A south radix isatidis polysaccharide, a preparation method and application thereof

Abstract

This invention belongs to the field of biomedical technology and discloses a polysaccharide from Isatis indigotica root, its preparation method, and its applications. It comprises the following components in molar percentage: arabinose 26.17%, galactose 23.61%, rhamnose 23.01%, glucose 13.05%, xylose 6.69%, mannose 6.06%, and fucose 1.40%-1.41%. This polysaccharide is a homogeneous single component with a weight-average molecular weight of 38.114 kDa and a polydispersity index of 1.253. This invention employs a purification process combining water extraction and alcohol precipitation with enzymatic protein desorption, ion exchange, and gel column chromatography, which is stable and controllable, suitable for large-scale production. Experiments have confirmed that this polysaccharide can significantly enhance the activity of antioxidant enzymes such as superoxide dismutase at non-toxic concentrations, reduce reactive oxygen species levels, and alleviate oxidative stress damage in cells and zebrafish. It can be used to prepare antioxidant compositions for pharmaceuticals, health products, and functional foods.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polysaccharide from Isatis indigotica root, its preparation method, and its application. Background Technology

[0002] Southern Isatis root is the plant Strobilanthes cusia of the Acanthaceae family ( Baphicacanthus cusia The dried root and rhizome of *Nees.* Bremek. are one of my country's traditional medicinal herbs, possessing properties such as clearing heat and detoxifying, cooling the blood and eliminating rashes, and preventing epidemics and killing parasites. They are commonly used to treat febrile diseases, erysipelas, mumps, and rashes. Modern pharmacological studies have shown that *Nees.* Bremek. root possesses various biological activities, including antiviral, antibacterial, anti-inflammatory, antitumor, and antioxidant effects, and has high medicinal development value.

[0003] Currently, various chemical components have been isolated from Isatis indigotica, including alkaloids, lignans, phenylethanoid glycosides, sterols, terpenes, flavonoids, and polysaccharides. Among them, polysaccharides, as a class of natural high-molecular polymers, possess diverse structures, immunomodulatory, antioxidant, and antitumor biological activities, and have low toxicity, making them a hot topic in natural product research. However, existing research has mostly focused on small-molecule compounds in Isatis indigotica, with relatively few systematic studies on its polysaccharide components, especially lacking in-depth analysis of their structural characteristics, physicochemical properties, and biological activities.

[0004] Existing literature on the extraction of polysaccharides from Isatis indigotica root mainly employs conventional methods such as water extraction and alcohol precipitation, and enzyme-assisted extraction. The resulting polysaccharides are mostly crude polysaccharides with complex compositions, lacking systematic purification and structural characterization of specific active polysaccharide components. Furthermore, no studies have yet clearly revealed the precise monosaccharide composition, molecular weight distribution, glycosidic bond linkages, and spatial structure of Isatis indigotica root polysaccharides. Regarding functional studies, although some studies have reported the antioxidant activity of crude Isatis indigotica root polysaccharides, their specific mechanisms of action, such as their regulatory effects on the activities of antioxidant enzymes like superoxide dismutase, catalase, and glutathione peroxidase, as well as verification of their in vivo antioxidant effects, still lack systematic experimental data.

[0005] Therefore, providing a polysaccharide from Isatis indigotica with a well-defined structure, high purity, and significant antioxidant activity, and establishing an efficient and reproducible preparation method for it, thereby clarifying its application potential in the field of antioxidants, has important scientific research value and practical significance. Summary of the Invention

[0006] This invention provides a polysaccharide from Isatis indigotica root, its preparation method, and its applications. The polysaccharide is composed of seven monosaccharides, including arabinose, galactose, and rhamnose, in a specific molar ratio, with a weight-average molecular weight of 38.114 kDa. The preparation method includes steps such as water extraction and alcohol precipitation, enzymatic protein desorption, and purification by ion exchange column and gel column chromatography. This polysaccharide exhibits significant antioxidant activity, promoting the activity of superoxide dismutase, catalase, and glutathione peroxidase, and can be used to prepare antioxidant compositions.

[0007] On the one hand, the present invention provides a polysaccharide from Isatis indigotica root, which adopts the following technical solution: A polysaccharide from Isatis indigotica root has a weight-average molecular weight of 38.114 kDa and a polydispersity index of 1.253. The polysaccharide from Isatis indigotica root is composed of arabinose, galactose, rhamnose, glucose, xylose, mannose, and fucose in molar percentages of 26.17%, 23.61%, 23.01%, 13.05%, 6.69%, 6.06%, and 1.40%, respectively.

[0008] Preferably, the polysaccharide from Isatis indigotica root contains the following structure:

[0009] On the other hand, the present invention also provides a method for preparing polysaccharides from Isatis indigotica root, using the following technical solution: A method for preparing polysaccharides from Isatis indigotica root includes the following steps: Step 1: Preparation of crude polysaccharide extract: The roots of Isatis indigotica were dried and pulverized in sequence, sieved, and then extracted with anhydrous ethanol to defatt them. The precipitate was collected by centrifugation. Deionized water was added to the precipitate, and the supernatant was collected by centrifugation after water bath extraction. The extraction was repeated several times and the supernatants were combined. The supernatant was concentrated under vacuum and then anhydrous ethanol was added for alcohol precipitation. The precipitate was collected by centrifugation to obtain crude polysaccharide extract. Step 2: Preparation of crude polysaccharide solution: Dissolve the crude polysaccharide extract in water, add papain for enzymatic hydrolysis to obtain polysaccharide extract hydrolysate; deproteinize using the Sevag method and collect the upper aqueous phase; add petroleum ether to the aqueous phase for defatting and collect the lower aqueous phase; after adsorption with macroporous resin AB-8, dialyze to remove small molecule components using a dialysis bag, and freeze-dry to obtain the lyophilized crude polysaccharide solution; Step 3: Ion exchange column purification and desalting: Dissolve the lyophilized crude polysaccharide solution in water and load it onto a DEAEseplife FF anion exchange column. Elute sequentially with deionized water, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L sodium chloride solutions. Collect the corresponding components eluted with 0.1 mol / L sodium chloride solution, dialyze to remove salt, and then freeze-dry. Step 4, Gel column elution and purification: Dissolve the lyophilized product obtained in Step 3 in water, load it onto a Sephacryl S-400HR gel column, elute with deionized water, collect the fraction corresponding to the second elution peak, dialyze to remove salt, and then freeze-dry to obtain Isatis indigotica polysaccharide.

