A method for purifying and preparing caraway polysaccharides and their applications

Abstract

This invention discloses a purified caraway polysaccharide, its preparation method, and its applications. Using caraway seeds as raw material, the invention employs an ultrasound-assisted enzymatic extraction method to extract the polysaccharide, which is then further purified to obtain purified caraway polysaccharide. Analysis and identification show that this purified polysaccharide is a heteropolysaccharide composed of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid. The purified caraway polysaccharide extracted in this invention exhibits significant inhibitory effects on DPPH, OH, ABTS, α-glucosidase, and α-amylase, and can significantly inhibit the proliferation of HepG2 and SNU449 liver cancer cells and induce apoptosis. Therefore, it can be used to prepare functional products with antioxidant, hypoglycemic, or anti-liver cancer effects, thereby improving the utilization value of caraway.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide preparation and application technology, and in particular to a method for purifying caraway polysaccharide, its preparation, and its application. Background Technology

[0002] Seed spices are an important category of spices and seasonings, hailed as a veritable "nutritional treasure trove," rich in dietary fiber, protein, volatile oils, polysaccharides, minerals, and flavonoids. Spice polysaccharides not only exhibit powerful biological activities, such as regulating the digestive system, potent anti-inflammatory effects, and antioxidant properties, but also possess the advantages of being non-toxic, harmless, and leaving no residue, thus attracting increasing attention in fields such as biomedicine. Currently, research on polysaccharides mainly focuses on purifying the fine structure and complex biological activities of polysaccharides. Studies have shown that the biological activity of polysaccharides is closely related to their structural characteristics, such as monosaccharide composition, molecular weight, and glycosidic bonds.

[0003] Caraway ( Carum carvi Caraway (L.), belonging to the genus Carum of the family Umbelliferae, is also known as wild carrot, false angelica, and is a widely used seed-based spice and seasoning, mainly distributed in Northeast, North, Northwest, Tibet, and western Sichuan of my country. Caraway seeds are commonly used as a spice in cooking various foods. The volatile oils abundant in the seeds and fruits promote digestion, have antioxidant and antibacterial properties, and the residue remaining after extracting the volatile oils is often used as livestock feed. To date, research on the active components of caraway has mainly focused on volatile oils and flavonoids; reports on the preparation, structural identification, and bioactivity of caraway polysaccharides are virtually nonexistent. Therefore, it is necessary to provide a method for extracting and purifying caraway polysaccharides and to study and explore their antioxidant, hypoglycemic, and anti-hepatocellular carcinoma activities, thereby providing a theoretical basis for the development and application of caraway. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the invention aims to provide a method for purifying caraway polysaccharides, its preparation, and its application.

[0005] To achieve the above objectives, the invention adopts the following technical solution: A purified polysaccharide from Caraway, comprising a heteropolysaccharide composed of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid, wherein the molar ratio of each monosaccharide is 1.58:19.95:13.09:22.14:1.73:7.22:1.28:29.32:3.69; the weight-average molecular weight of the purified polysaccharide is 18043 Da.

[0006] This invention also provides a method for preparing purified polysaccharides from caraway seeds, the method comprising: S1 Extraction of crude polysaccharides Dry and pulverized caraway seeds were added to an ethanol solution and refluxed to remove impurities. The residue was dried, and then pure water and cellulase were added. After mixing, the mixture was ultrasonically extracted at 50 °C. The supernatant was collected and concentrated to obtain concentrate I. Next, activated macroporous resin AB-8 was mixed with concentrate I and magnetically stirred for decolorization at 50±5 °C. After decolorization, the macroporous resin was removed, and the protein was removed using the Sevag method. The supernatant was collected and concentrated to obtain concentrate II. Finally, ethanol solution was added for precipitation overnight at 4 °C. The precipitate was collected, dried, and the crude caraway polysaccharide CCP was obtained. S2 Purification After redissolving the crude caraway polysaccharide CCP, the mixture was chromatographically analyzed using a DEAE-52 anion exchange column with 0.3 mol / L NaCl solution as elution. The eluent was then concentrated and dialyzed using a dialysis bag with a molecular weight cutoff of 3000–8000 Da. The resulting product was freeze-dried to obtain caraway polysaccharide CCP-III. CCP-III was dissolved in pure water, and the supernatant was collected by centrifugation. The supernatant was then chromatographically analyzed using a Sephacryl S-400 molecular sieve gel column with pure water as elution. The main peak was collected, concentrated, dialyzed, and freeze-dried to obtain purified caraway polysaccharide CCP-1.

[0007] It should be noted that the concentration of the ethanol solution in step S1 is 95% by volume.

[0008] It should be noted that the molecular weight cutoff of the dialysis bag mentioned in step S2 is 3000 Da; the dialysis solution used in step S2 is deionized water.

[0009] It should be noted that in step S1, the ratio of caraway seeds to water is 1 g: 15-35 mL; the volume ratio of macroporous resin AB-8 to concentrated solution I is 1:1; the volume ratio of ethanol solution to concentrated solution II is 4:1; and the volume ratio of the n-butanol and chloroform mixed solution used in the Sevag method is 1:4.

