Extraction and purification method of polysaccharide from stems and leaves of Scutellaria baicalensis and its application

Abstract

The invention discloses a method for extracting and purifying polysaccharides from the stems and leaves of scutellaria baicalensis and its application, and relates to a method for extracting and purifying polysaccharides from the stems and leaves of scutellaria baicalensis and its application. The invention is to solve the problem that only the root of scutellaria baicalensis is used as a Chinese medicinal material, resulting in a large amount of scutellaria baicalensis stem and leaf parts being abandoned, resulting in a waste of medicinal resources. The method is to obtain scutellaria baicalensis stem and leaf polysaccharides through extraction of scutellaria baicalensis stem and leaf polysaccharides, deproteinization by Sevag method, DEAE-52 ion exchange column chromatography and dextran gel G-200 column chromatography. Scutellaria baicalensis stem and leaf polysaccharides promote the proliferation of RAW264.7 cells in a concentration-dependent manner and improve the phagocytic ability of RAW264.7 cells. The present invention extracts and purifies scutellaria baicalensis stem and leaf polysaccharides with immunomodulatory activity from scutellaria baicalensis stem and leaves for the first time, and completes the partial structural identification of scutellaria baicalensis stem and leaf polysaccharides, solving the problem that a large amount of scutellaria baicalensis stem and leaf parts are abandoned, resulting in a waste of medicinal resources. The present invention belongs to the technical field of extraction of effective ingredients of traditional Chinese medicine.

Description

Technical Field

[0001] The invention relates to a method for extracting and purifying polysaccharide from scutellaria baicalensis stems and leaves and application thereof. Background Art

[0002] Scutellaria baicalensis is the dried root of the Lamiaceae plant (Scutellaria baicalensis). It has the effects of clearing heat and dampness, purging fire and detoxifying. It is primarily produced in Heilongjiang, Liaoning, Inner Mongolia, Hebei, and Henan. The stems and leaves of Scutellaria baicalensis, a perennial herb in the Lamiaceae family, are the above-ground parts of the plant. As a traditional Chinese medicinal ingredient, Scutellaria baicalensis is commonly used in many Chinese patent medicines, such as Shuanghuanglian Oral Liquid and Qingfei Paidu Decoction. It also plays a key role in the treatment of the novel coronavirus infection that has recently emerged. For example, the eighth edition of the treatment plan clearly specifies the dosage of Scutellaria baicalensis. Currently, Scutellaria baicalensis is in high demand domestically and is one of the most exported medicinal herbs. This results in a large amount of Scutellaria baicalensis stems and leaves being discarded each year, resulting in a waste of medicinal resources. In recent years, with the research on the stems and leaves of Scutellaria baicalensis, people have found that the stems and leaves also contain a large number of active components and have extremely high utilization value. However, there has been no report on the extraction and identification of polysaccharides in the stems and leaves of Scutellaria baicalensis. Summary of the Invention

[0003] The purpose of the present invention is to solve the technical problem that only the root of Scutellaria baicalensis is used as a traditional Chinese medicine, resulting in a large amount of Scutellaria baicalensis stems and leaves being abandoned, causing a waste of medicinal resources, and provide a method for extracting and purifying polysaccharides from Scutellaria baicalensis stems and leaves and the application thereof.

[0004] The extraction and purification method of Scutellaria baicalensis stem and leaf polysaccharide is as follows:

[0005] 1. Polysaccharide Extraction from Scutellaria Baicalensis Stems and Leaves:

[0006] The stems and leaves of Scutellaria baicalensis were dried and crushed, and passed through a 60-mesh sieve. The fine powder was reflux defatted twice with 10 times the volume of anhydrous ethanol, each time for 2 hours. The defatted powder was naturally dried. 3 kg of defatted Scutellaria baicalensis stem and leaf powder was weighed each time, and extracted twice under the conditions of a solid-liquid ratio of 1g:20mL-1g:30mL, a temperature of 80°C-100°C, and an extraction time of 3h-5h. The aqueous extracts were combined and concentrated, and anhydrous ethanol was added to an ethanol volume fraction of 80%. After precipitation overnight, the extract was centrifuged at 4000rpm for 5 minutes, and the precipitate was collected and naturally air-dried to obtain crude polysaccharide.

[0007] 2. Sevage method for deproteinization:

[0008] Chloroform and n-butanol were mixed in a volume ratio of 4:1 to obtain a Sevage solution. The crude polysaccharide obtained in step 1 was mixed with the Sevage solution in a volume ratio of 4:1. The mixture was vigorously stirred for 30 minutes and then centrifuged at 4000 rpm for 10 minutes to remove free protein. This was repeated several times until no obvious protein was produced. The deproteinized polysaccharide solution was vacuum evaporated and decolorized with 4.5% H2O2 for 100 minutes at 60°C. The mixture was then freeze-dried to obtain a sample polysaccharide.

[0009] 3. DEAE-52 ion exchange column chromatography:

[0010] (1) DEAE-52 cellulose pretreatment

[0011] Take 100g of DEAE-52 cellulose and soak it in 10 volumes of ddH2O for 24 hours. After it is fully swollen, vacuum filter to remove water, soak it in 0.05mol / LNaOH for 1 hour, wash it with water until neutral, soak it in 0.1mol / LHCl for 30 minutes, wash it with water until neutral, and finally treat it with 0.1mol / LNaOH for 30 minutes, wash it with water until neutral. Mix the filler and water in a mass ratio of 2:1 and stir thoroughly to make a filler slurry for column loading;

[0012] (2) Column Packing

[0013] Fix the chromatography column vertically on the iron stand, slowly pour the filler slurry into the column bed while stirring to prevent bubbles from forming; after filling, select ultrapure water as the mobile phase to press the column, and let it stand overnight to stabilize the filler surface;

[0014] (3) Sample loading

[0015] Take 2g of sample polysaccharide, dissolve it in ultrapure water to prepare a 100mg / mL polysaccharide solution, filter it through a 0.45μm filter membrane, and slowly pour it into the column. Connect the peristaltic pump and prepare for elution.

[0016] (4) Elution

[0017] Ultrapure water, 0.1 mol / L, 0.3 mol / L, 0.5 mol / L, and 1 mol / L NaCl solutions were sequentially used for gradient elution. The flow rate was controlled at 1 mL / min, and 5 mL was collected in one tube using the automatic collector.

[0018] (5) Sugar content detection

[0019] The sugar content of the eluate in each tube was detected by the phenol-sulfuric acid method. 200 μL of eluate was drawn from each collection tube, and 200 μL of 6% phenol solution and 1 mL of concentrated sulfuric acid were added respectively. After oscillation and mixing, the tubes were allowed to stand for 20 minutes. The absorbance of each tube at 490 nm was measured by a microplate reader, and the elution curve was drawn according to the absorbance value. The same fractions were combined according to the elution results, and the cut-off molecular weight was 3.5×10 3 The mixture was dialyzed in a Da dialysis bag for 72 h and freeze-dried to obtain fraction SSP-3;

[0020] 4. Sephadex G-200 column chromatography:

[0021] (1) Sephadex G-200 gel pretreatment

[0022] Soak Sephadex G-200 gel in 10 times the volume of ultrapure water for 24 hours to allow the gel to fully swell, then wash and stir evenly to prepare a gel slurry;

[0023] (2) Column Packing

[0024] Fix the chromatography column vertically on the iron stand, slowly pour the filler slurry into the column bed while stirring to prevent bubbles from forming; after filling, select ultrapure water as the mobile phase to press the column, and let it stand overnight to stabilize the filler surface;

[0025] (3) Sample loading

[0026] Take 200 mg of the component SSP-3 obtained in step 3 and prepare a 5% solution by mass. After filtering through a 0.45 μm filter membrane, slowly pour it into the column and prepare for elution.

[0027] (4) Elution

[0028] Ultrapure water was used as the mobile phase, the flow rate was controlled at 15 mL / h, and the automatic collector was set to collect one tube every 5 minutes;

[0029] (5) Sugar content detection

[0030] The sugar content of the eluate in each tube was detected by the phenol-sulfuric acid method. 200 μL of eluate was drawn from each collection tube, and 200 μL of 6% phenol solution and 1 mL of concentrated sulfuric acid were added respectively. After oscillation and mixing, the tubes were allowed to stand for 20 minutes. The absorbance of each tube at 490 nm was measured by a microplate reader, and the elution curve was drawn according to the absorbance value. The same fractions were combined according to the elution results, and the cut-off molecular weight was 3.5×10 3 The mixture was dialyzed in a Da dialysis bag for 72 h and freeze-dried to obtain Scutellaria baicalensis stem and leaf polysaccharide SSP-3a.

[0031] In step 1, 3 kg of defatted Scutellaria baicalensis stem and leaf powder was weighed each time and extracted twice under the conditions of a solid-liquid ratio of 1 g:20 mL, a temperature of 100° C., and an extraction time of 4.5 h.

[0032] In step 1, 3 kg of defatted Scutellaria baicalensis stem and leaf powder was weighed each time and extracted twice under the conditions of a solid-liquid ratio of 1 g:25 mL, a temperature of 100° C., and an extraction time of 5 h.

[0033] In step 1, 3 kg of defatted Scutellaria baicalensis stem and leaf powder was weighed each time and extracted twice under the conditions of a solid-liquid ratio of 1 g:30 mL, a temperature of 100° C., and an extraction time of 5 h.

[0034] The scutellaria baicalensis stem and leaf polysaccharide promotes the proliferation of RAW264.7 cells in a concentration-dependent manner.

[0035] The scutellaria baicalensis stem and leaf polysaccharide is used for improving the phagocytic ability of RAW264.7 cells.

[0036] The present invention obtains Scutellaria baicalensis stem-leaf polysaccharide by hot water extraction. The extraction conditions are optimized using response surface methodology, and a solid-liquid ratio of 1:20, an extraction time of 4.5 hours, and an extraction temperature of 100°C are determined. Under these conditions, a polysaccharide extraction rate of 8.38% can be achieved. After obtaining crude polysaccharide (Scutellaria baicalensis stem-leaf polysaccharide, SSP) using these extraction conditions, the polysaccharide is deproteinized and depigmented using the Sevage method and H2O2. The polysaccharide is initially impurity-removed and then separated and purified using DEAE-52 cellulose column chromatography. Screening for immunoreactivity in RAW264.7 macrophages reveals that the SSP-3 component has the best effect on regulating the immune proliferation activity of the cells. The purity of SSP-3 is then identified using gel chromatography, and secondary purification is performed using Sephadex G-200 gel column chromatography to obtain a highly active, uniform molecular weight polysaccharide SSP-3a. The composition of SSP-3a was detected by phenol-sulfuric acid method, sulfuric acid-carbazole method and Bradford method. The sugar content of SSP-3a was 91.15%, the uronic acid content was 37.9%, and the protein content was undetectable, indicating that SSP-3a is an acidic polysaccharide.

[0037] SSP-3a was purified from the polysaccharide of Scutellaria baicalensis stems and leaves. The UV spectrum scanning results showed that SSP-3a did not contain other impurities such as proteins and nucleic acids. The weight average molecular weight of the polysaccharide was 183223 according to the HPGPC method. In the infrared spectrum, there was a clear stretching vibration peak of CO in pyranoside. 1H-NMR showed that the polysaccharide had a β-configuration of the anomeric carbon. HPLC analysis revealed that SSP-3a is composed of four monosaccharides: mannose, glucuronic acid, glucose, and xylose, with a molar ratio of 1.476:5.472:2.982:2.805.