[0010] Preferably, in step one, the sieve mesh size is 50-70 mesh; the extraction time with anhydrous ethanol is 10-14 h; the water bath extraction temperature is 50-70℃ and the extraction time is 3.5-4.5 h; the supernatant is concentrated to 8%-15% of the original volume; and the volume ratio of the concentrated extract to anhydrous ethanol during alcohol precipitation is 1:3-1:5.

[0011] Preferably, in step two, the volume ratio of chloroform to n-butanol used in the Sevag method is 3:1-5:1, the volume ratio of the chloroform and n-butanol mixture to the polysaccharide extract enzymatic hydrolysate is 1:3-1:5, the molecular weight cutoff of the dialysis bag is 2500-3500 Da, and the dialysis time is 30-60 h.

[0012] Preferably, in step three, the elution flow rate of the DEAE seplife FF anion exchange column is 3-5 mL / min, and one tube is collected every 13-17 mL; the molecular weight cutoff of the dialysis bag used for dialysis desalination is 2500-3500 Da, and the dialysis time is 40-85 h.

[0013] Preferably, in step four, the elution flow rate of the Sephacryl S-400HR gel separation column is 0.8-1.2 mL / min, eluting 1.5 times the column volume, and collecting one tube every 8-15 mL; the molecular weight cutoff of the dialysis bag used for dialysis desalting is 2500-3500 Da, and the dialysis time is 40-85 h.

[0014] On the other hand, the present invention also provides the application of Isatis indigotica polysaccharide in the preparation of an antioxidant composition, wherein the antioxidant composition is a pharmaceutical, health product or functional food.

[0015] Preferably, the antioxidant composition exerts its antioxidant effect by promoting the activity of superoxide dismutase, catalase and glutathione peroxidase, thereby reducing the level of reactive oxygen species in cells.

[0016] Preferably, the antioxidant composition is used to alleviate oxidative stress damage in cells and the body.

[0017] In summary, the beneficial effects of the present invention are as follows: The polysaccharide from Isatis indigotica root provided by this invention is a single polysaccharide component with a well-defined structure and high purity. Its monosaccharide composition, molecular weight, and glycosidic bond linkages are clearly defined, with a weight-average molecular weight of 38.114 kDa and a polydispersity index of 1.253. The preparation method of this invention employs a purification process combining water extraction and alcohol precipitation, enzymatic protein dissociation, DEAE anion exchange column chromatography, and Sephacryl gel column chromatography. This process efficiently obtains highly homogeneous active polysaccharides, is stable and controllable, and is suitable for large-scale production.

[0018] The polysaccharide of this invention exhibits significant antioxidant activity, effectively promoting the activity of superoxide dismutase, catalase, and glutathione peroxidase, while simultaneously reducing intracellular reactive oxygen species levels. Cellular experiments and in vivo zebrafish experiments have both confirmed that this polysaccharide can significantly alleviate oxidative stress damage within a non-toxic concentration range, demonstrating excellent antioxidant efficacy. It can be applied to the preparation of antioxidant compositions in pharmaceuticals, health products, or functional foods, and has broad prospects for clinical application and market development. Attached Figure Description