[0010] It should be noted that in step S1, the ultrasonic power is 150-350 Hz; the ultrasonic extraction time is 20-60 min; the cellulase concentration is 1.5-3.5%; the ultrasonic extraction temperature is 50-55 ℃; the ultrasonic extraction is performed more than once; the decolorization time is 1-5 h; and the alcohol precipitation time is 10-16 h.

[0011] The purified caraway polysaccharide obtained based on this invention can be used in the preparation of products with antioxidant, hypoglycemic, antitumor, or α-glucosidase inhibitory properties.

[0012] It should be noted that the tumor referred to in the application is liver cancer.

[0013] It should be noted that the products used include anti-tumor drugs, drugs that inhibit α-glucosidase or α-glucosidase inhibitors, as well as drugs, foods or health products with antioxidant activity.

[0014] Furthermore, the purified caraway polysaccharide of this invention can also be used in the preparation of drugs that inhibit the proliferation of liver cancer cells or induce apoptosis of liver cancer cells.

[0015] The beneficial effects of the invention are: 1. This invention isolates and purifies polysaccharides from caraway seeds, and analyzes and identifies their molecular weight, monosaccharide composition, and chemical structure, determining their weight-average molecular weight and structural composition. In vitro antioxidant activity experiments show that this polysaccharide has significant scavenging effects on DPPH, OH, and ABTS free radicals; in vitro hypoglycemic activity experiments show that this polysaccharide has significant inhibitory effects on α-glucosidase and α-amylase; tumor cell experiments show that this polysaccharide has a significant inhibitory effect on the proliferation of HepG2 and SNU449 liver cancer cells and can induce apoptosis.

[0016] 2. The purified caraway polysaccharide (CCP-1) extracted in this invention has the potential to prepare functional products with antioxidant, hypoglycemic, and anti-liver cancer properties. This purified caraway polysaccharide can be used alone or in combination with other active ingredients, showing promising application prospects and commercial value, and can improve the utilization value of caraway. Attached Figure Description

[0017] Figure 1 This is the HPLC-GPC spectrum of CCP-1.

[0018] Figure 2 The image shows the FT-IR spectrum of CCP-1.

[0019] Figure 3 This is the ion chromatogram of a monosaccharide standard.

[0020] Figure 4 This is the ion chromatogram of CCP-1.

[0021] Figure 5 This is the total ion chromatogram for CCP-1.

[0022] Figure 6 For CCP-1 1 H NMR spectrum.

[0023] Figure 7 For CCP-1 13 C NMR spectrum.

[0024] Figure 8 This is the COSY spectrum of CCP-1.

[0025] Figure 9 The HSQC spectrum of CCP-1.

[0026] Figure 10 The NOESY spectrum of CCP-1.

[0027] Figure 11 The HMBC spectrum of CCP-1.

[0028] Figure 12 Scanning electron microscope image of CCP-1 (A: 500×; B: 1000×).

[0029] Figure 13 This is an atomic force microscope image of CCP-1.

[0030] Figure 14 The graph shows the scavenging effects of different concentrations of CCP-1 on DPPH, OH, and ABTS.

[0031] Figure 15 The graph shows the inhibition of α-amylase and α-glucosidase by different concentrations of CCP-1.

[0032] Figure 16 The graph shows the inhibitory effect of different concentrations of CCP-1 on the proliferation of HepG2 and SNU449 liver cancer cells.

[0033] Figure 17 Figure 1 shows the induction of apoptosis in HepG2 and SNU449 liver cancer cells by different concentrations of CCP-1 (A: apoptosis measured by flow cytometry; B: apoptosis rate analysis). Detailed Implementation

[0034] The invention will be further described below with reference to the accompanying drawings. It should be noted that this embodiment is based on the technical solution and provides detailed implementation methods and specific operation processes, but the scope of protection of the invention is not limited to this embodiment.

[0035] It should be noted that, unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following examples that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the reagents and raw materials used in this invention are commercially available.

[0036] In this invention, "caraway ( Carum carvi "L." belongs to the genus *Carum* of the family Umbelliferae. The dried *Carum* seeds used in this embodiment of the invention were purchased from the Bozhou Traditional Chinese Medicine Wholesale Market in Anhui Province.

[0037] Example 1: A method for preparing caraway polysaccharide CCP-1 1. Extraction of crude polysaccharides from Caraway seeds using ultrasound-assisted enzymatic hydrolysis. 1.1 Weigh 100 g of dried caraway seeds, crush them, pass them through a 40-mesh sieve, and reflux them with 95% ethanol for 3 h to remove lipids and small molecules. Dry the powder. Add deionized water at a ratio of 1:30 (g / mL), then add 3% cellulase and mix well. Adjust the pH to 4.8, and sonicate at 50 ℃ with 300 W power for 40 min. Collect the supernatant and concentrate it. Repeat this process twice, and finally concentrate all the supernatant to 100 mL.

[0038] 1.2 Mix macroporous resin AB-8 (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) with the concentrate at a volume ratio of 1:1, stir in a 50 ℃ water bath for 3 h to remove pigments, filter and collect the supernatant, concentrate for later use.