[0038] Lactate dehydrogenase assay results showed that SSP-3a had no toxic side effects on RAW264.7 cells within a concentration range of 1600 μg / mL. Resazurin sodium was used to test the proliferation of RAW264.7 cells. SSP-3a significantly promoted the proliferation of RAW264.7 cells in a concentration-dependent manner, with the proliferation rate reaching 182.71% at 800 μg / mL. Neutral red assay revealed that SSP-3a had the strongest effect on regulating phagocytic activity at 800 μg / mL, with a phagocytic index of 180.44%. In summary, SSP-3a promoted the proliferation of RAW264.7 cells in a concentration-dependent manner and also enhanced the phagocytic capacity of the cells, exerting an immunomodulatory effect.

[0039] Griess assay results showed that SSP-3a promoted NO secretion, reaching a peak of 42.69±3.63μM at 800μg / mL. ELISA assay results showed that the relative expression of cytokines (IL-6, IL-1β, and TNF-α) increased with increasing SSP-3a concentrations between 200μg / mL and 800μg / mL, with the highest cytokine secretion observed at 800μg / mL. qRT-PCR assay results also showed that SSP-3a significantly upregulated the expression of iNOS mRNA, IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA at the gene level, indicating that SSP-3a possesses immunomodulatory activity.

[0040] ELISA results showed that pretreatment with TLR4 antibodies and PDTC (an NF-κB inhibitor) significantly reduced SSP-3a's ability to promote cytokine secretion compared to the experimental group, suggesting that SSP-3a enhances the immunomodulatory effects of RAW264.7 cells by activating the TLR4 receptor. qRT-PCR results showed that SSP-3a significantly increased p65 mRNA expression and downregulated IκBα mRNA expression, indicating that SSP-3a participates in NF-κB signaling. Furthermore, SSP-3a significantly upregulated JNK, ERK, and p38 mRNA expression, suggesting that SSP-3a may also regulate immunity through the MAPK pathway. In summary, SSP-3a activates RAW264.7 cells through multiple pathways, exerting its immunomodulatory effects. The invention extracts scutellaria stem and leaf polysaccharide from the scutellaria stem and leaf, thereby solving the problem of waste of medicinal resources caused by abandonment of a large amount of scutellaria stem and leaf parts. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a comparison chart of the effects of different factors on the SSP extraction rate in Experiment 1. In the figure, A shows the effect of extraction temperature on the crude polysaccharide extraction rate, B shows the effect of extraction time on the crude polysaccharide extraction rate, and C shows the effect of solid-liquid ratio on the crude polysaccharide extraction rate.

[0042] Figure 2 This is the response surface analysis diagram of different factors in Experiment 1;

[0043] Figure 3 This is the contour distribution map of different factors in Experiment 1;

[0044] Figure 4 This is the elution curve of the DEAE-52 cellulose ion column in Experiment 1;

[0045] Figure 5 This is a comparison chart showing the effects of various DEAE-52 column chromatography components on the proliferation activity and phagocytic ability of RAW264.7 cells in Experiment 1;

[0046] Figure 6 This is the gel permeation chromatogram of SSP-3 in Experiment 1;

[0047] Figure 7 This is the Sephadex G-200 elution curve in Experiment 1;

[0048] Figure 8 This is a comparison of the appearance of the polysaccharide SSP-3a from the stems and leaves of Scutellaria baicalensis before and after purification in Experiment 1. A is the appearance of the polysaccharide SSP-3a from the stems and leaves of Scutellaria baicalensis before purification, and B is the appearance of the polysaccharide SSP-3a from the stems and leaves of Scutellaria baicalensis after purification.

[0049] Figure 9 This is the standard curve graph in Experiment 1. A is the glucose standard curve graph; B is the uronic acid standard curve graph; C is the protein standard curve graph;

[0050] Figure 10 This is the ultraviolet spectrum scan of SSP-3a in Experiment 1;

[0051] Figure 11 This is the gel permeation chromatogram of SSP-3a in Experiment 1;

[0052] Figure 12 This is the infrared spectrum of SSP-3a in Experiment 1;

[0053] Figure 13 This is the SSP-3a NMR in Experiment 1 1 H spectrum;

[0054] Figure 14 This is the HPLC chromatogram of the monosaccharide standard in Experiment 1;

[0055] Figure 15 This is the HPLC chromatogram of the sample in Experiment 1;

[0056] Figure 16 is a comparison of the effects of SSP-3a on the cytotoxicity of RAW264.7 cells in Experiment 1. All results are expressed as mean ± SD, and the two groups were compared using an unpaired two-tailed t test, ns, not statistically significant (P>0.05), P<0.05 was considered statistically significant and is indicated as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0057] Figure 17 Figure 1 shows the effect of SSP-3a on RAW264.7 cell morphology in Experiment 1. A is the blank control group; B, C, and D are the SSP-3a treatment groups at 200 μg / mL, 400 μg / mL, and 800 μg / mL, respectively; E is the LPS group.

[0058] Figure 18 Figure 1 is the effect of SSP-3a on the proliferation of RAW264.7 cells in Experiment 1. All results are expressed as mean ± SD, and the two groups were compared using an unpaired two-tailed t test, ns, not statistically significant (P > 0.05), and P < 0.05 was considered statistically significant and is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0059] Figure 19is the effect of SSP-3a on the phagocytic ability of RAW264.7 cells in experiment 1. All results are expressed as mean ± SD, and the two groups were compared using an unpaired two-tailed t test, ns, not statistically significant (P>0.05), P<0.05 was considered statistically significant and is indicated as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0060] Figure 20 Figure 1 is the effect of SSP-3a on NO secretion in RAW264.7 cells in Experiment 1. All results are expressed as mean ± SD, and the two groups were compared using an unpaired two-tailed t test, ns, not statistically significant (P > 0.05), and P < 0.05 was considered statistically significant and is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0061] Figure 21 is the effect of SSP-3a on the expression of iNOS mRNA in RAW264.7 cells in Experiment 1. All results are expressed as mean ± SD, and the two groups were compared using the unpaired two-tailed t test, ns, not statistically significant (P>0.05), P<0.05 was considered statistically significant and is indicated as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0062] Figure 22 This is the effect of SSP-3a on the secretion of cytokines IL-6, IL-1β, and TNF-α mRNA by RAW264.7 cells in experiment 1. All results are expressed as mean ± SD, and the two groups were compared using an unpaired two-tailed t test. ns, not statistically significant (P>0.05), P<0.05 was considered statistically significant and is indicated as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0063] Figure 23 is the effect of SSP-3a on the expression of RAW264.7 cells in Experiment 1. All results are expressed as mean ± SD, and the comparison between the two groups was performed using an unpaired two-tailed t test, ns, not statistically significant (P>0.05), P<0.05 was considered statistically significant and is indicated as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0064] Figure 24This is the effect of SSP-3a on the mRNA secretion of cytokines IL-6, IL-1β, and TNF-α in RAW264.7 cells after inhibitor treatment in Experiment 1. All results are expressed as mean ± SD, and the two groups were compared using an unpaired two-tailed t test. ns, not statistically significant (P>0.05), P<0.05 was considered statistically significant and is indicated as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0065] Figure 25 is the effect of SSP-3a on the mRNA expression of NF-κB pathway in RAW264.7 cells in Experiment 1. All results are expressed as mean ± SD, and the two groups were compared using an unpaired two-tailed t test, ns, not statistically significant (P>0.05), P<0.05 was considered statistically significant and is indicated as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0066] Figure 26 The results are shown in Table 1. The results show the effect of SSP-3a on the mRNA expression of the MAPK pathway in RAW264.7 cells in Experiment 1. All the results are expressed as the mean ± SD. The two groups were compared with the unpaired two-tailed t-test. ns, no statistical significance (P>0.05). P<0.05 was considered statistically significant and is indicated as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0067] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0068] Specific embodiment 1: The extraction and purification method of Scutellaria baicalensis stem and leaf polysaccharide in this embodiment is as follows:

[0069] 1. Polysaccharide Extraction from Scutellaria Baicalensis Stems and Leaves:

[0070] The stems and leaves of Scutellaria baicalensis were dried and crushed, and passed through a 60-mesh sieve. The fine powder was reflux defatted twice with 10 times the volume of anhydrous ethanol, each time for 2 hours. The defatted powder was naturally dried. 3 kg of defatted Scutellaria baicalensis stem and leaf powder was weighed each time, and extracted twice under the conditions of a solid-liquid ratio of 1g:20mL-1g:30mL, a temperature of 80°C-100°C, and an extraction time of 3h-5h. The aqueous extracts were combined and concentrated, and anhydrous ethanol was added to an ethanol volume fraction of 80%. After precipitation overnight, the extract was centrifuged at 4000rpm for 5 minutes, and the precipitate was collected and naturally air-dried to obtain crude polysaccharide.

[0071] 2. Sevage method for deproteinization:

[0072] Chloroform and n-butanol were mixed in a volume ratio of 4:1 to obtain a Sevage solution. The crude polysaccharide obtained in step 1 was mixed with the Sevage solution in a volume ratio of 4:1. The mixture was vigorously stirred for 30 minutes and then centrifuged at 4000 rpm for 10 minutes to remove free protein. This was repeated several times until no obvious protein was produced. The deproteinized polysaccharide solution was vacuum evaporated and decolorized with 4.5% H2O2 for 100 minutes at 60°C. The mixture was then freeze-dried to obtain a sample polysaccharide.

[0073] 3. DEAE-52 ion exchange column chromatography:

[0074] (1) DEAE-52 cellulose pretreatment

[0075] Take 100g of DEAE-52 cellulose and soak it in 10 volumes of ddH2O for 24 hours. After it is fully swollen, vacuum filter to remove water, soak it in 0.05mol / LNaOH for 1 hour, wash it with water until neutral, soak it in 0.1mol / LHCl for 30 minutes, wash it with water until neutral, and finally treat it with 0.1mol / LNaOH for 30 minutes, wash it with water until neutral. Mix the filler and water in a mass ratio of 2:1 and stir thoroughly to make a filler slurry for column loading;

[0076] (2) Column Packing

[0077] Fix the chromatography column vertically on the iron stand, slowly pour the filler slurry into the column bed while stirring to prevent bubbles from forming; after filling, select ultrapure water as the mobile phase to press the column, and let it stand overnight to stabilize the filler surface;

[0078] (3) Sample loading

[0079] Take 2g of sample polysaccharide, dissolve it in ultrapure water to prepare a 100mg / mL polysaccharide solution, filter it through a 0.45μm filter membrane, and slowly pour it into the column. Connect the peristaltic pump and prepare for elution.

[0080] (4) Elution

[0081] Ultrapure water, 0.1 mol / L, 0.3 mol / L, 0.5 mol / L, and 1 mol / L NaCl solutions were sequentially used for gradient elution. The flow rate was controlled at 1 mL / min, and 5 mL was collected in one tube using the automatic collector.

