Preparation method of acanthopanax senticosus polysaccharide and application of acanthopanax senticosus polysaccharide in antitumor drugs

Abstract

The invention belongs to the technical field of bioactive substances, and particularly relates to a preparation method of acanthopanax senticosus polysaccharide and application of the acanthopanax senticosus polysaccharide in antitumor drugs. According to the preparation method of the acanthopanax senticosus polysaccharide, provided by the invention, the acanthopanax senticosus crude polysaccharide is extracted by adopting an alcohol precipitation method, and then the acanthopanax senticosus crude polysaccharide is subjected to deproteinization and decoloration treatment by adopting D941 type macroporous resin, so that the acanthopanax senticosus polysaccharide with high purity and remarkable antitumor activity can be prepared. Meanwhile, by evaluating the in-vivo colon cancer resisting effect and mechanism of the acanthopanax senticosus polysaccharide, it is proved that the acanthopanax senticosus polysaccharide can remarkably inhibit in-vivo tumor growth by adjusting the immunity of the body. Therefore, a novel purification strategy can be provided for preparation of the plant polysaccharide, the resource utilization rate of traditional Chinese medicines can be increased, and a technical basis can be provided for development of antitumor drugs based on the acanthopanax senticosus polysaccharide.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive substances, and particularly relates to a preparation method of acanthopanax senticosus polysaccharide and its application in anti-tumor drugs. Background Art

[0002] Acanthopanax senticosus is a member of the Araliaceae family, with both medicinal and edible uses, and is mainly distributed in the northeast, Hebei, Shanxi and other regions of China. The whole plant of Acanthopanax senticosus has application value. Among them, the young leaves can be used as vegetable raw materials, the mature leaves and fruits can be made into tea beverages, and the roots and rhizomes have extensive applications in the fields of health nutrition and traditional Chinese medicine. The extracts of the roots and rhizomes of Acanthopanax senticosus have significant anti-tumor, anti-fatigue, anti-inflammatory, anti-stress, immune-enhancing and other effects. The chemical components of Acanthopanax senticosus mainly include polysaccharides, glycosides, lignans, flavonoids and fatty acids, and the chemical components of drugs are the basis of drug efficacy.

[0003] Due to their significant bioactive characteristics, plant polysaccharides have gradually become an indispensable part of human medical applications and have been widely used in the food industry. However, the extraction process of polysaccharides is usually accompanied by the coprecipitation of a large amount of impurities, such as proteins and pigments, which poses a severe challenge to the purification and structural analysis of polysaccharides. Traditional deproteinization and decolorization techniques are not only time-consuming and laborious, but also require the use of a large amount of organic reagents, which are prone to pose potential risks to food and drug safety.

[0004] Tumor is one of the main causes of death worldwide and has become a major category of diseases that seriously endanger human life and health and restrict social and economic development. Plant polysaccharides have the advantages of multiple pathways, multiple targets, low toxicity, high efficiency, small side effects, etc., and have a synergistic effect when combined with drugs, so they have potential anti-tumor development value.

[0005] Therefore, how to develop a safe and efficient preparation method to obtain acanthopanax senticosus polysaccharide with high purity and good anti-tumor activity is of great significance for further developing the medicinal value of Acanthopanax senticosus and improving the resource utilization rate of plants. Summary of the Invention

[0006] To solve the above problems, the purpose of the present invention is to provide a preparation method of acanthopanax senticosus polysaccharide and its application in anti-tumor drugs. The preparation method of acanthopanax senticosus polysaccharide provided by the present invention first extracts crude acanthopanax senticosus polysaccharide by water extraction, pH adjustment and alcohol precipitation methods, and then uses a special macroporous adsorption resin to perform efficient deproteinization and decolorization treatment on the crude acanthopanax senticosus polysaccharide, and can prepare acanthopanax senticosus polysaccharide with high purity and excellent anti-tumor activity.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is:

[0008] A preparation method of acanthopanax senticosus polysaccharide, comprising the following steps:

[0009] (1) Crush the acanthopanax senticosus raw material, then perform water extraction and filtration to obtain an extract; adjust the pH of the extract to 10 - 12 first, then stir, then adjust the pH of the extract to 5 - 6, then stir and stand, take the supernatant for alcohol precipitation treatment, and obtain crude acanthopanax senticosus polysaccharide after drying;

[0010] (2) Dissolve the crude acanthopanax senticosus polysaccharide in water to obtain a crude acanthopanax senticosus polysaccharide solution; purify the crude acanthopanax senticosus polysaccharide solution in macroporous resin, then collect the purified solution, and obtain the purified acanthopanax senticosus polysaccharide after concentration, dialysis and drying; the macroporous resin is D941 macroporous weak - basic anion exchange resin.

[0011] As a preferred scheme, in step (1), the crushing is to crush the acanthopanax senticosus raw material into segments of 2 - 10 cm each.

[0012] As a preferred scheme, in step (1), the water extraction is to extract the crushed acanthopanax senticosus raw material with water; the extraction process is: soak for 20 - 40 min first, then decoct for 0.5 - 2 h, and then perform hot reflux for 3 - 6 h; the mass ratio of the acanthopanax senticosus raw material to water during extraction is 1∶5 - 10, more preferably 1∶7.

[0013] As a preferred scheme, in step (1), adjusting the pH of the extract to 10 - 12 is adjusted with lime milk; the mass concentration of the lime milk is 10% - 30%; adjusting the pH of the extract to 5 - 6 is adjusted with sulfuric acid solution; the mass concentration of the sulfuric acid solution is 10% - 30%. In this step, through the sequential adjustment of the pH value, the present invention can promote the extraction of subsequent polysaccharide substances and reduce the content of impurities.

