Preparation method and application of peanut shell polysaccharide

Abstract

The present invention discloses a method for preparing peanut shell polysaccharide and its application. A method for preparing peanut shell polysaccharide comprises the steps of raw material pretreatment, alkali extraction, enzymolysis, enzyme inactivation, concentrated alcohol precipitation, water re-dissolution, deproteinization, stirring centrifugation, dialysis freeze drying, etc., and finally obtains the peanut shell polysaccharide. The present invention adopts ultrasonic assisted alkali method to extract peanut shell polysaccharide, and the yield is more than 3.7%. Compared with the traditional water extraction method, the yield of peanut shell polysaccharide is increased several times; at the same time, ultrasonic assistance reduces the dissolution of impurities, and the purity of the obtained peanut shell polysaccharide is as high as more than 90%. The present invention improves the comprehensive utilization value of peanut shells. The extracted peanut shell polysaccharide can effectively improve the histopathological structure of the colon of DSS-induced colitis mice, inhibit inflammatory response, and provide a new solution for the prevention and treatment of colitis.

Description

Technical Field

[0001] The invention relates to a method for preparing peanut shell polysaccharide and application of peanut shell polysaccharide components extracted from peanut shells in improving chronic colitis. Background Art

[0002] Peanuts are mainly used to produce peanut oil or refined foods, but a large number of by-products are produced every year during the peanut processing and production process, mainly peanut shells (PS). By weight, peanut shells account for about 20% of dry peanut pods. Currently, most peanut shells are treated as waste or fuel, and only a small part is used in a few fields such as feed and mushroom matrix, with an overall low utilization rate.

[0003] Studies have shown that peanut shells are rich in polysaccharides, which have multiple biological functions and potential application values. In recent years, bioactive polysaccharides have attracted much attention due to their unique physiological functions. These high-molecular compounds can resist gastrointestinal digestion and be fermented and utilized by intestinal flora in the large intestine, thereby playing a role in regulating intestinal microecology. However, despite the rich resources of peanut shell polysaccharides, related research is relatively lagging, and there are the following prominent problems: First, in terms of structural characterization, existing studies have failed to fully analyze the fine chemical structure of peanut shell polysaccharides, including key information such as monosaccharide composition, glycosidic bond type, branching degree and molecular weight distribution, which seriously restricts the in-depth understanding of its structure-activity relationship; second, in terms of biological activity research, although polysaccharides generally have immunomodulatory, antioxidant, and anti-tumor effects, there are few specific biological activity studies on peanut shell polysaccharides, especially in improving colon inflammation. The efficacy and mechanism of action have not been systematically studied. This lack of research not only limits the application potential of peanut shell polysaccharides in the field of functional foods and medicine, but also hinders its development as a new biomaterial.

[0004] At the same time, there are also reports on the research of polysaccharides in intestinal diseases, such as inflammatory bowel disease. Since peanut shells are rich in polysaccharides and there are public literature on polysaccharides in the treatment of inflammatory bowel disease, it is possible to explore the extraction of peanut shell polysaccharides and apply the extracted peanut shell polysaccharides to certain intestinal diseases.

[0005] In terms of the prior art, although there have been some patent reports on the comprehensive utilization of peanut shells, there are still obvious defects. For example, the invention patent with application number 201010541330.3 proposes a method for the joint preparation of active polysaccharides, flavonoids and dietary fiber, and uses ultrasonic treatment and enzymatic hydrolysis technology to improve the leaching efficiency of functional components, but its extraction process still has the problems of low polysaccharide yield and insufficient purity. The invention patent with application number 201710054553.9 improved the enzymatic hydrolysis process through site-directed mutagenesis technology, and realized the simultaneous extraction of polysaccharides and flavonoid compounds, but there is still a lack of in-depth exploration in the purification and structural characterization of polysaccharides. At the same time, the efficiency of extracting peanut shell polysaccharides by traditional water extraction and alcohol precipitation is extremely low, with a yield of only 0.73%, and the purity of crude polysaccharides is only 60%. This inefficient extraction method not only leads to a large loss of polysaccharides, but also introduces impurities such as proteins and pigments, which seriously affects the quality and subsequent application of the product.

[0006] Based on the above background, this patent combines modern chemical and biological technologies to conduct a comprehensive and in-depth study of peanut shell polysaccharides, and studies the improvement effect of peanut shell polysaccharides (PSP) on dextran sulfate sodium (DSS)-induced colitis mice, hoping to provide new scientific basis and technical support for the development and utilization of peanut shell polysaccharides. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention proposes a method for preparing peanut shell polysaccharide, and discloses the application of the peanut shell polysaccharide component extracted by the method in improving chronic colitis.

