Bai-shaohua uniform polysaccharide, and preparation method and application thereof

Abstract

The application discloses uniform polysaccharide of radix paeoniae alba and a preparation method and application thereof, and the preparation method comprises the following steps: (1) hot extraction of radix paeoniae alba with hot water, concentration of the extraction liquid, and alcohol precipitation to obtain crude polysaccharide; (2) removal of starch and protein in the crude polysaccharide, and dialysis to obtain refined polysaccharide; (3) fractionated elution of the refined polysaccharide on a DEAE-52 anion exchange column with different concentrations of NaCl solution to obtain radix paeoniae alba polysaccharide components; and (4) elution of the polysaccharide components on a Sephadex G-75 chromatographic column with water, collection of the water eluate, and screening of the water eluate through an in-vitro anti-inflammatory activity to obtain uniform polysaccharide of radix paeoniae alba with high anti-inflammatory activity. The application is guided by the in-vitro activity screening test results to guide the separation and purification of the radix paeoniae alba polysaccharide, and the uniform polysaccharide of radix paeoniae alba with high anti-inflammatory activity is obtained through purposeful screening; and the in-vivo and in-vitro pharmacodynamic experiments prove that the prepared uniform polysaccharide has definite anti-inflammatory activity, and has application prospects in preparation of anti-inflammatory drugs and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to plant polysaccharides, in particular to Radix Paeoniae Alba uniform polysaccharide and a preparation method thereof, and further relates to application of the Radix Paeoniae Alba uniform polysaccharide in preparation of anti-inflammatory drugs, and belongs to the field of Radix Paeoniae Alba polysaccharides and anti-inflammatory application thereof. BACKGROUND

[0002] Radix Paeoniae Alba is the dried root of Paeonia lactiflora Pall. of Ranunculaceae, which has the effects of nourishing blood and liver, regulating menstruation and yin, and stopping sweating and pain. Radix Paeoniae Alba produced in Bozhou, Anhui, is known as “Radix Paeoniae Alba of Bozhou” due to its high quality, large quantity and high medicinal value, and is one of the “ten famous medicines of Anhui”. Modern pharmacological studies have shown that the main active components of Radix Paeoniae Alba include monoterpenes, triterpenes, flavonoids and polysaccharides, etc., which have anti-inflammatory, immunoregulatory, liver-protecting, analgesic and anti-tumor effects. Polysaccharides are one of the active components of Radix Paeoniae Alba, and the activity researches on Radix Paeoniae Alba polysaccharides mainly focus on antioxidant, immunoregulatory, hypoglycemic and anti-tumor effects, and there are few literatures on anti-inflammatory activity.

[0003] At present, the researches on crude polysaccharides of Radix Paeoniae Alba mostly follow the traditional natural product separation and purification method, that is, extraction, separation and purification are firstly performed, then structure identification is carried out, and finally biological activity evaluation is performed. This method is time-consuming and low in efficiency, and in addition, due to the last step of activity evaluation, the activity of the product obtained by the last separation may be lost. SUMMARY

[0004] One of the purposes of the present application is to provide Radix Paeoniae Alba uniform polysaccharide with high anti-inflammatory activity;

[0005] The second purpose of the present application is to provide a preparation method of the Radix Paeoniae Alba uniform polysaccharide with high anti-inflammatory activity;

[0006] The third purpose of the present application is to apply the Radix Paeoniae Alba uniform polysaccharide with high anti-inflammatory activity to preparation of anti-inflammatory drugs.

[0007] The above purposes of the present application are achieved by the following technical solutions:

[0008] In one aspect of the present application, Radix Paeoniae Alba uniform polysaccharide with high anti-inflammatory activity is provided, which is composed of arabinose, glucose and galactose, and has a molecular weight of 3×10 3 ~ 6×10 4 Da.

[0009] In a preferred embodiment of the present application, the contents of arabinose, glucose and galactose in the Radix Paeoniae Alba uniform polysaccharide are 1.0471 μg / mg, 328.8442 μg / mg and 1.6345 μg / mg, respectively; and the molecular weight of the Radix Paeoniae Alba uniform polysaccharide is 4.936 Kda.