[0014] As a preferred scheme, in step (1), the alcohol precipitation treatment is to add ethanol to precipitate the supernatant, and the volume fraction of the added ethanol is 80% - 95%; the time of the alcohol precipitation treatment is 10 - 16 h.

[0015] As a preferred scheme, in step (2), the purification uses macroporous resin as an adsorbent, and column chromatography technology is used to elute and separate the crude acanthopanax senticosus polysaccharide solution to obtain a purified solution; the concentration of the crude acanthopanax senticosus polysaccharide solution is 5 - 8 mg / mL; when performing elution and separation, the eluent used is 0.1 - 0.3 mol / L sodium chloride solution, the sample loading speed is 1.5 - 2.5 mL / min, the sample loading volume is 20 - 40 mL, the elution speed is 1.5 - 3.75 mL / min, and the adsorption time is 6 - 10 h.

[0016] Based on the consideration of further improving the purity of acanthopanax senticosus polysaccharide, as a further preferred scheme, the concentration of the crude acanthopanax senticosus polysaccharide solution is 6.75 mg / mL; the eluent is a 0.2 mol / L sodium chloride solution, the sample loading speed is 2 mL / min, the sample loading volume is 30 mL, the elution speed is 3 mL / min, and the adsorption time is 8 h.

[0017] As a preferred scheme, in step (2), the concentration is to concentrate the purified solution to a relative density of 1.02 - 1.05 g / cm 3 ; the cut-off molecular weight of the dialysis is 3000 - 4000 Da; the drying is freeze-drying.

[0018] An acanthopanax senticosus polysaccharide prepared by the above preparation method, the weight-average molecular weight of the acanthopanax senticosus polysaccharide is 1×10 4 ~1.2×10 4 Da.

[0019] An application of the above acanthopanax senticosus polysaccharide, the application is the application of acanthopanax senticosus polysaccharide in the preparation of anti-tumor drugs; the anti-tumor drug is an anti-colorectal cancer drug.

[0020] The technical scheme of the present invention has the following advantages and beneficial effects:

[0021] The preparation method of acanthopanax senticosus polysaccharide provided by the present invention first extracts crude acanthopanax senticosus polysaccharide by water extraction, pH adjustment and alcohol precipitation methods, and then uses D941 macroporous resin to deproteinize and decolorize the crude acanthopanax senticosus polysaccharide, which can efficiently purify acanthopanax senticosus polysaccharide and prepare acanthopanax senticosus polysaccharide with high purity.

[0022] Furthermore, the present invention tests the purity, structure, molecular weight and monosaccharide composition of acanthopanax senticosus polysaccharide, and confirms that the prepared acanthopanax senticosus polysaccharide has high purity (87.66±0.84%), and is an acidic heteropolysaccharide, and its weight-average molecular weight (Mw) is (1 - 1.2)×10 4 Da, and the molecular weight distribution index is a wide distribution.

[0023] Furthermore, the present invention constructs a CT26.WT tumor-bearing mouse model to evaluate the anti-cancer effect and mechanism of acanthopanax senticosus polysaccharide in vivo. Meanwhile, from multiple aspects such as tumor inhibition rate, the contents of IL-2, TNF-α, and IFN-γ in serum, organ index, and HE staining, the anti-tumor effect of acanthopanax senticosus polysaccharide on colon cancer is evaluated. The experiment confirms that after oral administration of acanthopanax senticosus polysaccharide, the tumor volume and weight of colon cancer mice both decrease significantly, and the highest tumor growth inhibition rate can reach 37.40%. At the same time, acanthopanax senticosus polysaccharide can protect the immune organs of tumor-bearing mice and improve the phenomena of spleen swelling and thymus atrophy. In addition, acanthopanax senticosus polysaccharide can also increase the levels of tumor-related cytokines (IL-2, TNF-α, and IFN-γ). Thus, it is confirmed that acanthopanax senticosus polysaccharide can significantly inhibit the growth of tumors in vivo by regulating the immune ability of the body.

[0024] Therefore, by improving the preparation process of acanthopanax senticosus polysaccharide, the present invention not only successfully prepares acanthopanax senticosus polysaccharide with high purity and significant anti-tumor activity, but also reveals the anti-tumor mechanism and anti-tumor activity of acanthopanax senticosus polysaccharide in vivo. Therefore, the present invention can provide a novel purification strategy for the preparation of plant polysaccharides, is also conducive to improving the resource utilization rate of traditional Chinese medicine, and can also provide a technical basis for the development of anti-tumor drugs based on acanthopanax senticosus polysaccharide. Description of the Drawings

[0025] Figure 1 It is the single-factor result diagram of purifying crude acanthopanax senticosus polysaccharide based on D941 macroporous resin in Test Example 2 of the present invention;

[0026] Figure 2 It is the ultraviolet absorption spectrum of the acanthopanax senticosus polysaccharide prepared in Example 1 in Test Example 3 of the present invention;

[0027] Figure 3 It is the molecular weight chromatogram of the acanthopanax senticosus polysaccharide prepared in Example 1 in Test Example 3 of the present invention;

[0028] Figure 4 It is the monosaccharide composition chromatogram of the acanthopanax senticosus polysaccharide prepared in Example 1 in Test Example 3 of the present invention;

[0029] Figure 5 It is the Fourier transform infrared spectroscopy (FT-IR) diagram of the acanthopanax senticosus polysaccharide prepared in Example 1 in Test Example 3 of the present invention;