[0019] Figure 1 The ion elution curve of polysaccharide from Isatis indigotica root in DEAE-52 cellulose; Figure 2 The gel elution curve of polysaccharide from Isatis indigotica root in Sephadex G100. Figure 3 The UV-Vis spectrum of polysaccharides from Isatis indigotica root; Figure 4 Infrared scan spectrum of polysaccharide from Isatis indigotica root; Figure 5 The absolute molecular weight analysis diagram of polysaccharides from Isatis indigotica root is shown. Figure 6 A diagram showing the monosaccharide composition of polysaccharides from Isatis indigotica root. Figure 7 The proton NMR spectrum of polysaccharides from Isatis indigotica root; Figure 8 The carbon NMR spectrum of polysaccharides from Isatis indigotica root; Figure 9 COSY spectrum of NMR for polysaccharides from Isatis indigotica root; Figure 10 HSQC spectrum of NMR detection for polysaccharides from Isatis indigotica root; Figure 11 NOESY spectrum of NMR detection for polysaccharides from Isatis indigotica root; Figure 12 The HMBC spectrum of polysaccharide from Isatis indigotica root was determined by nuclear magnetic resonance. Figure 13 The chemical structure of polysaccharide from Isatis indigotica root; Figure 14The results indicate the cytotoxicity of polysaccharides from Isatis indigotica root. Figure 15 The antioxidant effect of polysaccharide from Isatis indigotica root on RAW264.7 peritoneal macrophages in mice; Figure 16 The effect of polysaccharide from Isatis indigotica on ROS content in zebrafish. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] Example Example 1 A method for preparing polysaccharides from Isatis indigotica root, specifically including the following steps: Step 1: Preparation of crude polysaccharide extract 1) The roots of Isatis indigotica are dried and crushed in sequence, and then the fragments of Isatis indigotica with qualified particle size are screened through a 50-mesh filter screen. 2) Add anhydrous ethanol to the qualified fragments of Isatis indigotica, extract for 10 h, and then collect the precipitate by centrifugation. 3) Add deionized water to the collected precipitate, then place it in a 50 ℃ water bath for 3.5 h, and then centrifuge to collect the supernatant extract; 4) Repeat the process of steps 1)-3) on the precipitate residue after collecting the supernatant extract in step 3) to obtain multiple supernatant extracts; finally, combine the supernatant extracts collected multiple times to obtain the extract. 5) Vacuum rotary evaporation of the extract and concentration to 8% of the original volume to obtain concentrated extract; 6) Add anhydrous ethanol to the concentrated extract for alcohol precipitation. The volume ratio of concentrated extract to anhydrous ethanol is 1:3. After the alcohol precipitation is completed, centrifuge and collect the precipitated solid to obtain crude polysaccharide extract. Step 2: Preparation of crude polysaccharide solution 1) Add deionized water to dissolve the crude polysaccharide extract obtained in step one, and then add papain to hydrolyze it to obtain a polysaccharide extract hydrolysate. 2) Add a mixture of chloroform and n-butanol to the enzymatic hydrolysate of the polysaccharide extract, mix thoroughly, and collect the upper aqueous phase; wherein, the volume ratio of chloroform to n-butanol is 3:1, and the volume ratio of the chloroform and n-butanol mixture to the enzymatic hydrolysate of the polysaccharide extract is 1:3. 3) Add petroleum ether to the upper aqueous phase of step 2), mix thoroughly, and collect the lower aqueous phase; 4) Add macroporous resin AB-8 to the lower aqueous phase of step 2) for adsorption, collect the liquid; then use a dialysis bag with a molecular weight cutoff of 2500 Da for dialysis for 30 h to remove small molecule components, obtain crude polysaccharide solution, and freeze dry it. Step 3: Purification and desalting of crude polysaccharide solution 1) After dissolving the freeze-dried component obtained in step 4) of step 2 with deionized water, load it onto a DEAEseplife FF anion exchange column for elution; 2) During the elution process, deionized water and sodium chloride solutions with concentrations of 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L were used to sequentially elute the crude polysaccharide solution using a gradient. The DEAE Seplife FF anion exchange column was used at a flow rate of 3 mL / min, and one tube was collected every 13 mL. The resulting product was as follows: Figure 1 The components corresponding to the four elution peaks shown; 3) Collect the above four components separately, dialyze them for 40 h using a dialysis bag with a molecular weight cutoff of 2500 Da to remove salt, and then freeze-dry them; Step 4: Elution to obtain polysaccharides from Isatis indigotica root. 1) Take the fraction obtained from elution with 0.1 mol / L sodium chloride solution in step 2) of step 3, after desalting and freeze-drying, dissolve it in deionized water, and load it onto a Sephacryl S-400HR gel column for elution; use deionized water, elute at a flow rate of 0.8 mL / min for 1.5 column volumes, and collect one tube every 8 mL; the result is as follows. Figure 2 The components corresponding to the two elution peaks shown; after dialysis for 40 h using a dialysis bag with a molecular weight cutoff of 2500 Da to remove salt, they were freeze-dried. 2) Take the second component and prepare Isatis indigotica polysaccharide.

[0022] Example 2 A method for preparing polysaccharides from Isatis indigotica root differs from Example 1 in that: in step 1), the mesh size of the filter sieve is changed from 50 mesh to 60 mesh; in step 2), the extraction time is changed from 10 h to 12 h; in step 3), the water bath temperature is changed from 50℃ to 60℃ and the extraction time is changed from 3.5 h to 4 h; in step 5), the concentration degree is changed from 10% to 8%; and in step 6), the volume ratio of the concentrated extract to anhydrous ethanol is changed from 3:1 to 4:1. The remaining steps are the same as in Example 1.

[0023] Example 3 A method for preparing polysaccharides from Isatis indigotica root differs from Example 1 in that, in step 2), the volume ratio of chloroform to n-butanol is changed from 3:1 to 4:1, and the volume ratio of the chloroform and n-butanol mixture to the polysaccharide extract enzymatic hydrolysate is changed from 1:3 to 1:4; in step 4), the molecular weight cutoff of the dialysis bag is changed from 2500 Da to 3000 Da, and the dialysis time is changed from 30 h to 48 h. The remaining steps are the same as in Example 1.

[0024] Example 4 A method for preparing polysaccharides from Isatis indigotica root differs from that in Example 1 in that, in step 3, 2), the flow rate of the anion exchange column purification is changed from 3 mL / min to 4 mL / min, and the volume of each tube is changed from 13 mL to 15 mL; in step 3), the molecular weight cutoff of the dialysis bag is changed from 2500 Da to 3000 Da, and the dialysis time is changed from 30 h to 48-72 h. The remaining steps are the same as in Example 1.

[0025] Example 5 A method for preparing polysaccharides from Isatis indigotica root, the specific steps of which differ from those in Example 1, includes the following steps in step four, elution to obtain the polysaccharides from Isatis indigotica root: 1) Take the fraction obtained by elution with 0.1 mol / L sodium chloride solution in step 2) of step 3, after desalting and freeze-drying, dissolve it in deionized water, and load it onto a Sephacryl S-400HR gel separation column for elution; the elution flow rate is 1 mL / min; another fraction is eluted with 1.5 column volumes of deionized water, and one tube is collected for every 12 mL. Deionized water was used to elute 1.5 column volumes at a flow rate of 1 mL / min, and 12 mL was collected in one tube. The fractions corresponding to the two elution peaks were obtained, and after desalting by dialyzing with a dialysis bag with a molecular weight cutoff of 3000 Da for 48 h-72 h, they were freeze-dried. 2) Take the second component and prepare Isatis indigotica polysaccharide.

[0026] The remaining steps are the same as in Example 1.