[0008] The invention adopts ultrasound-assisted alkali solution extraction, which significantly improves the yield and purity of polysaccharides.

[0009] The invention adopts physical and chemical component determination, high-performance gel permeation chromatography, high-performance anion exchange chromatography and Fourier transform infrared spectroscopy to preliminarily characterize the structure of the extracted peanut shell polysaccharide.

[0010] The present invention also proposes the use of polysaccharide components extracted from peanut shells in improving chronic colitis, including the use of the prepared peanut shell polysaccharide in preventing and treating DSS-induced colitis to improve the histopathological structure of the colon and inhibit inflammatory response; and the use of the prepared peanut shell polysaccharide in drugs for preventing and treating chronic colitis.

[0011] The technical solution adopted by the present invention is:

[0012] A method for preparing peanut shell polysaccharide comprises the following steps:

[0013] S1, raw material pretreatment: select non-moldy peanut shells, wash and dry them, and then crush and sieve them to obtain peanut shell powder;

[0014] S2, alkali extraction: take the peanut shell powder obtained in step S1, put it into sodium hydroxide solution, then stir it in a constant temperature water bath at 90°C for 10-20 minutes, and then perform ultrasonic treatment for 10-20 minutes. After alternating water bath and ultrasonic treatment, filter and centrifuge to obtain the supernatant;

[0015] S3, enzymatic hydrolysis, enzyme inactivation and alcohol precipitation: the pH value of the supernatant obtained in step S2 is adjusted to neutral, and then α-amylase is added, and the supernatant is enzymatically hydrolyzed in a 100° C. water bath and then cooled to room temperature; papain is then added, and the supernatant is stirred in a 60° C. water bath for enzymatic hydrolysis, and the enzyme is inactivated in a 100° C. water bath; the supernatant is quickly cooled in an ice bath, centrifuged and the precipitate is discarded, and the supernatant is concentrated by rotary evaporation, and 95% ethanol is slowly added to the concentrated supernatant while slowly stirring, and the supernatant is precipitated with alcohol overnight to obtain peanut shell crude polysaccharide;

[0016] S4, adding water to dissolve: adding water to dissolve the crude peanut shell polysaccharide obtained in step S3 to obtain a crude polysaccharide aqueous solution;

[0017] S5, deproteinization: the crude polysaccharide aqueous solution is deproteinized by the Sevage method, a mixed reagent of chloroform and n-butanol is selected as the deproteinization reagent, the volume ratio of chloroform to n-butanol is 4:1, and the volume amount of the mixed reagent of chloroform and n-butanol is 1 / 5 of the volume of the crude polysaccharide aqueous solution;

[0018] S6, stirring, centrifuging, dialysis and freeze-drying: the crude polysaccharide aqueous solution after deproteinization by adding chloroform and n-butanol mixed reagent is stirred for 10-20 minutes, centrifuged for 10-20 minutes, dialyzed with running water for 48 hours, dialyzed with distilled water for 24 hours, and the reagent and water are removed by rotary evaporation. The solution is freeze-dried on an inverted plate to obtain peanut shell polysaccharide.

[0019] In the step S2, 4% sodium hydroxide solution is used for alkali extraction, and the solid-liquid ratio of the alkali extraction is 1:20. The peanut shell powder is dissolved and precipitated, and the mixture is stirred for 15 minutes in a constant temperature water bath at 90°C, and then ultrasonically treated for 15 minutes. The water bath stirring and ultrasonic treatment are alternately performed 3-5 times. After filtering with gauze, the filtrate is centrifuged at room temperature and 4500r / min for 20 minutes to obtain the supernatant.

[0020] In the step S1, after the peanut shells are washed and dried, they are crushed and sieved to obtain peanut shell powder, and the following decolorization and defatting steps are also included: the peanut shell powder is soaked in n-hexane for 24 hours, stirred intermittently in the middle, and centrifuged at 4500 rpm for 20 minutes at room temperature; the precipitate is collected and then soaked in 95% ethanol for 24 hours, stirred intermittently in the middle, and centrifuged at 4500 rpm for 20 minutes at room temperature, the precipitate is recovered, and evaporated in a fume hood to obtain decolorized and defatted peanut shell powder, and then alkali extraction is performed.

[0021] In the step S2, ultrasonic treatment is performed for 15 minutes, the ultrasonic frequency is 40 kHz, the ultrasonic power is 150 W, and water bath stirring and ultrasonic treatment are performed alternately for 4 times at room temperature. The mixture is filtered and centrifuged to obtain the supernatant.

