Rhodomyrtus tomentosa polysaccharide RTP2-3 as well as preparation method, identification method and application thereof

Abstract

The invention relates to the technical field of medicines and health foods, in particular to rhodomyrtus tomentosa polysaccharide RTP2-3 as well as a preparation method, an identification method and application thereof. According to the specific technical scheme, the rhodomyrtus tomentosa polysaccharide RTP2-3 is mainly composed of arabinose, galactose and rhamnose, and the molecular weight range of the rhodomyrtus tomentosa polysaccharide RTP2-3 is 1000-100000 Da. The preparation method is simple, reaction conditions are mild, and large-scale production can be achieved; the chemical structure of the obtained high-purity rhodomyrtus tomentosa polysaccharide is identified, and a structural basis is provided for exploring the pharmacological activity mechanism of the rhodomyrtus tomentosa polysaccharide. Meanwhile, the obtained rhodomyrtus tomentosa polysaccharide RTP2-3 lays a foundation for rhodomyrtus tomentosa polysaccharide medicines, health-care foods and functional foods, quality control of the rhodomyrtus tomentosa polysaccharide medicines, health-care foods and functional foods, and deep research of structure-function relationships and action mechanisms of the rhodomyrtus tomentosa

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine and health food, in particular to a Rhodomyrtus tomentosa polysaccharide RTP2-3, a preparation method, an identification method and an application thereof. BACKGROUND

[0002] Ulcerative colitis (UC) is also known as idiopathic coloproctitis or chronic nonspecific ulcerative colitis, which is an inflammatory bowel disease of unknown etiology occurring in the rectum and colon. In recent years, the incidence and prevalence of UC have been increasing globally, with the peak age of onset being between 30 and 40 years old. UC is prone to repeated attacks and is difficult to cure, and has become a global health problem that seriously affects the quality of life of patients. The main clinical symptoms of UC are diarrhea, abdominal pain, mucous and purulent blood stool, etc., which generally leads to emaciation, anemia, water and electrolyte imbalance, and hypoproteinemia. If not properly controlled, UC is prone to canceration and is accompanied by various extraintestinal complications. At present, the etiology and pathogenesis of UC are not fully understood, and therefore, the treatment of UC remains a clinical challenge worldwide.

[0003] Polysaccharide, also known as polyose, is a kind of biological information macromolecule widely existing in animals, plants and microorganisms. Natural polysaccharides play an important role in various diseases due to their outstanding antioxidant and immunomodulatory activities, including tumors, anti-inflammatory and cardiovascular diseases. More and more evidence shows that plant polysaccharides can exert various effects such as hypoglycemic, immunomodulatory and anti-inflammatory by affecting intestinal homeostasis. Therefore, searching for potential therapeutic drugs from natural polysaccharides is expected to become an effective strategy for preventing and treating ulcerative colitis.

[0004] Rhodomyrtus tomentosa (Ait.) Hassk is the fruit of Rhodomyrtus tomentosa of Myrtaceae, which is sweet and astringent in taste and neutral in nature. It is clinically used for nourishing blood to stop bleeding, astringing intestines and consolidating essence. It is mainly used for blood deficiency, hemoptysis, nasal bleeding, coughing blood, metrorrhagia, spermatorrhea, leukorrhagia, dysentery, prolapse of the rectum, scalding and traumatic bleeding, etc. The chemical components of Rhodomyrtus tomentosa mainly include flavonoids, phenolic components, amino acids and polysaccharides. The previous studies on the active components of Rhodomyrtus tomentosa mainly focused on flavonoids and phenolic compounds. The pharmacological effects of polysaccharides, as one of the main active components of Rhodomyrtus tomentosa, have not been widely studied. At present, there are few reports on the homogeneous polysaccharide structure and anti-inflammatory effect of Rhodomyrtus tomentosa polysaccharide. Therefore, it is necessary to provide a method for extracting and purifying Rhodomyrtus tomentosa polysaccharide, so as to lay a foundation for quality control and in-depth research on the activity of treating ulcerative colitis of Rhodomyrtus tomentosa polysaccharide. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a mountain twist sub-polysaccharide RTP2-3 and a preparation method, identification method and application thereof, and the present application adopts water extraction and alcohol precipitation method, and high-concentration ethanol can separate polysaccharides with large polarity and good water solubility from polysaccharides with small polarity and poor water solubility, thereby solving the problem of complicated and difficult separation in the later stage caused by traditional water boiling method for extracting polysaccharides.