[0027] Example 6 The purpose of this embodiment is to provide a method for preparing polysaccharides from Isatis indigotica root, including the following steps: Step 1: Preparation of crude polysaccharide extract 1) The roots of Isatis indigotica are dried and crushed in sequence, and then the fragments of Isatis indigotica with qualified particle size are screened through a 70-mesh filter screen. 2) Add anhydrous ethanol to the qualified fragments of Isatis indigotica, extract for 14 h, and then collect the precipitate by centrifugation. 3) Add deionized water to the collected precipitate, then place it in a 70 ℃ water bath for 4.5 h, and then centrifuge to collect the supernatant extract; 4) Repeat the process of steps 1)-3) on the precipitate residue after collecting the supernatant extract in step 3) to obtain multiple supernatant extracts; finally, combine the supernatant extracts collected multiple times to obtain the extract. 5) Vacuum rotary evaporation of the extract and concentration to 15% of the original volume to obtain concentrated extract; 6) Add anhydrous ethanol to the concentrated extract for alcohol precipitation. The volume ratio of concentrated extract to anhydrous ethanol is 1:5. After the alcohol precipitation is completed, centrifuge and collect the precipitated solid to obtain crude polysaccharide extract. Step 2: Preparation of crude polysaccharide solution 1) Add deionized water to dissolve the crude polysaccharide extract obtained in step one, and then add papain to hydrolyze it to obtain a polysaccharide extract hydrolysate. 2) Add a mixture of chloroform and n-butanol to the enzymatic hydrolysate of the polysaccharide extract, mix thoroughly, and collect the upper aqueous phase; wherein, the volume ratio of chloroform to n-butanol is 5:1, and the volume ratio of the chloroform and n-butanol mixture to the enzymatic hydrolysate of the polysaccharide extract is 1:5. 3) Add petroleum ether to the upper aqueous phase of step 2), mix thoroughly, and collect the lower aqueous phase; 4) Add macroporous resin AB-8 to the lower aqueous phase of step 2) for adsorption, collect the liquid; then use a dialysis bag with a molecular weight cutoff of 3500 Da for 60 h to remove small molecule components, obtain crude polysaccharide solution, and freeze dry it. Step 3: Purification and desalting of crude polysaccharide solution 1) After dissolving the freeze-dried component obtained in step 4) of step 2 with deionized water, load it onto a DEAEseplife FF anion exchange column for elution; 2) During the elution process, deionized water and sodium chloride solutions with concentrations of 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L were used to sequentially elute the crude polysaccharide solution using a gradient. The DEAE seplife FF anion exchange column was purified at a flow rate of 5 mL / min, and one tube was collected every 17 mL. The fractions corresponding to the four elution peaks were obtained. 3) At the same time, collect the above four components separately, dialyze them for 85 h using a dialysis bag with a molecular weight cutoff of 3500 Da to remove salt, and then freeze-dry them; Step 4: Elution to obtain polysaccharides from Isatis indigotica root. 1) Take the fraction obtained from elution with 0.1 mol / L sodium chloride solution in step 2) of step 3, after desalting and freeze-drying, dissolve it in deionized water, and load it onto a Sephacryl S-400HR gel column for elution; use deionized water, elute at a flow rate of 1.2 mL / min for 1.5 column volumes, and collect one tube every 15 mL; obtain the fractions corresponding to the two elution peaks, dialyze them with a dialysis bag with a molecular weight cutoff of 3500 Da for 85 h to remove salt, and then freeze-dry them; 2) Take the second component and prepare Isatis indigotica polysaccharide.

[0028] Example 7 The purpose of this embodiment is to provide a method for preparing polysaccharides from Isatis indigotica root, including the following steps: Step 1: Preparation of crude polysaccharide extract 1) The roots of Isatis indigotica are dried and crushed in sequence, and then the fragments of Isatis indigotica with qualified particle size are screened through a 60-mesh filter screen. 2) Add anhydrous ethanol to the qualified fragments of Isatis indigotica, extract for 12 h, and then collect the precipitate by centrifugation. 3) Add deionized water to the collected precipitate and then place it in a water bath at 60 ℃ for 4 h. After that, centrifuge to collect the supernatant extract. 4) Repeat the process of step 1) - step 3) on the precipitate residue after collecting the supernatant extract in step 3), and then combine the supernatant extracts collected in the two steps to obtain the extract. 5) Vacuum rotary evaporation of the extract and concentration to 10% of the original volume to obtain concentrated extract; 6) Add anhydrous ethanol to the concentrated extract for alcohol precipitation. The volume ratio of concentrated extract to anhydrous ethanol is 1:4. After alcohol precipitation, centrifuge and collect the precipitate to obtain the crude polysaccharide extract. Step 2: Preparation of crude polysaccharide solution 1) Add deionized water to dissolve the crude polysaccharide extract prepared in step one, and then add papain to hydrolyze it to obtain polysaccharide extract hydrolysate. 2) Add a mixture of chloroform and n-butanol to the enzymatic hydrolysate of the polysaccharide extract, mix thoroughly, and collect the upper aqueous phase; wherein, the volume ratio of chloroform to n-butanol is 4:1, and the volume ratio of the chloroform and n-butanol mixture to the enzymatic hydrolysate of the polysaccharide extract is 1:4. 3) Add petroleum ether to the upper aqueous phase of step 2), mix thoroughly, and collect the lower aqueous phase; 4) Add macroporous resin AB-8 to the lower aqueous phase of step 2) for adsorption, collect the liquid; then use a dialysis bag with a molecular weight cutoff of 3000 Da for dialysis for 48 h to remove small molecule components, prepare polysaccharide solution, and freeze dry it. Step 3: Purification and desalting of crude polysaccharide solution 1) After dissolving the freeze-dried component obtained in step 4) of step 2 with deionized water, load it onto a DEAEseplife FF anion exchange column for elution; 2) During the elution process, deionized water and sodium chloride solutions with concentrations of 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L were used to sequentially elute the crude polysaccharide solution using a gradient. The DEAE seplife FF anion exchange column was purified at a flow rate of 4 mL / min, and one tube was collected every 15 mL. The fractions corresponding to the four elution peaks were obtained. 3) Collect the above four components separately, dialyze them for 48 h-72 h using a dialysis bag with a molecular weight cutoff of 3000 Da to remove salt, and then freeze-dry them; Step 4: Elution to obtain polysaccharides from Isatis indigotica root. 1) Take the fraction obtained from elution with 0.1 mol / L sodium chloride solution in step 2) of step 3, after desalting and freeze-drying, dissolve it in deionized water, and load it onto a Sephacryl S-400HR gel column for elution; use deionized water, elute 1.5 column volumes at a flow rate of 1 mL / min, and collect one tube every 12 mL; obtain the fractions corresponding to the two elution peaks, dialyze them with a dialysis bag with a molecular weight cutoff of 3000 Da for 48 h-72 h to remove salt, and then freeze-dry them; 2) Take the second component and prepare Isatis indigotica polysaccharide.