[0039] 1.3 Add Sevag reagent (a mixture of n-butanol and chloroform in a volume ratio of 1:4) to the concentrate to remove protein substances, centrifuge to collect the supernatant, and concentrate for later use.

[0040] 1.4 Add four times the volume of 95% ethanol solution to the concentrate, precipitate overnight, collect the precipitate and dry it to obtain caraway crude polysaccharide CCP.

[0041] 2. Crude polysaccharides from Caraway seeds were separated and purified by DEAE-52 cellulose column and Sephacryl S-400 gel chromatography. 2.1 The crude caraway polysaccharide CCP was redissolved in a 10 mg / mL polysaccharide solution and loaded onto a DEAE-52 chromatography column (the packing material was purchased from Shanghai Yuanye Biotechnology Co., Ltd.). Gradient elution was performed with 0–0.5 mol / L NaCl solution. The elution curve was tracked using the phenol-sulfuric acid method. The 0.3 mol / L NaCl eluent was collected according to the elution curve. The eluent was concentrated, dialyzed, and freeze-dried to obtain a caraway polysaccharide CCP-III.

[0042] 2.2 The freeze-dried caraway polysaccharide CCP-III was dissolved in pure water, centrifuged, and the supernatant was loaded onto a Sephacryl S-400 column (GE, USA). It was eluted with pure water, and the elution curve was tracked by phenol-sulfuric acid method. A single symmetrical peak appeared. The main peak was collected, concentrated, dialyzed, and freeze-dried to obtain purified caraway polysaccharide, named CCP-1.

[0043] Example 2: Identification of the physicochemical properties of caraway polysaccharide CCP-1 1. The total sugar content of CCP-1 was determined to be 92.27% using the phenol-sulfuric acid method.

[0044] 2. The protein content of CCP-1 was determined to be 1.51% using the Coomassie Brilliant Blue assay.

[0045] 3. The uronic acid content of CCP-1 was determined to be 18.36% using the sulfuric acid carbazole method.

[0046] 4. Scan the CCP-1 sample solution using a full-band UV scanner in the range of 200–800 nm. Plot the full-band scan curve with wavelength on the x-axis and OD value on the y-axis. The absence of a signal peak near 260–280 nm suggests that CCP-1 may not contain nucleic acid or protein components.

[0047] Example 3: Structural analysis of caraway polysaccharide CCP-1 1. Molecular weight determination of caraway polysaccharide CCP-1 The caraway polysaccharide CCP-1 sample was dissolved in a 0.1 mol / L NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL. After filtration through a 0.45 μm filter, HPLC-GPC analysis was performed. A gel permeation chromatography-differential-multi-angle laser light scattering (GPS) system was used. The liquid chromatography system was a U3000 (Thermo, USA), the differential detector was an Optilab T-rEX (Wyatt Technology, CA, USA), and the laser light scattering detector was a DAWN HELEOS II (Wyatt Technology, CA, USA).

[0048] A gel size exclusion column consisting of an Ohpak SB-805 HQ (300×8 mm) and an Ohpak SB-803 HQ (300×8 mm) in series was used. The column temperature was 45 ℃, the injection volume was 100 μL, the mobile phase was 0.1 mol / L NaNO3 solution (containing 0.02% NaN3), the flow rate was 0.6 mL / min, and the analysis time for each sample was 75 min.

[0049] The chromatographic data were processed using ASTRA 6.1 software, and an absolute molecular weight analysis chromatogram was plotted with retention time (Time, min) on the x-axis and molar mass (g / mol) on the y-axis.

[0050] 2. Infrared spectral detection of caraway polysaccharide CCP-1 2.0 mg of dried caraway polysaccharide CCP-1 sample was ground with KBr, compressed into tablets, and analyzed using a Nicolet 6700 FT-IR spectrometer at 4000–400 cm⁻¹. -1 Scan within the range.

[0051] 3. Monosaccharide composition analysis of caraway polysaccharide CCP-1 Weigh 4 mg of polysaccharide sample, add 1 mL of 2 mol / L TFA acid solution, and hydrolyze at 121 °C for 2 h. Purge with nitrogen and dry. Wash with anhydrous methanol, then dry again, repeating this process three times. Dissolve in sterile water and transfer to a chromatographic vial for HPLC analysis. The monosaccharide components were analyzed and detected using a Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA) and an electrochemical detector.

[0052] The liquid chromatography column was a Dionex™ CarboPac™ PA20 (150 × 3.0 mm, 10 μm), the injection volume was 5 μL, and the column temperature was 30 ℃. Mobile phase A (H2O), mobile phase B (0.1 mol / L NaOH), and mobile phase C (0.1 mol / L NaOH, 0.2 mol / L NaAc) were used, and the flow rate was 0.5 mL / min.

[0053] Quantification was performed using the external standard method. Standard curves were obtained by preparing standards of different concentrations (fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), mannuronic acid (Man-UA), and guluronic acid (Gul-UA)) and fitting them using Chromeleon software.