[0082] (5) Sugar content detection

[0083] The sugar content of the eluate in each tube was detected by the phenol-sulfuric acid method. 200 μL of eluate was drawn from each collection tube, and 200 μL of 6% phenol solution and 1 mL of concentrated sulfuric acid were added respectively. After oscillation and mixing, the tubes were allowed to stand for 20 minutes. The absorbance of each tube at 490 nm was measured by a microplate reader, and the elution curve was drawn according to the absorbance value. The same fractions were combined according to the elution results, and the cut-off molecular weight was 3.5×10 3 The mixture was dialyzed in a Da dialysis bag for 72 h and freeze-dried to obtain fraction SSP-3;

[0084] 4. Sephadex G-200 column chromatography:

[0085] (5) Sephadex G-200 gel pretreatment

[0086] Soak Sephadex G-200 gel in 10 times the volume of ultrapure water for 24 hours to allow the gel to fully swell, then wash and stir evenly to prepare a gel slurry;

[0087] (6) Column Packing

[0088] Fix the chromatography column vertically on the iron stand, slowly pour the filler slurry into the column bed while stirring to prevent bubbles from forming; after filling, select ultrapure water as the mobile phase to press the column, and let it stand overnight to stabilize the filler surface;

[0089] (7) Sample loading

[0090] Take 200 mg of the component SSP-3 obtained in step 3 and prepare a 5% solution by mass. After filtering through a 0.45 μm filter membrane, slowly pour it into the column and prepare for elution.

[0091] (8) Elution

[0092] Ultrapure water was used as the mobile phase, the flow rate was controlled at 15 mL / h, and the automatic collector was set to collect one tube every 5 minutes;

[0093] (5) Sugar content detection

[0094] The sugar content of the eluate in each tube was detected by the phenol-sulfuric acid method. 200 μL of eluate was drawn from each collection tube, and 200 μL of 6% phenol solution and 1 mL of concentrated sulfuric acid were added respectively. After oscillation and mixing, the tubes were allowed to stand for 20 minutes. The absorbance of each tube at 490 nm was measured by a microplate reader, and the elution curve was drawn according to the absorbance value. The same fractions were combined according to the elution results, and the cut-off molecular weight was 3.5×10 3 The mixture was dialyzed in a Da dialysis bag for 72 h and freeze-dried to obtain Scutellaria baicalensis stem and leaf polysaccharide SSP-3a.

[0095] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that in step 1, 3 kg of defatted Scutellaria baicalensis stem and leaf powder is weighed each time and extracted twice at a solid-liquid ratio of 1 g:20 mL, a temperature of 100° C., and an extraction time of 4.5 h. Other aspects are the same as specific embodiment 1.

[0096] Specific embodiment 3: This embodiment differs from specific embodiments 1 or 2 in that in step 1, 3 kg of defatted Scutellaria baicalensis stem and leaf powder is weighed each time and extracted twice at a solid-liquid ratio of 1 g:25 mL, a temperature of 100° C., and an extraction time of 5 h. Other aspects are the same as specific embodiments 1 or 2.

[0097] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that in step 1, 3 kg of defatted Scutellaria baicalensis stem and leaf powder is weighed each time and extracted twice at a solid-liquid ratio of 1 g:30 mL, a temperature of 100° C., and an extraction time of 5 h. Other aspects are the same as specific embodiments 1 to 3.

[0098] Specific embodiment 5: The polysaccharide from the stems and leaves of Scutellaria baicalensis as described in any one of specific embodiments 1 to 4 promotes the proliferation of RAW264.7 cells in a concentration-dependent manner.

[0099] Specific embodiment six: The polysaccharide from the stems and leaves of Scutellaria baicalensis described in this embodiment and any one of specific embodiments one to four is used to improve the phagocytic ability of RAW264.7 cells.

[0100] The following experiments were used to verify the effects of the present invention:

[0101] Experiment 1:

[0102] 2.1 Test materials

[0103] 2.1.1 Test samples

[0104] Scutellaria baicalensis stems and leaves are produced in Boli County, Heilongjiang Province.

[0105] 2.1.2 Test cells

[0106] Macrophages (RAW264.7) were purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai.

[0107] 2.1.3 Test reagents

[0108]

[0109]

[0110] 2.1.4 Test instruments and equipment

[0111]

[0112] 2.2 Test methods

[0113] 2.2.1 Optimization of hot water extraction conditions

[0114] 2.2.1.1 Single-factor experiment of SSP extraction

[0115] This experiment selected three factors: solid-liquid ratio, extraction temperature, and extraction time for a single-factor experiment. The single-factor extraction conditions are listed in the table below. Each time, 10 g of powder was weighed, and the parameters were fixed at an ethanol precipitation concentration of 80%. The number of extractions was two. The effects of different extraction factors on the extraction yield of Scutellaria stem and leaf polysaccharides (SSP) were investigated using solid-liquid ratios of (1:10, 1:15, 1:20, 1:25, and 1:30 g / mL), extraction temperature of (100°C), and extraction time of (2 h); extraction temperatures of (60, 70, 80, 90, and 100°C), solid-liquid ratio of (1:20), and extraction time of (2 h); and extraction time of (1, 2, 3, 4, and 5 h), solid-liquid ratio of (1:20), and extraction temperature of (100°C). The factor levels are shown in Table 2-1.

[0116] Table 2-1 Level factor table of single factor experiment

[0117]

[0118] 2.2.1.2 Determination of SSP sugar content and calculation of extraction rate

[0119] To create a standard curve: Accurately weigh 10 mg of glucose and dilute to the mark in a 10 mL volumetric flask. Prepare a stock solution with a concentration of 1 mg / mL. Pipette 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the stock solution, respectively, and add distilled water to make up to 10 mL. Pipette 1 mL of the prepared glucose solution into a test tube. For the blank control group, add 1 mL of distilled water. Add 1 mL of 6% phenol solution and 5 mL of concentrated sulfuric acid to each test tube. Mix thoroughly, heat in a boiling water bath for 20 minutes, remove from the tube, and cool to room temperature. Measure the absorbance of glucose solutions of varying concentrations at 490 nm. Draw a glucose standard curve with glucose concentration as the horizontal axis and absorbance as the vertical axis.

[0120] Determination of polysaccharide sample concentration: Accurately weigh 10 mg of crude polysaccharide sample and prepare a 0.1 mg / mL solution. Pipette 1 mL of polysaccharide solution and measure the absorbance according to the above method. Calculate the sugar content in the sample according to the glucose standard curve, and calculate the SSP extraction rate according to the following formula, where Y is the SSP extraction rate; C is the sugar concentration in the sample; V is the volume of the SSP solution; F is the dilution factor; W is the weight of the drug powder; and 1000 is the conversion factor.

[0121]

[0122] 2.2.2.3 Box-Behnken response surface method optimization experiment

[0123] Based on the results of the single-factor experiments, a response surface experiment was designed using Box-Behnken software. The three factors were solid-liquid ratio (A), extraction temperature (B), and extraction time (C). The SSP extraction rate was used as the response value for the analysis. The experimental levels and factors are shown in the table below.

[0124] Table 2-2Box-Behnken design factorlevel table

[0125]

[0126] 2.2.2 Extraction of polysaccharides from Scutellaria baicalensis stems and leaves

[0127] The stems and leaves of Scutellaria baicalensis were sun-dried and ground, then passed through a 60-mesh sieve. The fine powder was then reflux-defatted twice with 10 volumes of anhydrous ethanol, each time for 2 hours. The defatted powder was then air-dried. 3 kg of defatted powder was prepared and subjected to hot water extraction at a material-liquid ratio of 1:20, an extraction temperature of 100°C, and an extraction time of 4.5 hours. The extractions were combined and concentrated to an appropriate volume. The ethanol concentration was adjusted to 80%. After precipitation overnight, the mixture was centrifuged at 4000 rpm for 5 minutes. The precipitate was collected and air-dried to obtain crude polysaccharide.

[0128] 2.2.3 Purification of polysaccharides from Scutellaria baicalensis stems and leaves

[0129] 2.2.3.1 Deproteinization by Sevage Method

[0130] A Sevage solution (composed of chloroform and n-butanol in a 4:1 volume ratio) was prepared. The polysaccharide solution and Sevage solution were mixed in a 4:1 volume ratio. After vigorous stirring for 30 minutes, the mixture was centrifuged at 4000 rpm for 10 minutes to remove free protein. This process was repeated several times until no significant protein was produced. The deproteinized polysaccharide solution was vacuum evaporated to an appropriate volume and then decolorized with 4.5% H2O2 for 100 minutes at 60°C. The solution was then freeze-dried. The protein content was determined by the Bradford method, and the deproteinization rate was calculated.

[0131] 2.2.3.2DEAE-52 ion exchange column chromatography

[0132] (1) DEAE-52 cellulose pretreatment

[0133] Soak 100g of DEAE-52 cellulose in 10 volumes of ddH2O for 24 hours. Once fully swollen, remove the water by vacuum filtration. Soak in 0.05mol / L NaOH for 1 hour, wash with water until neutral, then soak in 0.1mol / L HCl for 30 minutes, wash with water until neutral, and finally treat with 0.1mol / L NaOH for 30 minutes, wash with water until neutral. Mix the filler with water in a 2:1 mass ratio and stir thoroughly to make a homogenous slurry for column loading.

[0134] (2) Column Packing

[0135] Fix the chromatography column vertically on an iron stand and slowly pour the filler slurry into the column bed while stirring to prevent bubbles. After filling, use ultrapure water as the mobile phase and press the column. Let it stand overnight to allow the filler surface to stabilize.

[0136] (3) Sample loading

[0137] Take 2g of sample polysaccharide, dissolve it in an appropriate amount of ultrapure water to prepare a 100mg / mL polysaccharide solution, pass it through a 0.45μm filter membrane, slowly pour it into the column, connect the peristaltic pump, and prepare for elution.

[0138] (4) Elution

[0139] Ultrapure water, 0.1 mol / L, 0.3 mol / L, 0.5 mol / L, and 1 mol / L NaCl solutions were sequentially used for gradient elution. The flow rate was controlled at 1 mL / min, and 5 mL was collected in one tube using the automatic collector.

[0140] (5) Sugar content detection

[0141] The sugar content of the eluate in each tube was detected by the phenol-sulfuric acid method. 200 μL of eluate was drawn from each collection tube, and 200 μL of 6% phenol solution and 1 mL of concentrated sulfuric acid were added respectively. After oscillation and mixing, the tubes were allowed to stand for 20 minutes. The absorbance of each tube at 490 nm was measured using a microplate reader, and the elution curve was drawn based on the absorbance. The same fractions were combined according to the elution results, and the cut-off molecular weight was 3.5×10 3 The product was dialyzed in a Da dialysis bag for 72 h and freeze-dried to obtain fraction SSP-3.

[0142] 2.2.3.3 Sephadex G-200 column chromatography

[0143] (9) Sephadex G-200 gel pretreatment

[0144] Soak an appropriate amount of gel in 10 times the volume of ultrapure water for 24 h to allow the gel to fully swell, wash it, and stir it evenly to prepare a gel homogenate.

[0145] (10) Column Packing

[0146] Wet column packing, the operation is the same as "2.2.3.2".

[0147] (11) Sample loading

[0148] Take 200 mg of the component SSP-3 obtained in "2.2.3.2", prepare it into a 5% solution, filter it through a 0.45 μm filter membrane, and slowly pour it into the column to prepare for elution.

[0149] (12) Elution

[0150] Ultrapure water was used as the mobile phase, and the flow rate was controlled at 15 mL / h. An automatic collector was set to collect one tube every 5 minutes. The sugar content was detected in the same way as in "2.2.3.2". The elution curve was drawn, and the corresponding fractions were combined, collected and freeze-dried, and named polysaccharide SSP-3a.