[0029] Test case Test Example 1 The polysaccharide from Isatis indigotica prepared in Example 1 was subjected to ultraviolet spectral scanning analysis. The specific steps are as follows: A small amount of polysaccharide sample was weighed and dissolved in deionized water to prepare a 5 mg / mL polysaccharide solution. The polysaccharide solution was analyzed using a Thermo Fisher Scientific (USA) multi-functional microplate reader. Deionized water was used as a blank control and the results were measured under the same conditions.

[0030] like Figure 3 As shown, the sample showed no significant absorption in the wavelength range of 200 nm to 400 nm, indicating that the sample contained almost no impurities such as pigments, nucleic acids, and proteins.

[0031] Test Example 2 The polysaccharide from Isatis indigotica prepared in Example 1 was subjected to infrared spectroscopy analysis. The specific steps are as follows: A small amount of polysaccharide sample was weighed and mixed with 200 mg of potassium bromide, then pressed into a 1 mm thick sheet and analyzed. The sample was scanned using a Nicolet iZ-10 Fourier transform infrared spectrometer with a resolution of 4.00 cm⁻¹. -1 The scanning range is 4000-400 cm. -1 ; The results are as follows Figure 4 As shown, at 3409.09 cm -1 The strong, broad absorption bands around the left and right are related to the stretching vibration of OH; at 2934.09 cm⁻¹ -1 The absorption peak at 1634.97 cm⁻¹ was assigned to the stretching vibration of CH, indicating that it is a typical polysaccharide; -1 The strong absorption peak at 1740 cm⁻¹ is due to the C=O stretching vibration; -1 The absence of a peak at 1401.27 cm⁻¹ indicates the absence of uronic acid in the polysaccharide structure; -1 The absorption peak at 1071.39 cm⁻¹ represents the CN stretching and NH bending vibrations; -1 The significant absorption at the point indicates the presence of bending vibrations of the CO bond in the COH or COC structure.

[0032] Test Example 3 The molecular weight of the polysaccharide from Isatis indigotica prepared in Example 1 was determined, and the specific steps are as follows: The 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, filtered through a 0.45 μm filter membrane, and then analyzed. The chromatographic system used was a gel permeation chromatography-differential chromatography-multi-angle laser light scattering system. 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). Two gel size exclusion columns, Ohpak SB-805 HQ (300 × 8 mm) and Ohpak SB-803 HQ (300 × 8 mm), were used in series. The column temperature was 45 °C, the injection volume was 100 µL, the mobile phase was A (0.02% NaN3, with a concentration of 0.1 mol / L NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min.

[0033] The results are as follows Figure 5As shown, the weight-average molecular weight (Mw) of BcP-b2 is 38.114 kDa, and the polydispersity index (Mw / Mn) is 1.253.

[0034] Test Example 4 The monosaccharide composition of the polysaccharide from Isatis indigotica prepared in Example 1 was analyzed, and the specific steps are as follows: The sample was hydrolyzed in a sealed tube at 121 °C for 2 h with 2 mol / L trifluoroacetic acid. The sample was then dried under nitrogen and washed three times with methanol. After redissolving the sample in deionized water, the monosaccharide composition of the polysaccharides from *Isatis indigotica* root was analyzed by high-performance anion exchange chromatography (HPAEC) on a CarboPac PA-20 anion exchange column. A Dionex ... TM CarboPac TM PA20 (150×3.0 mm, 10 μm) liquid chromatography column; injection volume: 5 µL. Mobile phase A (H2O), mobile phase B (0.1 mol / L NaOH), mobile phase C (0.1 mol / L NaOH, 0.2 M NaAc), flow rate: 0.5 mL / min; column temperature: 30℃. The gradient elution conditions for the mobile phase were as follows: 0 min, A phase / B phase / C phase (95:5:0, V / V); 26 min, A phase / B phase / C phase (85:5:10, V / V); 42 min, A phase / B phase / C phase (85:5:10, V / V); 42.1 min, A phase / B phase / C phase (60:0:40, V / V); 52 min, A phase / B phase / C phase (60:40:0, V / V); 52.1 min, A phase / B phase / C phase (95:5:0, V / V); 60 min, A phase / B phase / C phase (95:5:0, V / V).

[0035] The results are as follows Figure 6 As shown, the polysaccharide from Isatis indigotica root is composed of arabinose, galactose, rhamnose, glucose, xylose, mannose, and fucose, with contents of 26.17%, 23.61%, 23.01%, 13.05%, 6.69%, 6.06%, and 1.40%, respectively.

[0036] Test Example 5 The polysaccharide from Isatis indigotica prepared in Example 1 was subjected to methylation analysis, and the specific steps are as follows: The dried sample was dissolved in DMSO, and methylation was carried out in DMSO / NaOH solution using CH3I. The methylated polysaccharide was hydrolyzed at 121 °C for 1.5 h, reduced with sodium borohydride, and acetylated with acetic anhydride at 100 °C for 2.5 h. GC-MS was performed using an Agilent 6890A-5975C column equipped with an Agilent BPX70 column (30 m × 0.25 mm × 0.25 µm, SGE, Australia) and high-purity helium (split ratio 10:1). The temperature was set at 140 °C for 2.0 min, then increased at 3 °C / min to 230 °C for 3 min. The scan mode was SCAN, and the scan range (m / z) was 50 to 350. The results are shown in Table 1.