[0054] 4. Methylation analysis of caraway polysaccharide CCP-1 Weigh 5 mg of dried caraway polysaccharide CCP-1 sample and dissolve it in 1 mL of pure water. Add 1 mL of 100 mg / mL 1-cyclohexyl-2-morpholinoethyl carbodiimide methyl p-toluenesulfonate and react for 2 h. Add 1 mL of 2 mol / L imidazole. Divide the sample into two equal portions and add 1 mL of 30 mg / mL NaBH4 and 1 mL of 30 mg / mL NaBD4 to each portion, reacting for 3 h. Terminate the reaction with 100 μL of glacial acetic acid. Dialyze for 48 h and then freeze-dry. Dissolve the sample in 500 μL of DMSO, add 1 mg of NaOH and incubate for 30 min, then react with 50 μL of iodomethane for 1 h. Add 1 mL of pure water and 2 mL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and wash with water three times. Pipette the lower dichloromethane phase and dry under nitrogen to obtain methylated caraway polysaccharide CCP-1.

[0055] Methylated caraway polysaccharide CCP-1 was placed in a stoppered test tube and hydrolyzed with 100 μL of 2 mol / L TFA at 121 °C for 90 min, then evaporated to dryness at 30 °C. 50 μL of 2 mol / L ammonia and 50 μL of 1 mol / L NaBD4 were added, and the reaction was allowed to proceed at room temperature for 2.5 h to reduce the hydrolysis product. The reaction was terminated with 20 μL of glacial acetic acid, dried under nitrogen, washed twice with 250 μL of methanol, and dried under nitrogen. 250 μL of acetic anhydride was added, and the reaction was carried out at 100 °C for 2.5 h. After standing for 10 min in 1 mL of pure water, 500 μL of dichloromethane was added, vortexed, and the aqueous phase was discarded by centrifugation. The washing was repeated three times. The lower dichloromethane phase was collected and analyzed by GC-MS.

[0056] Chromatographic conditions: Agilent gas chromatography system (Agilent 6890A; Agilent Technologies, USA), TG-200 column (30 m × 0.25 mm × 0.25 µm, SGE, Australia), autosampler model G4567A. Injection volume 1 μL, split ratio 10:1, carrier gas high-purity helium, flow rate 1.5 mL / min; column oven initial temperature 150 ℃ held for 1 min, programmed temperature increase to 210 ℃ at 2 ℃ / min, held for 2 min, programmed temperature increase to 240 ℃ at 2 ℃ / min, held for 3 min.

[0057] Mass spectrometry conditions: An Aigilent quadrupole mass spectrometry system (Agilent 5977B; Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. Ion source temperature: 200 °C; MS quadrupole temperature: 110 °C; ionization energy: 50 EV; transfer line temperature: 210 °C; mass scan range (m / z): 50–350.

[0058] 5. Nuclear magnetic resonance analysis of caraway polysaccharide CCP-1 Dissolve an appropriate amount of caraway polysaccharide CCP-1 completely in D2O to prepare a 40 mg / mL polysaccharide solution. Add 0.5 mL of this solution to an NMR tube. Place the NMR tube into a Bruker AVANCE NEO-600 NMR spectrometer to scan one-dimensionally. 1 H spectrum and 13 C-spectrum, two-dimensional COSY, HSQC, NOESY, and HMBC spectra.

[0059] 6. Scanning electron microscopy analysis of caraway polysaccharide CCP-1 Using tweezers, small amounts of caraway polysaccharide CCP-1 were carefully picked up and attached to the sample stage covered with conductive adhesive. A conductive layer was deposited using a vacuum spraying apparatus, and then the surface morphology was observed at 500x and 1000x magnification under an accelerating voltage of 3 kV.

[0060] 7. Atomic force microscopy analysis of caraway polysaccharide CCP-1 Disperse caraway polysaccharide CCP-1 in a small amount of water, then drop it onto a mica substrate, dry it, and place it on a sample stage for scanning.

[0061] 8. Structural analysis results of caraway polysaccharide CCP-1 8.1 Molecular weight results of caraway polysaccharide CCP-1 The HPLC-GPC spectrum of CCP-1 is as follows: Figure 1 As shown, the weight-average molecular weight (Mw) and number-average molecular weight (Mn) of CCP-1 are 18043 Da and 13163 Da, respectively, and the Mw / Mn ratio is 1.371.

[0062] 8.2 Infrared Spectroscopic Analysis of Caraway Polysaccharide CCP-1 The infrared spectrum of CCP-1 is as follows: Figure 2 As shown. CCP-1 is at 3419.58 cm. -1 The strong and broad absorption peak at 2936.07 cm⁻¹ is a characteristic absorption peak of the OH stretching vibration of intermolecular association in polysaccharides. -1 The absorption peak at 1414.80 cm⁻¹ corresponds to the stretching vibrations of the pyran ring and CH₂. The absorption peaks in the range of 1414.80–1143.33 cm⁻¹ are due to these vibrations. -1 The absorption peaks observed within the range may be related to the variable-angle vibration of CH or the symmetric stretching vibration of C=O in -COO. The characteristic absorption peaks of these three types of carbohydrates confirm that CCP-1 is a polysaccharide.

