Preparation method of safflower seed meal polysaccharide

Abstract

The invention discloses a preparation method of safflower seed meal polysaccharide, which is characterized by comprising the following steps: mixing safflower seed meal powder with distilled water; boiling the mixture, continuously stirring, extracting the precipitate, adding distilled water again, and repeating the operation; filtering the mixture with filter paper, and collecting filtrate; adding 95% ethanol, and standing for 24 hours to precipitate the polysaccharide at 4 DEG C; separating to obtain an upper-layer supernatant and a lower-layer precipitate; drying the precipitate by using a rotary evaporator until a concentrated solution is obtained, and removing protein by using a Sevag method; removing pigments in the concentrated solution by using AB-8 macroporous column chromatography to obtain a crude polysaccharide sample;

Description

Technical Field

[0001] The present invention belongs to the technical field of plant polysaccharides, and specifically relates to a preparation method of safflower seed meal polysaccharide and the application of the obtained safflower seed meal polysaccharide. Background Art

[0002] Safflower seeds are widely used for oil production, but their seed meal has not been fully utilized. If the safflower seed meal resources can be fully utilized, it can not only bring benefits to farmers, but also bring benefits to oil enterprises, and at the same time promote the development of the safflower planting industry. The polysaccharides extracted from safflower seed meal have the potential to be used as novel, sustainable and natural anti-tumor agents. With the increasing demand for natural low-toxic anti-cancer agents, the polysaccharides extracted from safflower seed meal have attracted much attention due to their diverse biological activities and anti-tumor characteristics.

[0003] However, the current technologies for extracting, purifying and analyzing polysaccharides lack efficiency, and the characterization of their biological activities is still not perfect. The present invention aims to overcome these limitations and provide a systematic method to obtain bioactive polysaccharides with high purity and strong anti-tumor activity. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of safflower seed meal polysaccharide and the obtained safflower seed meal polysaccharide. The present invention uses safflower seed meal as raw material, extracts and purifies safflower seed meal polysaccharides (SSRP1, SSRP2, SSRP3) through hot water extraction method, DEAE-52 and Sephadex-G 100 chromatographic purification technologies, and analyzes their component structures and anti-tumor activities.

[0005] The present invention provides a preparation method of safflower seed meal polysaccharide, including the following steps: Step S1. Mix safflower seed meal powder with distilled water; boil the mixture and continuously stir, add distilled water again after extracting the precipitate and repeat the above operation; then filter the mixture with filter paper and collect the filtrate; add 95% ethanol, let stand for 24 hours to precipitate the polysaccharide at 4°C; separate to obtain the upper supernatant and the lower precipitate; use a rotary evaporator to dry the precipitate until a concentrated solution is obtained, use the Sevag method to remove proteins; use AB-8 macroporous column chromatography to remove pigments in the concentrated solution to obtain a crude polysaccharide sample.

[0006] Step S2. Rinse the DEAE-52 cellulose resin with NaOH to precipitate it. Wash it with distilled water until a neutral pH value is reached. Rinse the column with HCl. Then wash it with distilled water until the pH value is neutral. Gently pour it into the chromatography column without introducing air bubbles. Let the resin precipitate under gravity. After the packing is compacted, drain the excess water. Equilibrate the chromatography column with 3 bed volumes of the starting buffer and NaCl solutions of different concentrations. Load the crude polysaccharide sample into the chromatography column. Use NaCl solutions with continuously increasing concentrations. Collect 10 mL volumes of the eluate, analyze the carbohydrate content, and determine the elution curve. By monitoring the results of the phenol-sulfuric acid test of the eluate, three eluted peak polysaccharides are pooled and named SSRP1, SSRP2, and SSRP3 respectively; Step S3. Assemble a Sephadex-G-100 chromatography column as described above. Ensure that the chromatography column is equilibrated overnight with the selected equilibration buffer before loading the sample. Start collecting the sample when the eluate begins to clear. Continue to collect equal volumes of the eluate, generally 10 mL. Extract the polysaccharide with distilled water and NaCl solutions of different concentrations. Collect the eluate by the phenol-sulfuric acid method, and obtain three purified polysaccharides of SSRP1, SSRP2, and SSRP3 by measuring the results by the phenol-sulfuric acid method.

[0007] Furthermore, in step S1, the liquid-solid ratio of safflower seed meal to distilled water is 10:1.

[0008] Furthermore, the NaOH and HCl for rinsing the DEAE-52 cellulose resin in step S2 are: 3 - 4 bed volumes of 0.5 M NaOH and 3 bed volumes of 0.5 M HCl.

[0009] Furthermore, the NaCl solutions of different concentrations in step S2 are: gradient elution is carried out successively with 0.05, 0.1, 0.15, and 0.2 mol / L NaCl solutions.

[0010] Furthermore, the separation and purification conditions of Sephadex-G-100 in step S3 are: the flow rate is 0.5 - 1 mL / min; the NaCl solutions of different concentrations are: 0.05, 0.1, 0.15, and 0.2 mol / L.

[0011] With the increasing demand for natural low-toxic anticancer agents, the polysaccharides extracted from safflower seed meal have attracted much attention due to their diverse biological activities and antitumor properties. Safflower seeds are widely used for oil production, but their seed meal has not been fully utilized. The polysaccharides extracted from safflower seed meal have the potential to be used as novel, sustainable, and natural antitumor agents.