[0037] Table 1. Glycosidic bond composition of BcP-b2

[0038] Test Example 6 Nuclear magnetic resonance analysis was performed on the polysaccharide from Isatis indigotica prepared in Example 1. The specific steps are as follows: The dried BcP-b2 sample was dissolved in 0.5 mL of D2O to a final concentration of 40 mg / mL. 1 D-NMR and 2 D-NMR was recorded at 25 ℃ 1 H-NMR, 13 C-NMR, COSY, NOESY, HMBC, and HSQC spectra.

[0039] One-dimensional 1 H spectrum, 13 The C spectrum, and the two-dimensional COSY, HSQC, HMBC, and NOESY spectra are shown below. Figure 7-12 As shown. 1 ¹H NMR is primarily used to distinguish the conformations of glycosidic bonds in polysaccharide structures. The proton signal of BcP-b2 is distributed in a narrow region of 3.0–5.5 ppm, a typical characteristic of polysaccharides. The NMR signal in the 4.3–5.8 ppm range is attributed to anodic hydrogen atom signals; 4.3–4.8 ppm indicates a β-configuration glycoside, and 4.8–5.8 ppm indicates an α-configuration. Preliminary assessment indicates the presence of both α and β glycosidic bond conformations in BcP-b2, such as at 4.4, 4.42, 4.47, 5.02, 5.05, 5.17, and 5.21 ppm. A clear chemical shift signal was observed at 1.18 ppm, corresponding to H6 of the α-L-Rhap unit. An H signal corresponding to O-CH3 was observed, with a chemical shift of 3.65 ppm. Multiple anodic carbon signals were found in the structure of BcP-b2. 13Cross peaks in the anodic region of C NMR and HSQC spectra identified anodic signals of 5.02 / 107.5, 4.4 / 101.64, 5.17 / 109.23, 5.05 / 101.08, 5.21 / 98.39, 4.47 / 103.14, and 4.42 / 103.38 ppm, which are represented by glycosyl residues AG.

[0040] Referring to Table 2, based on the methylation results and anodic signals, the sugar residues A and B can be deduced as follows: →2)-α-D-Glcp1→,→3)-β-D-Glcp(1→,α-L-Araf(1→,→3)-α-L- →(1→,α-D-→(1→,→6)-β-→(1→,→3,6)-β-D-Galp(1→).

[0041] The signal peak at approximately 16.57 ppm was identified as the C6 signal of rhamnose, and the signal peak at approximately 59.42 ppm was the carbon signal of O-CH3. From 1 H- 13 The signal was obtained from the C HMBC spectrum. A diagnostic signal for BcP-b2, showing a 1,6-linkage between α-D-Galp and β-D-Galp units, was observed. The ch-cross peak between the keto isomers C6 (δ 66.57 ppm) and H1 (δ 5.02 ppm) corresponds to the relationship between C6 of the β-D-Gal residue and H1 of the α-D-Galp residue. NOESY spectral signals yielded AH1 / GH6 (δ 5.02 / 3.82 ppm), FH1 / GH3 (δ 4.47 / 4.35 ppm), and GH1 / BH3 (δ 4.42 / 4.02 ppm), suggesting that C6 is the major branch point. The signal at δ 5.17 / 84.03 ppm indicates a 1,3-linkage between α-L-Araf and β-D-Galp. Therefore, the possible structure of this polysaccharide is speculated as follows: Figure 13 As shown.

[0042] Table 2 Sugar residues of BcP-b2 1 H and 13 Chemical shift of C

[0043] Test Example 7 The cytotoxicity experiment of the polysaccharide from Isatis indigotica prepared in Example 1 was carried out, and the specific steps are as follows: Mouse peritoneal macrophages RAW264.7 were cultured in TMEM medium (containing 100 U / mL penicillin, 100 mg / mL streptomycin, and 10% fetal bovine serum) in a 5% CO2 incubator. When cell confluence reached 80%-90%, the cells were digested with 0.25% trypsin to release them from the adherent state. After resuspending the collected cells, they were counted using a hemocytometer and seeded at 10⁴ cells / well in 100 µL of each well, and cultured for 24 h. The old medium was aspirated, and the cells were treated with different concentrations of Isatis indigotica polysaccharide (0, 1, 10, 100, 1000 μg / mL) for 24 h. Then, 10 µL of LCK-8 reagent was added to each well, and the cells were incubated at 37°C for 4 h. The absorbance was measured at 450 nm to quantify the relative cell viability.

[0044] The results are as follows Figure 14 As shown, within the concentration range of 0-1000 μg / mL, Isatis indigotica polysaccharide had no toxic effect on RAW264.7 macrophages and significantly promoted the viability of these cells.

[0045] Test Example 8 Nuclear magnetic resonance analysis was performed on the polysaccharide from Isatis indigotica prepared in Example 1 to study its antioxidant activity. The cell culture method was as shown in Test Example 8.

[0046] (1) Detection of superoxide dismutase (SOD) activity. After cell digestion, the cells were subjected to a concentration of 1.5 × 10⁻⁶. 6 Cells were seeded per well in 6-well plates and cultured for 24 h. Cells were treated with different concentrations of Isatis indigotica polysaccharide (0, 10, 50, and 100 μg / mL) for 24 h, washed twice with PBS, and collected. SOD activity was measured using a SOD activity assay kit (WST-8 method; Shanghai Beyotime). Cells were lysed using the SOD sample preparation solution in the kit, and protein concentration was determined by the BCA method for sample quantification. A WST-8 / enzyme working solution was prepared (151 μL of SOD detection buffer, 8 μL of WST-8, and 1 μL of enzyme solution were uniformly mixed for each reaction to obtain 160 μL of WST-8 / enzyme working solution). The reaction initiation solution (40×) in the kit was dissolved and mixed thoroughly, and then diluted with 39 μL of SOD detection buffer per 1 μL of reaction initiation solution (40×). The resulting solution was the reaction initiation working solution. Table 3 shows the sample assay system.