[0151] 2.2.4 Determination of SSP-3a composition

[0152] 2.2.4.1 Determination of sugar content by phenol-sulfuric acid method

[0153] The method for drawing the standard curve is the same as that in "2.2.1.2". Take 10 mg of freeze-dried polysaccharide SSP-3a, dissolve it in 10 mL of ddH2O to prepare a 1 mg / mL solution, measure 1 mL, and treat it according to the method in "2.2.1.2" to calculate the sugar content of SSP-3a.

[0154] 2.2.4.2 Determination of protein content by Bradford method

[0155] Prepare BSA standard solutions of varying concentrations according to the kit instructions. After treatment as directed, add G250 staining solution, measure absorbance at 595 nm, and plot a standard curve. Treat SSP-3a similarly, measure absorbance, and calculate protein content in the samples based on the standard curve.

[0156] 2.2.4.3 Determination of uronic acid content by carbazole sulfate method

[0157] (1) Preparation of sodium tetraborate solution

[0158] Accurately weigh 0.478 g of Na2B4O7·10H2O and dilute to 100 mL with concentrated sulfuric acid.

[0159] (2) Preparation of carbazole solution

[0160] Weigh 0.075 g of carbazole and dilute to 50 mL with anhydrous ethanol to prepare a carbazole reagent with a mass fraction of 0.15%.

[0161] (3) Drawing of standard curve

[0162] Galacturonic acid (Gal UA) was selected as the standard and prepared as a 1 mg / mL stock solution. Pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the stock solution into test tubes and add water to 1 mL. Add 5.0 mL of sodium tetraborate solution in an ice bath, mix thoroughly, and heat in a 100°C water bath for 20 minutes. Cool to room temperature, then add 0.2 mL of 0.15% carbazole solution to each tube, mix thoroughly, and let stand at room temperature for 2 hours. Measure the absorbance of each tube at 530 nm and plot a standard curve.

[0163] (4) Determination of uronic acid content in SSP-3a

[0164] 3 mg of polysaccharide SSP-3a was prepared into 1 mg / mL solution with 3 mL of distilled water. After the above experimental method was followed, the uronic acid content in SSP-3a was calculated according to the standard curve.

[0165] 2.2.5 Purity Identification of SSP-3a

[0166] SSP-3a was identified by gel permeation chromatography using the following chromatographic conditions: a Tskgel G2500PWXL column (250 × 4.6 mm), a RID-20A(+) differential refractive index detector, ultrapure water as the mobile phase, a flow rate of 0.8 mL / min, an injection volume of 20 μL, and a column temperature of 40°C. A 2 mg sample was accurately weighed and dissolved in 2 mL of mobile phase to prepare a 1 mg / mL solution. The purity of SSP-3a was then determined.

[0167] 2.2.6 Determination of uniform molecular weight of SSP-3a

[0168] Standard curve drawing: Inject different molecular weight dextran standards (12600, 63300, 126000, 287000, 556000) in descending order according to their relative molecular weight, and test according to the chromatographic conditions in "2.2.5". MW ) and retention time (t R )Draw the standard curve and calculate the regression equation.

[0169] Sample solution preparation: Weigh 2 mg of polysaccharide SSP-3a sample, dissolve it in 2 mL of ultrapure water, and inject it under the same chromatographic conditions as the standard. R ) was substituted into the regression equation to calculate its relative molecular mass Mw value.

[0170] 2.2.7 Infrared Spectroscopy (IR) Scanning

[0171] Weigh 3.0 mg of polysaccharide SSP-3a sample, grind it evenly with an appropriate amount of dried KBr (the mass ratio of polysaccharide SSP-3a sample to KBr is 1:100-1:200), press it into a tablet, and observe the result on an infrared spectrometer at 4000 cm -1 ~400cm -1 Scan at the wavelength, record the data, and save the spectrum.

[0172] 2.2.8 Nuclear magnetic resonance (NMR) determination

[0173] 20 mg of polysaccharide SSP-3a was dissolved in 0.5 mL of D2O and placed in a nuclear magnetic resonance tube. 1 H-NMR analysis, 1 The H-NMR analysis frequency was 600 MHz.

[0174] 2.2.9 Determination of monosaccharide composition by HPLC

[0175] Preparation of monosaccharide standard solution: Accurately weigh the standard substances mannose (Man), rhamnose (Rha), xylose (Xyl), arabinose (Rha), glucose (Glc), galactose (Gal UA), glucuronic acid (Glc UA), and galacturonic acid (Gal UA), and prepare them into a 3 mmol / L aqueous solution with ultrapure water to prepare a mixed monosaccharide reference aqueous solution. Use the same treatment method to prepare each monosaccharide into a 3 mmol / L aqueous solution.

[0176] Sample hydrolysis: Accurately weigh 10.0 mg of polysaccharide SSP-3a into an ampoule, add 3 mL of 2.0 mol / L trifluoroacetic acid (TFA), seal the bottle mouth, and hydrolyze at 110 °C for 180 min to obtain the sample hydrolyzate. Add methanol and repeatedly concentrate until there is no sour taste. Dissolve the hydrolyzate in 2 mL of ultrapure water and set aside.

[0177] Preparation of PMP (1-phenyl-3-methyl-5-pyrazolone) solution: Take 4.268 g of refined PMP powder and dissolve it in methanol in a 50 mL volumetric flask to make a 0.5 mol / mL PMP solution for later use.

[0178] PMP derivatization: 1.2 mL of each standard monosaccharide, mixed monosaccharide reference aqueous solution and polysaccharide SSP-3a hydrolyzed sample solution were taken in a 5.0 mL centrifuge tube, and 600 μL of 0.5 mol / L PMP methanol solution and 0.3 mol / L NaOH solution were added in sequence. After mixing, the mixture was reacted in a 75°C water bath for 60 min, cooled to room temperature, neutralized with 600 μL of 0.3 mol / L hydrochloric acid solution, extracted with equal volumes of isoamyl acetate and chloroform solution in sequence, and centrifuged (5000 rpm, 5 min). This process was repeated several times to remove residual PMP, and the target layer was aspirated and filtered through a 0.22 μm microporous filter membrane for later use.

[0179] HPLC injection analysis: The chromatographic conditions were determined as follows: Diamonsil C-18 column, 250×4.6 mm; mobile phase (acetonitrile-phosphate buffered saline solution 17:83); column temperature (40°C); detection wavelength (245 nm); flow rate (1.0 mL / min); and injection volume (20 μL). The sample was injected and analyzed under these chromatographic conditions.

[0180] 2.2.10 Macrophage (RAW264.7) Culture

[0181] RAW264.7 cells are a mouse-derived mononuclear macrophage cell line that grows adherently and has a rounded appearance. They are a commonly used inflammatory cell model. Culture conditions include: Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 0.1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. After cell recovery, observe cell morphology and growth daily. Subculture every 1-2 days, and begin experiments after cells reach the logarithmic growth phase.

[0182] 2.2.11 Cytotoxicity test

[0183] The lactate dehydrogenase (LDH) kit was used to detect the effect of polysaccharide SSP-3a on the toxicity of macrophage RAW264.7.

[0184] (1) Transfer RAW264.7 cells in the logarithmic growth phase to a 96-well plate and count them with a counting plate, with 1 to 3 × 10 cells per well. 5 Culture in an incubator for 24 h.

[0185] (2) Different concentrations of SSP-3a (25-1600 μg / mL) were added to the sample wells, and culture medium was added to the control wells. Three parallel groups were set up in each well and cultured for 24 h.

[0186] (3) Remove the culture medium, add LDH releasing solution, incubate in the incubator for 1 hour, take the supernatant and place it in a new 96-well plate for testing.

[0187] (4) Add the prepared LDH detection working solution to a new 96-well plate according to the kit instructions, mix well, and incubate at room temperature in the dark for 30 minutes.

[0188] (5) The OD value of each well was measured at 490 nm, and the relative toxicity (%) of SSP-3a to RAW264.7 cells was calculated according to the following formula.

[0189]

[0190] 2.2.12 Cell morphology observation

[0191] RAW264.7 cells in the logarithmic growth phase were seeded in 6-well plates and different concentrations of SSP-3a (200, 400, and 800 μg / mL) were added. Culture medium was added to the blank control group, and 1 μg / mL lipopolysaccharide (LPS) was selected as the positive control group. After 24 hours of action, the culture medium was discarded, and the cells were rinsed with PBS three times. The morphology and number changes of RAW264.7 cells were observed under a 200× inverted microscope, and random photos were taken. Each group was repeated three times.

[0192] 2.2.13 Cell proliferation assay

[0193] The effect of SSP-3a on the proliferation of RAW264.7 cells was determined using resazurin sodium.

[0194] (1) RAW264.7 cells in the logarithmic growth phase were transferred to a 96-well plate and cultured in an incubator for 24 h.

[0195] (2) SSP-3a was added to the sample wells at concentrations of 200 μg / mL, 400 μg / mL, and 800 μg / mL, respectively; culture medium was added to the blank control wells, and 1 μg / mL of LPS was added to the positive control wells. Three replicate wells were set up for each group, and the cells were cultured in the incubator for 36 h.

[0196] (3) Dissolve 2 mg of resazurin sodium in 2 mL of PBS to prepare a 1 mg / mL reagent. Add 10 μL of resazurin sodium to each well and incubate in an incubator for 30 minutes.

[0197] (4) The absorbance of each well was measured at 570 nm, and the survival rate of RAW264.7 cells was calculated according to the formula.

[0198]

[0199] 2.2.14 Cell phagocytosis assay

[0200] Neutral red assay was used to detect the effect of SSP-3a on the phagocytic ability of RAW264.7 cells.

[0201] (1) RAW264.7 cells in the logarithmic growth phase were transferred to a 96-well plate and cultured in an incubator for 24 h.

[0202] (2) SSP-3a was added to the sample wells at concentrations of 200 μg / mL, 400 μg / mL, and 800 μg / mL, respectively; culture medium was added to the blank control wells, and 1 μg / mL of LPS was added to the positive control wells. Three replicate wells were set up for each group, and the cells were cultured in the incubator for 48 h.

[0203] (3) Discard the culture medium, add 100 μL of neutral red solution to each well, and incubate for 1 h.

[0204] (4) Discard the supernatant and wash 2-3 times with PBS. Add 100 μL of cell lysis buffer to each well and incubate overnight.

[0205] (5) Detect the absorbance of each well at 540 nm and calculate the phagocytic rate of RAW264.7 cells according to the formula.

[0206]

[0207] 2.2.15 Griess test

[0208] The Griess method was used to detect the effect of SSP-3a on the NO secretion ability of RAW264.7 cells.

[0209] (1) RAW264.7 cells in the logarithmic growth phase were transferred to a 96-well plate and cultured in an incubator for 24 h.

[0210] (2) SSP-3a was added to the sample wells at concentrations of 200 μg / mL, 400 μg / mL, and 800 μg / mL, respectively; culture medium was added to the blank control wells, and 1 μg / mL of LPS was added to the positive control wells. Three replicate wells were set up for each group, and the cells were cultured in the incubator for 48 h.

[0211] (3) Take 50 μL of the supernatant from each well and add it to a new 96-well plate. Add 50 μL of Griess Reagent I and Griess Reagent II respectively according to the instructions of the kit and incubate at room temperature for 30 min.

[0212] (4) The absorbance of each well was measured at 540 nm, and the effect of different concentrations of SSP-3a on the NO secretion level of RAW264.7 cells was calculated based on the NO standard curve.