[0022] In the step S2, the residue after filtering and centrifugation is extracted again with distilled water, and the material-liquid ratio is 1:10. After the residue is dissolved, it is stirred in a constant temperature water bath at 90°C with ultrasonic assistance. After the water bath ends, it is centrifuged at 4500 rpm for 10 minutes, and the supernatant is taken and combined with the supernatant in step S2, and then step S3 is entered.

[0023] In step S3, the amount of α-amylase added is 1-2% of the mass of the supernatant in step S2 after adjusting the pH value to neutral, and the supernatant is stirred in a water bath at 100° C. for 2 hours and then cooled to room temperature, and then papain is added according to 0.3-0.5% of the mass of the liquid after cooling to room temperature.

[0024] In step S3, the enzymatic hydrolysis time of papain in a 60° C. water bath with stirring is 1.5 h, and the enzyme inactivation time of papain is 30 min.

[0025] The invention also discloses the use of peanut shell polysaccharide prepared by the preparation method in preventing and treating DSS-induced colitis, so as to improve the histopathological structure of the colon and inhibit inflammatory response.

[0026] The invention also discloses application of peanut shell polysaccharide prepared by the preparation method in medicine for preventing and treating chronic colitis.

[0027] Beneficial effects of the invention:

[0028] 1. The peanut shell polysaccharide preparation method of the present invention adopts ultrasound-assisted alkali extraction to significantly improve the yield and purity of polysaccharides. The yield of peanut shell polysaccharides extracted according to the method of the present invention is more than 3.7%, which is several times higher than that of the traditional water extraction method; at the same time, the use of ultrasound to assist in reducing the dissolution of impurities improves the purity of polysaccharides, and obtains a polysaccharide with high sugar purity (≥90%).

[0029] 2. The present invention has found through research that the polysaccharide component significantly improved the inflammation level of mice with DSS-induced chronic colitis, and the high-dose treatment of chronic colitis had a better therapeutic effect, indicating that the polysaccharide component can effectively improve the histopathological structure of the colon, inhibit the inflammatory response, and provide a new solution for the prevention and treatment of colitis.

[0030] 3. The preparation method of peanut shell polysaccharide of the present invention improves the comprehensive utilization value of peanut shell. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A high performance gel permeation chromatography calibration curve table of peanut shell polysaccharide obtained by the method for preparing peanut shell polysaccharide of the present invention;

[0032] Figure 2 for Figure 1 The sample molecular weight of peanut shell polysaccharide;

[0033] Figure 3 The figure shows the Fourier infrared spectrum of peanut shell polysaccharide obtained by the method for preparing peanut shell polysaccharide of the present invention;

[0034] Figure 4 The figure shows the high-performance anion exchange chromatography of peanut shell polysaccharide obtained by the method for preparing peanut shell polysaccharide of the present invention;

[0035] Figure 5 for Figure 4 The molar ratio of each monosaccharide in the

[0036] Figure 6 The effects of different feeding methods on the body weight of mice in each group;

[0037] Figure 7 The weight change rate of mice in each group on the 28th day compared with the initial weight;

[0038] Figure 8 This is a graph showing the relationship between time and the disease activity index of each group of mice under different feeding methods;

[0039] Fig. 9 This is a relationship diagram of the disease activity index of mice in each group under different feeding methods on the 28th day;

[0040] Fig.10 The morphological diagrams of the colon after dissection of the five groups of mice;

[0041] Fig.11 Cross-sectional images of the colon after dissection of the three groups of mice;

[0042] Fig.12 Shown is a picture of a peanut shell polysaccharide sample prepared by the preparation method of Example 1 of the present invention. DETAILED DESCRIPTION

[0043] In order to make the technical concept and advantages of the present invention more clearly understood, the following is a brief description of the present invention in conjunction with the attached Figure 1-Figure 12 The technical solution of the present invention is further described in detail. It should be understood that the following embodiments are only used to explain and illustrate the preferred implementation of the present invention, and should not be regarded as and do not constitute a limitation on the scope of patent protection claimed by the present invention.

[0044] Example 1

[0045] The invention discloses a method for preparing peanut shell polysaccharide, comprising the following steps:

[0046] S1, raw material pretreatment: select non-moldy peanut shells, wash, dry in an oven, and crush through an 80-mesh sieve to obtain peanut shell powder;

[0047] S2, alkali extraction: take 20g of the peanut shell powder obtained in step S1, put it into 400ml of 4% sodium hydroxide solution for dissolution and extraction, then stir it in a constant temperature water bath at 90℃ for 15min, then perform ultrasonic treatment for 15min, the ultrasonic frequency is 40kHz, the ultrasonic power is 150W, and the water bath and ultrasonic treatment are alternately performed four times in sequence, and the feed liquid is filtered through gauze and centrifuged at 4500 rpm for 20min at room temperature to take the supernatant; the residue after filtration and centrifugation is dissolved and extracted again with 200ml of distilled water, stirred in a constant temperature water bath at 90℃ for 2h, assisted by ultrasound, centrifuged at 4500 rpm for 20min at room temperature to retain the supernatant, and the two supernatants are combined.