[0010] Another aspect of the present application is to provide a method for preparing the uniform polysaccharide of Radix Paeoniae Alba with high anti-inflammatory activity, comprising:

[0011] (1) performing hot extraction on Radix Paeoniae Alba with hot water and then performing alcohol precipitation to obtain crude polysaccharide of Radix Paeoniae Alba;

[0012] (2) removing starch and protein in the crude polysaccharide of Radix Paeoniae Alba and then performing dialysis to obtain refined polysaccharide of Radix Paeoniae Alba;

[0013] (3) loading the refined polysaccharide of Radix Paeoniae Alba on a DEAE-52 anion exchange column for preliminary separation, and collecting water eluate to obtain polysaccharide component of Radix Paeoniae Alba with high anti-inflammatory activity;

[0014] (4) loading the polysaccharide component of Radix Paeoniae Alba with high anti-inflammatory activity on a Sephadex G-75 column for water elution, collecting water eluate, and obtaining uniform polysaccharide PLPN-2 of Radix Paeoniae Alba with high anti-inflammatory activity through in vitro anti-inflammatory activity screening.

[0015] In a preferred embodiment of the present application, in step (1), the Radix Paeoniae Alba is crushed and sieved before being subjected to defatting by refluxing with ethanol, wherein the volume-to-mass ratio of ethanol to Radix Paeoniae Alba powder is 1:3-1:7, and the defatting temperature is 75-95℃; the defatted powder is mixed with hot water for hot extraction; the volume-to-mass ratio of hot water to Radix Paeoniae Alba is 3-15; the hot extraction temperature is 60-100℃, the hot extraction time is 1-3h, and the extraction is performed 1-4 times.

[0016] In a preferred embodiment of the present application, in step (1), the volume concentration of ethanol in the alcohol precipitation process is 50%-90%.

[0017] In step (2), the method for removing starch in the crude polysaccharide can be any method for removing starch in the art, including adding α-amylase to the filtrate after the crude polysaccharide is dissolved for starch removal; for reference, the present application provides a specific embodiment for removing starch in the crude polysaccharide, including adding α-amylase to the filtrate after the crude polysaccharide of Radix Paeoniae Alba is dissolved for enzymatic starch removal; wherein the ratio of α-amylase to the crude polysaccharide of Radix Paeoniae Alba is (1:2)-(5:1) in terms of mL / Kg; the enzymatic hydrolysis temperature is 30-60℃, and the enzymatic hydrolysis time is 0.5-10h.

[0018] The method for removing protein in the crude polysaccharide can be any conventional method for removing protein in the art, including adding Sevag reagent for protein removal.

[0019] In a preferred embodiment of the present application, the step (3) is performed by using 0M NaCl, 0.1M NaCl, 0.2M NaCl, 0.3M NaCl, 0.4M NaCl, 0.5M NaCl solution for stepwise elution, the elution rate is 5mL / min, and the eluate is collected according to 10mL / tube, and the water eluate (0M NaCl) is collected to obtain the high anti-inflammatory activity of the bai shao polysaccharide component; further preferably, the 0M NaCl eluate is combined and concentrated under reduced pressure, and then dialyzed with a dialysis bag with a molecular weight cut-off of 3500Da, and then dialyzed with deionized water, and the concentrated solution is vacuum freeze-dried to obtain the high anti-inflammatory activity of the bai shao polysaccharide component.

[0020] In a preferred embodiment of the present application, the step (4) is performed by loading the high anti-inflammatory activity of the bai shao polysaccharide component into a Sephadex G-75 column for water elution, the elution rate is 0.2mL / min, and the eluate is collected according to 2mL / tube, and the eluate of the 30th tube to the 55th tube is combined to obtain the high anti-inflammatory activity of the bai shao uniform polysaccharide; further preferably, the eluate of the 30th tube to the 55th tube is combined and concentrated under reduced pressure, and the concentrated solution is vacuum freeze-dried to obtain the high anti-inflammatory activity of the bai shao uniform polysaccharide PLPN-2.