[0213] 2.2.16 ELISA test

[0214] The ELISA double antibody sandwich method was used to detect the effect of SSP-3a on the secretion of inflammatory factors in RAW264.7 cells.

[0215] (1) Culture: RAW264.7 cells in the logarithmic growth phase were transferred to a 6-well plate and cultured in an incubator for 24 h.

[0216] (2) Sample addition: SSP-3a was added to the sample wells at concentrations of 200 μg / mL, 400 μg / mL, and 800 μg / mL, respectively; culture medium was added to the blank control wells, and 1 μg / mL of LPS was added to the positive control wells. The cells were cultured in an incubator for 36 h. The cell culture supernatants of the different groups were collected and stored for later use.

[0217] (3) Coating: Absorb the antibody onto the solid phase carrier, dilute the specific antibody to a certain concentration with coating solution, add 50 μL to each well of the reaction plate, and incubate at 37°C for 2 h or 4°C overnight.

[0218] (4) Washing: Pour off the coating solution, pipette PBST to fill each well, let it stand for 1 minute, then pour it out, and repeat 3 times.

[0219] (5) Sample addition: Add 150 μL of cell culture supernatant from the sample group and control group, respectively, with three replicates per group. Incubate at 37°C for 2 h or at 4°C overnight, discard the supernatant, and wash three times according to step (5).

[0220] (6) Blocking: Add 200 μL of 5% BSA blocking solution to each well, incubate at 37°C for 2 h or at 4°C overnight, and discard the supernatant.

[0221] (7) Add enzyme: Add 100 μL of enzyme-labeled antibody diluted with 5% BSA to each well, incubate at 37°C for 2 h or at 4°C overnight, discard the supernatant, and wash three times according to step (5).

[0222] (8) Color development: Add 70 μL of HRP color development solution, let it stand for 30 min, and observe the color change.

[0223] (9) Termination: Add 30 μL of stop solution and let it stand for 15 min.

[0224] (10) Detection: Detect the absorbance of each well at 450 nm.

[0225] 2.2.17 qRT-PCR detection of mRNA expression

[0226] 2.2.17.1 Extraction of total RNA from RAW264.7 cells

[0227] (1) Culture: RAW264.7 cells in the logarithmic growth phase were transferred to a 6-well plate and cultured in an incubator for 24 h.

[0228] (2) Sample addition: SSP-3a was added to the sample wells at concentrations of 200 μg / mL, 400 μg / mL, and 800 μg / mL, respectively; culture medium was added to the blank control wells, and 1 μg / mL of LPS was added to the positive control wells. The cells were cultured in an incubator for 24 h.

[0229] (3) Lysis: Remove the supernatant, rinse the treated cells three times with PBS, add 500 μL TRIZOL, pipette repeatedly, and transfer to a centrifuge tube.

[0230] (4) Precipitation of pollutants: Add 200 μL RNase Free dH2O to the lysate, shake thoroughly, incubate at room temperature for 15 min, centrifuge at 12,000 rpm at room temperature for 15 min, and transfer the supernatant to a new tube.

[0231] (5) Precipitate RNA: Add 500 μL of isopropanol to the supernatant, mix well, incubate at room temperature for 10 min, centrifuge at 12,000 rpm for 10 min at room temperature, and discard the supernatant.

[0232] (6) Washing RNA: Add 500 μL of 75% ethanol and centrifuge at 8000 rpm for 3 min at room temperature.

[0233] (7) Resuspend RNA: Add 30 μL RNase-Free ddH2O to dissolve RNA and store at -20°C until ready for use.

[0234] 2.2.17.2 Fluorescence quantitative PCR

[0235] (1) Preparation of reverse transcription reaction system (total volume 10 μL)

[0236] Table 2-3 Preparation of reverse transcription reaction system

[0237]

[0238] (2) Reverse transcription reaction conditions: 37°C for 5 min, 85°C for 5 sec, and 4°C for 10 min.

[0239] (3) Preparation of qRT-PCR reaction system (total volume 20 μL)

[0240] Table 2-4 Preparation of qRT-PCR reaction system

[0241]

[0242] (4) Amplification reaction conditions were set as specified in the instructions, and PCR reaction was performed.

[0243] (5) The primer sequences for qRT-PCR experiments are shown in Tables 2-5.

[0244] Table 2-5 Primer sequences

[0245]

[0246] 2.2.18 Statistical analysis

[0247] All results are expressed as mean ± SD. The comparison between the two groups was performed with the unpaired two-tailed t test, ns, not statistically significant (P>0.05). P<0.05 was considered statistically significant and is represented as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. GraphPad software was used for statistical analysis.

[0248] 3.1 Optimization of extraction conditions

[0249] 3.1.1 Single-factor experiment

[0250] The results of the single-factor experiment are as follows Figure 1 As shown in the figure, the extraction rate increased with increasing material-liquid ratio. The maximum increase in polysaccharide extraction rate occurred between 1:15 and 1:20. The maximum extraction rate reached at a material-liquid ratio of 1:25, after which the extraction rate declined. The best extraction time for polysaccharides was between 3 and 5 hours. The increase in extraction rate plateaued after 4 hours, indicating that polysaccharide extraction was essentially complete. The extraction rate increased with increasing temperature, but the upward trend slowed at temperatures between 80 and 100°C. This suggests that while increasing temperature can increase polysaccharide extraction, high temperatures may disrupt the sugar structure, thereby reducing the upward trend in extraction rate. Based on the results of the single-factor experiments, the material-liquid ratios (1:20, 1:25, and 1:30 g / mL), extraction temperatures (80, 90, and 100°C), and extraction times (3, 4, and 5 hours) were determined for the response surface experimental design.

[0251] 3.1.2 Response surface optimization of the optimal process for SSP extraction

[0252] The three factors and three levels in the experiment are shown in Table 3-1. A total of 17 groups of experiments were designed using Design Expert 10.0 software, as shown in Table 3-2.

[0253] Table 3-1 Response surface test factor level table

[0254]

[0255] Table 3-2 Response surface analysis scheme and test results

[0256]

[0257] 3.1.3 Establishment of regression model and variance analysis

[0258] The regression equation obtained by Box-Behnken analysis is: Y = 8.14 + 0.35A + 0.094B - 0.16C + 0.57AB - 0.059AC - 0.59BC - 0.23A 2 -0.89B 2 -0.42C 2 The data in Table 3-3 show that the model group P value is less than 0.05, indicating that the model is significant. The lack-of-fit term P = 0.4452 is greater than 0.05, indicating that the regression equation fit of the data model obtained in this experiment is high and the experimental results are relatively feasible. Furthermore, the data in the table also show that the degree of influence of each factor on the SSP extraction rate is A (extraction temperature) > C (material-liquid ratio) > B (extraction time), which corresponds to the results of the previous single-factor experiment.

[0259] Table 3-3Box-Behnken test design and regression analysis results

[0260]

[0261] *Significant difference (P<0.05), **Extremely significant difference (P<0.01)

[0262] 3.1.4 Response surface analysis and optimization

[0263] Effects of interaction of different process conditions on the extraction rate of polysaccharides from Scutellaria baicalensis stems and leaves Figure 2 、 Figure 3 shown. Figure 2 As shown in Figures A and 3A, in the interaction between temperature and liquid-to-liquid ratio, the fluctuation amplitude of the surface under temperature conditions is greater than that of the liquid-to-liquid ratio, making it a sensitive factor in the interaction between the two. Furthermore, as the temperature increases, the extraction rate of stem and leaf polysaccharides also increases. Figure 2 B. Figure 3 As shown in Figure B, the contour distribution diagram of the interaction between time and temperature is elliptical, indicating that the two have a significant impact on the polysaccharide extraction rate; and in the interaction between the two, the fluctuation amplitude of the temperature direction surface is larger. When the extraction time is fixed, the polysaccharide extraction rate tends to increase with the increase of extraction temperature. Figure 2 C. Figure 3 The contour distribution of time and material-liquid ratio in Figure C is approximately circular, indicating that the interaction between the two factors on polysaccharide extraction efficiency is not significant. In comparison, the material-liquid ratio has a greater impact on polysaccharide extraction efficiency in the interaction between the two factors. In summary, the influence of each factor on extraction efficiency is in the order of A > C > B, that is, extraction temperature > material-liquid ratio > extraction time.

[0264] 3.1.5 Box-Behnken regression model validation

[0265] After analyzing the experimental data using Design Expert 10 software, the optimal extraction conditions for polysaccharides from the stems and leaves of Scutellaria baicalensis were determined to be a solid-liquid ratio of 1:21.29, an extraction time of 4.44 hours, and an extraction temperature of 98.74°C. The predicted total extraction yield was 8.41637%. Based on the feasibility of actual operation, the experimental conditions were changed to a solid-liquid ratio of 1:20, an extraction time of 4.5 hours, and an extraction temperature of 100°C. The experiment was repeated three times, and the calculated SSP extraction yield was 8.38%, which is basically consistent with the model's prediction. Therefore, subsequent extraction experiments will be carried out under these optimized conditions.

[0266] 3.2 Purification of crude polysaccharides from Scutellaria baicalensis stems and leaves

[0267] 3.2.1 Sevage deproteinization

[0268] The crude polysaccharide from the stems and leaves of Scutellaria baicalensis was extracted by water extraction and alcohol precipitation with a yield of 7.35%. After deproteinization by the Sevage method, the polysaccharide was tested for protein and sugar content, and the deproteinization rate was 72.5% and the polysaccharide loss rate was 12.83%.

[0269] 3.2.2 DEAE-52 cellulose column chromatography

[0270] The deproteinized stem and leaf polysaccharides were separated and purified using DEAE-52 cellulose. After gradient elution, four different polysaccharide fractions were obtained. The corresponding tube numbers were combined and named SSP-1, SSP-2, SSP-3, and SSP-4. The elution curve is shown in Figure 2. Figure 4 The activity of the four polysaccharide components was determined, and RAW264.7 cells were used to detect the regulatory effects of each component on their proliferation and phagocytic ability. Figure 5 As shown in the results, compared with the blank control group, the four fractions obtained by column chromatography of crude polysaccharide from the stems and leaves of Scutellaria baicalensis significantly improved the proliferation and phagocytic ability of RAW264.7 cells, among which SSP-3 at 800 μg / mL had the best effect in promoting cell proliferation and improving phagocytic ability, and the immune activity of SSP-3 was also significantly improved compared with the polysaccharide before purification, so the SSP-3 fraction was subsequently selected for further purification.

[0271] The purity of SSP-3a was tested by gel permeation chromatography. Figure 6 As shown, there are two polysaccharides with different molecular weights in SSP-3. The relative molecular weights of the two polysaccharides are calculated based on the dextran standard curve. Sephadex G-200 will be used as the column chromatography filler for the secondary purification of SSP-3.

[0272] 3.2.3 Sephadex G-200 column chromatography

[0273] Sephadex column chromatography can achieve purification effects based on the difference in molecular size and is widely used in the purification of biomacromolecules such as polysaccharides and proteins. The elution curve is consistent with the gel permeation chromatography results, and two molecular weight polysaccharides were successfully separated. Figure 7 Tubes 20 to 43 and 61 to 68 were combined and named SSP-3a and SSP-3a, respectively. The two polysaccharides were collected and freeze-dried. The mass and sugar content of the two polysaccharides were compared. Since the mass and sugar content of the purified SSP-3a were higher, SSP-3a was selected for structural characterization and investigation of its immunological activity mechanism in subsequent experiments.