[0048] S3, enzymatic hydrolysis, enzyme inactivation and alcohol precipitation: adjust the pH value of the supernatant obtained in step S2 to neutral, then add α-amylase at a mass ratio of 2% to the neutralized feed solution, stir in a water bath at 100°C for 2 hours, and then cool to room temperature; add papain at a mass ratio of 0.5% to the feed solution cooled to room temperature, stir in a water bath at 60°C for 1.5 hours, inactivate the enzyme in a water bath at 100°C for 30 minutes, quickly cool in an ice bath, and then centrifuge at 4°C , the centrifugal condition is 10000rpm, after centrifugation for 10min, the precipitate is discarded, the supernatant is collected, and concentrated by rotary evaporation; 95% ethanol is slowly added to the concentrate while slowly stirring, the volume ratio of the concentrate to ethanol is 1:5.33, and the concentrate is precipitated overnight in a refrigerator at 4℃, centrifuged at 4500rpm for 20min at room temperature, the remaining precipitate is re-dissolved in 300ml pure water, stirred in a constant temperature water bath at 90℃ for 2h, the ethanol is removed by rotary evaporation and concentrated, dialyzed with distilled water, the water is changed every 2h, and dialyzed in a refrigerator at 4℃ for 2d;

[0049] After dialysis, the sugar was freeze-dried to obtain 742 mg of peanut shell crude polysaccharide, of which the neutral sugar content was 70%, the acidic sugar content was 15.92%, and the protein content was 11.73% (all by mass fraction);

[0050] S4, adding water to dissolve: adding water to dissolve the crude peanut shell polysaccharide obtained in step S3 to obtain a crude polysaccharide aqueous solution;

[0051] S5, deproteinization: In order to further reduce the protein content in the peanut shell crude polysaccharide, it is necessary to perform deproteinization treatment on it. The crude polysaccharide aqueous solution is deproteinized by the Sevage method, and the process is repeated three times. A mixed reagent of chloroform and n-butanol is used as the deproteinization reagent. The volume ratio of chloroform to n-butanol is 4:1, and the volume amount of the mixed reagent of chloroform and n-butanol is 1 / 5 of the volume of the crude polysaccharide aqueous solution.

[0052] S6, stirring, centrifuging, dialysis and freeze-drying: the crude polysaccharide aqueous solution after deproteinization treatment with the addition of chloroform and n-butanol mixed reagents was stirred for 15 minutes, centrifuged for 15 minutes, dialyzed with running water for 48 hours, dialyzed with distilled water for 24 hours, and the reagents and water were removed by rotary evaporation. The peanut shell polysaccharide was obtained by freeze-drying with an inverted plate, and the yield was 3.71%, of which the protein content was reduced from 11.73% to 5%. Fig.12 This is a sample picture of peanut shell polysaccharide prepared by the method of the present invention.

[0053] Figure 4 The high-performance anion exchange chromatography of peanut shell polysaccharide obtained by the preparation method of peanut shell polysaccharide of the present invention is shown. The monosaccharide composition of peanut shell polysaccharide is analyzed by high-performance anion exchange chromatography. The obtained crude peanut shell polysaccharide and the main monosaccharides of peanut shell polysaccharide are xylose, rhamnose and galactose.

[0054] Example 2

[0055] The preparation method of peanut shell polysaccharide in this embodiment is similar to that in Example 1 and is not described in detail. The differences are briefly described as follows:

[0056] In this embodiment, in step S1, the peanut shell powder obtained by raw material pretreatment is subjected to a decolorization and defatting step and then enters step S2 for alkali extraction:

[0057] Take 20g of peanut shell powder, soak it in 100ml of n-hexane for 24h, stir it intermittently, and centrifuge it at 4500 rpm for 20min at room temperature; collect the precipitate and soak it in 100ml of 95% ethanol for 24h, stir it intermittently, and centrifuge it at 4500 rpm for 20min at room temperature, recover the precipitate, and evaporate it in a fume hood to obtain decolorized and defatted peanut shell powder, that is, use decolorized and defatted peanut shell powder as the raw material for alkali extraction.

[0058] The optimal solid-liquid ratio of peanut shell powder to n-hexane or 95% ethanol is 1:5 = W / V. The purpose of n-hexane immersion and stirring is to degrease the sample, and the purpose of ethanol immersion and stirring is to decolorize the sample.

[0059] In step S3, the added amount of α-amylase is 1%, and the added amount of papain is 0.3%.