[0063] CCP-1 is 1612.59 cm -1 The absorption peak at 1096.84 cm⁻¹ corresponds to the C=O vibration peak of the acetylamino group in the polysaccharide, proving that the purified polysaccharide contains an amino group; -1 A strong absorption peak is observed at this location, which is a characteristic region of the polysaccharide pyranose backbone, indicating the presence of COC bonds on the glycosidic bond and stretching vibrations related to C-OH side groups; at 1400 cm⁻¹... -1 The nearby absorption peak indicates the presence of a carboxyl group (-COOH) in the sugar chain, suggesting the possible presence of uronic acid; 640.14 cm⁻¹ -1 The absorption peak at that location indicates the presence of α-glycosides.

[0064] 8.3 Monosaccharide composition analysis of caraway polysaccharide CCP-1 Figure 3 This is an ion chromatogram of a monosaccharide standard. Figure 4 The ion chromatogram of CCP-1 shows that, by comparison, the main monosaccharide composition and its molar ratio are fucose:arabinose:rhamnose:galactose:glucose:xylose:mannose:galacturonic acid:glucuronic acid, which are 1.58:19.95:13.09:22.14:1.73:7.22:1.28:29.32:3.69.

[0065] 8.4 Methylation Analysis of Caraway Polysaccharide CCP-1 The methylated GC-MS report, combined with GC-MS spectral databases and previously reported spectral results, was analyzed to determine the retention times of each peak and the main characteristic fragments. The methylation analysis results of CCP-1 are summarized in Table 1, and the total ion chromatogram is shown below. Figure 5 As shown.

[0066] Table 1. Methylation analysis results of CCP-1

[0067] 8.5 NMR Spectrum Analysis of Caraway Polysaccharide CCP-1 CCP-1 1 H and 13 C NMR spectrum as shown Figure 6 and Figure 7 As shown, the proton NMR signal of CCP-1 is mainly concentrated between δ 3.0 and 5.5 ppm. Multiple coupled signal peaks were identified in the anodic signal region between δ 4.3 and 5.4 ppm, indicating the presence of various sugar residues. The corresponding chemical shifts of the anodic hydrogens are δ 4.34, 4.4, 4.53, 4.58, 4.89, 4.95, 4.98, 5.01, 5.04, and 5.24 ppm, respectively.

[0068] CCP-1 detected multiple signal peaks in the anodic carbon region, combined with 13 Cross-peaks in the anodic regions of the C10 NMR and HSQC spectra identified the anodic signals in this polysaccharide as: δ 4.95 / 98.98, 4.98 / 107.44, 4.34 / 103.65, 4.53 / 103.73, 4.89 / 98.51, 5.24 / 97.51, 5.04 / 107.03, 5.01 / 99.73, 4.4 / 103.12, and 4.58 / 103.57 ppm, respectively denoted as sugar residues A, B, C, D, E, F, G, H, I, and J. Combined with the methylation information and anodic signals of this polysaccharide (see...), Figure 8 , 9 Based on a comprehensive review of literature reports (10, 11), it is inferred that sugar residue A is →4)-α-D-Gal pA-(1→, sugar residue B is α-L-Ara) f -(1→, sugar residue C is →3,6)-β-D-Gal p -(1→, sugar residue D is β-D-Gal p -(1→, sugar residue E is α-D-Gal p A-(1→, sugar residue F is →2,4)-α-L-Rha p -(1→, sugar residue G is →5)-α-L-Ara f -(1→, sugar residue H is →2)-α-L-Rha p -(1→, sugar residue I is β-D-Xyl) p -(1→, sugar residue J is →4,6)-β-D-Gal p -(1→。and its 1 H and 13 The C chemical shifts were assigned, and the results are shown in Table 2.

[0069] Table 2 NMR chemical shifts of CCP-1

[0070] Based on the analysis of one-dimensional and two-dimensional NMR information and methylation results, it is inferred that this polysaccharide is mainly composed of →4)-α-D-Gal p A-(1→,→3,6)-β-D-Gal p -(1→,→2,4)-α-L-Rha p -(1,→2)-α-L-Rha p -(1→and→4,6)-β-D-Gal p -(1→ etc. are interconnected to form the main chain, and the branches are mainly composed of β-D-Gal p -(1→linked to sugar residues→3,6)-β-D-Gal p -(1→) O -3 positions, etc.

[0071] 8.6 Scanning electron microscopy analysis of caraway polysaccharide CCP-1 Scanning electron microscope image of caraway polysaccharide CCP-1 is shown below. Figure 12 As shown, CCP-1 exhibits a loose and porous network structure, with its surface mainly consisting of small fragment structures that are relatively dispersed, and there are many gaps between the fragments, giving it a nearly soft texture.

[0072] 8.7 Atomic force microscopy analysis of caraway polysaccharide CCP-1 Atomic force microscopy has gradually become an important tool for characterizing plant polysaccharides, allowing direct observation of the morphology and structure of polysaccharides at the molecular level. For example... Figure 13As shown, CCP-1 exhibits spherical and irregular mass structures of different sizes, with a height mainly ranging from 1 to 2 nm, indicating that CCP-1 has molecular chains that aggregate together.

