharides are shown in Table 2.

[0141] Further utilize HMBC long-range correlation spectrum ( Figure 9 F) and NOESY spectrum ( Figure 9G), the sequence of interconnection of each sugar residue was deduced. (1) The correlation peaks AE H-1 / AE C-4 (5.31 / 76.76), AE H-4 / AE C-1 (3.57 / 99.63) and AE H-1 / AE H-4 (5.31 / 3.57) indicate the presence of →4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→ connection; (2) The correlation peaks show that AE H-1 / BE C-4 (5.31 / 77.28) and AE H-1 / BE H-4 (5.31 / 3.54) indicate the presence of O-4 position connection between →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→); (3) The correlation peaks BE H-1 / AE C-4 (5.26 / 76.76) and BE H-1 / AE C-4 (5.26 / 76.76) indicate the presence of O-4 position connection between →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→. H-4 (5.26 / 3.57) indicates that there is a connection between →4,6)-α-D-Glcp-(1→ and →4)-α-D-Glcp-(1→) at position O-4; (4) Correlation peaks A' H-1 / B C-6 (4.88 / 67.54) and A' H-1 / B H-6 (4.88 / 3.80 and 3.84) indicate that there is a connection between →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→) at position O-6; (5) Correlation peak A 1,3,5 H-1 / A 1,5 C-5 (5.03 / 66.44) indicates that there is a connection between →3,5)-α-L-Araf-(1→ and →5)-α-L-Araf-(1→ on O-5; (6) Related peak A 1,5 H-1 / A C-4 ​​C-5 (5.00 / 76.76) indicates that there is a connection between →5)-α-L-Araf-(1→4)-α-D-Glcp-(1→).

[0142] Preliminary speculation suggests that the polysaccharide sample's structure is mainly composed of glucan with →4)-α-D-Glcp-(1→ as the main chain. Side chains are attached to the O-6 positions of certain →4)-α-D-Glcp-(1→ units, and it contains sugar structural fragments such as 1,5-Araf, 1,3,5-Araf, and t-Araf.

[0143] Figure 10 The results of the Congo red experiment on ginseng polysaccharide obtained in Example 1 are as follows: After ginseng polysaccharide forms a stable complex with Congo red, the maximum absorption wavelength exhibits a red shift within a concentration range below 0.2 mol / L NaOH. As the NaOH concentration continues to increase, the stable complex formed by ginseng polysaccharide and Congo red is destroyed and gradually degrades, resulting in a blue shift of the maximum absorption wavelength. Microscopically, it is speculated that the ginseng polysaccharide molecular chain has the potential to form a triple helix structure.

[0144] Figure 11The images shown are scanning electron microscope (SEM) images of ginseng polysaccharides obtained in Example 1, where A: 800x, B: 2000x, C: 5000x, and D: 10000x. The morphology of the ginseng polysaccharides was observed under magnifications of 800x, 2000x, 5000x, and 10000x. Under 800x magnification, the polysaccharide surface was relatively rough, containing rod-shaped, plate-shaped, spherical, and filamentous structures. Under 2000x magnification, the polysaccharide surface was relatively smooth, mainly consisting of filamentous structures with a small number of plate-shaped structures. Under 5000x magnification, a large sphere and numerous small spherical structures were visible in the center of the polysaccharide filamentous structure. Under 10000x magnification, the small spherical structures were elliptical and semi-circular.

[0145] Table 2

[0146]

[0147] Figure 12 The image shows an atomic force microscopy (AFM) image of ginseng polysaccharide obtained in Example 1. The AFM image of ginseng polysaccharide depicts the presence of multiple island-like structures ranging in size from -7.1 nm to 9.1 nm. The R0 of ginseng polysaccharide... max R q R a The values ​​were 25.6 nm, 1.38 nm, and 0.636 nm, respectively. This result indicates that the aggregation structure of ginseng polysaccharides is due to the entanglement of molecular chains and the interaction of intermolecular and intramolecular hydrogen bonds.

[0148] Figure 13 The DSC-TGA results for the ginseng polysaccharide obtained in Example 1 show three significant mass losses within the temperature range of 30°C to 600°C. The first mass loss occurred between 39.12°C and 111.45°C, with a loss rate of 7.13%, which can be attributed to the evaporation of moisture in the sample. The second mass loss occurred between 111.45°C and 324.24°C, with a loss rate of 53.84%, corresponding to a significant reduction in mass, which may be a result of polysaccharide depolymerization. The mass loss rate between 324.24°C and 600°C was 14.02%, indicating oxidative decomposition of organic matter. At a temperature of 304°C, the maximum decomposition rate of the DTG curve was 10.21% per minute.