[0021] The present application analyzes the inhibition of the release of inflammatory factor IL-6 by the prepared bai shao uniform polysaccharide PLPN-2, and the results show that PLPN-2 can significantly reduce the release amount of IL-6, indicating that it has strong anti-inflammatory activity; the in vivo and in vitro pharmacodynamic tests show that the bai shao uniform polysaccharide PLPN-2 provided by the present application has significant anti-inflammatory activity and can be applied to prepare anti-inflammatory drugs.

[0022] Another aspect of the present application is to apply the provided bai shao uniform polysaccharide to prepare anti-inflammatory drugs.

[0023] The bai shao uniform polysaccharide provided by the present application can be added to various excipients and pharmaceutically acceptable excipients or carriers required for the preparation of different dosage forms, and then prepared into any suitable clinical preparation by conventional pharmaceutical preparation methods, and the dosage forms of the drugs include tablets, sprays, lyophilized powders, granules, pills, capsules or oral liquids.

[0024] The excipient or carrier of the present application refers to the conventional excipient or carrier in the field of pharmacy, for example: diluent, disintegrant, lubricant, excipient, binder, glidant, filler, surfactant and the like; in addition, other auxiliary agents such as flavoring agent and sweetening agent can also be added in the composition. The diluent can be one or more components for increasing the weight and volume of the tablet, and the commonly used diluents include lactose, starch, pregelatinized starch, microcrystalline cellulose, sorbitol, mannitol and inorganic calcium salt and the like; among them, the most commonly used are lactose, starch and microcrystalline cellulose. The disintegrant can be one or a mixture of several of cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, alginic acid and microcrystalline cellulose. The lubricant can include one or a mixture of several of stearic acid, sodium stearate, magnesium stearate, calcium stearate, polyethylene glycol, talc and hydrogenated vegetable oil. The binder can be one or several components conducive to granulation; it can be starch paste, hydroxypropyl methyl cellulose and polyvinylpyrrolidone. The glidant can be one or a mixture of several of micronized silica, talc and magnesium trisilicate. The surfactant can be one or several components capable of improving wettability and increasing drug dissolution, and the commonly used is sodium dodecyl sulfate.

[0025] The present application is guided by the results of in vitro activity screening test, and the separation and purification of Radix Paeoniae Alba polysaccharide is guided, and the Radix Paeoniae Alba uniform polysaccharide with high anti-inflammatory activity is obtained by targeted screening; the pharmacodynamic experiment in vivo and in vitro proves that the Radix Paeoniae Alba uniform polysaccharide provided in the present application has exact anti-inflammatory activity, and has application prospect in the preparation of anti-inflammatory drug preparations. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Protective effect of DEAE elution components on LPS-induced macrophage inflammation model; RAW264.7 macrophages were pre-stimulated with 1 μg / mL LPS for 2 hours, and then treated with different concentrations (25, 100 and 200 μg / mL) of elution components (PLPN, PLPA-1, PLPA-2, PLPA-3) for 10 hours; (a) IL-6, (b) IL-1β and (c) TNF-α levels in the culture medium were measured using ELISA kit; the values are represented as the mean ± SD of three independent experiments. ## p<0.01 compared with Control, *p<0.05, **p<0.01 compared with LPS group.

[0027] Figure 2 The content of IL-6 in the culture medium was determined using ELISA kit after two gel elution components PLPN-1 and PLPN-2 were given different concentrations (50, 100 and 200 μg / mL) and continued to be cultured for 10 hours; the values are represented as the mean ± SD of three independent experiments; ## p<0.01 compared with Control group, *p<0.05, **p<0.01 compared with LPS group.

[0028] Figure 3 The figure is the morphology characteristic of the high anti-inflammatory activity uniform polysaccharide of Radix Paeoniae Alba in the embodiment of the present application.

[0029] Figure 4 The figure is the UV spectrum of the high anti-inflammatory activity uniform polysaccharide of Radix Paeoniae Alba in the embodiment of the present application.

[0030] Figure 5 The figure is the IR spectrum of the high anti-inflammatory activity uniform polysaccharide of Radix Paeoniae Alba in the embodiment of the present application.