[0073] Example 4: Identification of the antioxidant activity of caraway polysaccharide CCP-1 1. Scavenging activity of caraway polysaccharide CCP-1 against DPPH CCP-1 prepared in Example 1 and the positive drug vitamin C (V) were used. C Sample solutions of different concentrations (50, 100, 200, 400, 800 μg / mL) were prepared. 2.0 mL of the polysaccharide solution of each concentration was mixed with 1.0 mL of 0.08 mg / mL DPPH solution and recorded as solution s; 2.0 mL of the sample solution was mixed with 1.0 mL of methanol and recorded as solution b; 2.0 mL of methanol was mixed with 1.0 mL of DPPH solution and recorded as solution c. All solutions were placed at room temperature in the dark and in a shaker for 30 min. The absorbance values ​​As, Ab, and Ac were measured at 517 nm. Each group had three replicates, and the experiment was repeated three times. The DPPH free radical scavenging rate was calculated using the formula: Scavenging rate (%) = 100 × [1 - (As - Ab) / Ac]. Simultaneously, the inhibition rate was plotted against the inhibitor concentration. Based on the inhibition curve, the levels of caraway polysaccharide CCP-1 and the positive antioxidant V were determined. C IC 50 value.

[0074] 2. Scavenging activity of caraway polysaccharide CCP-1 against OH Take five stoppered test tubes and add 1.0 mL of 2.0 mmol / L FeSO4 and 1.0 mL of 6.0 mmol / L salicylic acid-ethanol solution to each, then shake and mix well. Add different concentrations of caraway polysaccharide CCP-1 solution (50, 100, 200, 400, 800 μg / mL) to each, then add 1.0 mL of 1.0 mmol / L H2O2 solution to each, shake and mix well. Heat in a 37 ℃ water bath for 30 min, zero the tube with distilled water, and measure the absorbance at 510 nm using a UV spectrophotometer. Calculate the OH scavenging rate using the following formula: Scavenging rate (%) = 100 × [1 - (As - Ab) / Ac], where Ac is the absorbance value with 1 mL of distilled water added, As is the absorbance value with different concentrations of sample added, and Ab is the absorbance value without H2O2 added. Simultaneously, the inhibition rate was plotted against the inhibitor concentration, and the levels of caraway polysaccharide CCP-1 and the positive antioxidant V were determined from the inhibition curve. C IC 50 value.

[0075] 3. Scavenging activity of caraway polysaccharide CCP-1 against ABTS Prepare the ABTS working solution. Before use, dilute the ABTS working solution with phosphate buffer (pH=7.0) to an absorbance of 0.70±0.02 at 734 nm. Mix 4 mL of the ABTS working solution with 0.4 mL of different concentrations of Caraway polysaccharide CCP-1 solution (50, 100, 200, 400, 800 μg / mL), react in the dark for 6 min, and measure the absorbance at 734 nm. Use distilled water instead of the sample solution as a blank control and vitamin C as a positive control. Calculate the ABTS free radical scavenging rate using the formula: Scavenging rate (%) = 100 × (1 - As / Ab), where Ab represents the absorbance of the blank control and As represents the absorbance of the sample reaction solution. Simultaneously, plot the inhibition rate against the inhibitor concentration, and determine the IC50 values ​​of Caraway polysaccharide CCP-1 and the positive antioxidant vitamin C based on the inhibition curve. 50 value.

[0076] 4. Results of antioxidant activity of caraway polysaccharide CCP-1 The results are as follows Figure 14 As shown in the results, the scavenging ability of caraway polysaccharide CCP-1 against DPPH, OH, and ABTS free radicals increases with increasing concentration, exhibiting a clear linear relationship. IC50... 50 The values ​​were 142.71, 237.72, and 78.23 μg / mL, respectively; when the concentration was 800 μg / mL, the scavenging ability of caraway polysaccharide against DPPH, OH, and ABTS free radicals reached its maximum; indicating that caraway polysaccharide has good antioxidant activity and can be further developed into a natural antioxidant.

[0077] Example 5: Identification of the hypoglycemic activity of caraway polysaccharide CCP-1 1. Inhibition experiment of caraway polysaccharide CCP-1 on α-amylase Take 500 μL of different concentrations of caraway polysaccharide CCP-1 (125, 250, 500, 1000, 2000 μg / mL) and an equal volume of α-amylase solution (1.0 U / mL), mix them evenly, and incubate at 37 ℃ for 10 min. Then add 500 μL of 1% soluble starch solution and continue incubation at 37 ℃ for 10 min. Then add 1 mL of 3,5-dinitrosalicylic acid color indicator to terminate the reaction. After boiling in a water bath for 5 min, add 10 mL of distilled water to dilute the mixture. Use acarbose as a positive control and an equal volume of PBS as a background group. Measure the absorbance at 540 nm. Calculate the inhibition rate (%) of the sample against α-amylase according to the following formula: = 100 × [1 - (As - Ab) / Ac], where As represents the absorbance value of the polysaccharide, enzyme, and starch mixture, Ab represents the absorbance value of PBS instead of the enzyme solution, and Ac represents the absorbance value of PBS instead of the sample solution. Simultaneously, the inhibition rate was plotted against the inhibitor concentration, and the IC50 values ​​of caraway polysaccharide CCP-1 and positive acarbose were determined from the inhibition curves. 50 value.