[0006] To achieve the above object, the present application is implemented by the following technical solutions:

[0007] The present application discloses a mountain twist sub-polysaccharide RTP2-3, which is mainly composed of arabinose, galactose and rhamnose, and has a molecular weight range of 1000-100000 Da.

[0008] Preferably, the mountain twist sub-polysaccharide RTP2-3 is mainly composed of t-alpha-L-Araf, t-alpha-L-Rhap, 1)-alpha-L-Araf-(3 1)-alpha-L-Araf-(5 t-alpha-D-Galp, 1)-alpha-L-Araf-(3,5 1)-beta-D-Galp(4 1)-alpha-L-Araf-(2,3,5 1)-beta-D-Galp-(3 1)-alpha-D-Galp-(3,6 and 1)-beta-D-Galp-(3,4,6.

[0009] Correspondingly, a preparation method of the mountain twist sub-polysaccharide RTP2-3 comprises the following steps:

[0010] (1) After the dried fruit of the mountain twist is crushed into powder, water is added and heated for extraction to obtain an extraction liquid;

[0011] (2) The extraction liquid of step (1) is subjected to multiple alcohol precipitation, and after each alcohol precipitation, the supernatant obtained after alcohol precipitation is subjected to re-alcohol precipitation; the crude polysaccharide RT1, the crude polysaccharide RT2 and the crude polysaccharide RT3 are obtained after each alcohol precipitation, respectively;

[0012] (3) The crude polysaccharide RT2 is purified to obtain the mountain twist polysaccharide RT2, and the mountain twist polysaccharide RT2 is subjected to ion exchange column chromatography, gradient elution is performed with 0-2M NaCl solution, and then the sugar part is collected according to the elution curve and concentrated, freeze-dried to obtain the mountain twist polysaccharide RT2-3;

[0013] (4) The mountain twist polysaccharide RT2-3 is added into an aqueous solution and centrifuged, the supernatant is subjected to molecular sieve gel column chromatography, eluted with water, and the sugar part is collected according to the elution curve and concentrated, freeze-dried to obtain the mountain twist polysaccharide RTP2-3.

[0014] Preferably, in step (1), the amount of water added is 6-10 times the weight of the *Solanum nigrum* powder, the heating temperature is 60-80℃, the heating extraction time is 1-3h, and the number of heating extractions is 2-4.

[0015] Preferably, in step (2), the multiple alcohol precipitation process is as follows: the extract from step (1) is concentrated under reduced pressure to obtain concentrate 1; ethanol is added to concentrate 1 until the ethanol volume concentration is a%, and the mixture is allowed to stand. The precipitate and supernatant are collected to obtain crude polysaccharide RT1 and supernatant 1; supernatant 1 is concentrated under reduced pressure to obtain concentrate 2; ethanol is added to concentrate 2 until the ethanol volume concentration is b%, and the mixture is allowed to stand. The precipitate and supernatant are collected to obtain crude polysaccharide RT2 and supernatant 2; supernatant 2 is concentrated under reduced pressure to obtain concentrate 3; ethanol is added to concentrate 3 until the ethanol volume concentration is c%, and the mixture is allowed to stand. The precipitate is collected to obtain crude polysaccharide RT3.

[0016] Where 10 ≤ a < b < c < 100.

[0017] Preferably, the temperature for vacuum concentration is 40-70℃, and the settling time is 10-15h.

[0018] Accordingly, a method for identifying RTP2-3, a polysaccharide from *Solanum nigrum*, includes the following steps:

[0019] (1) Take the polysaccharide RTP2-3 from the mountain pine and hydrolyze it with 1.8 mL of 0.2 M TFA. The hydrolysis product was detected by high performance liquid chromatography.

[0020] (2) Take the polysaccharide RTP2-3 from the mountain thorn, dry it, compress it into tablets, and detect it by infrared spectroscopy;

[0021] (3) Take the polysaccharide RTP2-3 from the mountain pine, methylate it, hydrolyze it, reduce it, acetylate it, and then perform GC-MS detection.

[0022] Preferably, in step (3), the methylation process is as follows: add the polysaccharide RTP2-3 of *Solanum nigrum* and anhydrous DMSO into a reaction flask, then add dry sodium hydroxide, sonicate for 20-40 min, add iodomethane in three portions under ice bath conditions and in the dark, sonicate for 20-40 min each time, add distilled water to decompose the residual iodomethane after the reaction, add chloroform for extraction, centrifuge and take the chloroform layer to obtain the methylated polysaccharide RTP2-3 of *Solanum nigrum*.