[0274] 3.3 Physicochemical properties and composition determination of SSP-3a

[0275] SSP-3a appearance Figure 8 B, white in appearance, fluffy in appearance, light in texture, easily soluble in water, insoluble in organic reagents such as methanol and acetone. The relative contents of total sugar, uronic acid and protein of SSP-3a were determined by phenol-sulfuric acid method, carbazole sulfate method and Bradford method respectively. The corresponding standard curves of the three methods are shown in Figure 2. Figure 9 The sugar content of SSP-3a was 92.15%, the uronic acid content was 37.9%, and the protein content was undetectable, indicating that SSP-3a is an acidic polysaccharide.

[0276] 3.4 SSP-3a structure identification

[0277] 3.4.1 SSP-3a UV Spectrum Scan

[0278] The UV spectrum scanning results of SSP-3a are as follows Figure 10 As shown in the figure, according to the results, SSP-3a has no absorption peaks at 260nm and 280nm, indicating that SSP-3a does not contain nucleic acids and proteins, which is consistent with the protein content result detected by the Bradford method, indicating that the purification result of SSP-3a is good and does not contain other impurities.

[0279] 3.4.2 Purity Identification of SSP-3a

[0280] The purity of SSP-3a was determined by gel permeation chromatography. Figure 11 As shown in the chromatogram, the peak shape is single and symmetrical, indicating that the molecular weight of SSP-3a is uniformly distributed, has been completely purified, and is a polysaccharide with uniform molecular weight.

[0281] 3.4.3 Determination of relative molecular weight of SSP-3a

[0282] By analyzing the GPC data of dextran standards with different molecular weights, the regression equation was calculated as log M W=-0.2566383t R +6.849402, R 2 =R 2 =0.9901. The retention time t R Substitute to obtain the weight average relative molecular weight M of ASPF W It is 183223.

[0283] 3.4.4SSP-3a Infrared Spectrum Scan

[0284] In the infrared spectrum scanning diagram of SSP-3a, we can see the characteristic absorption peaks of polysaccharides in SSP-3a. Figure 12 As shown, 3422.88cm -1 The strong broad peak at 2934.37 cm is the OH stretching vibration peak of OH. -1 The absorption peak at 1744.35 cm is the asymmetric stretching vibration peak of CH2. -1 The absorption peak at 1614.56 cm is caused by the asymmetric stretching vibration of C=O in the carboxyl group. -1 The absorption peak at 1416.40 cm is the stretching vibration peak of uronic acid -COO-. These two vibration peaks indicate that SSP-3a contains acidic groups. -1 The absorption peaks at 1101.77 cm are caused by OH bending vibration or CH2 bending vibration. -1 、1019.17cm -1 The stretching peak at 763.37 cm is the stretching vibration of CO in COH or COC (pyranoside), proving that the sugar group in SSP-3a is a pyranose ring. -1 The absorption peak at indicated that SSP-3a has an α-glycoside configuration.

[0285] 3.4.5 SSP-3a NMR 1 H spectrum

[0286] In general, the chemical shift of the proton at the C1 position of α-type pyranose is usually greater than 5.0 ppm, while the chemical shift of the proton of β-type sugar is less than 5.0 ppm, so it can be 1 This was distinguished by H-NMR spectral data. Figure 13 As shown in H4.5-5.0 ppm is usually the chemical shift of the proton on the anomeric carbon of β-pyranose, so it can be inferred that the terminal carbon of SSP-3a is β-configuration. The hydrogen proton chemical shifts at 4.10 ppm and 4.18 ppm indicate β-glucosylpyranose, and the hydrogen signals at 4.34 ppm and 4.78 ppm indicate the terminal hydrogen signals of glucose and mannose, respectively, which are consistent with the monosaccharide determination results of HPLC. The signals at 3.38-4.27 ppm are the proton signals on the oxygen-linked carbon.

[0287] 3.4.6 Analysis of Monosaccharide Composition of SSP-3a

[0288] The retention time of monosaccharide standards and SSP-3a hydrolyzate was compared. Figure 14 、 Figure 15 Table 3-4. It was finally determined that SSP-3a is composed of four monosaccharides: mannose, glucuronic acid, glucose, and xylose, with a molar ratio of 1.476:5.472:2.982:2.805.

[0289] Table 3-4 Comparison of monosaccharide retention times

[0290]

[0291] Effects of 3.5SSP-3a on the activity of RAW264.7 cells

[0292] 3.5.1 Detection of SSP-3a cytotoxicity to RAW264.7 cells

[0293] First, a cytotoxicity test was conducted on SSP-3a against RAW264.7 cells. Cell necrosis causes structural damage to the cell membrane, which releases stable lactate dehydrogenase (LDH) in the cell. Therefore, by detecting the content of LDH, the toxicity to RAW264.7 cells can be determined. Figure 16 The experimental results showed that LDH secretion gradually increased with increasing SSP-3a concentration, but there was no significant difference compared to the control group, indicating that SSP-3a was non-cytotoxic to RAW264.7 cells within a concentration range of 1600 μg / mL. Excessively high drug concentrations may affect the osmotic balance within cells, so subsequent cell-based experiments were conducted using SSP-3a at concentrations ranging from 200 μg / mL to 800 μg / mL.

[0294] 3.5.2 Observation of RAW264.7 Cell Morphology

[0295] In order to study the effect of SSP-3a on the morphology of RAW264.7 cells, the cells were treated with different concentrations for 24 hours and then observed under an inverted microscope at a magnification of 200 times. Figure 17 As shown, Figure 17 A is the group without drug treatment, in which the cells are round and bright. Figure 17 E is the positive control group. After the cells were treated with 1 μg / mL LPS, typical post-activation characteristics appeared: cell volume increased, cell morphology changed to polygonal and long spindle-shaped, and cell activity increased. Figure 17 Figures BD and B show microscopic observations of RAW264.7 cells treated with different concentrations of SSP-3a. Compared with the positive control group, cell morphology changed, demonstrating activation characteristics. At 800 μg / mL of SSP-3a, a long carboxylate morphology was clearly observed, indicating that SSP-3a can activate RAW264.7 cells.

[0296] 3.5.3 Effect of SSP-3a on RAW264.7 Cell Proliferation

[0297] Resazurin sodium is a water-soluble, easily redox-sensitive blue fluorescent dye that changes from a blue / colorless fluorescent state to a pink / bright fluorescent state under resorufin reducing conditions in many biological processes. Resazurin sodium is quickly reduced when added to living cells and is often used as a redox indicator in cell proliferation analysis. In this experiment, resazurin sodium was used as an indicator to detect the effect of SSP-3a on the proliferation activity of RAW264.7 cells. The experimental results are shown in the figure. Figure 18 As shown in the results, when the concentration of SSP-3a was 200 μg / mL-800 μg / mL, it significantly promoted the proliferation activity of RAW264.7 cells in a concentration-dependent manner, and at 800 μg / mL, the proliferation promoting effect was the best, with the proliferation rate reaching 182.71%, indicating that SSP-3a can promote the proliferation of macrophages to regulate immunity.

[0298] 3.5.4 Effect of SSP-3a on the phagocytic ability of RAW264.7 cells

[0299] Macrophages in the body mainly play the role of phagocytizing foreign matter, so the investigation of macrophage phagocytosis is an important means to study its immune activity. Neutral red solution is a commonly used live cell staining solution. When cell proliferation accelerates and the number of cells increases, the amount of neutral red that can be taken in will increase. Therefore, it can be used to detect the phagocytic ability of RAW264.7 cells. The test results are as follows Figure 19 As shown, at a concentration of 200 μg / mL, SSP-3a significantly enhanced the phagocytic capacity of RAW264.7 cells; at 800 μg / mL, it showed the strongest effect, with a phagocytic rate reaching 180.44%. SSP-3a significantly increased the phagocytic rate of RAW264.7 cells in a concentration-dependent manner at concentrations ranging from 200 μg / mL to 800 μg / mL. These experimental results demonstrate that SSP-3a regulates the phagocytic capacity of RAW264.7 cells and possesses immunomodulatory properties.

[0300] 3.5.5 Effect of SSP-3a on NO secretion in RAW264.7 cells

[0301] NO is an important inflammatory mediator that can be produced by activated macrophages. It can inactivate ribonucleases and eliminate pathogens and tumor cells. Therefore, NO is an important indicator for examining the immune activity of macrophages. At the same time, NO is produced by nitric oxide synthase (iNOS) when cells are stimulated. Therefore, the amount of NO secreted can also be used to indirectly infer the expression of iNOS. The Griess method was used to detect the NO content after treatment with different concentrations of SSP-3a. The results are as follows: Figure 20 As shown, the NO concentration in the negative control group was 27.08±0.72μM. After treatment with 1μg / mL LPS, the NO secretion concentration increased to 45.39±1.84μM. At a SSP-3a concentration of 200μg / mL, NO secretion reached 33.71±1.47μg / mL, showing a highly significant difference compared to the negative control group. Furthermore, the effect of promoting NO secretion by RAW264.7 cells increased with increasing concentrations, reaching a maximum of 42.69±3.63μM at 800μg / mL. These results suggest that SSP-3a can significantly promote NO production in RAW264.7 cells in a concentration-dependent manner, thereby enhancing immunomodulatory effects.

[0302] 3.5.6 Effect of SSP-3a on iNOS mRNA Expression in RAW264.7 Cells

[0303] iNOS can regulate the production of NO, and the expression of iNOS can reflect the state of macrophage activation. Therefore, the effect of SSP-3a on the expression of iNOS mRNA in RAW264.7 cells was detected to reflect the regulatory effect of SSP-3a on RAW264.7 cells. Cells were treated with different concentrations of SSP-3a for 24 hours, and the expression of iNOS mRNA was detected by qRT-PCR. The results are shown in Figure 2. Figure 21 As shown in the results, compared with the blank control group, SSP-3a concentrations ranging from 200 μg / mL to 800 μg / mL significantly upregulated iNOS mRNA expression. This trend is consistent with the experimental results of NO secretion, indicating that SSP-3a can significantly increase NO secretion and iNOS expression, thereby exerting an immunomodulatory effect.

[0304] 3.5.7 Effect of SSP-3a on Cytokine Secretion in RAW264.7 Cells

[0305] Activated macrophages participate in and regulate immune defense and immune regulation by secreting immune active factors. External stimuli can activate RAW264.7 cells, which can promote the production of various cytokines, such as inflammatory factors, interferons and chemokines, thereby mediating and regulating immune active responses. ELISA test results are as follows Figure 22 As shown, after treatment with 1 μg / mL of LPS, the relative expression levels of IL-6, IL-1β, and TNF-α in RAW264.7 cells were significantly increased. Compared with the blank control group, the relative expression levels of all three cytokines were significantly upregulated after treatment with different concentrations of SSP-3a, with the highest effect at a concentration of 800 μg / mL. These experimental results indicate that SSP-3a can activate the immune response of RAW264.7 cells and induce the cells to produce immune factors.

[0306] 3.5.8 Effects of SSP-3a on the mRNA Expression of Cytokines in RAW264.7 Cells

[0307] The effect of SSP-3a on cytokine mRNA levels was detected by qRT-PCR. The experimental results are as follows Figure 23 As shown in the results, after 24 hours of treatment, both the LPS and SSP-3a groups significantly upregulated the expression of IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA compared to the blank control group. These experimental results indicate that SSP-3a can promote cytokine secretion in RAW264.7 cells by enhancing the expression of IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA, thereby exerting an immune effect.