[0012] However, the current techniques for extracting, purifying, and analyzing polysaccharides lack efficiency, and the characterization of their biological activities remains imperfect. The present invention aims to overcome these limitations and provide a systematic method to obtain bioactive polysaccharides with high purity and strong anti-tumor activity.

[0013] To better solve the problem of resource waste of safflower seed meal, through a large number of exploratory experiments, the inventors proposed a suitable extraction method by optimizing factors such as the temperature of water extraction and alcohol precipitation, the liquid-solid ratio, and the extraction time. The present invention uses the water extraction and alcohol precipitation method to extract polysaccharides from safflower seed meal, which can also maintain the biological activity of the polysaccharides and is an ideal method for extracting polysaccharides from safflower seed meal. Brief Description of the Drawings

[0014] Figure 1 It is the elution curve of safflower seed meal polysaccharide on a DEAE-52 cellulose column.

[0015] Figure 2 It is the total sugar standard curve of SSRP.

[0016] Figure 3 It is the elution curve of SSRP1 on a Sephadex-G100 chromatographic column.

[0017] Figure 4 It is the elution curve of SSRP2 on a Sephadex-G100 chromatographic column.

[0018] Figure 5 It is the elution curve of SSRP3 on a Sephadex-G100 chromatographic column.

[0019] Figure 6 The zeta potential of the safflower seed meal polysaccharide SSRP1 nanoemulsion is -21.2 mV.

[0020] Figure 7 The zeta potential of the safflower seed meal polysaccharide SSRP2 nanoemulsion is -32. mV.

[0021] Figure 8 The zeta potential of the safflower seed meal polysaccharide SSRP3 nanoemulsion is -16.4 mV.

[0022] Figure 9 It is the high-performance liquid chromatography (HPLC) chromatogram of standard monosaccharides.

[0023] Figure 10 It is the HPLC chromatogram of SSRP1.

[0024] Figure 11 It is the HPLC chromatogram of SSRP2.

[0025] Figure 12 It is the HPLC chromatogram of SSRP3.

[0026] Figure 13 It is the Fourier transform infrared spectrum of SSRP.

[0027] Figure 14-a It is the nuclear magnetic resonance spectrum of SSRP1 ( 1 H).

[0028] Figure 14-b It is the nuclear magnetic resonance spectrum of SSRP1 ( 13 C).

[0029] Figure 14-c It is the nuclear magnetic resonance spectrum of SSRP1 (HSQC).

[0030] Figure 14-d It is the nuclear magnetic resonance spectrum of SSRP1 (COSY).

[0031] Figure 14-e It is the nuclear magnetic resonance spectrum of SSRP1 (HMBC).

[0032] Figure 14-f It is the nuclear magnetic resonance spectrum of SSRP1 (NOESY).

[0033] Figure 15-a It is the scanning electron microscope of SSRP1.

[0034] Figure 15-b It is the scanning electron microscope of SSRP2.

[0035] Figure 15-c It is the scanning electron microscope of SSRP3.

[0036] Figure 16-a It is the inhibitory effect of SSRP1 on the cell proliferation of HepG2 cell line.

[0037] Figure 16-b It is the inhibitory effect of SSRP2 on the cell proliferation of HepG2 cell line.

[0038] Figure 16-c It is the inhibitory effect of SSRP3 on the cell proliferation of HepG2 cell line. Specific implementation manners

[0039] The above content of the present invention will be further described in detail through the following examples. Obviously, the described examples are only a part of the examples of the present invention, rather than all the examples. All other examples obtained by those of ordinary skill in the art based on the examples in the present invention without creative efforts belong to the scope of protection of the present invention.

[0040] Example 1. Preparation method of crude polysaccharide from safflower seed meal Step S1. Mix 50 g of safflower seed meal powder with 500 ml of distilled water in a flask. After boiling the mixture at 100 °C with continuous stirring for 2 hours, extract the precipitate, add another 500 ml of distilled water and repeat the above operation. Then filter the mixture with filter paper to remove the seed meal and collect the filtrate. Add 95% ethanol and let it stand for 24 hours to precipitate the polysaccharide at 4 °C. Separate to obtain the upper supernatant and the lower precipitate. Dry the precipitate using a rotary evaporator until a concentrated solution is obtained. Use the Sevag method to remove proteins and use AB-8 macroporous column chromatography to remove pigments in the concentrated solution to obtain a crude polysaccharide sample.

[0041] Example 2. Preparation method of crude polysaccharide from safflower seed meal Step S1. Mix 50 g of safflower seed meal powder with 500 ml of distilled water in a flask. After boiling the mixture at 90 °C with continuous stirring for 2 hours, extract the precipitate, add another 500 ml of distilled water and repeat the above operation. Then filter the mixture with filter paper to remove the seed meal and collect the filtrate. Add 95% ethanol and let it stand for 24 hours to precipitate the polysaccharide at 4 °C. Separate to obtain the upper supernatant and the lower precipitate. Dry the precipitate using a rotary evaporator until a concentrated solution is obtained. Use the Sevag method to remove proteins and use AB-8 macroporous column chromatography to remove pigments in the concentrated solution to obtain a crude polysaccharide sample.