[0030] Figure 6 It is the result of the effect of acanthopanax senticosus polysaccharide intervention on the tumor mass of each group of CT26.WT colorectal cancer tumor-bearing mice in Test Example 4 of the present invention;

[0031] Figure 7In Experimental Example 4 of the present invention, the results of the effects of Eleutherococcus senticosus polysaccharide intervention on the thymus and spleen indices of CT26.WT colorectal cancer-bearing mice in each group;

[0032] Figure 8 In Experimental Example 4 of the present invention, the results of the effects of Eleutherococcus senticosus polysaccharide intervention on the contents of IL-2, TNF-α, and IFN-γ in CT26.WT colorectal cancer-bearing mice in each group. Specific Embodiments

[0033] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0034] In the following embodiments of the present invention, the dialysis bag was purchased from Hunan Yibo Biotechnology Co., Ltd. The D941 macroporous resin is specifically the D941 macroporous weakly basic anion exchange resin, which was purchased from Zhengzhou Hecheng New Material Technology Co., Ltd. Other types of macroporous resins (D101 type, S-8 type, HPD-100 type, AB-8 type, X-5 type, H103 type, XDA-7 type, HP20 type, ADS-F8 type) were also purchased from Zhengzhou Hecheng New Material Technology Co., Ltd. Before use, the above macroporous resins were pretreated to remove the monomers and pore-forming agents adsorbed in the pores during the resin synthesis process. The pretreatment process of the macroporous resin was as follows: Different polar macroporous resins (S-8, ADS-F8, HP20, AB-8, X-5, H103, D101, XAD-7, HPD100, D941) were respectively added to ultrapure water, stirred and left to stand, the upper suspended matter was removed, and then filtered; then anhydrous ethanol with a height 2-3 cm higher than its surface was added and soaked for 24 h, then washed with water until there was no alcohol smell, 4% NaOH solution by mass concentration was added, stirred, left to stand for 4 h, washed with water until neutral, 4% HCl solution by mass concentration was added, stirred, left to stand for 4 h, and washed with water until neutral, and then reserved. Other raw materials and the like used in the following examples, unless otherwise specified, are commonly used in the art, publicly available, or can be obtained through commercial channels.

[0035] In the following embodiments of the present invention, the diameter of the chromatography column used for purification is 20 cm, the ratio of diameter to height is 1:5, and the macroporous resin is used as the filler of the chromatography column, and its dosage accounts for 30% - 60% of the total volume of the chromatography column. In other embodiments, the chromatography column can also be of other sizes, and those skilled in the art can make routine selections according to the filler dosage and the target dosage, and the present invention does not make special restrictions.

[0036] Example 1

[0037] This embodiment provides a method for preparing Acanthopanax senticosus polysaccharide, which specifically comprises the following steps:

[0038] (1) Extraction of Acanthopanax senticosus polysaccharide: Weigh the Acanthopanax senticosus medicinal material that has been crushed (2-10 cm segments) and dried to a constant weight, add 7 times the mass of pure water of the Acanthopanax senticosus feed, soak for 30 min, then boil for 1 h, reflux for 4 h, filter, and wait until the temperature of the medicinal solution drops below 50°C, adjust the pH to 11 with 20% by mass lime milk, stir thoroughly and let stand for 30 min, re-measure the pH value and find it is between 10 and 12, then slowly add 20% by mass sulfuric acid solution to the medicinal solution, adjust the pH to 5, stir thoroughly, stop stirring when the re-measured pH value is between 5 and 6, let the medicinal solution stand for 5 h, separate the supernatant, slowly add 95% by volume ethanol aqueous solution, stirring while adding, so that the alcohol content of the medicinal solution reaches 85%, stir thoroughly and let stand in a sealed container for 13 h, then dry the obtained precipitate to obtain Acanthopanax senticosus crude polysaccharide (ASP).

[0039] (2) Purification of Acanthopanax senticosus crude polysaccharide: Weigh the Acanthopanax senticosus crude polysaccharide powder obtained in the previous step, dissolve it completely in water, and prepare an Acanthopanax senticosus crude polysaccharide solution. 30 mL of D941 macroporous resin was used as filler and loaded into a chromatography column. The Acanthopanax senticosus crude polysaccharide solution was adsorbed and purified on a chromatography column filled with D941 macroporous resin, using 0.2 mol / L sodium chloride solution as an eluent, controlling the concentration of the Acanthopanax senticosus crude polysaccharide solution to be 6.75 mg / mL, the loading speed to be 2 mL / min, the loading volume to be 30 mL, the elution speed to be 3 mL / min, the adsorption time to be 8 h, and collecting the components eluted by the eluent, and then concentrating the resulting eluate to a relative density of 1.02 g / cm 3 , placed in a 3500Da dialysis bag for 48 hours (diffused water was changed every 2 to 3 hours during the dialysis process), the dialyzed polysaccharide solution was collected, and freeze-dried at -80°C using a Modulyo D-230 vacuum freeze dryer for 36 hours to obtain purified Acanthopanax senticosus polysaccharide (PASP). After testing, the purity of the purified Acanthopanax senticosus polysaccharide was 87.66±0.84%.

[0040] Test Example 1: Selection of Macroporous Resin

[0041] Decolorization rate test: Weigh 3 g of each of the 10 pretreated macroporous resins (D941 type, D101 type, S-8 type, HPD-100 type, AB-8 type, X-5 type, H103 type, XDA-7 type, HP20 type, ADS-F8 type), and place them in 100 mL conical flasks respectively. Add 25 mL of the Acanthopanax senticosus polysaccharide solution with a concentration of 12.5 mg / mL (prepared by dissolving the Acanthopanax senticosus polysaccharide obtained in step (1) of Example 1 in water). Place it in a constant temperature oscillator, set the conditions to 100 r / min, oscillate at 30 °C for 12 h, filter, take the supernatant, measure the absorbance at the absorption wavelength of 300 nm, and then calculate the decolorization rate.