[0060] In this embodiment, the yield of peanut shell polysaccharide is 3.76%, the percentage of neutral sugar is 73%, the percentage of acidic sugar is 20%, and the percentage of protein is 5%.

[0061] Figure 1 , Figure 2They are respectively a high performance gel permeation chromatography calibration curve table and a sample molecular weight of peanut shell polysaccharide obtained by a method for preparing peanut shell polysaccharide of the present invention. The molecular weight of the peanut shell polysaccharide sample is determined to be about 40-60 kDa by high performance gel permeation chromatography (HPGPC).

[0062] Figure 3 The figure shows the Fourier infrared spectrum of peanut shell polysaccharide obtained by the method for preparing peanut shell polysaccharide of the present invention; the structural characteristics of the peanut shell polysaccharide sample were recorded using a Nicolet 5700 FTIR spectrometer, and the results are as follows: 3400cm -1 and 2900cm -1 The absorption bands at 1620cm and 1630cm are typical characteristics of polysaccharides, which are related to the OH-stretching vibration and CH-stretching vibration in polysaccharide molecules. -1 The absorption peak at 1050 cm-1 is attributed to the OH bending vibration. -1 The strong absorption peak at 650 cm-1 is attributed to the CO bending vibration caused by COH or COC, indicating the presence of a pyranose ring. -1 There is an absorption peak at , indicating the presence of β-glycosidic bond.

[0063] Based on the electrochemical activity of sugar molecules and their ionization characteristics in strong alkaline solution, high performance anion exchange chromatography (HPAEC) and pulsed amperometric detection (PAD) were used to analyze the high performance anion exchange chromatography of peanut shell polysaccharide. Figure 4 As shown in the figure, peanut shell polysaccharide has xylose, rhamnose and galactose as the main monosaccharide components. Figure 5 The molar ratio of each monosaccharide composition is shown.

[0064] The experimental process for characterizing the structure of peanut shell polysaccharide is as follows:

[0065] 1) Phenol-sulfuric acid method to measure neutral sugar content:

[0066] Accurately weigh 5 mg of standard dextran (or glucose) into a 50 ml volumetric flask, add water to the scale, draw 0, 0.2, 0.4, 0.6, 0.8, 1.0 ml from the volumetric flask into test tubes respectively, make up to 1.0 ml with distilled water respectively, then add 1.0 ml of 3% phenol to each test tube in turn and mix well, slowly add 4.0 ml of concentrated sulfuric acid, shake well and let stand for 30 minutes, then measure the absorbance at 490 nm, calibrate the instrument with 1.0 ml of water according to the same color development, the horizontal axis is the micrograms of polysaccharide, the vertical axis is the optical density value, and draw a standard curve of micrograms of polysaccharide and optical density value.

[0067] Prepare 0.1 mg / ml peanut shell polysaccharide sample, take 1 ml, add 1.0 ml of 3% phenol, quickly add 4.0 ml of concentrated sulfuric acid, shake on a vortex, mix thoroughly, let stand for 30 minutes, measure the absorbance at 490 nm, and bring the measured absorbance into the standard curve to obtain the polysaccharide concentration.

[0068] 2) Carbazole-sulfuric acid method for uronic acid: Use 0.1mg / ml galacturonic acid standard solution, pipette 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0mL into each test tube, add distilled water to 1ml, slowly add 6ml pure sulfuric acid in an ice bath, shake while adding, then cool to room temperature after 20min in a water bath at 85℃, add 0.2ml of 0.1% carbazole-ethanol to each test tube, keep at room temperature for 2h, and measure absorbance at 530nm. Use 1.0ml water as blank in the same color development operation.

[0069] Prepare 0.1 mg / ml peanut shell polysaccharide sample, take 1 ml, then add 6 ml pure sulfuric acid, shake on a vortex, mix thoroughly, water bath at 85°C for 20 minutes, take out and cool to room temperature, then add 0.1% carbazole-ethanol 0.2 ml, keep at room temperature for 2 hours, and measure the absorbance at 530 nm.

[0070] 3) Coomassie brilliant blue method for protein content: bovine serum albumin was used as the standard and the Bradford protein concentration assay kit was used to determine the protein content. The operation method was carried out according to the instructions.

[0071] 4) BCA method for protein content determination: The protein content was determined using the BCA protein detection kit, and all samples were measured three times in parallel.

[0072] 5) Molecular weight determination:

[0073] The molecular weight of PCP samples was determined by high performance gel permeation chromatography (HPGPC) using an Agilent 1260HPL liquid chromatography system equipped with a differential refractive index detector, an Ultrahydrogel TM-1000 column (300 mm × 7.8 mm), and a UV detector. The mobile phase was prepared with 0.02% (w:v) NaN and ultrapure water, and a standard curve was drawn using dextran T standards (molecular weight of 10 kDa, 40 kDa, 50 kDa, 70 kDa, 500 kDa, 2000 kDa) and glucose. PSP samples and standards (concentration of 1 mg / mL) were prepared with the mobile phase and analyzed after complete dissolution and filtration through a 0.22 μm membrane injection system.