[0042] Example 3. Preparation method of crude polysaccharide from safflower seed meal Step S1. Mix 50 g of safflower seed meal powder with 500 ml of distilled water in a flask. After boiling the mixture at 80 °C with continuous stirring for 2 hours, extract the precipitate, add another 500 ml of distilled water and repeat the above operation. Then filter the mixture with filter paper to remove the seed meal and collect the filtrate. Add 95% ethanol and let it stand for 24 hours to precipitate the polysaccharide at 4 °C. Separate to obtain the upper supernatant and the lower precipitate. Dry the precipitate using a rotary evaporator until a concentrated solution is obtained. Use the Sevag method to remove proteins and use AB-8 macroporous column chromatography to remove pigments in the concentrated solution to obtain a crude polysaccharide sample.

[0043] Example 4. Preparation method of crude polysaccharide from safflower seed meal Step S1. Mix 50 g of safflower seed meal powder with 500 ml of distilled water in a flask. Boil the mixture at 60 °C while stirring continuously for 2 hours. After extracting the precipitate, add 500 ml of distilled water again and repeat the above operation. Then filter the mixture with filter paper to remove the seed meal and collect the filtrate. Add 95% ethanol and let it stand for 24 hours to precipitate the polysaccharide at 4 °C. Separate to obtain the upper supernatant and the lower precipitate. Dry the precipitate using a rotary evaporator until a concentrated solution is obtained. Use the Sevag method to remove proteins and use AB-8 macroporous column chromatography to remove pigments in the concentrated solution to obtain a crude polysaccharide sample.

[0044] Experimental Example 1: Detection Experiment on the Extraction Rate of Safflower Seed Meal Polysaccharide 1. Experimental Method Determine the polysaccharide extraction rates of the crude polysaccharide extraction methods in Example 1, Example 2, Example 3, and Example 4. Among them, for the polysaccharide determination method, glucose is used as the standard sample, and the phenol-sulfuric acid method is used to determine the polysaccharide content. Calculate the yield of the crude polysaccharide according to the formula: Crude polysaccharide yield (%) = (mass of the extracted crude polysaccharide / mass of safflower seed meal after pretreatment) 2. Experimental Results The experimental results are shown in Table 1, Table 2, and Table 3.

[0045] Table 1. Yields of Crude Safflower Seed Meal Polysaccharides at Different Temperatures Table 2. Statistical Analysis of Process Parameters of Crude Safflower Seed Meal Polysaccharide As can be seen from Table 1 and Table 2, the optimal temperature for the extraction method of safflower seed meal polysaccharide provided by the present invention is 100 °C, and its purity is 10% ± 0.88.

[0046] Experimental Example 2: Physical and Chemistry of Safflower Seed Meal Polysaccharide 1. Experimental Materials: The safflower seed meal polysaccharides SSRP1, SSRP2, and SSRP3 prepared as described in the specification.

[0047] 2. Experimental Method: Determine the component analysis, molecular weight analysis, thermal property analysis, and recovery rate determination of safflower seed meal polysaccharide.

[0048] 2.1 Monosaccharide Composition: The monosaccharide composition was evaluated by high performance liquid chromatography (HPLC) derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP). A total of 5.0 mg of safflower seed residue polysaccharide (SSRP) was hydrolyzed with 5.0 mL of 3.0 mol / L trifluoroacetic acid (TFA) at 110 °C for 8 hours. Here, the resulting solution was evaporated to dryness under nitrogen and redissolved in 300 µL of distilled water. We selected 250 µL of the SSRP hydrolyzate (1.0 mg / mL) and mixed it with 250 µL of 0.6 mol / L NaOH and 500 µL of 0.4 mol / L pmp - methanol solution. Incubate for 60 minutes in the dark at 70 °C. After cooling for 10 minutes, 100 µL of 0.3 mol / L HCl was added to terminate the reaction. Then 1.0 mL of chloroform was added and vortexed Extracted three times. After centrifugation at 10000 rpm for 5 minutes, the supernatant was detected by HPLC. The analysis was performed using an Ultimate 3000 high performance liquid chromatography system equipped with a diode array detector and an Ultimate C18 column (4.6 mm × 200 mm, 5 µm). The analysis was carried out at a temperature of 30 °C and the detection wavelength was 250 nm. The mobile phase was 83% phosphate buffer (pH 6.7, 50 mmol / L) and 17% acetonitrile, and the flow rate was 1.0 mL / min. The external standard method was used to determine the monosaccharide composition, referring to the monosaccharide mixed standard with a concentration of 2.0 mg / mL.

[0049] 2.2 Molecular weight: The molecular weight (Mw) of safflower seed meal polysaccharide (SSRP) was determined by high performance gel permeation chromatography (HPGPC) using a Waters 1515 system. The system was equipped with a refractive index detector (1260 RID, Agilent Technologies, CA, USA) and a G4000-swxl column (4.6 mm x 300 mm, Tosoh Biosep, USA)). HPGPC analysis was carried out under constant temperature conditions of 30 °C to produce stable elution conditions. During the analysis, 10 µL of the SSRP sample with a concentration of 2 mg / mL was injected into the system. The substance was eluted with a KH2PO4 solution (0.1 mol / L) as the mobile phase at a flow rate of 1.0 mL / min. To determine the molecular weight, the elution curve of SSRP was compared with the elution curve of dextran standards with known molecular weights to obtain a calibration curve.