[0042] Polysaccharide retention rate test: Take 1.0 mL of the supernatant obtained above, then add 0.5 mL of 5% phenol solution, mix well, then add 2.5 mL of concentrated sulfuric acid, quickly shake evenly, let it stand and cool to room temperature, use a UV-visible spectrophotometer to measure and record the absorbance value at 490 nm, calculate the polysaccharide content according to the pre-determined glucose standard curve, and calculate the polysaccharide retention rate.

[0043] Protein removal rate test: Take 1.0 mL of the supernatant obtained above and place it in a glass test tube. Add 5.0 mL of Coomassie Brilliant Blue solution to the test tube, quickly shake evenly, let it stand for 15 min, then measure and record the absorbance value at 595 nm, calculate the protein content according to the pre-determined protein standard curve, and calculate the protein removal rate.

[0044] Among them, decolorization rate (%) = (absorbance before decolorization - absorbance after decolorization) / absorbance before decolorization × 100%.

[0045] Protein removal rate (%) = (protein content before purification - protein content after purification) / protein content before purification × 100%.

[0046] Polysaccharide retention rate (%) = polysaccharide content after purification / polysaccharide content before purification × 100%.

[0047] Furthermore, calculate the membership degree values of pigment clearance rate, protein clearance rate, and sugar retention rate according to the decolorization rate, protein removal rate, and polysaccharide retention rate, sum the three to obtain the comprehensive membership degree value (Y), and judge the comprehensive adsorption performance of the macroporous resin according to its size. The larger this value is, the stronger its comprehensive adsorption ability. Among them, the comprehensive membership degree value j = 1, 2, 3; where: x j is the measured value of the experimental index in this experiment, x maxj is the maximum measured value of the experimental index in this experiment, x minj(which is the minimum measured value of the experimental indicators for this time). j = 1, 2, 3, representing the decolorization rate (j = 1), protein removal rate (j = 2), and polysaccharide retention rate (j = 3), respectively.

[0048] The measurement results of the polysaccharide retention rate, protein removal rate, decolorization rate, and comprehensive membership degree value after the purification of Acanthopanax senticosus polysaccharide by different macroporous resins are shown in Table 1.

[0049] Table 1. Comparison of the purification effects of different macroporous resins on Acanthopanax senticosus polysaccharide solution

[0050]

[0051]

[0052] As can be seen from Table 1, after the treatment of Acanthopanax senticosus polysaccharide solution with D941 macroporous resin, the polysaccharide retention rate is the highest (up to 66.89%), and at the same time, the protein and decolorization rates are the highest (94.74% and 95.08% respectively), and the comprehensive membership degree value is the largest (2.30). Therefore, D941 macroporous resin was selected for the subsequent purification experiment of Acanthopanax senticosus polysaccharide.

[0053] Experimental Example 2. Selection of purification conditions

[0054] Select D941 macroporous resin with the best performance in the static experiment of Experimental Example 1 as the adsorbent, and fill the solution of Acanthopanax senticosus polysaccharide prepared in step (1) of Example 1 into a chromatography column containing 30 mL of D941 macroporous resin (diameter-height ratio 1:5) for subsequent dynamic adsorption tests to evaluate the purification effect. The designed factor parameters include: ① eluent type (water, 25% v / v ethanol, NaCl solutions with concentrations of 0.1 M, 0.2 M, and 0.3 M); ② contact time between the sample solution and the macroporous resin, that is, adsorption time (0, 1, 2, 4, and 8 h); ③ sample volume (25 mL to 50 mL); ④ concentration of Acanthopanax senticosus polysaccharide solution (3.375 mg / mL to 27 mg / mL); ⑤ sample loading flow rate (3 to 12 mL / min); ⑥ elution flow rate (1.5 to 15 mL / min). Measure the polysaccharide retention rate, protein removal rate, and decolorization rate under the influence of each factor, and then calculate the comprehensive membership degree according to the comprehensive membership degree value calculation method in part of Experimental Example 1. Use the comprehensive membership degree value to evaluate the selected factor range, and the results are as Figure 1 shown.

[0055] From Figure 1It can be seen that when using 0.2 mol / L NaCl solution as the elution solvent, the retention rate of polysaccharide is the highest, the protein removal rate is the highest, the decolorization rate is medium, and the comprehensive membership degree value is the highest. Therefore, the optimal eluent is 0.2 mol / L NaCl solution. With the increase of adsorption time, the protein removal rate and decolorization rate first increase and then tend to be stable. Adsorption reaches equilibrium after 8 h, so the adsorption time is selected as 8 h. When the sample loading volume is greater than 30 mL, the protein removal rate drops sharply, and the protein adsorption of D941 resin reaches saturation. Therefore, the sample loading volume is selected as 30 mL. When the concentration of Acanthopanax senticosus polysaccharide solution is higher than 6.75 mg / mL, the protein removal rate drops sharply, and the protein adsorption of D941 resin reaches saturation. Therefore, the concentration of Acanthopanax senticosus polysaccharide solution is selected as 6.75 mg / mL. When the sample loading rate is greater than 3 mL / min, the protein removal rate and decolorization rate drop sharply. Therefore, the sample loading rate is selected as 3 mL / min. When the elution flow rate is greater than 3 mL / min, the protein removal rate and decolorization rate drop sharply. Therefore, the elution flow rate is selected as 3 mL / min.