[0308] Effects of 3.6SSP-3a on NF-κB and MAPK pathways in RAW264.7 cells

[0309] 3.6.1 Effect of SSP-3a on cytokine secretion in RAW264.7 cells after inhibitor treatment

[0310] Macrophage activation can be initiated by stimulation of pattern recognition receptors, among which TLR4 and TLR2 are the main receptors for polysaccharide-activated macrophage immune responses; when the corresponding receptors on the cell surface are activated, intracellular signal transduction can be initiated, activating signal pathways such as NF-κB. PDTC is a commonly used NF-κB inhibitor. Figure 24As shown in the results, compared with the blank control group, the 800 μg / mL SSP-3a treatment group significantly increased the relative expression of IL-6, IL-1β, and TNF-α in RAW264.7 cells. The TLR4 antibody inhibitor group and the PDTC treatment group significantly reduced the relative expression of IL-6, IL-1β, and TNF-α induced by SSP-3a in RAW264.7 cells compared with the SSP-3a treatment group. The TLR4 treatment group had a highly significant inhibitory effect on SSP-3a-induced IL-6 and IL-1β secretion. Compared with the SSP-3a treatment group, the TLR2 antibody inhibitor treatment group significantly downregulated the SSP-3a-induced IL-6 and TNF-α expression in RAW264.7 cells, but had no inhibitory effect on the SSP-3a-induced increase in IL-1β expression. Although TLR2 antibodies were able to reduce the relative expression of IL-6 and TNF-α (**P<0.01), this was significantly different from the inhibitory effect of TLR4 antibodies (****P<0.0001). Therefore, it is speculated that SSP-3a exerts its immunomodulatory effects largely by activating the TLR4 receptor.

[0311] The experimental results showed that treatment with specific TLR4 antibodies blocked signal transduction and reduced SSP-3a-induced cytokine secretion in RAW264.7 cells, indicating that SSP-3a can activate the TLR4 receptor on the surface of RAW264.7 cells to activate the cell's immune response and thereby produce cytokines; while the TLR2 antibody group had no significant difference in IL-1β expression compared with the experimental group. Although the expression of IL-6 and TNF-α was significantly reduced, there was a big difference compared with the effect of TLR4 antibodies, indicating that SSP-3a may not stimulate cell activation through the TLR2 receptor; and the effect of PDTC inhibitors can indicate that SSP-3a may regulate the secretion of immune factors by participating in signal transduction of the NF-κB pathway.

[0312] 3.6.2 Effect of SSP-3a on NF-κB pathway mRNA expression in RAW264.7 cells

[0313] p65 is a subunit of the NF-κB family, and its presence can activate the NF-κB pathway; IκBα is a key member of the IκB (inhibitory protein) family. In resting cells, NF-κB protein exists in the cytoplasm as a homodimer or heterodimer, binding to the p65 subunit and IκB. Upon external stimulation, IκBα becomes phosphorylated and degraded, inducing the translocation of the p65 subunit to the cell nucleus, where it binds to sites on target gene promoters and induces the transcription of immune-related genes to exert its effects.

[0314] The effect of SSP-3a on the expression of p65 mRNA and IκBα mRNA in the NF-κB signaling pathway was detected by qRT-PCR. Figure 25 As shown, compared with the blank control group, SSP-3a concentrations ranging from 200 μg / mL to 800 μg / mL significantly upregulated p65 mRNA expression in a dose-dependent manner. Furthermore, increasing SSP-3a concentration significantly downregulated IκBα mRNA expression. The regulatory trends of SSP-3a on p65 and IκBα mRNA expression were consistent with those observed in the LPS group. These results suggest that SSP-3a can participate in signal transduction within the NF-κB pathway and activate immune responses.

[0315] 3.6.3 Effect of SSP-3a on MAPK pathway mRNA expression in RAW264.7 cells

[0316] When the TLR4 receptor is activated, it can amplify the stimulation signal by recruiting downstream adaptor proteins, transmit the signal through MyD88-dependent and independent pathways, and activate the NF-κB and MAPK signaling pathways, thereby upregulating the expression of inflammatory genes. The results of previous experiments showed that SSP-3a can activate cells by activating the TLR4 receptor on the surface of RAW264.7 cells, and can participate in signal transduction in the NF-κB pathway. In order to verify whether SSP-3a can regulate the MAPK pathway, the qRT-PCR method was used to detect the effect of SSP-3a on the expression of JNKmRNA, ERK mRNA and p38 mRNA in the MAPK signaling pathway. The results are shown in Figure 2. Figure 26 As shown in the results, compared with the blank control group, both LPS and SSP-3a significantly increased the expression of JNK mRNA, ERK mRNA, and p38 mRNA, and the expression levels were significantly upregulated with increasing SSP-3a concentration. These experimental results suggest that SSP-3a can upregulate the expression of genes involved in the MAPK signaling pathway, thereby exerting an immunomodulatory effect.

[0317] The invention obtains Scutellaria baicalensis stem-leaf polysaccharide by hot water extraction. The extraction conditions are optimized using response surface methodology, and a solid-liquid ratio of 1:20, an extraction time of 4.5 hours, and an extraction temperature of 100°C are determined. Under these conditions, a polysaccharide extraction rate of 8.38% can be achieved. After obtaining crude polysaccharide (SSP) by extraction using the above conditions, the polysaccharide is deproteinized by the Sevage method (the crude polysaccharide is repeatedly treated with the Sevage method, and the deproteinization rate is 72.5% as determined by the Bradford method), depigmented by H2O2, and initially impurities removed. The polysaccharide is then separated and purified using DEAE-52 cellulose column chromatography. Screening for immunoreactivity in RAW264.7 macrophages reveals that the SSP-3 component has the best effect on regulating the immune proliferation activity of the cells. The purity of the SSP-3 is then identified by gel chromatography, and secondary purification is performed using Sephadex G-200 gel column chromatography, ultimately obtaining a highly active, uniform molecular weight polysaccharide SSP-3a. The composition of SSP-3a was detected by phenol-sulfuric acid method, sulfuric acid-carbazole method and Bradford method. The sugar content of SSP-3a was 91.15%, the uronic acid content was 37.9%, and the protein content was undetectable, indicating that SSP-3a is an acidic polysaccharide.

[0318] SSP-3a was purified from a polysaccharide extracted from the stems and leaves of Scutellaria baicalensis. Ultraviolet (UV) spectroscopy revealed that SSP-3a was free of impurities such as proteins and nucleic acids. HPGPC analysis revealed a weight-average molecular weight of 183,223. The infrared spectrum revealed two distinct peaks indicating acidic group vibrations, as well as CO stretching peaks and the presence of an α-glycosidic configuration within the pyranoside. The 1H-NMR spectrum revealed a β-configuration of the anomeric carbon and the presence of terminal hydrogen signals for glucose and mannose, consistent with HPLC analysis of monosaccharides. HPLC analysis confirmed that SSP-3a is composed of four monosaccharides: mannose, glucuronic acid, glucose, and xylose, with a molar ratio of 1.476:5.472:2.982:2.805.

[0319] SSP-3a's immunomodulatory effect on RAW264.7 cells: Lactate dehydrogenase assay results showed that SSP-3a had no toxic effects on RAW264.7 cells within a concentration range of 1600 μg / mL. Resazurin sodium was used to assess the proliferation of RAW264.7 cells. SSP-3a significantly promoted the proliferation of RAW264.7 cells in a concentration-dependent manner, with the proliferation rate reaching 182.71% at 800 μg / mL. Neutral red assay revealed that SSP-3a exhibited the strongest phagocytic activity at 800 μg / mL, with a phagocytic index of 180.44%. In summary, SSP-3a promoted the proliferation of RAW264.7 cells in a concentration-dependent manner and also enhanced the phagocytic capacity of the cells, exerting an immunomodulatory effect.

[0320] When RAW264.7 cells are stimulated by external factors, they become activated and release a variety of cytokines, such as IL-6, IL-1β, TNF-α, and interferon, thereby exerting an immunomodulatory effect. NO is an important inflammatory mediator secreted by immune cells that can eliminate pathogens. Its production is regulated by nitric oxide synthase (iNOS), and therefore indirectly reflects the expression of iNOS.

[0321] RAW264.7 cells were treated with different concentrations of SSP-3a to investigate their effects on NO and cytokine secretion. Griess assay results revealed that SSP-3a promoted NO secretion, reaching a peak of 42.69±3.63μM at 800μg / mL. ELISA assays revealed that the relative expression of cytokines (IL-6, IL-1β, and TNF-α) increased with increasing SSP-3a concentrations between 200μg / mL and 800μg / mL, with the highest cytokine secretion observed at 800μg / mL. qRT-PCR assays also revealed that SSP-3a significantly upregulated the expression of iNOS mRNA, IL-6 mRNA, IL-1β mRNA, and TNF-α mRNA at the gene level, indicating that SSP-3a possesses immunomodulatory activity.

[0322] The mechanism of SSP-3a activating RAW264.7 cells: The results of ELISA experiments showed that compared with the experimental group, the TLR4 antibody and PDTC (NF-κB inhibitor) pretreatment group were both able to significantly reduce the ability of SSP-3a to promote cytokine secretion, indicating that SSP-3a enhances the immunoregulatory effect of RAW264.7 cells by activating the TLR4 receptor. qRT-PCR results showed that SSP-3a significantly increased p65 mRNA expression and downregulated IκBα mRNA expression. This regulatory trend was consistent with the results in the LPS group, indicating that SSP-3a participates in NF-κB signaling. Furthermore, SSP-3a significantly upregulated JNK, ERK, and p38 mRNA expression. SSP-3a activates the TLR4 receptor, stimulating macrophage proliferation and phagocytosis. It also participates in the NF-κB and MAPK signaling pathways, regulating the expression of key genes in these pathways and inducing macrophage secretion of NO, TNF-α, IL-6, and IL-1β. This suggests that SSP-3a can also regulate immunity through the MAPK pathway. In summary, SSP-3a can activate RAW264.7 cells through multiple pathways, exerting an immunomodulatory effect.