[0031] Figure 6 The figure is the in vitro anti-inflammatory activity research of the polysaccharide of Radix Paeoniae Alba, wherein A, B, C are respectively the influence of PLPN, PLPA-1, PLPA-2, PLPA-3 on the release of IL-6, IL-1β, TNF-α in the macrophage inflammation model; D is the comparison of the inhibitory effect of PLPN-1, PLPN-2 on the release of IL-6 after the gel chromatography separation of PLPN; E, F, G are respectively the inhibition of the high anti-inflammatory activity uniform polysaccharide of Radix Paeoniae Alba on the release of IL-6, IL-1β, TNF-α of LPS-induced RAW264.7 macrophages.

[0032] Figure 7 The figure is the influence result of PLPN-2 on the body weight, colon and serum inflammatory factors of colitis mice; A: the change of the body weight of mice, B: the colon length of mice, C-E: the content of TNF-α, IL-1β, IL-6 in the serum of mice; #p<0.05, ##p<0.01 compared with the Control group; *p<0.05, **p<0.01 compared with the DSS group.

[0033] Figure 8 The figure is the colon HE section staining chart; wherein A, B, C, D, E are respectively the colon HE section staining chart of the Control group, the DSS group, the positive drug group (MES group), the low-dose PLPN-2 group, the medium-dose PLPN-2 group, the high-dose PLPN-2 group. DETAILED DESCRIPTION

[0034] The present application will be further described in conjunction with specific embodiments. The advantages and features of the present application will become more apparent with the description. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements fall within the protection scope of the present application.

[0035] Example 1 Preparation of high anti-inflammatory activity uniform polysaccharide of Radix Paeoniae Alba, anti-inflammatory screening and determination of physical and chemical properties

[0036] (1) Take the Baishao decoction pieces, crush, pass through a No. 4 sieve. The volume to mass ratio of ethanol to Baishao powder is 1:5; the reflux extraction temperature is 85°C, the time is 2h, and the extraction is performed twice. Filter, and after the medicinal material powder is evaporated at room temperature until there is no alcohol smell, dry at 60°C, to obtain Baishao defatted powder.

[0037] Add 10.65 times the amount of water to Baishao defatted powder 100g, heat and extract twice, 2h each time, and combine the extract; reduce to 100mL at 60°C under reduced pressure, add ethanol to a final concentration of 80%, stand for 12h, filter the precipitate to obtain crude Baishao polysaccharide;

[0038] (2) Dissolve the crude Baishao polysaccharide in distilled water and filter; reduce the solvent to a certain volume under reduced pressure, and add α-amylase according to a volume ratio of α-amylase (μL) to polysaccharide solution (mL) of 1:3, water bath at 60°C, stir for 1h, and water bath at 90°C for 10min to obtain a concentrated solution; centrifuge the concentrated solution (at a centrifugal speed of 10000xg for 15min), and after vigorous shaking for 30min according to a volume ratio of water solution: chloroform: n-butanol of 30:5:1, centrifuge (at a centrifugal speed of 10000xg for 15min), deproteinize 5 times after centrifugation, reduce the residual organic solvent at 60°C under reduced pressure, and use a dialysis bag with a molecular weight cut-off of 3500Da to water dialysis for 48h, then continue to use deionized water to water dialysis for 24h, and vacuum freeze-dry the concentrated solution to obtain refined Baishao polysaccharide 1.76g.

[0039] (3) Take 200mg of refined Baishao polysaccharide, dissolve in a small amount of distilled water, filter, and load the filtrate onto a DEAE-52 anion exchange column that has been equilibrated, and use NaCl solutions of different concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5M) to perform stepwise elution, at an elution speed of 5mL / min, and collect the eluate according to 10mL / tube, detect the absorbance value of each tube by the phenol-sulfuric acid method, and draw a DEAE elution curve. According to the elution curve, collect the eluate containing polysaccharide, reduce under pressure, dialyze in a dialysis bag with a molecular weight cut-off of 3500Da for 48h, then continue to dialyze in deionized water for 24h, and vacuum freeze-dry the concentrated solution to obtain the following four polysaccharide components: PLPN (0M NaCl elution), PLPA-1 (0.1M NaCl elution), PLPA-2 (0.2M NaCl elution), and PLPA-3 (0.3M NaCl elution).