[0047] Table 3. Superoxide dismutase activity assay system

[0048] After incubating the above reaction solution at 37℃ for 30 min, the absorbance was measured at 450 nm. Inhibition percentage = (A blank control 1 - A sample) / (A blank control 1 - A blank control 2) × 100%. SOD enzyme activity units in the test sample = SOD enzyme activity units in the detection system = inhibition percentage / (1 - inhibition percentage) units. Results are as follows... Figure 15 As shown in Figure A, LPS significantly inhibited the activity of cellular SOD; while 10, 50, and 100 μg / mL of BcP-b2 significantly increased intracellular SOD activity.

[0049] (2) Catalase (CAT) activity assay. After cell digestion, the CAT activity was measured at 1.5 × 10⁻⁶. 6 Cells were seeded per well in 6-well plates and cultured for 24 h. Cells were treated with different concentrations of Isatis indigotica polysaccharide (0, 10, 50, and 100 μg / mL) for 24 h, washed twice with PBS, and collected. Cells were lysed using Western blotting and IP cell lysis buffer, and protein concentration was determined by the BCA method for sample quantification. Catalase activity was detected using a catalase assay kit (Shanghai Beyotime). Standard curve solutions with concentrations of 250 mmol / L, 0 mmol / L, 0.625 mmol / L, 1.25 mmol / L, 2.5 mmol / L, and 3.75 mmol / L were prepared using the hydrogen peroxide solution provided in the kit. The sample assay system is shown in Table 4.

[0050] Table 4 Catalase Assay System

[0051] The above system was reacted at 25°C for 3 min. 450 μL of catalase reaction termination solution was added, and the reaction was terminated by inverting the tube. 40 μL of catalase detection buffer was added to a clean centrifuge tube, followed by 10 μL of the terminated and mixed reaction mixture. 10 μL of the 50 μL mixture from the previous step was added to one well of a 96-well plate. Simultaneously, 4 μL of each of the following hydrogen peroxide standard curve solutions (0 mmol / L, 0.625 mmol / L, 1.25 mmol / L, 2.5 mmol / L, and 3.75-0.1 mmol / L) were added to one well of a 96-well plate. 200 μL of chromogenic working solution was added, and the mixture was incubated at 25°C for 15 min before measuring A520. A standard curve was plotted: A520 = k [hydrogen peroxide micromoles] + b. The values ​​of k and b were calculated from the standard curve. Residual hydrogen peroxide micromoles = (A520-b) / k. For calculating catalase activity in cell or tissue samples: [Sample catalase activity] = [[Residual hydrogen peroxide micromoles in blank control] - [Residual hydrogen peroxide micromoles in sample]] × [Dilution factor] / ([Reaction time in minutes] × [Sample volume] × [Protein concentration]).

[0052] The results are as follows Figure 15 As shown in Figure B, LPS significantly inhibited CAT activity in cells; while BcP-b2 at concentrations of 10 μg / mL, 50 μg / mL, and 100 μg / mL significantly increased intracellular CAT activity.

[0053] (3) Glutathione peroxidase (GSH-Px) activity assay. Cells were digested and then subjected to a concentration of 1.5 × 10⁻⁶ g / L. 6 Cells were seeded per well in 6-well plates and cultured for 24 h. Cells were treated with different concentrations of Isatis indigotica polysaccharide (0, 10, 50, and 100 μg / mL) for 24 h, washed twice with PBS, and collected. Cells were lysed using Western blotting and IP cell lysis buffer, and protein concentration was determined by the BCA method for sample quantification. Glutathione peroxidase (GSH) activity was detected using a kit from Beyotime. 62.5 mmol / L NADPH solution, 75 mmol / L GSH solution, GPx detection working solution, and 30 mmol / L peroxidase reagent solution were prepared according to the kit instructions and incubated at 25 °C. The sample determination system is shown in Table 5 below. Table 5 Glutathione peroxidase detection system

[0054] After reacting the samples at room temperature for 15 min, add 10 μL of 30 mmol / L peroxide reagent solution to each well and mix well. Immediately measure A using a microplate reader. 412 At this point, the reading is recorded as 0 min, and A is measured for 15 min. 412 value.

[0055] Glutathione peroxidase activity in sample = Glutathione peroxidase activity in detection system × dilution factor / protein concentration in sample = [(ΔA)] 412 / min) / (ε μM [×L (cm))]×[dil×(V (mL) / V sample (mL))] / protein concentration in the sample.

[0056] The results are as follows Figure 15 As shown in Figure C, LPS significantly inhibited the activity of cellular GSH-Px; while 10, 50, and 100 μg / mL of BcP-b2 significantly increased intracellular GSH-Px activity.

[0057] (4) Measurement of intracellular reactive oxygen species (ROS) levels. After cell digestion, the ROS levels were measured at 10... 4 Cells were seeded at 100 μL per well in 96-well plates and cultured for 24 h. Cells were treated with different concentrations of Isatis indigotica polysaccharide (0, 10, 50, and 100 μg / mL) and 1 μg / mL LPS for 24 h. ROS levels were detected using a reactive oxygen species (ROS) assay kit. The probe DCFH-DA provided in the kit was diluted 1:1000 with serum-free culture medium to a final concentration of 10 μM. The cell culture medium was removed, and 100 µL of diluted DCFH-DA was added. The cells were incubated at 37°C for 20 min. Cells were washed three times with serum-free cell culture medium and photographed under a fluorescence microscope. Results are shown below. Figure 15 As shown in Figure D, LPS significantly increased intracellular ROS levels; while 10 μg / mL, 50 μg / mL and 100 μg / mL BcP-b2 significantly reduced intracellular ROS levels.