[0078] 2. Inhibitory effect of caraway polysaccharide CCP-1 on α-glucosidase The inhibitory effect of caraway polysaccharide CCP-1 on α-glucosidase was detected by the pNPG method. 100 μL of α-glucosidase solution (0.5 U / mL) was mixed with 100 μL of polysaccharide solution (125, 250, 500, 1000, 2000 μg / mL) and incubated at 37 °C for 10 min. Then, 100 μL of pPNG solution was added to initiate the reaction. After mixing, the mixture was incubated at 37 °C for 60 min. Finally, 1 mL of 1 M sodium carbonate solution was added to the reaction solution to stop the reaction. Acarbose was used as a positive control, and an equal volume of PBS was used as a background. The absorbance was measured at 405 nm. The inhibition rate (%) of the sample against α-glucosidase was calculated using the following formula: 100 × [1 - (As - Ab) / Ac], where As represents the absorbance value of the mixture of polysaccharide, enzyme, and pPNG, Ab represents the absorbance value of the mixture after PBS was used to replace the enzyme solution, and Ac represents the absorbance value of the sample solution after PBS was used to replace the sample solution. Simultaneously, an inhibition rate was plotted against the inhibitor concentration, and the IC50 values ​​of caraway polysaccharide CCP-1 and positive acarbose were determined based on the inhibition curve. 50 value.

[0079] 3. Results of the hypoglycemic activity of caraway polysaccharide CCP-1 The results are as follows Figure 15 As shown in the results, the inhibitory effect of caraway polysaccharide CCP-1 on α-amylase and α-glucosidase exhibits a dose-response relationship with concentration; as the concentration of CCP-1 increases, the inhibitory effect on α-amylase and α-glucosidase gradually strengthens, and the IC50 value increases.50 The values ​​were 378.97 and 226.81 μg / mL, respectively. Caraway polysaccharide CCP-1 can delay starch digestion, thereby effectively regulating postprandial blood glucose levels from starchy foods. This may be related to the α-(1→4) glycosidic bonds and uronic acid in its structure. The results indicate that caraway polysaccharide has good hypoglycemic activity and can be further developed into a natural hypoglycemic product.

[0080] Example 6: Identification of the anti-hepatocellular caraway polysaccharide CCP-1 1. Inhibitory effect of caraway polysaccharide CCP-1 on the growth of HepG2 and SNU449 liver cancer cells. The inhibitory effect of Caraway polysaccharide CCP-1 on the growth of HepG2 and SNU449 cells was determined using the MTT assay. Under dark conditions, 50 mg of MTT powder was accurately weighed, added to 10 mL of PBS buffer, mixed thoroughly, filtered through a 0.22 µm filter for sterilization, and stored at 4 °C in the dark. HepG2 and SNU449 cells in the logarithmic growth phase were collected, and cell density was adjusted with culture medium. 100 µL of cell suspension was added to each well of a 96-well cell culture plate. Different concentrations of polysaccharide solution (50, 100, 200, 400, 800 μg / mL) were added to each well in the experimental groups. 5-fluorouracil (5-Fu, 100 μg / mL) was used as a positive control, and the control group contained an equal volume of culture medium. Each group had three replicates. After 48 h of culture, 20 µL of MTT working solution was added to each well of the 96-well plate. After 4 h of culture, 150 µL of DMSO solution was added to each well and shaken thoroughly to completely dissolve the blue-purple formazan crystals. The absorbance at 570 nm was measured using a microplate reader. The change in cell growth relative to the control group is expressed as the growth inhibition rate (%), calculated using the formula: Growth inhibition rate (%) = 100 × (1 - As / Ac), where As represents the absorbance of the polysaccharide experimental group and Ac represents the absorbance of the control group. Simultaneously, an inhibition rate versus concentration curve was plotted, and the IC50 of caraway polysaccharide CCP-1 in inhibiting liver cancer cell growth was determined based on the inhibition curve. 50 value.

[0081] 2. The effect of caraway polysaccharide CCP-1 on inducing apoptosis in HepG2 and SNU449 liver cancer cells. HepG2 and SNU449 cells were seeded in 12-well plates and cultured overnight. Afterward, the cells were treated with different concentrations of polysaccharide solutions (100, 200, 400 μg / mL), followed by digestion with 0.25% EDTA-free trypsin, centrifugation, washing with pre-cooled PBS, and cell counting. 1×10⁻⁶ cells were collected. 5Transfer each cell to a new EP tube, centrifuge at 500 g for 5 min, and resuspend in 100 μL of flow cytometry staining buffer. Counterstain with Annexin V and PI staining solutions, incubate on ice in the dark for 15 min, then add 400 μL of flow cytometry staining buffer, mix thoroughly by pipetting, filter, and then detect apoptosis in HepG2 and SNU449 cells using a flow cytometer.