[0050] 2.3 Thermal properties: Thermogravimetric (TG) and differential scanning calorimetry (DSC) tests were carried out using a thermogravimetric analyzer (STA 449 F3, Netzsch, Germany) to evaluate the thermal properties of safflower seed residue polysaccharide (SSRP). In the experiment, 10 mg of the SSRP sample was placed in an Al2O3 crucible, and the temperature was raised from 25 °C to 700 °C at a regulated heating rate of 10 °C / min. The study was conducted in a nitrogen atmosphere with a stable flow rate of 50 mL / min to provide an inert environment to avoid oxidative damage during the heat treatment.

[0051] 3. Experimental results: The experimental results are as Figure 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and shown in Tables 3, 4 and 5.

[0052] Table 3 Purity, chemical composition and zeta potential of crude polysaccharide and SSRP Table 4 Molecular weight of SSRP Sample Weight-average molecular weight (kDa) Number-average molecular weight (kDa) Peak molecular weight (kDa) Polydispersity index (PDI) (Mw / Mn) SSRP1 0.707 0.393 0.185 1.798 SSRP2 2.033 0.903 0.368 2.252 SSRP3 3.942 1.551 0.949 2.542 Table 5 Monosaccharide composition of SSRP Serial number Peak name Retention time (min) 1 Fructose 1.961 2 Glucuronic acid 8.838 3 Flucose 11.240 4 Galactose 13.813 3.1 Recovery rate: The SSRP solution was separated and purified by DEAE-52 cellulose fast flow to obtain three independent elution peaks ( Figure 1 ). And the total sugar standard curve ( Figure 2 ). These three polysaccharide elution peaks were named SSRP1, SSRP2 and SSRP3. Based on the content of crude polysaccharide, the recovery rates of SSRP1, SSRP2 and SSRP3 were 10.22%, 8.5% and 6.33% respectively. According to the different molecular weights, these three elution peaks were further purified by a Sephadex-G-100 chromatographic column ( Figures 3 - 5 ). The final purified three elution peaks were abbreviated as SSRP1, SSRP2 and SSRP3 respectively, and the recovery rates were 72.15%, 65.60% and 61.77%.

[0053] 3.2 Molecular weight: HPGPC analysis showed the homogeneity and molecular weight of SSRP, and the results are shown in Table 4. The molecular weights of SSRP1, SSRP2 and SSRP3 were approximately 0.707 kDa, 2.033 kDa and 3.942 kDa respectively. The broadband peaks and low aggregation index values (0.707, 2.033 and 3.942 respectively) of these three elution peaks confirmed that they were homogeneous polysaccharides.

[0054] 3.3 Monosaccharide composition: Chemical composition and zeta potential of SSRP. As shown in Table 3, the zeta potentials of different SSRPs vary greatly ( Figures 6 - 8 ). Among them, SSRP1, SSRP2, and SSRP3 have higher negative charges, which are SSRP1: -17.60 mV, SSRP2: -18.67 mV, and SSRP3: -16.40 mV, respectively. Generally speaking, the zeta potential affects the stability of polysaccharides, and polysaccharides with more negative charges exhibit a more stable molecular structure. The results show that SSRP3 is more likely to aggregate and is also more stable.

[0055] In this study, the monosaccharide composition of SSRP was determined by PMP derivatization - high performance liquid chromatography. According to the retention time and peak area of each standard product ( Figures 9 - 12 ), it can be seen that SSRP is a neutral heteropolysaccharide composed of fructose, glucuronic acid, glucose (Glc), and galactose (Gal), with a molar ratio of 1.00:1.89:2.83. Obviously, the main monosaccharide in SSRP is galactose. This result is slightly different from previous studies (Table 5). For example, the acidic polysaccharide of N. tangutorum Bobr. is composed of Gal, Man, Glc, Ara, and Rha, with a molar ratio of 4.63:2.63:2.36:2.19:1.

[0056] 3.4 Polysaccharide purity: The purity of SSRP1 was measured to be 90.91% ± 1.07, the purity of SSRP2 was 88.90% ± 0.97, and the purity of SSRP1 was 77.78% ± 0.59. Experimental Example 3: Structural Characterization of Safflower Seed Meal Polysaccharide 1. Experimental materials: The safflower seed meal polysaccharides SSRP1, SSRP2, and SSRP3 prepared as described in the specification.

[0057] 2. Experimental methods: Fourier transform infrared spectroscopy analysis, NMR spectroscopy analysis, and SEM scanning analysis were performed on the safflower seed meal polysaccharide.

[0058] 2.1 Fourier transform infrared spectroscopy analysis of safflower seed meal polysaccharide: According to the KBr disk method, the infrared spectrum of SSRP powder was prepared. The dried SSRP (1.0 mg) was mixed with KBr powder (100 mg) and ground and pressed into a pellet. Using a Nicolet 5700 FT - IR spectrometer (Thermo Electron, Madison, WI, USA), the Fourier transform infrared spectrum of SSRP was collected in the frequency range of 4000 - 400 cm −1 .