[0056] On the basis of the above single-factor tests, the elution solvent was fixed as 0.2 mol / L NaCl solution, the adsorption time was fixed as 8 h, and the concentration of Acanthopanax senticosus polysaccharide solution was fixed as 6.75 mg / mL. A response surface optimization design with three parameters and three levels was used to optimize the three parameters for the purification of Acanthopanax senticosus polysaccharide, including the sample loading flow rate (A, mL / min), the sample loading volume (B, mL), and the elution flow rate (C, mL / min). Each variable was coded at three levels (-1, 0, 1) for statistical analysis, and the factor level design is shown in Table 2. At the same time, Table 3 gives the range and levels of the independent variables, as well as the results of the overall design. The overall design included 17 experimental points and was carried out in a random order. Table 4 shows the comprehensive membership degree values and the results of variance analysis. The optimal parameters for the purification of Acanthopanax senticosus polysaccharide were optimized from Table 4, and three verification experiments were carried out.

[0057] Table 2. Factors and levels of the response surface experiment

[0058]

[0059] Table 3. Response surface experiment design and results

[0060]

[0061] Table 4. Variance analysis of the regression model

[0062]

[0063] As can be seen from the results in Tables 2 to 4 above, the optimal sample loading speed finally determined by the purification test is 2 mL / min, the sample loading volume is 30 mL, and the elution speed is 3 mL / min. Further, under the above-preferred optimal experimental conditions, the purification of acanthopanax senticosus polysaccharide was carried out (i.e., using the preparation method and process parameters of Example 1), and the content of acanthopanax senticosus polysaccharide in the finally obtained product was measured to be 87.66 ± 0.84%. This shows that the method of the present invention can obtain acanthopanax senticosus polysaccharide with high purity.

[0064] Test Example III, Physicochemical Properties and Structural Characterization of Acanthopanax Senticosus Polysaccharide (PASP)

[0065] (1) Ultraviolet Absorption in the Range of 200 - 400 nm

[0066] Accurately weigh 10 mg of acanthopanax senticosus polysaccharide (PASP) purified and prepared in Example 1, dissolve it in distilled water, and make it up to 10 mL in a volumetric flask to prepare a solution. Place it in a UV-visible spectrophotometer and scan in the wavelength range of 200 - 400 nm. The results are as Figure 2 shown. As can be Figure 2 seen, the purified acanthopanax senticosus polysaccharide has high purity and few impurities.

[0067] (2) Molecular Weight Determination

[0068] Use a high-performance gel permeation chromatograph to detect the molecular weight of the acanthopanax senticosus polysaccharide purified in Example 1. The test steps are as follows:

[0069] ① Sample Preparation: Accurately weigh 10 mg of the freeze-dried powder of acanthopanax senticosus polysaccharide prepared in Example 1, add 0.1 mol / L sodium nitrate solution, mix gently, and swell at 4°C for 24 h to prepare a 1.0 mg / mL sample solution. Filter through a 0.45 μm filter membrane and then inject the sample for detection.

[0070] ② Chromatographic Conditions: High-performance gel permeation chromatograph (Agilent 1260, USA); Detector: Multi-angle laser light scattering detector (MALLs, DAWN HELEOS II, USA) - refractive index detector (RID, G1362A, 40°C); Chromatographic column is TSKgel G3000pwxl (7.8 × 300 mm, 5 μm); Mobile phase is 0.1 mol / L NaNO 3 , isocratic elution; Column temperature 35°C; Flow rate 0.5 mL / min; Injection volume 30 μL; Detection time 40 min. RID temperature 40°C; MALLS laser wavelength 664 nm; Use Astra 6.1 software to collect and process data.

[0071] The detection results of the molecular weight of the acanthopanax senticosus polysaccharide detected by the high-performance gel permeation chromatograph are as Figure 3 shown. As can be Figure 3It can be known that the weight-average molecular weight (Mw) of PASP is 11460 Da, the number-average molecular weight (Mn) is 5186 Da, and the molecular weight distribution index (D = Mw / Mn) is 2.211 (>2), indicating a wide distribution.

[0072] (3) Monosaccharide composition analysis

[0073] The monosaccharide composition of the Acanthopanax senticosus polysaccharide purified in Example 1 was determined by the PMP pre-column derivatization HPLC method. The specific steps are as follows:

[0074] ① Acid hydrolysis: Weigh accurately 25 mg of the freeze-dried powder of Acanthopanax senticosus polysaccharide, place it in a 5 mL volumetric flask, add water to the mark, shake well to prepare a 5 mg / mL polysaccharide solution. Accurately pipette 1.0 mL of the polysaccharide solution and place it in a thick-walled pressure-resistant bottle. Add 1.0 mL of 4 mol / L trifluoroacetic acid, immediately tighten the screw cap after filling with nitrogen, and hydrolyze in a 120 °C forced-air drying oven for 2 h. Take it out and cool to room temperature. Add 6 mL of pure methanol to the bottle, and then each time when it is concentrated under reduced pressure to half of the original volume, add 4 mL of pure methanol again. Repeat the operation 6 times to remove the residual trifluoroacetic acid. Finally, concentrate the solution to dryness under reduced pressure, dry it in an oven at 60 °C for 2 h, and dissolve it with 1.0 mL of water for standby.