[0074] 6) Monosaccharide composition analysis

[0075] The monosaccharide composition of PSP was analyzed using high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) (Dionex-5500, Dionex Corporation, Sunnyvale, California, USA) based on the electrochemical activity of sugar molecules and their ionization in strong alkaline solutions. Determination of monosaccharide composition (ordinary sugar) by sulfuric acid method: accurately weigh 5.0 mg of sample into a stoppered test tube, add 0.5 ml of 12 M concentrated sulfuric acid (sulfuric acid: water = 2:1) with a pipette under ice bath conditions, stir under a magnetic stirrer for half an hour, then add 2.5 ml of distilled water, cool in a 100 degrees Celsius oil bath for 2-4 hours, pour the sample solution into a 50 ml volumetric flask, rinse the stoppered test tube containing the sample solution with distilled water, and finally make up to 50 ml, shake well, then take 2 ml to dilute and make up to 10 ml, shake well, use a 2 ml syringe to absorb the dilution, pass through a 0.22 μm membrane, rinse the injection bottle first, and then fill it with sample solution (you can first add a little sample solution to cover the bottom of the bottle, flick the bottom of the bottle with your hand until there are no bubbles, and then slowly add until it overflows the bottle mouth to ensure that there are no bubbles in the injection bottle), stopper it, and inject the sample.

[0076] 7) Fourier transform infrared (FT-IR) spectroscopy

[0077] The structural characteristics of peanut shell polysaccharide samples were recorded using a Nicolet 5700 FTIR spectrometer from the United States. Briefly, freeze-dried peanut shell polysaccharide was thoroughly mixed with KBr at a ratio of 1:100 (w / w) and ground into powder. The powder was pressed into thin sheets and the mixture was immediately placed in the light path at 4000–400 cm -1 Scanning can be performed within the range of 64 times with a resolution of 4cm -1 .

[0078] Example 3

[0079] This example shows the research on the improvement effect of peanut shell polysaccharide on DSS-induced inflammatory mice.

[0080] Test method:

[0081] 1. Fifty five-week-old healthy male mice (SPF grade C57BL / 6J, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were selected and randomly divided into five groups, with 10 mice in each group, named control group, DSS model group, low-dose peanut shell polysaccharide group, medium-dose peanut shell polysaccharide group, and high-dose peanut shell polysaccharide group. Each group was adaptively fed for one week, and SPF mouse feed (Jiangxi Qianchong Scientific Instrument Co., Ltd.) was used during the adaptive feeding period.

[0082] Adaptive feeding conditions: All mice were housed under specific pathogen-free (SPF) conditions with a 12-h light-dark cycle and provided with the above-mentioned maintenance mouse feed and water ad libitum.

[0083] 2. Modeling:

[0084] Control group: blank group, using the previous adaptive feeding conditions to continue feeding until the 28th day;

[0085] DSS model group: The mice were fed with the previous adaptive feeding conditions until day 28, during which 3% (w / v) dextran sulfate sodium (DSS) was added to the drinking water for 7 days starting from day 22, and the drinking water was infused at 0.1 ml / 10 g according to the weight of the mice until day 28;

[0086] Low-dose peanut shell polysaccharide group: The mice were fed with the previous adaptive feeding conditions for 28 days. During this period, the mice were infused with peanut shell polysaccharide solution at a dose of 75 mg / kg BW daily from the 8th day until the 28th day. From the 22nd day, 3% (w / v) dextran sulfate sodium (DSS) was added to the drinking water until the 28th day.

[0087] Peanut shell polysaccharide medium-dose group: The mice were fed with the previous adaptive feeding method until the 28th day. During this period, the mice were infused with peanut shell polysaccharide solution at 150 mg / kg BW daily from the 8th day until the 28th day. From the 22nd day, 3% (w / v) dextran sulfate sodium (DSS) was added to the drinking water until the 28th day.

[0088] High-dose peanut shell polysaccharide group: The mice were fed with the previous adaptive feeding method until the 28th day. During this period, peanut shell polysaccharide solution was infused daily at 300 mg / kg BW starting from the 8th day until the 28th day. From the 22nd day, 3% (w / v) dextran sulfate sodium (DSS) was added to the drinking water until the 28th day.

[0089] For the above groups of mice, the body weight, feces status and rectal bleeding of the mice were recorded from day 22 to day 28 to track the progression of colitis.