[0059] 2.2 NMR Spectral Analysis of Polysaccharides from Safflower Seed Meal: For NMR analysis, SSRP (40 mg) was suspended in 1 ml (99.96%) of D2O, freeze-dried twice, and then dissolved in high-quality D2O (1 ml). 1 1H NMR spectra were acquired at 25 °C using an AVANCE III 400 MHz spectrometer (Bruker, Germany). Nuclear magnetic resonance technology can effectively characterize the detailed structure of complex polysaccharides, including α- or β-anomeric configurations, glycosidic bond types, and the sequence of monosaccharide units.

[0060] Nuclear magnetic resonance spectroscopy is the most convenient and effective technique for understanding the detailed structural information of polysaccharides, such as monosaccharide composition, α- or β-anomeric configurations, glycosidic bond types, and sugar sequences. In this paper, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy were used to analyze the chemical shifts of repeating sugar residues in polysaccharides. The structures of SSRP1 and SSRP2 were analyzed by 1D NMR ( 1 H, 13 C) and 2D NMR ( 1 H- 1 HCOSY, HSQC, HMBC).

[0061] 2.3 SEM Scanning Analysis of Polysaccharides from Safflower Seed Meal: The microstructure of polysaccharides from safflower seed meal was studied using a high-resolution thermal field emission scanning electron microscope (QUANTA 400FEG, FEI Company, USA). A layer of gold was deposited by sputter coating to make the sample conductive, and then imaging was performed under high vacuum at 10.0 kV.

[0062] 3. Experimental Results: 3.1. The Fourier transform infrared spectroscopy results of polysaccharides from safflower seed meal are as Figure 13 , shown in Table 6.

[0063] Table 6 Fourier transform infrared absorption rates of SSRP Prominent absorption peaks at 3370 cm -1 and 2930 cm -1 are characteristic peaks of polysaccharides. The broad absorption peak near 3370 cm -1 is attributed to O-H stretching vibration, while the peak near 2930 cm -1 is attributed to -CH stretching vibration, including -CH, -CH2, and -CH3. The prominent frequency band at 1648 cm -1 comes from the vibrations of O-H and -COOR. The prominent frequency band at 1451 cm -1The absorption peak at [[]] also indicates the bending vibrations of C-H, C-O, and C=O, confirming the presence of uronic acid. This conclusion is consistent with the monosaccharide composition. In addition, the signal at 1250 cm -1 shows a typical asymmetric stretching vibration of sulfate radical S=O. The absorption signal near 1035 cm -1 is attributed to C-O-H and C-O-C, indicating the presence of pyranose. The region between 1200 cm -1 and 800 cm -1 is the characteristic fingerprint of carbohydrates, including C-O-C glycosidic bond vibration and C-O-H bond. The signal in the range of 1200 - 1100 cm -1 is generated by the stretching vibration of glycosidic bonds. The band at 868 cm -1 proves the existence of α-structure. In addition, the peaks at 763 cm -1 and 674 cm -1 can be attributed to the β configuration of pyranose, and the unique absorption at 868 cm -1 indicates the possible existence of α-pyranose. The unique absorption at 868 cm -1 shows the possible existence of α-pyranose.

[0064] 3.2. The NMR spectra of safflower seed meal polysaccharide are shown in Figure 14 and Table 7.

[0065] Table 7 Chemical shifts of 1 H and 13 C of the SSP1 backbone Seed meal monosaccharide C1 / H1 C2 / H2 C3 / H3 C4 / H4 C5 / H5 C6 / H6 AnGalp 2-sulphate 105.7 / 4.61 103.9 / 4.62 103.6 / 4.42 100.8 / 5.30 96 / 5.19 NA α- 2,3-Fucp 99.9 / 5.05 75.40 / 3.59 71.53 / 4.02 75.59 / 3.80 61.25 / 3.42, 3.30 NA α-1-Galp 98.43 / 5.00 NA NA NA NA NA α-3-Fucp 100.6 / 5.06 NA NA NA NA NA In the isomeric region (δ 4.3 - 5.5 ppm) of the H-1 1 H NMR spectrum (Figure 14 - a), there are two strong signals at 4.99 (d, J = 3.7 Hz) and 5.42 (d, J = 3.9 Hz) ppm, and four weak signals at δ 4.41, 5.09, 5.14, and 5.37. These signals are considered to be the C1 proton signals of glucose residues, indicating that the polysaccharide is also composed of α-glycosidic bonds (δ > 5.0 ppm) and β-glycosidic bonds (δ < 5.0 ppm). The overlapping signals in the region of δ 3.48 - 4.22 ppm belong to the protons of sugar residues C2, C3, C4, C5, and C6.

[0066] In the isomeric region (δ 92 - 105 ppm) of the 13C NMR spectrum (Figure 14 - b), six main signals were observed, including three strong signals (δ 98.0, 98.4, 103.7 ppm) and three weak signals (δ 100.3, 100.6, 101.7 ppm), all of which belong to C1 of glucose residues. The signals also indicate the presence of α - glycosidic bonds (δ 98 - 103 ppm) and β - glycosidic bonds (δ 103 - 106 ppm). The C2 - C6 signals of sugar residues are distributed in the range of δ 61.1 - 81.3 ppm, among which δ 61.9 - 69.6 ppm are secondary carbon signals. In addition, 1 1H NMR front - field signals (δ1.17, 1.19, 1.79, 2.32 ppm) and 13C NMR front - field signals (δ19.9, 22.6, 32.5 ppm) show the presence of rhamnose residues and acetyl groups in SSRP1.