[0040] The results of the comparison of the anti-inflammatory activities of the four polysaccharide components are as follows:

[0041] 1x10 6Cell suspension of 1 μg / mL was seeded into 96-well plates. After overnight culture, RAW264.7 macrophages were induced with 1 μg / mL LPS to establish an inflammation model. After 2 hours, different concentrations of PLPN, PLPA-1, PLPA-2, and PLPA-3 solutions (25 μg / mL, 100 μg / mL, and 200 μg / mL) were administered. After 10 hours of further culture, the levels of IL-6, IL-1β, and TNF-α in the cell supernatant were measured.

[0042] The results showed that PLPN had the strongest inhibitory effect on three inflammatory factors in macrophages, indicating that PLPN may have strong anti-inflammatory activity. Figure 1 ).

[0043] (4) Dissolve 50 mg of the obtained PLPN in a small amount of distilled water and filter. Load the filtrate onto a equilibrated Sephadex G-75 column and elute with deionized water at a rate of 0.2 mL / min. Collect 2 mL of eluent per tube. Detect the absorbance of each tube using the phenol-sulfuric acid method and plot the gel elution curve. Combine the eluents containing polysaccharides according to the elution curve. Combine tubes 12 to 17, concentrate under reduced pressure, and freeze-dry the concentrate under vacuum to obtain PLPN-1; combine tubes 30 to 55, concentrate under reduced pressure, and freeze-dry the concentrate under vacuum to obtain polysaccharide PLPN-2.

[0044] The results of the in vitro anti-inflammatory activity comparison of PLPN-1 and PLPN-2 are as follows:

[0045] With 1×10 6 Cell suspension of 1 μg / mL was seeded into 96-well plates. After overnight culture, RAW264.7 macrophages were induced with 1 μg / mL LPS to establish an inflammation model. Two hours later, different concentrations of PLPN-1 and PLPN-2 solutions (50 μg / mL, 100 μg / mL, and 200 μg / mL) were administered. After 10 hours of further culture, the levels of NO and IL-6 in the cell supernatant were measured. The inhibitory effects of the two PLPN components, PLPN-1 and PLPN-2, separated by gel chromatography on the release of the inflammatory factor IL-6 were compared. The results showed that PLPN-2 significantly reduced the release of IL-6. Figure 2 This indicates that it may have strong anti-inflammatory activity.

[0046] Figure 3 The image shows the morphological characteristics of PLPN-2. Scanning electron microscopy was used to observe the polysaccharide morphology and particle size. Figure 3 In A, PLPN-2 is a white powder; Figure 3 The SEM scan image in B shows that its shape is an irregular sheet with a smooth surface, and some are spherical particles.

[0047] Figure 4 The UV spectrum of PLPN-2 is shown. The solution with a concentration of 0.1 mg / mL was prepared and scanned by UV-Vis spectrophotometer in the wavelength range of 200-400 nm to obtain the UV spectrum. Figure 4 It can be seen that the absorption curve is smooth, indicating that the protein content in PLPN-2 is low.

[0048] Figure 5 The infrared spectrum of PLPN-2 is shown. About 2 mg of PLPN-2 was weighed and added with 100 mg of dry KBr powder, which was ground in an agate mortar and pressed into a KBr tablet. The tablet was placed in a Fourier infrared spectrum scanner and scanned in the range of 4000 cm -1 -400 cm -1 . The absorption peak wave numbers were 3380 cm -1 , 2937 cm -1 , 1645 cm -1 , 1415 cm -1 , 1150 cm -1 , 1080 cm -1 , 1022 cm -1 , 930 cm -1 , and 845 cm -1 , respectively. It can be seen from Figure 5 that the molecular structure contains hydroxyl, methylene, methine, carbonyl, ether oxygen, and pyranose ring.