[0082] 3. Results of the anti-hepatocellular caraway polysaccharide CCP-1 The MTT test results are as follows Figure 16 As shown, the inhibitory effect of caraway polysaccharide CCP-1 on the growth of HepG2 and SNU449 cells exhibited a concentration-dependent effect; the higher the polysaccharide concentration, the stronger the inhibitory effect on liver cancer cell growth. The IC50 values ​​of CCP-1 on the growth inhibition rate of HepG2 and SNU449 cells were also shown. 50 The values ​​were 277.17 μg / mL and 475.93 μg / mL, respectively, and CCP-1 had a more significant inhibitory effect on the growth of HepG2 cells.

[0083] In addition, flow cytometry results such as Figure 17 As shown, the apoptosis rate of liver cancer cells was significantly increased under CCP-1 treatment compared to the control group. The apoptosis rate of liver cancer cells increased significantly with increasing polysaccharide concentration. The highest apoptosis rates were observed in HepG2 and SNU449 cells at a CCP-1 concentration of 400 μg / mL, at 18.05% and 11.66%, respectively, with CCP-1 inducing apoptosis in HepG2 cells more effectively.

[0084] The above results indicate that caraway polysaccharide has good anti-liver cancer activity and can be further developed into a natural anti-liver cancer product.

[0085] For those skilled in the art, various corresponding changes and modifications can be made based on the above technical solutions and concepts, and all such changes and modifications should be included within the scope of protection of the invention claims.

Claims

1. A purified polysaccharide from Caraway seeds, characterized in that, The purified polysaccharide comprises a heteropolysaccharide composed of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid, wherein the molar ratio of each monosaccharide is 1.58:19.95:13.09:22.14:1.73:7.22:1.28:29.32:3.69; the weight-average molecular weight of the purified polysaccharide is 18043 Da.

2. A method for preparing the purified caraway polysaccharide as described in claim 1, characterized in that, The method includes: S1 Extraction of crude polysaccharides Dry and pulverized caraway seeds were added to an ethanol solution and refluxed to remove impurities. The residue was dried, and then pure water and cellulase were added. After mixing, the mixture was ultrasonically extracted at 50 °C. The supernatant was collected and concentrated to obtain concentrate I. Next, activated macroporous resin AB-8 was mixed with concentrate I and magnetically stirred for decolorization at 50±5 °C. After decolorization, the macroporous resin was removed, and the protein was removed using the Sevag method. The supernatant was collected and concentrated to obtain concentrate II. Finally, ethanol solution was added for precipitation overnight at 4 °C. The precipitate was collected, dried, and the crude caraway polysaccharide CCP was obtained. S2 Purification After redissolving the crude caraway polysaccharide CCP, the mixture was chromatographically analyzed using a DEAE-52 anion exchange column with 0.3 mol / L NaCl solution added for elution. The eluent was then concentrated and dialyzed using a dialysis bag with a molecular weight cutoff of 3000–8000 Da. The resulting product was freeze-dried to obtain caraway polysaccharide CCP-III. CCP-III was dissolved in pure water, and the supernatant was collected by centrifugation. The supernatant was then chromatographically analyzed using a Sephacryl S-400 molecular sieve gel column with pure water elution. The main peak was collected, concentrated, dialyzed, and freeze-dried to obtain purified caraway polysaccharide CCP-1.

3. The method for preparing purified polysaccharides from Caraway seeds according to claim 2, characterized in that, The concentration of the ethanol solution in step S1 is 95% by volume.

4. The method for preparing purified polysaccharides from Caraway seeds according to claim 2, characterized in that, The molecular weight cutoff of the dialysis bag in step S2 is 3000 Da; the dialysis solution used in step S2 is deionized water.

5. The method for preparing purified polysaccharides from Caraway seeds according to claim 2, characterized in that, In step S1, the ratio of caraway seeds to water is 1 g: 15-35 mL; the volume ratio of macroporous resin AB-8 to concentrated solution I is 1:1; the volume ratio of ethanol solution to concentrated solution II is 4:1; and the volume ratio of the n-butanol and chloroform mixed solution used in the Sevag method is 1:

4.

6. The method for preparing purified polysaccharides from Caraway seeds according to claim 2, characterized in that, In step S1, the ultrasonic power is 150–350 Hz; the ultrasonic extraction time is 20–60 min; the cellulase concentration is 1.5–3.5%; the ultrasonic extraction temperature is 50–55 °C; the ultrasonic extraction is performed more than once; the decolorization time is 1–5 h; and the alcohol precipitation time is 10–16 h.

7. The use of the purified caraway polysaccharide according to any one of claims 1-6 in the preparation of products with antioxidant, hypoglycemic, antitumor, or α-glucosidase inhibitory properties.

8. The application of the purified caraway polysaccharide according to claim 7 in the preparation of products with antioxidant, hypoglycemic, antitumor, or α-glucosidase inhibitory properties, characterized in that... The tumor is liver cancer.

9. The application of the purified caraway polysaccharide according to claim 7 in the preparation of products with antioxidant, hypoglycemic, antitumor, or α-glucosidase inhibitory properties, characterized in that... The products include antitumor drugs, drugs that inhibit α-glucosidase or α-glucosidase inhibitors, as well as drugs, foods or health products with antioxidant activity.

10. The use of the purified caraway polysaccharide according to any one of claims 1-6 in the preparation of a drug for inhibiting the proliferation of liver cancer cells or inducing apoptosis of liver cancer cells.

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