[0149] Based on the above structural analysis of ginseng polysaccharides, the structural formula of ginseng polysaccharides is as follows:

[0150] In the structural formula, n and m are positive integers and n = 6m; ​​x, y and z are positive integers and x + y + z = 4.

[0151] Inhibitory Effects of Ginseng Polysaccharides on Tumor Growth of Human Hepatocellular Carcinoma (HepG2), Human Gastric Carcinoma (SGC7901), and Human Breast Cancer Cells (MCF-7): Using human hepatocellular carcinoma (HepG2), human gastric carcinoma (SGC7901), and human breast cancer cells (MCF-7) as research subjects, the inhibitory effects of ginseng polysaccharides on these three cells were detected using the CCK8 assay. Experimental subjects: Human hepatocellular carcinoma (HepG2), human gastric carcinoma (SGC7901), and human breast cancer cells (MCF-7), provided by the National Research Center for Anti-tumor Drugs of Harbin University of Commerce.

[0152] The specific steps are as follows:

[0153] Collect cells in the logarithmic growth phase, wash three times with 3 mL PBS, then digest with 1 mL trypsin. Stop digestion by adding 1 mL of serum-containing 1640 medium. Adjust the cell concentration to 1 × 10⁻⁶ cells using serum-free 1640 medium, depending on the number of plates to be plated. 4 Cells / mL were added to each well of a 96-well plate, with 100 μL of cell culture added to each well. The blank group was replaced with cell-free and serum-free medium, and the control group was replaced with cell-containing but serum-free medium. Five concentrations were set for the drug treatment groups (25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL). To avoid solution evaporation and edge effects, 100 μL of PBS was added to each well around the perimeter of the 96-well cell culture plate for fixation. After culturing in a 37°C incubator with 5% CO2 for 24 h, the medium was changed once with fresh 1640 medium without serum before cell culture. After 48 h of drug treatment and culture, 10 μL of CCK8 was added to each well of the 96-well cell culture plate and incubated in an incubator for 2 h. The absorbance was then measured at a wavelength of 450 nm.

[0154] Cell inhibition rate (%) = [(OD 对照组 -OD 给药组 ) / (OD 对照组 -OD 空白组 )]×100%

[0155] Data processing and analysis were performed using SPSS software. Results for each group are expressed as (…). The mean ± standard deviation (±s) is used to represent the mean of multiple samples. One-way ANOVA is used to compare the means of multiple samples. A p < 0.05 is considered statistically significant.

[0156] The results showed that ginseng polysaccharide had inhibitory activity against human hepatocellular carcinoma cells HepG2, human gastric carcinoma cells SGC7901, and human breast cancer cells MCF-7 in a dose-dependent manner within the concentration range of 25-400 μg / mL.

[0157] Figure 14 The inhibition rate of different concentrations of ginseng polysaccharide on human hepatocellular carcinoma cells HepG2; Figure 15 The inhibition rate of different concentrations of ginseng polysaccharide on human gastric cancer cells SGC7901; Figure 16 The inhibition rate of different concentrations of ginseng polysaccharide on human breast cancer cells MCF-7. Figures 14-16 The results indicate that ginseng polysaccharide has an inhibitory effect on the proliferation of HepG2 cells, SGC7901 cells and MCF-7 cells. The maximum inhibition rates of proliferation of HepG2 cells, SGC7901 cells and MCF-7 cells were 22.87%, 24.68% and 39.45% respectively in the concentration range of 25-400 μg / mL.

Claims

1. A ginseng polysaccharide, characterized in that: The structural formula of ginseng polysaccharide is: In the structural formula, n and m are positive integers and n = 6m; ​​x, y and z are positive integers and x + y + z = 4.

2. The method for preparing ginseng polysaccharide as described in claim 1, characterized in that: The preparation method of ginseng polysaccharides is carried out according to the following steps:

1. Weigh out ginseng slices and soak them in distilled water for 8-12 hours to obtain a mixture; the content of ginseng slices in the mixture is 100g / L; 2. The soaked ginseng slices and solution are extracted using a hot water extraction process to obtain the extract; The hot water extraction method is as follows: under constant temperature conditions of 85~95℃, distilled water is used for extraction 2~4 times, each extraction lasting 2~4 hours, and the extracts are combined and collected. The ratio of distilled water to ginseng slices added in each extraction is 3-8L: 400-500g; 3. Centrifuge the collected extract to remove large particulate impurities; Small particulate impurities are then removed by vacuum filtration to obtain a purified extract; 4. Concentrate the purified extract to obtain a concentrated solution, add ethanol solution to the concentrated solution and carry out alcohol precipitation at a low temperature of 2~8℃; The concentration process is as follows: a rotary evaporation process is used, with a temperature of 55~65℃ and a time of 4~8 hours, to concentrate to 10~20% of the volume of the purified extract; 5. Centrifuge the solution after alcohol precipitation to separate the precipitate. After freeze-drying the precipitate, the crude ginseng polysaccharide product is obtained.