[0049] The molecular weight of PLPN-2 was determined using a gel chromatography-differential-multiple angle laser light scattering system. The liquid phase system was U3000 (Thermo, USA), the differential detector was Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was DAWN HELEOS II (Wyatt technology, CA, USA). The gel exclusion chromatography columns Ohpak SB-805 HQ (300x8 mm) and Ohpak SB-803 HQ (300x8 mm) were connected in series. The column temperature was 45℃, the injection volume was 100 μL, the mobile phase was 0.02% NaN3 and 0.1 M NaNO3, and the flow rate was 0.6 mL / min. The weight average molecular weight (Mw) was 4.936 KDa.

[0050] Take PLPN-2 about 5 mg, 0.05M trifluoroacetic acid 10 mL in a sealed ampoule, hydrolysis at 100 ℃ for 30 minutes, the reaction mixture is dried with nitrogen, evaporated with methanol to eliminate excess acid. The sample is dissolved in water, filtered through a microporous membrane. The Thermo ICS5000 ion chromatography system (ICS5000, Thermo Fisher Scientific, USA) was used to analyze and detect the monosaccharide components, and 100 mM sodium hydroxide aqueous solution was used as the mobile phase, 0.5 mL / min flowed through Dionex TM CarboPac TM PA20 (150x3.0mm, 10um) chromatographic column was used to analyze the monosaccharide composition of PLPN-2, and the results are shown in Table 1.

[0051] Table 1 Analysis results of monosaccharide composition

[0052] Monosaccharide composition Arabinose Glucose Galactose Content (pg / mg) 1.0471 328.8442 1.6345

[0053] Test Example 1 In vitro pharmacodynamics test of PLPN-2

[0054] 1. Cell culture and grouping

[0055] RAW264.7 macrophage cells were cultured in DMEM high-glucose medium containing 1% double antibody and 10% fetal bovine serum in a 37°C, 5% CO2 constant temperature incubator.

[0056] Cell grouping: Control group, LPS group; LPS + different concentrations of polysaccharide solution group, after 2h of LPS action, different concentrations of polysaccharide solution were added for continuous culture for 10h, and the cell supernatant was collected for detection of inflammatory indicators.

[0057] 2. Determination of inflammatory factors in cell supernatant

[0058] Enzyme-linked immunoassay (ELISA) was used for detection, and the kit was purchased from Beijing Solabio Technology Co., Ltd. The specific operation process was carried out according to the kit instruction manual.

[0059] 3. Data processing

[0060] SPSS 26.0 software was used for one-way ANOVA, and the results were expressed as mean ± SD. Figure 6 A, 6B, 6C are the effects of PLPN, PLPA-1, PLPA-2, PLPA-3 on the release of IL-6, IL-1β, TNF-α in macrophage inflammatory model, and the results show that PLPN has the strongest inhibitory effect on the three inflammatory factors in macrophages, indicating that PLPN may have strong anti-inflammatory activity. Figure 6D The inhibitory effect of PLPN-2 on the release of IL-6 was compared with that of PLPN-1 after separation by gel chromatography. The results showed that PLPN-2 could reduce the release of IL-6, indicating that it might have strong anti-inflammatory activity. Figure 6 E, 6F, 6G are the effects of PLPN-2 on the release of IL-6, IL-1β, and TNF-α in LPS-induced RAW264.7 macrophages, respectively. The results showed that PLPN-2 could inhibit the release of inflammatory factors in LPS-induced RAW264.7 macrophages, indicating that it had anti-inflammatory activity.

[0061] Test Example 2 In vivo pharmacodynamic test of PLPN-2

[0062] 1 Test method

[0063] 1.1 Animal grouping, model establishment, and dosing regimen

[0064] 36 6-8 week-old C57 / BL6 mice were adapted for one week, during which they were allowed to drink water and eat freely. After the adaptation period, the mice were randomly divided into a normal group, a DSS group, a positive drug group (MES group), a low-dose PLPN-2 group, a medium-dose PLPN-2 group, and a high-dose PLPN-2 group. Except for the normal group, the DSS model group and the PLPN-2 dose groups were allowed to drink 3% DSS solution freely to establish an acute ulcerative colitis mouse model. At the same time, the low-, medium-, and high-dose groups were given 40 mg / kg, 80 mg / kg, and 160 mg / kg of PLPN-2 solution by gavage once a day for 7 consecutive days. On the 8th day, the mice were sacrificed after anesthesia. The body weight of the mice was measured, the colon length was determined, the serum and colon tissue of all mice were collected, and stored at -80°C, or part of the colon tissue was fixed in 4% neutral formalin for further study.