[0023] Preferably, in step (3), the hydrolysis and reduction process is as follows: take methylated senna polysaccharide RTP2-3, then add TFA, hydrolyze in a constant temperature oil bath at 110-120℃ for 4-6 hours, evaporate under reduced pressure to dryness, then repeatedly add methanol to evaporate to neutral pH, and then use NaBH4 to react at 35-45℃ for 25-35 minutes to reduce the hydrolysis product;

[0024] The acetylation process is: adding glacial acetic acid to the reduced hydrolysate to terminate the reaction and spin-drying the product, then adding acetic anhydride and pyridine to obtain the acetylated product.

[0025] Correspondingly, the application of the Shanzha polysaccharide RTP2-3 in the preparation of a drug or health care product for treating colitis.

[0026] The present application has the following beneficial effects:

[0027] 1. The present application uses water extraction and alcohol precipitation to preliminarily separate the Shanzha polysaccharide, which has a significant effect, and the preparation method is simple, the reaction conditions are mild, and large-scale production is possible.

[0028] 2. The present application uses column chromatography to secondarily separate and purify the Shanzha crude polysaccharide, which has a significant effect, and a Shanzha polysaccharide RTP2-3 pure product is prepared for the first time.

[0029] 3. The structure of the purified Shanzha polysaccharide RTP2-3 pure product is identified, and the physicochemical properties of each polysaccharide component are determined, which provides structural basis for exploring the pharmacological activity mechanism.

[0030] 4. The Shanzha polysaccharide RTP2-3 pure product obtained in the present application has complete components, clear structure and controllable quality, can restore the intestinal length of zebrafish, inhibit neutrophil infiltration in zebrafish, and down-regulate the ROS and NO levels in zebrafish, thereby playing a role in treating ulcerative colitis, and provides a basis for the application of Shanzha polysaccharide in the fields of medicine, health care products and the like.

[0031] 5. The present application lays a foundation for the drug, quality control and in-depth study of the structure-activity relationship and mechanism of action of Shanzha polysaccharide. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a high-performance liquid chromatogram of monosaccharide composition of the Shanzha polysaccharide RTP2-3;

[0033] Figure 2 It is an infrared spectrum of the Shanzha polysaccharide RTP2-3;

[0034] Figure 3 It is a graph showing the effect of the Shanzha polysaccharide RTP2-3 on the intestinal length of zebrafish;

[0035] Figure 4 It is a graph showing the effect of the Shanzha polysaccharide RTP2-3 on the neutrophils in the intestinal tract of zebrafish;

[0036] Figure 5 It is a graph showing the effect of the Shanzha polysaccharide RTP2-3 on the ROS and NO in the intestinal tract of zebrafish. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of protection of the present application. If not specifically indicated, the technical means used in the examples is the conventional means known to those of ordinary skill in the art.

[0038] The present application discloses a kind of mountain twist sub polysaccharide RTP2-3, the mountain twist sub polysaccharide RTP2-3 is mainly composed of arabinose, galactose and rhamnose, its molecular weight range is 1000-100000Da.The mountain twist sub polysaccharide RTP2-3 is significantly effective to ulcerative colitis treatment.

[0039] Specifically, the mountain twist sub polysaccharide RTP2-3 is mainly composed of t-α-L-Araf, t-α-L-Rhap, →1)-α-L-Araf-(3→, →1)-α-L-Araf-(5→, t-α-D-Galp, →1)-α-L-Araf-(3,5→, →1)-β-D-Galp(4→, →1)-α-L-Araf-(2,3,5→, →1)-β-D-Galp-(3→, →1)-α-D-Galp-(3,6→ and →1)-β-D-Galp-(3,4,6→.

[0040] The present application discloses a kind of mountain twist sub polysaccharide RTP2-3 preparation method, comprising the following steps:

[0041] (1) Pulverization

[0042] The dried fruit of mountain twist is pulverized, washed clean with water and dried to obtain mountain twist powder.

[0043] (2) Water extraction

[0044] The mountain twist powder obtained in step (1) is added to water and heated to extract to obtain an extract; wherein the amount of water added is 6-10 times the weight of the mountain twist powder, the heating temperature is 60-80°C, the heating extraction time is 1-3h, and the heating extraction times is 2-4 times.