6. Ginseng crude polysaccharide was redissolved in 15-25 times its volume of water in a beaker to obtain a polysaccharide aqueous extract. The extract was then deproteinized using the Sevag method and finally freeze-dried to obtain deproteinized ginseng polysaccharide. The volume ratio of the crude ginseng polysaccharide to distilled water is 1:15~25; The process of deproteinizing ginseng crude polysaccharide using the Sevag method is as follows: the polysaccharide aqueous extract and Sevag reagent are mixed and extracted for 30 min, centrifuged at 4000 rpm for 10 min, the precipitate is removed, the solution is collected, and one deproteinization process is completed; the deproteinization process is repeated 5-7 times.

7. Swell DEAE-52 cellulose in ultrapure water for 3 to 7 days, then sequentially perform alkali activation, acid activation, and alkali activation. After each activation, wash DEAE-52 cellulose with ultrapure water until the pH reaches 6-8. Pack DEAE-52 cellulose into an anion exchange chromatography column. Mix the deproteinized ginseng polysaccharide obtained in step 6 with distilled water to obtain a 10 mg / mL ginseng polysaccharide solution, then centrifuge and filter sequentially. Load the resulting solution into an anion exchange chromatography column and let it stand for 12-24 hours. After standing, inject eluent into the anion exchange chromatography column and collect the eluent discharged from the column. Dialyze the collected eluent with running water. Finally, freeze-dry to obtain purified ginseng polysaccharide. Collect 2 mL of eluent in 100 tubes. The ratio of DEAE-52 cellulose to ultrapure water is 50g: 500~800mL; The alkaline activation uses 0.4~0.6 mol / L NaOH, and the acid activation uses 0.4~0.6 mol / L HCl; 8. Sephacryl S-300 propylene dextran gel (preserved in anhydrous ethanol) was washed with ultrapure water to remove the anhydrous ethanol and packed into an anion exchange chromatography column. The purified ginseng polysaccharide obtained in step 7 was mixed with distilled water to obtain a 10 mg / mL ginseng polysaccharide solution. The solution was then centrifuged and filtered sequentially. The resulting solution was loaded into an anion exchange chromatography column and allowed to stand for 12–24 h. After standing, ultrapure water was injected into the anion exchange chromatography column as the eluent. The eluent discharged from the anion exchange chromatography column was collected. The collected eluent was dialyzed with running water and finally freeze-dried to obtain ginseng polysaccharide (GPS). 100 tubes of eluent were collected, with 1 mL collected from each tube. The centrifugation process was carried out at a speed of 1500 rpm for 10 min. The filtration uses a 0.22μm aqueous filter membrane; The flow rate during sample loading is 1 mL / min; The running water dialysis uses a 3.5kDa dialysis bag, with a dialysis time of 24 hours and distilled water changed every 4 hours.

3. The method for preparing ginseng polysaccharide according to claim 2, characterized in that: The centrifugation process in step three is carried out at a speed of 4000 rpm for 10 minutes.

4. The method for preparing ginseng polysaccharide according to claim 2, characterized in that: The ethanol solution in step four has a mass fraction of 99%, the volume ratio of ethanol solution to concentrate is (5~6):1, and the alcohol precipitation time is 12~24h.

5. The method for preparing ginseng polysaccharide according to claim 2, characterized in that: The centrifugation process in step five is carried out at a speed of 3000 rpm for 15 minutes.

6. The method for preparing ginseng polysaccharide according to claim 2, characterized in that: The centrifugation process described in step seven is carried out at a speed of 4000 rpm for 10 minutes.

7. The method for preparing ginseng polysaccharide according to claim 2, characterized in that: The filtration described in step seven uses a 0.45μm aqueous filter membrane.

8. The method for preparing ginseng polysaccharide according to claim 2, characterized in that: The flow rate for loading the sample in step seven is 1 mL / min.

9. The method for preparing ginseng polysaccharide according to claim 2, characterized in that: The flushing dialysis described in step seven uses a 3.5 kDa dialysis bag and lasts for 24 hours.

10. The application of ginseng polysaccharide as described in claim 1, characterized in that: Ginseng polysaccharides are used to prepare antitumor drugs with bioactivity that inhibits human liver cancer cells and human gastric cancer cells.

Images