[0065] 1.2 Determination of inflammatory markers

[0066] The whole blood was collected in a clean 1.5 mL centrifuge tube, left at room temperature for 2 h, and then placed in a 4°C refrigerator overnight. After the blood clot solidified, it was centrifuged at 3500 x g for 10 min to separate the serum. The levels of interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the mouse serum were determined according to the instructions of the kit.

[0067] 1.3 Histological examination of colon tissue

[0068] To evaluate the severity of colitis, a portion of the distal colon was washed with pre-cooled PBS and immediately fixed in 10% paraformaldehyde solution for 24 h. After dehydration and paraffin embedding, the tissue was cut into 5 mm slices using a tissue slicer. The changes in colon tissue morphology were detected by hematoxylin and eosin staining (H&E). The colon tissue HE staining section is shown inFigure 8 As shown.

[0069] 1.4 Statistical Methods

[0070] One-way ANOVA was performed using SPSS 17.0 software. Results are expressed as mean ± SD; a p-value less than 0.05 was considered statistically significant.

[0071] 2. Experimental Results

[0072] (1) Effects of PLPN-2 on mouse body weight and colon length

[0073] like Figure 7 As shown in A and 7B, after modeling, the body weight of mice in all experimental groups decreased to varying degrees. However, compared with the DSS group, the decrease in body weight was alleviated in mice in different PLPN-2 dosage groups and the positive drug group (MES group). After 7 days of experimentation, the body weight of mice in the model group decreased to 69.87% of their initial body weight, a significant reduction (p<0.01). The body weight of mice in the L-PLPN-2, M-PLPN-2, H-PLPN-2, and positive drug groups (MES group) decreased (p<0.01) to 76.64%, 76.94%, 81.59%, and 77.02% of their initial body weight, respectively. Compared with the DSS group, the shortening of the colon in mice in the L-PLPN-2, M-PLPN-2, H-PLPN-2, and positive drug groups (MES group) was significantly improved (p<0.05). BSDF-1 showed a dose-response relationship in improving colon shortening in colitis mice.

[0074] (2) Effects of PLPN-2 on serum IL-6, IL-1β and TNF-α

[0075] like Figure 7 As shown in C, 7D, and 7E, compared with the Control group, the DSS group showed a significant increase in the levels of IL-6, IL-1β, and TNF-α (p<0.01). Compared with the DSS group, the levels of L-PLPN-2, M-PLPN-2, H-PLPN-2 groups and the positive control group (MES group) all showed varying degrees of decrease. This indicates that both high- and medium-dose PLPN-2 groups can significantly inhibit the release of IL-6, IL-1β, and TNF-α, indicating that PLPN-2 has anti-inflammatory activity, and both high- and medium-dose PLPN-2 groups have significant anti-inflammatory activity.

[0076] (3) Effects of PLPN-2 on mouse colon tissue

[0077] like Figure 8As shown, normal group mice colon morphology, can see the closely arranged intestinal epithelial cells, clear structure and a large number of goblet cells, no inflammatory cells infiltration phenomenon. For the model group, due to the induction of DSS, the colon epithelial mucosa appears inflammation, goblet cells decrease, crypt structure is damaged to a great extent, and a large area of inflammatory cell infiltration appears. L-PLPN-2, M-PLPN-2 and H-PLPN-2 can alleviate the inflammatory cell infiltration symptoms of cells, the number of goblet cells and crypt structure are repaired to a certain extent. H-PLPN-2 group and positive drug group can make the colon mucosa structure tend to normal, inflammatory cell infiltration decreases significantly, restores the epithelial cell structure, the number of goblet cells increases, and the crypt structure basically recovers to normal state.