[0045] (3) Fractionation and alcohol precipitation

[0046] The extract of step (2) is concentrated under reduced pressure (vacuum degree-0.1MPa) to obtain concentrated solution 1; ethanol is added to concentrated solution 1 until the volume concentration of ethanol is a%, and then it is left to stand, the precipitate and supernatant are collected to obtain crude polysaccharide RT1 and supernatant 1;

[0047] The supernatant 1 is concentrated under reduced pressure to obtain concentrated solution 2; ethanol is added to the concentrated solution 2 until the volume concentration of ethanol is b%, and then the mixture is left to stand, the precipitate and supernatant are collected, and the crude polysaccharide RT2 and supernatant 2 are obtained;

[0048] The supernatant 2 is concentrated under reduced pressure to obtain concentrated solution 3; ethanol is added to the concentrated solution 3 until the volume concentration of ethanol is c%, and then the mixture is left to stand, and the precipitate is collected to obtain the crude polysaccharide RT3;

[0049] wherein 10≤a

[0050] (4) Purification

[0051] (4.1) Primary purification

[0052] The crude polysaccharide RT2 obtained in step (3) is subjected to protein removal, dialysis, and freeze-drying to obtain the polysaccharide RT2 from Yanzhizi;

[0053] (4.2) Secondary purification

[0054] The polysaccharide RT2 from Yanzhizi after primary purification is subjected to ion exchange column chromatography, gradient elution is performed using a NaCl solution with a concentration of 0-2 M, and the elution curve is tracked using the phenol-sulfuric acid method; according to the elution curve, the sugar part is collected and concentrated, and freeze-drying is performed to obtain the polysaccharide RT2-3 from Yanzhizi.

[0055] The polysaccharide RTP from Yanzhizi is dissolved in water, centrifuged, and the supernatant is discarded; the supernatant is subjected to molecular sieve gel column chromatography, eluted with water, and the elution curve is detected using the phenol-sulfuric acid method; according to the elution curve, the sugar part is collected and concentrated, and freeze-drying is performed to obtain the polysaccharide RTP2-3 from Yanzhizi.

[0056] The application discloses a method for identifying polysaccharide RTP2-3 from Yanzhizi, which comprises the following steps:

[0057] (1) The polysaccharide RTP2-3 from Yanzhizi is hydrolyzed with 1.8 mL of 0.2 M TFA, and the hydrolyzate is detected by high performance liquid chromatography;

[0058] (2) The polysaccharide RTP2-3 from Yanzhizi is dried, tabletted, and detected by infrared spectroscopy;

[0059] (3) The polysaccharide RTP2-3 from Yanzhizi is methylated, hydrolyzed, reduced, and acetylated, and then subjected to GC-MS detection;

[0060] The methylation process is: adding Shan Zhanzi polysaccharide RTP2-3 and anhydrous DMSO into a reaction bottle, then adding dry sodium hydroxide, ultrasonic for 20-40 min, adding iodomethane in three times under ice bath condition and avoiding light, ultrasonic for 20-40 min each time, adding distilled water to decompose residual iodomethane after the reaction is completed, adding chloroform for extraction, centrifuging to take chloroform layer, and obtaining the methylated Shan Zhanzi polysaccharide RTP2-3.

[0061] The hydrolysis and reduction process is: taking the methylated Shan Zhanzi polysaccharide RTP2-3, then adding TFA, hydrolyzing for 4-6 h under a constant temperature oil bath of 110-120 DEG C, evaporating to dryness under reduced pressure, repeatedly adding methanol and spin-drying to neutral pH, then reducing the hydrolysis product by using NaBH4 at 35-45 DEG C for 25-35 min;

[0062] The acetylation process is: adding glacial acetic acid to the reduced hydrolysis product to terminate the reaction and spin-drying the product, then adding acetic anhydride and pyridine to obtain the acetylated product.

[0063] The present application uses Shan Zhanzi dry powder as raw material, separates the crude polysaccharide by water extraction and alcohol precipitation, deproteins the extracted crude polysaccharide, then purifies the Shan Zhanzi crude polysaccharide by ion exchange chromatography and gel molecular sieve column chromatography, first prepares a Shan Zhanzi polysaccharide pure product, systematically analyzes and identifies the physical and chemical properties, molecular weight and monosaccharide composition of the polysaccharide pure product, and successfully obtains the structure information of the Shan Zhanzi polysaccharide, wherein the Shan Zhanzi polysaccharide RTP2-3 is a heteropolysaccharide, mainly composed of t-alpha-L-Araf, t-alpha-L-Rhap, 1)-alpha-L-Araf-(3 1)-alpha-L-Araf-(5 1)-alpha-D-Galp, 1)-alpha-L-Araf-(3,5 1)-beta-D-Galp(4 1)-alpha-L-Araf-(2,3,5 1)-beta-D-Galp-(3 1)-alpha-D-Galp-(3,6 and 1)-beta-D-Galp-(3,4,6.