[0090] There were no special circumstances in the mice in the control group. They were active, had shiny and smooth hair, and had normal urination and defecation.

[0091] Figure 6 The effects of different feeding methods on the body weight of mice in each group. Figure 7 The weight change rate of mice in each group on the 28th day compared with the initial weight change. Except for the control group, the other groups all showed weight loss; Figure 8 This is a graph showing the relationship between time and the disease activity index of each group of mice under different feeding methods; Fig. 9This is a relationship diagram of the disease activity index of each group of mice under different feeding methods on the 28th day; except for the control group, the mice in the other groups all showed weight loss, reduced activity, and varying degrees of diarrhea and bloody stools. Figure 6 , Figure 7 , Figure 8 , Fig. 9 It means that DSS successfully induced an acute ulcerative colitis model. Compared with the control group, mice in the DSS model group had slow behavior, reduced activity, messy and dull hair, and obvious diarrhea and bloody stools. Mice in the low-dose, medium-dose, and high-dose peanut shell polysaccharide groups all had varying degrees of diarrhea and bloody stools, but the bloody stools in the high-dose peanut shell polysaccharide group were the least severe. The mice in the high-dose peanut shell polysaccharide group had the smallest weight loss, and the weight loss caused by the low-dose and medium-dose peanut shell polysaccharide groups was less than that in the DSS model group.

[0092] Fig.10 The figure shows the colon morphology of five groups of mice after dissection. After dissection, it was found that compared with the control group, the mice in the other groups showed varying degrees of colon shortening. The colon length of the mice in the DSS model group was significantly shortened, the intestinal wall was thickened, and varying degrees of congestion and edema were observed. The shortening of the colon in the low-dose peanut shell polysaccharide group, the medium-dose peanut shell polysaccharide group, and the high-dose peanut shell polysaccharide group was inhibited to a certain extent. PSP can prevent and inhibit the shortening of the colon caused by colitis, among which the high-dose peanut shell polysaccharide group can significantly alleviate the shortening of the colon (p<0.05), and the congestion and edema of the colon in this group of mice was relatively mild.

[0093] Fig.11 The cross-sections of the colons of the three groups of mice after dissection are shown. The colon tissue structure of the mice in the control group was intact and there was no pathological phenomenon. Compared with the control group, the colon tissue of the mice in the DSS model group underwent obvious pathological changes, the colon muscle layer was significantly thinned, the mucosa was eroded, a large number of inflammatory cells were infiltrated in the mucosa, the crypt structure was damaged and disordered, and the submucosal layer was obviously edematous. The high-dose peanut shell polysaccharide group can alleviate the tissue damage caused by colitis, but a small amount of inflammatory cells infiltrated, a small amount of crypt tissue was destroyed, but the intestinal villi were intact and the intestinal mucosal structure was intact.

[0094] Therefore, the peanut shell polysaccharide prepared by the present invention can be used to prevent and treat DSS-induced colitis to improve the histopathological structure of the colon and inhibit inflammatory response.

[0095] The preparation method of peanut shell polysaccharide of the present invention uses ultrasonic assisted alkali method to extract peanut shell polysaccharide with a yield of more than 3.7%. Compared with the traditional water extraction method, the yield of peanut shell polysaccharide is increased by four times; at the same time, ultrasonic assistance reduces the dissolution of impurities to obtain a polysaccharide with high purity (≥90%). The present invention improves the comprehensive utilization value of peanut shells. The peanut shell polysaccharide component of the present invention can effectively improve the histopathological structure of the colon, inhibit inflammatory response, and provide a new solution for the prevention and treatment of colitis.

[0096] Of course, the above description is only a preferred embodiment of the present invention and does not constitute a limitation of the present invention. Under the guidance of the prior art, those skilled in the art can make other modifications to the implementation of the present invention without creative work, and any modification made within the spirit and principle of the present invention or simple replacement or equivalent replacement using conventional technical means in the field should be included in the protection scope of the present invention.