[0058] Example 13 Animal experiments were conducted on the polysaccharide from Isatis indigotica prepared in Example 1. The specific steps are as follows: Zebrafish with a 3-dpf melanin allele mutation were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. A blank control group, a model control group, a positive control group, and experimental groups (low, medium, and high concentrations) were set up. The positive control was N-acetylcysteine ​​(NAC) at a concentration of 62.5 μg / mL. The experimental groups were treated with BcP-b2 at concentrations of 500 μg / mL, 1000 μg / mL, and 2000 μg / mL, with a volume of 3 mL per well. After treatment at 28℃ for 3 h, except for the normal control group, all other groups were treated with menadione at 28℃ to establish a zebrafish oxidative damage model. After another day of treatment at 28℃, the zebrafish in each experimental group were stained with CellROX in the dark. After staining, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Images were then saved, and data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The fluorescence intensity of the zebrafish yolk sac was analyzed and statistically analyzed, and the antioxidant efficacy of the samples was evaluated based on the statistical analysis results of this index. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. p A value <0.05 indicates that the difference is statistically significant.

[0059] Figure 16 The results showed that ROS production was significantly increased in the hydrogen peroxide treatment group compared to the control group. However, at BcP-b2 concentrations of 6.25 µg / mL, 12.5 µg / mL, and 25 µg / mL, the extract significantly reduced ROS production, demonstrating the in vivo antioxidant activity of BcP-b2.

[0060] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A polysaccharide from Isatis indigotica root, characterized in that, The weight-average molecular weight of the polysaccharide from Isatis indigotica root is 38.114 kDa, and the polydispersity index is 1.

253. The polysaccharide from Isatis indigotica root is composed of arabinose, galactose, rhamnose, glucose, xylose, mannose, and fucose in molar percentages of 26.17%, 23.61%, 23.01%, 13.05%, 6.69%, 6.06%, and 1.40%, respectively.

2. The polysaccharide from Isatis indigotica root according to claim 1, characterized in that, The polysaccharide from Isatis indigotica root contains the following structure: 。 3. A method for preparing polysaccharide from Isatis indigotica root as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1: Preparation of crude polysaccharide extract: The roots of Isatis indigotica were dried and pulverized in sequence, sieved, and then extracted with anhydrous ethanol to defatt them. The precipitate was collected by centrifugation. Deionized water was added to the precipitate, and the supernatant was collected by centrifugation after water bath extraction. The extraction was repeated several times and the supernatants were combined. The supernatant was concentrated under vacuum and then anhydrous ethanol was added for alcohol precipitation. The precipitate was collected by centrifugation to obtain crude polysaccharide extract. Step 2: Preparation of crude polysaccharide solution: Dissolve the crude polysaccharide extract in water, add papain for enzymatic hydrolysis to obtain polysaccharide extract hydrolysate; deproteinize using the Sevag method and collect the upper aqueous phase; add petroleum ether to the aqueous phase for defatting and collect the lower aqueous phase; after adsorption with macroporous resin AB-8, dialyze to remove small molecule components using a dialysis bag, and freeze-dry to obtain the lyophilized crude polysaccharide solution; Step 3: Ion exchange column purification and desalting: Dissolve the lyophilized crude polysaccharide solution in water and load it onto a DEAE seplife FF anion exchange column. Elute sequentially with deionized water, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L sodium chloride solutions. Collect the corresponding components eluted with 0.1 mol / L sodium chloride solution, dialyze to remove salt, and then freeze-dry. Step 4, Gel column elution and purification: Dissolve the lyophilized product obtained in Step 3 in water, load it onto a Sephacryl S-400HR gel column, elute with deionized water, collect the fraction corresponding to the second elution peak, dialyze to remove salt, and then freeze-dry to obtain the polysaccharide from Isatis indigotica root.

4. The method for preparing polysaccharide from Isatis indigotica root according to claim 3, characterized in that, In step one, the sieve mesh size is 50-70 mesh; the extraction time with anhydrous ethanol is 10-14 h; the water bath extraction temperature is 50-70℃ and the extraction time is 3.5-4.5 h; the supernatant is concentrated to 8%-15% of the original volume; and the volume ratio of the concentrated extract to anhydrous ethanol during alcohol precipitation is 1:3-1:

5.

5. The method for preparing polysaccharide from Isatis indigotica root according to claim 3, characterized in that, In step two, the volume ratio of chloroform to n-butanol used in the Sevag method is 3:1-5:1, and the volume ratio of the chloroform and n-butanol mixture to the polysaccharide extract enzymatic hydrolysate is 1:3-1:5; the molecular weight cutoff of the dialysis bag is 2500-3500 Da, and the dialysis time is 30-60 h.

6. The method for preparing polysaccharide from Isatis indigotica root according to claim 3, characterized in that, In step three, the elution flow rate of the DEAEseplife FF anion exchange column is 3-5 mL / min, and one tube is collected every 13-17 mL; the molecular weight cutoff of the dialysis bag used for dialysis desalination is 2500-3500 Da, and the dialysis time is 40-85 h.

7. The method for preparing polysaccharide from Isatis indigotica root according to claim 3, characterized in that, In step four, the elution flow rate of the Sephacryl S-400HR gel separation column is 0.8-1.2 mL / min, eluting 1.5 times the column volume, and collecting one tube every 8-15 mL; the molecular weight cutoff of the dialysis bag used for dialysis desalting is 2500-3500 Da, and the dialysis time is 40-85 h.

8. The application of the polysaccharide from Isatis indigotica root as described in claim 1 or 2 in the preparation of an antioxidant composition, wherein the antioxidant composition is a pharmaceutical, health product, or functional food.

9. The application according to claim 8, characterized in that, The antioxidant composition exerts its antioxidant effect by promoting the activity of superoxide dismutase, catalase and glutathione peroxidase, thereby reducing the level of reactive oxygen species in cells.

10. The application according to claim 8, characterized in that, The antioxidant composition is used to alleviate oxidative stress damage in cells and the body.

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