[0067] The carbon - proton correlations can be revealed from the HSQC spectrum. As shown in Figure 14, cross - peaks of 96 / 5.19, 105.7 / 4.61, 103.9 / 4.62, 103.6 / 4.42 and 100.8 / 5.30 were clearly observed in the low - field region of the HSQC spectrum. By comparing with the previously reported data, we deduced that the anomeric carbon (C1) / anomeric hydrogen (H1) signals of 92.03 / 5.42, 95.09 / 5.14, 98.43 / 5.00, 99.9 / 5.05 and 100.6 / 5.06 are 4 - linked AnGalp 2 - sulphate residue, α - 2,3 - Fucp, α - 1 - Galp and α - 3 - Fucp, respectively.

[0068] In addition, 1 H– 13The sugar residue sequence of SSRP1 was determined by HMBC. Taking the most abundant sugar residue as an example, the chemical shifts of the anomeric carbon (C1) / anomeric hydrogen (H1) signals were 92.03 ppm and 5.42 ppm, respectively (Figure 14-a-c). In the ¹H-¹H COSY spectrum, the chemical shifts of the H1 / H2 signals were 5.42 ppm and 3.59 ppm, respectively (Figure 14-d). From the HSQC spectrum, the chemical shift of C2 associated with H2 was determined to be 75.40 ppm (Figure 14-c). Similarly, the chemical shifts of the corresponding carbon-hydrogen signals on sugar residue A were as follows: C3 / H3 (4.02 / 71.53 ppm), C4 / H4 (3.80 / 75.59 ppm), C5 / H5 (3.42, 3.30 / 61.25 ppm) (Figure 14-d-f). Through one-dimensional nuclear magnetic resonance ( 1 H, 13 C) and two-dimensional nuclear magnetic resonance, chemical shift (COSY), heteronuclear multiple-carbon correlation spectroscopy (HMBC), and distortionless enhancement by polarization transfer (DEPT) techniques were used to analyze the structure of SSRP. The nuclear magnetic resonance spectrum of SSRP1 is shown. In 1 H as shown in the NMR spectrum (Figure 14-a), some peaks overlapped in the resonance regions of 4.3 - 4.8 ppm and 4.8 - 5.5 ppm. The strong signal at 4.52 ppm was the C1 proton signal of glucose. In addition, seven strong signal peaks were detected in the anomeric region, with chemical shifts of 5.13 ppm, 4.98 ppm, 4.43 ppm, 4.37 ppm, 5.05 ppm, 4.85 ppm, and 4.54 ppm, respectively, and a weak signal peak with a chemical shift of 5.26 ppm. The research results indicate that SSRP1 has α and β configuration glycosidic bonds.

[0069] 3.3. SEM analysis of the polysaccharide from safflower seed meal is shown in Figure 15.

[0070] The surface ultrastructure of SSRP can be observed from the scanning electron microscope images (Figure 15). The surface of SSRP shows a high degree of plant cell wall rupture and a huge pore structure, presenting a spongy, loose, folded, and rich porous structure. This phenomenon is likely the result of ultrasonic-induced cavitation bubble formation. The three-dimensional shape of safflower seed meal polysaccharide (SSRP) is closely related to its physical properties, and its structural morphology can be characterized by scanning electron microscopy. The scanning electron micrograph in Figure 15 shows SSRPs at different magnifications (20 million times and 200 million times). The surface of SSRP is rough, showing a porous flake structure, accompanied by some debris and branched structures. This phenomenon indicates that the high-frequency vibration of ultrasonic waves has a significant impact on the surface structure of SSRPs. The structures of SSRP1, SSRP2, and SSRP3 exhibit similar microstructural characteristics, presenting an irregular flaky aggregated surface morphology and existing in an aggregated state. Therefore, they may have a relatively large specific surface area. It is worth noting that SSRP3 has a smooth and complete structural feature, which is similar to the polysaccharide extracted from Morinda citrifolia (Noni). This unique structure may endow SSRP3 with special physicochemical properties. The rupture of bubbles generated by these structures during subsequent processes will form an extremely strong mechanical shear force, which helps to destroy the cell wall. In addition, some structures in SSRPs show fractures, which may be due to the complex cross-linking between the main chain and the branched chain.

[0071] Experimental Example 4: Inhibitory Effect of Safflower Seed Meal Polysaccharide on HL60 Cells 1. Experimental Materials: The crude safflower seed meal polysaccharide prepared as described in the specification, SSRP1, SSRP2, and SSRP3.

[0072] 2. Experimental Methods: Preparation of HL60 Cell Culture HL60 cells (human promyelocytic leukemia cell line) were used. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were kept in a humidified incubator at 37°C with a carbon dioxide concentration of 5%. The cells were passaged every 2 - 3 days to maintain exponential growth.