[0075] ② Pre-column derivatization: Accurately pipette 50 μL of the solution after acid hydrolysis above and place it in a centrifuge tube. Add 50 μL each of 0.3 mol / L NaOH solution and 0.5 mol / L methanol solution of 1-phenyl-3-methyl-5-pyrazolone (PMP), mix well and react in a 70 °C water bath for 100 min. Take out the centrifuge tube and cool to room temperature, then add 50 μL of 0.3 mol / L HCl solution to neutralize NaOH. Add water to make up to 2 mL, then add 2 mL of chloroform, vortex and mix well for 3 min, centrifuge at 5000 r / min for 5 min, discard the lower chloroform layer, collect the upper aqueous solution, vortex and extract 3 times to remove the excess PMP, pass the water layer through a 0.22 μm microporous filter membrane, and use high-performance liquid chromatography for injection analysis.

[0076] ③ HPLC detection: The chromatographic conditions are as follows: The chromatographic column is a Dikma C18 chromatographic column (4.6 mm × 250 mm × 5 μm), the injection volume is 10 μL, the column temperature is 30 °C, the mobile phase velocity is 1 mL / min, and the mobile phase A is 0.1 M Na 2 HPO 4 and NaH 2 PO 4The buffer solution, mobile phase B is acetonitrile, isocratic elution is carried out according to the ratio of A:B = 83:17, and the detector used is an ultraviolet detector. Finally, the absorbance values of the standard product and the sample are detected at 250 nm, and signal conversion is carried out. According to the different peak emergence times and peak areas, the monosaccharide composition and ratio in the polysaccharide sample can be judged. The determination results of the monosaccharide composition of acanthopanax senticosus polysaccharide are as Figure 4 shown.

[0077] It can be Figure 4 seen that acanthopanax senticosus polysaccharide contains 7 monosaccharides, namely mannose, rhamnose, galacturonic acid, glucose, galactose, xylose and arabinose, and the molar ratio is mannose∶rhamnose∶galacturonic acid∶glucose∶galactose∶xylose∶arabinose = 9.75∶8.40∶4.25∶34.53∶25.84∶5.44∶11.80. It can be seen from this that the purified acanthopanax senticosus polysaccharide is an acidic heteropolysaccharide.

[0078] (4) Infrared spectrum determination to identify characteristic functional groups

[0079] Accurately weigh 2 mg of the acanthopanax senticosus polysaccharide purified in Example 1 and mix it with 150 mg of potassium bromide (KBr). After grinding, press it into a tablet with a thickness of 1.5 mm. Subsequently, use a Fourier transform infrared spectrometer (Nicolet iS5, Thermo) to scan the pressed sample and analyze the characteristic absorption peaks in the polysaccharide sample. The scanning range is 4,000 - 400 cm -1 , and the number of scans is 32 times. The infrared spectrum determination results are as Figure 5 shown.

[0080] It can be Figure 5 seen from the infrared spectrum diagram that acanthopanax senticosus polysaccharide has a strong absorption peak near 3402 cm -1 , which is the stretching vibration of the hydroxyl group (-OH). The broad and smooth peak shape indicates the existence of hydrogen bonds within or between polysaccharide molecules. The absorption peak at 2930 cm -1 is formed by the stretching vibration of the carbon-hydrogen bond (C-H) of the methylene group (-CH 2 -) in the sugar. The peaks near 1414 cm -1 and 1384 cm -1 are respectively the C-H bending vibration absorption peaks of -CH 2 - and methyl (-CH 3 ) in the sugar. The absorption peak near 1640 cm -1 is caused by the stretching vibration of the carbonyl group (C=O). The peak is strong and narrow, indicating that acanthopanax senticosus polysaccharide contains a carboxyl group (-COOH) and there is uronic acid. The special band in the region of 1200 - 1000 cm -1 mainly corresponds to the stretching vibration of the carbon-oxygen-hydrogen bond (C-O-H) side group and the glycosidic bond (C-O-C) vibration; 1152 cm-1 and 1078 cm -1 The absorption peaks at correspond to the stretching vibration absorption peaks of the carbon-oxygen bond (C-O) on the pyran ring. The absorption at 1040 cm -1 and 1020 cm -1 is due to the bending vibration of the alcohol hydroxyl group, indicating that the Acanthopanax senticosus polysaccharide exists in the form of pyranose. The peak near 917 cm -1 is the characteristic peak of β-pyranose, and the peak near 865 cm -1 is the characteristic peak of α-pyranose, indicating the coexistence of α- and β-glycosidic bonds in the Acanthopanax senticosus polysaccharide, which needs to be further explored by other methods. At the same time, in the fingerprint characteristic region within the range of 1000 - 500 cm -1 , it is mainly the stretching vibration of C-O and C-O-C of the polysaccharide.

[0081] Experimental Example 4: Antitumor Activity Test

[0082] The antitumor activity of the Acanthopanax senticosus polysaccharide purified and prepared in Example 1 was tested, and the specific steps are as follows:

[0083] (1) Grouping and Administration

[0084] Male BALB / c mice at 6 weeks of age (weighing 18 ± 2 g, purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were housed in a standard experimental environment (relative humidity: 50 ± 5%; environmental temperature: 23 ± 2°C; light: 12L:12D). The test mice underwent a one-week pre-experiment acclimation phase, including administering 0.2 mL of normal saline to the test mice by daily gavage. After 7 days of adapting to the test conditions, these mice were randomly divided into 6 groups, with 8 mice in each group.