Claims

1. A use of peanut shell polysaccharide in the preparation of a drug for preventing and treating chronic colitis, characterized in that: The method for preparing peanut shell polysaccharide comprises the following steps: S1, raw material pretreatment: select non-moldy peanut shells, wash and dry them, and then crush and sieve them to obtain peanut shell powder; S2, alkali extraction: take the peanut shell powder obtained in step S1, put it into sodium hydroxide solution, then stir it in a constant temperature water bath at 90°C for 10-20 minutes, and then perform ultrasonic treatment for 10-20 minutes. After alternating water bath and ultrasonic treatment, filter and centrifuge to obtain the supernatant; S3, enzymatic hydrolysis, enzyme inactivation and alcohol precipitation: the pH value of the supernatant obtained in step S2 is adjusted to neutral, and then α-amylase is added, and the supernatant is enzymatically hydrolyzed in a 100° C. water bath and then cooled to room temperature; papain is then added, and the supernatant is stirred in a 60° C. water bath for enzymatic hydrolysis, and the enzyme is inactivated in a 100° C. water bath; the supernatant is quickly cooled in an ice bath, centrifuged and the precipitate is discarded, and the supernatant is concentrated by rotary evaporation, and 95% ethanol is slowly added to the concentrated supernatant while slowly stirring, and the supernatant is precipitated with alcohol overnight to obtain peanut shell crude polysaccharide; S4, adding water to dissolve: adding water to dissolve the crude peanut shell polysaccharide obtained in step S3 to obtain a crude polysaccharide aqueous solution; S5, deproteinization: the crude polysaccharide aqueous solution is deproteinized by the Sevage method, a mixed reagent of chloroform and n-butanol is selected as the deproteinization reagent, the volume ratio of chloroform to n-butanol is 4:1, and the volume amount of the mixed reagent of chloroform and n-butanol is 1 / 5 of the volume of the crude polysaccharide aqueous solution; S6, stirring, centrifuging, dialysis and freeze-drying: the crude polysaccharide aqueous solution after deproteinization by adding chloroform and n-butanol mixed reagent is stirred for 10-20 minutes, centrifuged for 10-20 minutes, dialyzed with running water for 48 hours, dialyzed with distilled water for 24 hours, and the reagent and water are removed by rotary evaporation. The solution is freeze-dried on an inverted plate to obtain peanut shell polysaccharide.

2. The use of peanut shell polysaccharide according to claim 1 in the preparation of a drug for preventing and treating chronic colitis, characterized in that: In the step S2, 4% sodium hydroxide solution is used for alkali extraction, and the solid-liquid ratio of the alkali extraction is 1:

20. The peanut shell powder is dissolved and precipitated, and the mixture is stirred for 15 minutes in a constant temperature water bath at 90°C, and then ultrasonically treated for 15 minutes. The water bath stirring and ultrasonic treatment are alternately performed 3-5 times. After filtering with gauze, the filtrate is centrifuged at room temperature and 4500r / min for 20 minutes to obtain the supernatant.

3. The use of peanut shell polysaccharide according to claim 1 in the preparation of a drug for preventing and treating chronic colitis, characterized in that: In the step S1, after the peanut shells are washed and dried, they are crushed and sieved to obtain peanut shell powder, and the following decolorization and defatting steps are also included: the peanut shell powder is soaked in n-hexane for 24 hours, stirred intermittently in the middle, and centrifuged at 4500 rpm for 20 minutes at room temperature; the precipitate is collected and then soaked in 95% ethanol for 24 hours, stirred intermittently in the middle, and centrifuged at 4500 rpm for 20 minutes at room temperature, the precipitate is recovered, and evaporated in a fume hood to obtain decolorized and defatted peanut shell powder, and then alkali extraction is performed.

4. The use of peanut shell polysaccharide according to claim 1 or 2 in the preparation of a drug for preventing and treating chronic colitis, characterized in that: In the step S2, ultrasonic treatment is performed for 15 minutes, the ultrasonic frequency is 40 kHz, the ultrasonic power is 150 W, and water bath stirring and ultrasonic treatment are performed alternately for 4 times at room temperature. The mixture is filtered and centrifuged to obtain the supernatant.

5. The use of peanut shell polysaccharide according to claim 4 in the preparation of a drug for preventing and treating chronic colitis, characterized in that: In the step S2, the residue after filtering and centrifugation is extracted again with distilled water, and the material-liquid ratio is 1:

10. After the residue is dissolved, it is stirred in a constant temperature water bath at 90°C with ultrasonic assistance. After the water bath ends, it is centrifuged at 4500 rpm for 10 minutes, and the supernatant is taken and combined with the supernatant in step S2, and then step S3 is entered.

6. The use of peanut shell polysaccharide according to claim 1 in preparing a drug for preventing and treating chronic colitis, characterized in that: In step S3, the amount of α-amylase added is 1-2% of the mass of the supernatant in step S2 after adjusting the pH value to neutral, and the supernatant is stirred in a water bath at 100° C. for 2 hours and then cooled to room temperature, and then papain is added according to 0.3-0.5% of the mass of the liquid after cooling to room temperature.

7. The use of peanut shell polysaccharide according to claim 1 in the preparation of a drug for preventing and treating chronic colitis, characterized in that: In step S3, the enzymatic hydrolysis time of papain in a 60° C. water bath with stirring is 1.5 h, and the enzyme inactivation time of papain is 30 min.

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