[0073] Preparation of Polysaccharide Solution SSRP1, SSRP2, and the crude polysaccharide were weighed. Each polysaccharide was dissolved in sterile distilled water to prepare stock solutions of 1000 µg / mL, 500 µg / mL, 250 µg / mL, 125 µg / mL, 62.5 µg / mL, 31.25 µg / mL, and 15.625 µg / mL.

[0074] Count HL60 cells using an automated cell counter. The optimal density is 5000 - 10000 cells per well on a 96-well plate. Add 100 µL of cell suspension to each well.

[0075] Polysaccharide treatment Add 100 µL of polysaccharide diluent to the corresponding wells. This brings the final volume in each well to 200 µL. Ensure a wide concentration range (1000 µg / mL to 15.625 µg / mL) to obtain a complete dose-response curve. Incubate at 37 °C and 5% CO2 for 72 hours.

[0076] Cell viability assay (MTT) Prepare an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution with PBS (5 mg / mL). After incubating for 72 hours, add 20 µL of MTT solution to each well. Incubate at 37 °C for 3 - 4 hours, and then add 100 µL of DMSO (dimethyl sulfoxide) to each well.

[0077] Absorbance measurement Measure the absorbance at 570 nm (reference wavelength: 630 nm) using a microplate reader. Record the absorbance value for each well.

[0078] Data analysis Calculate the percentage of cell viability for each well using the following formula: Cell viability (%) = ((Absorbance of treated well - Absorbance of positive control) / (Absorbance of negative control - Absorbance of positive control)) × 100 3. Experimental results: Crude polysaccharide: The IC50 value is 357.48 ± 63.33 μg / mL. This indicates that a relatively high concentration of this compound is required to inhibit 50% of the HL60 cell activity. The large standard deviation indicates variability in the effectiveness of this compound.

[0079] SSRP-1: The IC50 value is 294.10 ± 8.17 μg / mL. This compound is more effective than the crude polysaccharide and requires a lower concentration to achieve the same inhibitory effect. The smaller the standard deviation, the more consistent the results.

[0080] SSRP-2: The IC50 value is 155.87 ± 25.75 μg / mL. This compound is the most effective among the three because it requires the lowest concentration to inhibit 50% of the HL60 cell activity. The moderate standard deviation indicates some variability, but less than that of the crude polysaccharide.

[0081] In summary, under experimental conditions, SSRP-2 showed the strongest inhibitory effect on HL60 cell activity, followed by SSRP-1, and finally the crude polysaccharide. The results indicate that SSRP-2 may be a promising candidate for further anti-cancer research or development.

[0082] Experimental Example Five: Anti-tumor Effect of Safflower Seed Meal Polysaccharide on HepG2 Cancer Cells 1. Experimental Materials: The safflower seed meal polysaccharides SSRP1, SSRP2, and SSRP3 prepared as described in the specification.

[0083] 2. Experimental Method: Culturing Cells: HepG2 cells were cultured in complete medium RPMI-1640 containing 100 μg / mL streptomycin, 100 units / mL penicillin, and 10% inactivated fetal bovine serum (FBS), and placed in a humidified incubator at 37°C and 5% CO2. When the cells grew to confluence at 70 - 80% and were in the logarithmic growth phase, the cells were passaged into the same medium and continued to be cultured under the same conditions for subsequent experiments.

[0084] Determining Anti-cancer Activity: The cytotoxicity of SSRP was evaluated using the CCK-8 assay. Cells from the HepG2 cancer cell line in the logarithmic growth phase were prepared into a cell suspension, and the cell concentration was adjusted to 1×10 4 cells / mL using a cell counting chamber. After homogenizing the cell suspension with a pipette, the cell suspension was introduced into a 96-well plate at a density of 1000 cells per well (100 μL) using a multi-channel pipette. Subsequently, it was placed in an incubator at 37°C and 5% CO2 for 24 hours. After the cells adhered completely, the culture medium was aspirated, and the cells were treated with SSRP solutions (100, 200, 300, 400 mg / mL) dissolved in fresh medium for 48 h. The cells treated with complete culture medium (0 mg / mL SSRP) were used as the blank control, and 10% DMSO was used as the positive control. After the incubation ended, the culture medium was removed, 10% CCK-8 reagent was added, and the 96-well plate was placed back in the incubator for another 4 hours. The determination of CCK-8 was carried out according to the manufacturer's protocol. The experiment was repeated three times, the absorbance was measured at a wavelength of 450 nm, and then the cell viability was calculated according to the following formula Cell viability (%) = "Ai" / "As" × 100% where Ai and As are the optical density (OD) values of the experimental group and the control group, respectively.

[0085] 3. Experimental Results: The proliferation ability of the HepG2 cell line decreased significantly in a dose-dependent manner under the action of SSRP.

[0086] When studying the inhibitory effect of SSRP1 on HepG2 cells (Figure 16-A), after 48 hours of culture, the survival rate of HepG2 cells decreased from 100% to 52.3%. The inhibitory effect on the proliferation of HepG2 cells was the greatest at a concentration of 100 mg / mL, with an inhibition rate of 47.7%, which was significantly different from that of the blank group (p < 0.001). In addition, compared with the control group, the inhibition rate of the positive group was also highly statistically significant (p < 0.0001).