[0085] The specific grouping is as follows: blank group, model group, and three Acanthopanax senticosus polysaccharide PASP treatment groups of Example 1 (PASPL group: 50 mg / kg of PASP; PASPM group: 100 mg / kg of PASP; PASPH group: 200 mg / kg of PASP, all prepared with normal saline), and 5-fluorouracil positive control group (5-Fu, 20 mg / kg). The CT26.WT mouse colon cancer cells were inoculated subcutaneously in the right anterior axilla of the mice in the model group, PASP treatment groups, and positive control group (about 5×10 6(number / unit, 0.15 mL). Intervention was started on the second day after inoculation. In the PASP group, gavage administration was performed daily according to the dose. The positive control group was treated with 5-Fu at a concentration of 20 mg / kg, and the intraperitoneal injection volume was 0.2 mL / unit, once every two days, lasting until the end. The blank group and the model group were gavaged with normal saline throughout the study period, with a volume of 0.2 mL / unit. The change in tumor volume was measured every 2 days. On the 18th day after administration, the mice were anesthetized with 20% urethane, and tissues and blood were extracted for subsequent research.

[0086] (2) Determination of tumor inhibition rate and immune organ index

[0087] The tumor length (L, mm) and width (W, mm) were measured with a vernier caliper every other day during the experiment, and the tumor volume was calculated (tumor volume (mm 3 ) = LW 2 / 2). Further, the blood remaining on the surface of the cancer tissue was rinsed three times with cold saline. After the operation, the exact weight of each tumor was recorded, and the tumor inhibition rate was calculated. At the same time, after the experiment ended, the weights of the immune organs thymus and spleen were accurately recorded. Among them, the tumor inhibition rate TIR (%) = (WM - WT) / WM × 100%; WM is the average tumor weight of the mice in the model control group, and WT is the average tumor mass of the mice in the treatment group. The immune organ index (mg / g) = immune organ weight / mouse weight.

[0088] Among them, the measurement results of the tumor mass of CT26.WT colorectal cancer-bearing mice in each group are as Figure 6 shown. The measurement results of the thymus and spleen indices of CT26.WT colorectal cancer-bearing mice in each group are as Figure 7 shown. The comprehensive results of each index are shown in Table 5.

[0089] Table 5. Effects of polysaccharide of Acanthopanax senticosus on tumor inhibitory activity of CT26.WT colorectal cancer-bearing mice

[0090]

[0091] Note: * , P < 0.05 compared with the blank group, ** , P < 0.01 compared with the blank group; # , P < 0.05 compared with the model group, ## , P < 0.01 compared with the model group.

[0092] In this experiment, to evaluate the anti-tumor effect of PASP, a mouse colorectal cancer model (CT26.WT) was used for experimental research. As Figure 6As shown in Table 5, oral administration of Acanthopanax senticosus polysaccharide at doses of 50, 100, and 200 mg / kg significantly inhibited tumor growth and reduced tumor weight in a dose-dependent manner (p<0.05). The tumor growth inhibition rates (TIR) were 5.18%, 17.87%, and 37.40%, respectively, indicating that Acanthopanax senticosus polysaccharide has a significant inhibitory effect on tumor growth.

[0093] As Figure 7 As shown in Table 5, compared with the blank group, the thymus weight and spleen weight of CT26.WT tumor-bearing mice were significantly increased (p<0.05). These results suggest that the proliferation of CT26.WT tumor cells may damage the function of immune organs. Compared with the model group, significant changes occurred in the thymus and spleen indices of mice after PASPH administration (p<0.05), preliminarily indicating that PASP has the potential to reduce splenomegaly and thymic atrophy, and thus may improve the immune dysfunction of CT26.WT tumor-bearing mice. The improvement effect of 5-FU on splenomegaly and thymic atrophy was lower than that of PASP, because 5-Fu is a chemotherapeutic drug, and the spleen and thymus are immune tissues, and chemotherapeutic drugs cause damage to immune tissues.

[0094] (3) Histopathological changes of tumors

[0095] The solid tumors after the above experiments were fixed with neutral paraformaldehyde, embedded in paraffin and further processed. Then the tissues were sectioned into 4-μm thick sections. HE staining was performed, and the pathological changes of tumor tissues were observed under a Nikon Eclipse Ci-L optical microscope (Nikon, Japan).

[0096] In this study, hematoxylin-eosin (H&E) staining on tumor tissue sections was used to analyze the organizational structure and morphological changes. The cell nuclei of tumor cells in tumor tissues were dark blue-purple, and the cytoplasm was pink-purple. The tumor tissue cells in the model group were dense, with intact cell nuclei, clear nucleoli, uniform cytoplasm, and dense chromatin, suggesting a proliferative environment for tumor cell growth. Compared with the model group, tumor cells in the 5-Fu group and PASP treatment group showed necrosis, nuclear fragmentation or dissolution, and blurred nuclear membranes and nucleoli; the necrotic areas in tumor tissues increased significantly, the cell volume decreased, the cell arrangement was loose, the lymphocyte infiltration increased, and the interstitial space expanded. With the increase of the PASP administration dose, the damage effect on tumor tissues was more obvious, and the cell gap was also more obvious. Morphological observations showed that Acanthopanax senticosus polysaccharide may inhibit tumor cell proliferation by regulating the host immune response.

[0097] (4) Effects on serum cytokines of tumor-bearing mice

[0098] Collect the peripheral blood of the mice in each group above, and centrifuge at 4000 r / min for 15 min at 4°C to prepare serum. Use an ELISA kit to detect the serum concentrations of TNF-α, IL-2, and IFN-γ according to the manufacturer's instructions. All measurements were performed using a FlexA-200 enzyme-linked immunosorbent assay analyzer (Allsheng, China). The results are as Figure 8 shown.

[0099] As Figure 8 shown, compared with the blank group, the levels of anti-tumor related cytokines (IL-2, TNF-α, and IFN-γ) in the model group of mice decreased significantly (p < 0.05). Compared with the model group, the levels of TNF-α, IL-2, and IFN-γ increased significantly after administration of acanthopanax polysaccharide (p < 0.05), and showed a dose-dependence. The cytokines in the 5-FU group were significantly lower than those in the model group. The results indicate that acanthopanax polysaccharide may enhance the immune function and inhibit tumor progression by promoting the release of cytokines in the anti-tumor response of tumor-bearing mice, and the effect is better than that of 5-FU.