Claims

1. The application of Scutellaria baicalensis stem and leaf polysaccharide is characterized by The scutellaria stem and leaf polysaccharide promotes the proliferation of RAW264.7 cells in a concentration-dependent manner; The extraction and purification method of the polysaccharide from the stems and leaves of Scutellaria baicalensis is as follows:

1. Extraction of polysaccharides from stems and leaves of Scutellaria baicalensis: The stems and leaves of Scutellaria baicalensis are dried and crushed, and passed through a 60-mesh sieve. The fine powder is refluxed and defatted twice with 10 times the volume of anhydrous ethanol, each time for 2 hours. The defatted powder is naturally dried, and 3 kg of defatted stem and leaf powder of Scutellaria baicalensis is weighed each time, and extracted twice under the conditions of a solid-liquid ratio of 1g:20mL-1g:30mL, a temperature of 80℃-100℃, and an extraction time of 3h-5h. After the water extracts are combined, they are concentrated, anhydrous ethanol is added to a volume fraction of 80% of ethanol, and after precipitation overnight, centrifuged at 4000rpm for 5min, the precipitate is collected, and naturally air-dried to obtain crude polysaccharides; 2. Sevage method for deproteinization: Chloroform and n-butanol were mixed at a volume ratio of 4:1 to obtain a Sevage solution, the crude polysaccharide obtained in step 1 was mixed with the Sevage solution at a volume ratio of 4:1, and the mixture was vigorously stirred for 30 minutes and then centrifuged at 4000 rpm for 10 minutes to remove free protein, and the process was repeated for several times until no obvious protein was produced, the deproteinized polysaccharide solution was vacuum evaporated, and decolorized with 4.5% H2O2 for 100 minutes at a decolorization temperature of 60°C, and then freeze-dried to obtain a sample polysaccharide; 3. DEAE-52 ion exchange column chromatography: (1) DEAE-52 cellulose pretreatment Take 100g DEAE-52 cellulose, soak it in 10 times the volume of ddH2O for 24h, wait for it to swell fully, remove water by vacuum filtration, soak it in 0.05mol / LNaOH for 1h, wash it with water until neutral, soak it in 0.1mol / LHCl for 30min, wash it with water until neutral, and finally treat it with 0.1mol / LNaOH for 30min, wash it with water until neutral, mix the filler and water in a mass ratio of 2:1, stir it well, make a filler slurry, and prepare it for column loading; (2) Column Packing Fix the chromatography column vertically on the iron frame, slowly pour the filler slurry into the column bed, and stir at the same time to prevent bubbles from forming; after filling, select ultrapure water as the mobile phase to press the column, and let it stand overnight to stabilize the filler surface; (3) Sample loading Take 2g of sample polysaccharide, dissolve it in ultrapure water to prepare a 100mg / mL polysaccharide solution, filter it through a 0.45μm filter membrane, slowly pour it into the column, connect the peristaltic pump, and prepare for elution; (4) Elution Ultrapure water, 0.1mol / L, 0.3mol / L, 0.5mol / L, and 1mol / L NaCl solutions were used in sequence for gradient elution. The flow rate was controlled at 1mL / min, and the automatic collector collected one tube every 5mL. (5) Sugar content detection The sugar content of the eluate in each tube was tested by the phenol-sulfuric acid method. 200 μL of the eluate was taken from each collection tube, and 200 μL of 6% phenol solution and 1 mL of concentrated sulfuric acid were added respectively. After oscillation and mixing, the mixture was allowed to stand for 20 min. The absorbance of each tube at 490 nm was measured by an enzyme-labeled instrument, and the elution curve was drawn according to the absorbance. The same fractions were combined according to the elution results and the molecular weight cut-off was 3.5 × 10 3 The mixture was dialyzed in a Da dialysis bag for 72 h and freeze-dried to obtain component SSP-3; 4. Sephadex G-200 column chromatography: (1) Sephadex G-200 gel pretreatment Soak the Sephadex G-200 gel in 10 times the volume of ultrapure water for 24 hours to allow the gel to fully swell, and then stir evenly after washing to prepare a gel homogenate; (2) Column Packing Fix the chromatography column vertically on the iron frame, slowly pour the filler slurry into the column bed, and stir at the same time to prevent bubbles from forming; after filling, select ultrapure water as the mobile phase to press the column, and let it stand overnight to stabilize the filler surface; (3) Sample loading Take 200 mg of the component SSP-3 obtained in step 3, prepare it into a 5% solution by mass, filter it through a 0.45 μm filter membrane, and slowly pour it into the column to prepare for elution; (4) Elution Ultrapure water was used as the mobile phase, the flow rate was controlled at 15 mL / h, and the automatic collector was set to collect one tube every 5 min; (5) Sugar content detection The sugar content of the eluate in each tube was tested by the phenol-sulfuric acid method. 200 μL of the eluate was taken from each collection tube, and 200 μL of 6% phenol solution and 1 mL of concentrated sulfuric acid were added respectively. After oscillation and mixing, the mixture was allowed to stand for 20 min. The absorbance of each tube at 490 nm was measured by an enzyme-labeled instrument, and the elution curve was drawn according to the absorbance. The same fractions were combined according to the elution results and the molecular weight cut-off was 3.5 × 10 3 The mixture was dialyzed in a Da dialysis bag for 72 h and freeze-dried to obtain Scutellaria baicalensis stem and leaf polysaccharide SSP-3a.

2. The use of the polysaccharide from the stems and leaves of Scutellaria baicalensis according to claim 1, characterized in that In step 1, 3 kg of defatted Scutellaria baicalensis stem and leaf powder was weighed each time, and extracted twice under the conditions of a solid-liquid ratio of 1 g:20 mL, a temperature of 100° C., and an extraction time of 4.5 h.

3. The use of the polysaccharide from the stems and leaves of Scutellaria baicalensis according to claim 1, characterized in that In step 1, 3 kg of defatted Scutellaria baicalensis stem and leaf powder was weighed each time, and extracted twice under the conditions of a solid-liquid ratio of 1 g:25 mL, a temperature of 100° C., and an extraction time of 5 h.

4. The use of the polysaccharide from the stems and leaves of Scutellaria baicalensis according to claim 1, characterized in that In step 1, 3 kg of defatted Scutellaria baicalensis stem and leaf powder was weighed each time, and extracted twice under the conditions of a solid-liquid ratio of 1 g:30 mL, a temperature of 100° C., and an extraction time of 5 h.

5. The application of Scutellaria baicalensis stem and leaf polysaccharide is characterized by The scutellaria stem and leaf polysaccharide is used to improve the phagocytic ability of RAW264.7 cells; The extraction and purification method of the polysaccharide from the stems and leaves of Scutellaria baicalensis is as follows:

1. Extraction of polysaccharides from stems and leaves of Scutellaria baicalensis: The stems and leaves of Scutellaria baicalensis are dried and crushed, and passed through a 60-mesh sieve. The fine powder is refluxed and defatted twice with 10 times the volume of anhydrous ethanol, each time for 2 hours. The defatted powder is naturally dried, and 3 kg of defatted stem and leaf powder of Scutellaria baicalensis is weighed each time, and extracted twice under the conditions of a solid-liquid ratio of 1g:20mL-1g:30mL, a temperature of 80℃-100℃, and an extraction time of 3h-5h. After the water extracts are combined, they are concentrated, anhydrous ethanol is added to a volume fraction of 80% of ethanol, and after precipitation overnight, centrifuged at 4000rpm for 5min, the precipitate is collected, and naturally air-dried to obtain crude polysaccharides; 2. Sevage method for deproteinization: Chloroform and n-butanol were mixed at a volume ratio of 4:1 to obtain a Sevage solution, the crude polysaccharide obtained in step 1 was mixed with the Sevage solution at a volume ratio of 4:1, and the mixture was vigorously stirred for 30 minutes and then centrifuged at 4000 rpm for 10 minutes to remove free protein, and the process was repeated for several times until no obvious protein was produced, the deproteinized polysaccharide solution was vacuum evaporated, and decolorized with 4.5% H2O2 for 100 minutes at a decolorization temperature of 60°C, and then freeze-dried to obtain a sample polysaccharide; 3. DEAE-52 ion exchange column chromatography: (1) DEAE-52 cellulose pretreatment Take 100g DEAE-52 cellulose, soak it in 10 times the volume of ddH2O for 24h, wait for it to swell fully, remove water by vacuum filtration, soak it in 0.05mol / LNaOH for 1h, wash it with water until neutral, soak it in 0.1mol / LHCl for 30min, wash it with water until neutral, and finally treat it with 0.1mol / LNaOH for 30min, wash it with water until neutral, mix the filler and water in a mass ratio of 2:1, stir it well, make a filler slurry, and prepare it for column loading; (2) Column Packing Fix the chromatography column vertically on the iron frame, slowly pour the filler slurry into the column bed, and stir at the same time to prevent bubbles from forming; after filling, select ultrapure water as the mobile phase to press the column, and let it stand overnight to stabilize the filler surface; (3) Sample loading Take 2g of sample polysaccharide, dissolve it in ultrapure water to prepare a 100mg / mL polysaccharide solution, filter it through a 0.45μm filter membrane, slowly pour it into the column, connect the peristaltic pump, and prepare for elution; (4) Elution Ultrapure water, 0.1mol / L, 0.3mol / L, 0.5mol / L, and 1mol / L NaCl solutions were used in sequence for gradient elution. The flow rate was controlled at 1mL / min, and the automatic collector collected one tube every 5mL. (5) Sugar content detection The sugar content of the eluate in each tube was tested by the phenol-sulfuric acid method. 200 μL of the eluate was taken from each collection tube, and 200 μL of 6% phenol solution and 1 mL of concentrated sulfuric acid were added respectively. After oscillation and mixing, the mixture was allowed to stand for 20 min. The absorbance of each tube at 490 nm was measured by an enzyme-labeled instrument, and the elution curve was drawn according to the absorbance. The same fractions were combined according to the elution results and the molecular weight cut-off was 3.5 × 10 3 The mixture was dialyzed in a Da dialysis bag for 72 h and freeze-dried to obtain component SSP-3; 4. Sephadex G-200 column chromatography: (5) Sephadex G-200 gel pretreatment Soak the Sephadex G-200 gel in 10 times the volume of ultrapure water for 24 hours to allow the gel to fully swell, and then stir evenly after washing to prepare a gel homogenate; (6) Column Packing Fix the chromatography column vertically on the iron frame, slowly pour the filler slurry into the column bed, and stir at the same time to prevent bubbles from forming; after filling, select ultrapure water as the mobile phase to press the column, and let it stand overnight to stabilize the filler surface; (7) Sample loading Take 200 mg of the component SSP-3 obtained in step 3, prepare it into a 5% solution by mass, filter it through a 0.45 μm filter membrane, and slowly pour it into the column to prepare for elution; (8) Elution Ultrapure water was used as the mobile phase, the flow rate was controlled at 15 mL / h, and the automatic collector was set to collect one tube every 5 min; (5) Sugar content detection The sugar content of the eluate in each tube was tested by the phenol-sulfuric acid method. 200 μL of the eluate was taken from each collection tube, and 200 μL of 6% phenol solution and 1 mL of concentrated sulfuric acid were added respectively. After oscillation and mixing, the mixture was allowed to stand for 20 min. The absorbance of each tube at 490 nm was measured by an enzyme-labeled instrument, and the elution curve was drawn according to the absorbance. The same fractions were combined according to the elution results and the molecular weight cut-off was 3.5 × 10 3 The mixture was dialyzed in a Da dialysis bag for 72 h and freeze-dried to obtain Scutellaria baicalensis stem and leaf polysaccharide SSP-3a.

6. The use of the polysaccharide from the stems and leaves of Scutellaria baicalensis according to claim 5, characterized in that In step 1, 3 kg of defatted Scutellaria baicalensis stem and leaf powder was weighed each time, and extracted twice under the conditions of a solid-liquid ratio of 1 g:20 mL, a temperature of 100° C., and an extraction time of 4.5 h.

7. The use of the polysaccharide from the stems and leaves of Scutellaria baicalensis according to claim 5, characterized in that In step 1, 3 kg of defatted Scutellaria baicalensis stem and leaf powder was weighed each time, and extracted twice under the conditions of a solid-liquid ratio of 1 g:25 mL, a temperature of 100° C., and an extraction time of 5 h.

8. The use of the polysaccharide from the stems and leaves of Scutellaria baicalensis according to claim 5, characterized in that In step 1, 3 kg of defatted Scutellaria baicalensis stem and leaf powder was weighed each time, and extracted twice under the conditions of a solid-liquid ratio of 1 g:30 mL, a temperature of 100° C., and an extraction time of 5 h.

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