Claims

1. A uniform polysaccharide from Radix Paeoniae Alba having anti-inflammatory activity, characterized in that, Arabinose, glucose and galactose consist; wherein, arabinose, glucose and galactose content is 1.0471 ug / mg, 328.8442 ug / mg and 1.6345 ug / mg respectively; the weight average molecular weight of the uniform polysaccharide of bai shao is 4.936 Kda; The preparation method of the uniform polysaccharide of bai shao comprises: (1) the bai shao is heated with hot water and then alcohol precipitation is carried out to obtain bai shao crude polysaccharide; (2) the starch and protein in the bai shao crude polysaccharide are removed, and dialysis is carried out to obtain bai shao refined polysaccharide; (3) the bai shao refined polysaccharide is loaded on a DEAE-52 anion exchange column for preliminary separation, and the water eluate is collected to obtain the bai shao polysaccharide component with high anti-inflammatory activity; (4) the bai shao polysaccharide component with high anti-inflammatory activity is loaded on a Sephadex G-75 chromatographic column for elution with water, the elution speed is 0.2 mL / min, the eluate is collected according to 2 mL / tube, and the eluate of the 30th tube to the 55th tube is combined to obtain the uniform polysaccharide of bai shao with high anti-inflammatory activity.

2. The uniform polysaccharides of Radix Paeoniae Alba according to claim 1, characterized in that, In step (1), the bai shao is crushed and sieved, and then ethanol reflux degreasing is carried out, wherein the volume to mass ratio of ethanol to bai shao powder is 1:3-1:7, and the reflux degreasing temperature is 75°C-95°C; the degreased powder is mixed with hot water for heat extraction; the volume to mass ratio of hot water to bai shao is 3-15; the heat extraction temperature is 60-100°C, the heat extraction time is 1-3 h, and the extraction is carried out 1-4 times.

3. The uniform polysaccharides of Radix Paeoniae Alba according to claim 1, characterized in that, In step (1), the volume concentration of ethanol in the alcohol precipitation process is 50%-90%.

4. The uniform polysaccharides of Radix Paeoniae Alba according to claim 1, characterized in that, The method for removing starch in the crude polysaccharide of Radix Paeoniae Alba in step (2) comprises adding α - starch is removed by enzymolysis of amylase; wherein, the ratio of amylase and crude polysaccharide of Radix Paeoniae Alba is (1:2)~(5:1); the enzymolysis temperature is 30~60ºC, and the enzymolysis time is 0.5~10h. α - starch is removed by enzymolysis of amylase; wherein, the ratio of amylase and crude polysaccharide of Radix Paeoniae Alba is (1:2)~(5:1); the enzymolysis temperature is 30~60ºC, and the enzymolysis time is 0.5~10h. The method for removing the protein in the crude polysaccharide comprises adding Sevag reagent to remove the protein.

5. The uniform polysaccharides of Radix Paeoniae Alba according to claim 1, characterized in that, In step (3), the collected water eluate is combined and concentrated under reduced pressure, and then dialyzed with a dialysis bag with a molecular weight cut-off of 3500 Da; then, deionized water dialysis is carried out, the concentrated solution is vacuum freeze-dried to obtain the bai shao polysaccharide component with high anti-inflammatory activity.

6. The uniform polysaccharides of Radix Paeoniae Alba according to claim 1, characterized in that, In step (4), the eluate of the 30th tube to the 55th tube is combined and concentrated under reduced pressure, and then the concentrated solution is vacuum freeze-dried to obtain the uniform polysaccharide of bai shao with high anti-inflammatory activity.

7. The use of the uniform polysaccharide of bai shao in the preparation of anti-inflammatory drugs according to any one of claims 1-6.

8. An anti-inflammatory pharmaceutical composition consisting of a therapeutically effective amount of an active ingredient and a pharmaceutical preparation adjuvant or carrier, wherein, The active ingredient is the uniform polysaccharide of bai shao according to any one of claims 1-6.

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