[0087] When studying the inhibitory effect of SSRP2 on the proliferation of HepG2 cells (Figure 16-B), 100 - 400 mg / mL SSRP2 could reduce the growth rate of HepG2 cells by 4.5 - 46.3%. When studying the inhibitory effect of SSRP2 on the proliferation of HepG2 cells (Figure 16-B), 100 - 400 mg / mL SSRP2 could reduce the growth of HepG2 cells by 4.5 - 46.3%. At the maximum administration concentration, compared with the blank control, the cell viability was significantly reduced to 53.7% (p < 0.001). When studying the inhibitory effect of SSRP3 on the proliferation of HepG2 cells (Figure 16-C), after treating with 100 mg / ml SSRP3 for 48 hours, compared with the control group, the survival rate of cancer cells decreased significantly to 74.4% (p < 0.05). However, this decrease in cell viability was still significantly weaker than the inhibitory effect of the positive control group (DMSO). Nevertheless, compared with the control group, this result was still significant (p < 0.05). These results indicate that SSRP1 and SSRP2 may have the potential to inhibit cancer cell proliferation. However, at a high concentration of 400 mg / mL, all three SSRPs showed weak activity, and we will further explore other activities of SSRPs in subsequent studies.

[0088] This result is consistent with the findings in existing studies on the inhibition of tumor cell proliferation by natural polysaccharides through multiple mechanisms. Natural polysaccharides may exert anti-tumor effects by directly acting on the tumor cell membrane, inducing apoptosis, altering tumor gene expression, or enhancing immune function. In addition, the structural characteristics and physicochemical properties of SSRPs may also affect their efficacy in inhibiting cancer cell proliferation.

Claims

1. A method for preparing safflower seed meal polysaccharide, characterized in that, It includes the following steps: Mix safflower seed meal powder with distilled water; boil the mixture while constantly stirring, extract the precipitate, add distilled water again and repeat the above operation; then filter the mixture with filter paper and collect the filtrate; add 95% ethanol, let it stand for 24 hours to precipitate the polysaccharide at 4°C; separate to obtain the upper supernatant and the lower precipitate; Dry the precipitate using a rotary evaporator until a concentrated solution is obtained, and use the Sevag method to remove proteins; Use AB-8 macroporous column chromatography to remove pigments in the concentrated solution to obtain a crude polysaccharide sample.

2. The preparation method of the safflower seed meal polysaccharide according to claim 1, wherein, The crude polysaccharide prepared in Claim 1 is fractionated into three safflower seed meal polysaccharides through the following steps, and the specific steps are as follows: Rinse the DEAE-52 cellulose resin with 3 - 4 bed volumes of 0.5M NaOH; let it precipitate; wash with distilled water until the pH value reaches neutral; rinse with 3 bed volumes of 0.5M HCl; then wash with distilled water until the pH value is neutral; let the resin be mixed and suspended in distilled water; Gently pour it into the chromatographic column without introducing air bubbles; let the resin precipitate under the action of gravity; after the packing is compacted, drain the excess water; equilibrate the chromatographic column with 3 bed volumes of the starting buffer and NaCl solutions with different concentrations (0.05, 0.1, 0.15, and 0.2 mol / L); load the crude polysaccharide sample into the chromatographic column; use NaCl solutions with continuously increasing concentrations; collect 10 - milliliter eluates, analyze the carbohydrate content, and determine the elution curve; by monitoring the results of the phenol-sulfuric acid test of the eluate, pool to obtain three elution peak polysaccharides, named SSRP1, SSRP2, and SSRP3 respectively.

3. The preparation method of the safflower seed meal polysaccharide according to claim 2, characterized in that, The three safflower seed meal polysaccharides obtained in Claim 2 are purified through the following steps, and the specific steps are as follows: Install a Sephadex-G-100 chromatographic column; ensure that the chromatographic column is equilibrated overnight with the selected equilibrium buffer before loading the sample; start collecting the sample when the eluate begins to clear; continue to collect eluates of equal volume, generally 10 milliliters; extract the polysaccharide with distilled water and NaCl solutions with different concentrations (0.05, 0.1, 0.15, and 0.2 mol / L); collect the eluate by measuring the results through the phenol-sulfuric acid method to obtain three purified SSRP1, SSRP2, and SSRP3 polysaccharides.

4. The preparation method of the safflower seed meal polysaccharide according to claim 1, wherein, In step S1, the extraction temperature is optimized to 100°C.

5. The preparation method of the safflower seed meal polysaccharide according to claim 1, characterized in that, In step S1, the concentration of the distilled water solvent and the liquid-solid ratio are 10:

1.

6. The preparation method of the safflower seed meal polysaccharide according to claim 1, wherein, In step S1, continuous stirring is carried out for 2 hours.

7. The preparation method of the polysaccharide according to claim 1, wherein, In step S2, column chromatography techniques, namely DEAE-52 cellulose chromatography and Sephadex-G-100 chromatography, are used to purify the polysaccharide.

8. The safflower seed meal polysaccharide prepared by the preparation method of the safflower seed meal polysaccharide according to any one of Claims 1 to 7.

9. The use of the polysaccharide, which is the safflower seed meal polysaccharide prepared by the preparation method of the safflower seed meal polysaccharide according to any one of Claims 1 to 7, in the preparation of anticancer drugs.

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