[0100] Based on the above results, it can be seen that in this invention, a CT26.WT tumor-bearing mouse model was constructed to evaluate the anti-cancer effect and mechanism of acanthopanax polysaccharide in vivo. The experiments showed that after oral administration of acanthopanax polysaccharide, the volume and weight of the tumors in the mice decreased significantly, and the highest tumor growth inhibition rate reached 37.40%. At the same time, combined with the organ index, it was found that acanthopanax polysaccharide could protect the immune organs of tumor-bearing mice and improve the phenomena of spleen swelling and thymus atrophy. In addition, acanthopanax polysaccharide could also increase the levels of tumor-related cytokines (IL-2, TNF-α, and IFN-γ). Thus, it can be seen that acanthopanax polysaccharide can significantly inhibit the growth of tumors in vivo by regulating the immune ability of the body.

[0101] In summary, in this invention, by improving the preparation process of acanthopanax polysaccharide, not only was acanthopanax polysaccharide with high purity and significant anti-tumor activity successfully prepared, but also the anti-tumor mechanism and anti-tumor activity of acanthopanax polysaccharide in vivo were revealed. Therefore, this invention can provide a novel purification strategy for the preparation of plant polysaccharides, is also beneficial to improving the resource utilization rate of traditional Chinese medicine, and can also provide a technical basis for the development of anti-tumor drugs based on acanthopanax polysaccharide.

[0102] Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing Acanthopanax senticosus polysaccharide, characterized in that: The following steps are involved: (1) crushing the Acanthopanax senticosus raw material, then extracting with water and filtering to obtain an extract; adjusting the pH of the extract to 10-12, then stirring, then adjusting the pH of the extract to 5-6, then stirring, standing, taking the supernatant for alcohol precipitation, and drying to obtain a crude Acanthopanax senticosus polysaccharide; (2) Dissolving the Acanthopanax senticosus crude polysaccharide in water to obtain an Acanthopanax senticosus crude polysaccharide solution; purifying the Acanthopanax senticosus crude polysaccharide solution in a macroporous resin, and then collecting the purified solution, concentrating, dialyzing, and drying to obtain purified Acanthopanax senticosus polysaccharide; the macroporous resin is a D941 macroporous weakly alkaline anion exchange resin.

2. The method for preparing Acanthopanax senticosus polysaccharide according to claim 1, characterized in that: In step (1), the crushing is to crush the Acanthopanax senticosus raw material into segments of 2 to 10 cm each.

3. The method for preparing Acanthopanax senticosus polysaccharide according to claim 1, characterized in that: In step (1), the water extraction is to extract the crushed Acanthopanax senticosus raw material with water; the extraction process is: first soaking for 20-40 minutes, then boiling for 0.5-2 hours, and then reflux for 3-6 hours; during the extraction, the mass ratio of the Acanthopanax senticosus raw material to water is 1:5-10.

4. The method for preparing Acanthopanax senticosus polysaccharide according to claim 1, characterized in that: In step (1), the pH of the extract is first adjusted to 10-12 by using lime milk; the mass concentration of the lime milk is 10%-30%; the pH of the extract is adjusted to 5-6 by using sulfuric acid solution; the mass concentration of the sulfuric acid solution is 10%-30%.

5. The method for preparing Acanthopanax senticosus polysaccharide according to claim 1, characterized in that: In step (1), the alcohol precipitation treatment is to add ethanol to precipitate the supernatant, and the volume fraction of the added ethanol is 80% to 95%; the time of the alcohol precipitation treatment is 10 to 16 hours.

6. The method for preparing Acanthopanax senticosus polysaccharide according to any one of claims 1 to 5, characterized in that: In step (2), the purification is performed by using a macroporous resin as an adsorbent and adopting a column chromatography technique to elute and separate the Acanthopanax senticosus crude polysaccharide solution to obtain a purified solution; the concentration of the Acanthopanax senticosus crude polysaccharide solution is 5-8 mg / mL; During the elution separation, the eluent used is a 0.1-0.3 mol / L sodium chloride solution, the loading speed is 1.5-2.5 mL / min, the loading volume is 20-40 mL, the elution speed is 1.5-3.75 mL / min, and the adsorption time is 6-10 h.

7. The method for preparing Acanthopanax senticosus polysaccharide according to claim 6, characterized in that: The concentration of the Acanthopanax senticosus polysaccharide solution is 6.75 mg / mL; the eluent is 0.2 mol / L sodium chloride solution, the loading speed is 2 mL / min, the loading volume is 30 mL, the elution speed is 3 mL / min, and the adsorption time is 8 h.

8. The method for preparing Acanthopanax senticosus polysaccharide according to any one of claims 1 to 5, characterized in that: In step (2), the concentration is to concentrate the purified solution to a relative density of 1.02-1.05 g / cm 3 ; The molecular weight cutoff of the dialysis is 3000~4000Da; and the drying is freeze-drying.

9. An Acanthopanax senticosus polysaccharide prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The weight average molecular weight of the Acanthopanax senticosus polysaccharide is 1×10 4 ~1.2×10 4 Da.

10. A use of Acanthopanax senticosus polysaccharide as claimed in claim 9, characterized in that: The application is the application of Acanthopanax senticosus polysaccharide in the preparation of anti-tumor drugs; the anti-tumor drug is an anti-colon cancer drug.

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