[0064] The application discloses application of Shan Zhanzi polysaccharide RTP2-3 in preparation of a drug or health care product for treating colonitis.

[0065] The application will be further described in combination with specific examples.

[0066] Example 1

[0067] A preparation method of Shan Zhanzi polysaccharide RTP2-3 comprises the following steps:

[0068] S1, crushing

[0069] 1kg of dried fruits of Rubus corchorifolius was crushed, washed quickly with cold water, and dried to obtain Rubus corchorifolius powder;

[0070] S2, water extraction

[0071] To the Rubus corchorifolius powder obtained in step S1, 8 times the weight of water was added, and heated to 70°C for extraction for 1 hour. The extract was collected, and the residue was dried to obtain the extract and residue;

[0072] S3, fractional alcohol precipitation

[0073] The extract obtained in step S2 was concentrated under reduced pressure (vacuum degree -0.1 MPa) to obtain concentrated solution 1. Ethanol was added to the concentrated solution 1 to a volume concentration of 30%, and the precipitate and supernatant were collected to obtain crude polysaccharide RT1 and supernatant 1;

[0074] The supernatant 1 was concentrated under reduced pressure to obtain concentrated solution 2. Ethanol was added to the concentrated solution 2 to a volume concentration of 60%, and the precipitate and supernatant were collected to obtain crude polysaccharide RT2 and supernatant 2;

[0075] The supernatant 2 was concentrated under reduced pressure to obtain concentrated solution 3. Ethanol was added to the concentrated solution 3 to a volume concentration of 90%, and the precipitate was collected to obtain crude polysaccharide RT3;

[0076] wherein 10≤a

[0077] S4, purification

[0078] S4-01, primary purification

[0079] The crude polysaccharide RT2 obtained in step S3 was subjected to protein removal by Sevage method. After protein removal, the crude polysaccharide was subjected to dialysis using a dialysis bag (molecular weight cut-off 1000 Da) and freeze-drying to obtain Rubus corchorifolius polysaccharide RT2;

[0080] S4-02, secondary purification

[0081] 150 mg of Rubus corchorifolius polysaccharide RT2 was dissolved in 5 mL of deionized water and loaded onto a DEAE-FF column. Gradient elution was performed using 0-2 M NaCl solution, and one elution peak appeared, in which the elution peak was the 0.1 M NaCl elution part (phenol-sulfuric acid method was used to track the elution curve during the elution process, and the sugar part was collected according to the elution curve). After concentration and freeze-drying, Rubus corchorifolius polysaccharide RT2-3 was obtained;

[0082] The above freeze-dried polysaccharide RT2-3 of Yanzhizi was dissolved with water, centrifuged, and the supernatant was taken and subjected to Sephadex G-100 column and eluted with water. The elution curve was tracked by phenol-sulfuric acid method, and a single symmetrical peak was obtained. The main peak was collected, concentrated, frozen and dried to obtain polysaccharide RTP2-3 of Yanzhizi.

[0083] Example 2

[0084] Structure analysis of polysaccharide RTP2-3 of Yanzhizi

[0085] (I) Test material: polysaccharide RTP2-3 of Yanzhizi in Example 1.

[0086] (II) Test method:

[0087] 1. Monosaccharide composition analysis

[0088] Sample processing:

[0089] 4mg of polysaccharide RTP2-3 of Yanzhizi was precisely weighed and hydrolyzed with 2M TFA (trifluoroacetic acid) at 120°C for 4 hours. After the reaction, the hydrolysis product of polysaccharide RTP2-3 of Yanzhizi was concentrated with anhydrous methanol three times to completely remove the remaining TFA. It was dissolved with deionized water to 1mL, centrifuged, and 100μL of sample solution was taken, 100μL of 0.3M NaOH solution was added, and 100μL of 0.5M PMP methanol solution was added, mixed, reacted in a 70°C water bath for 30min, cooled, neutralized with 105μL of 0.3M HCl solution, added with deionized water to 1mL, and added with an equal volume of chloroform solution, shaken vigorously, centrifuged, and the chloroform phase was removed. The water phase was extracted twice, filtered with a 0.22μm filter membrane, and used for HPLC sample analysis.

[0090] Chromatographic column:

[0091] Kromasil 100-5-C18, 4.6x250mm, 5μm; mobile phase: 0.1M phosphate (pH=6.9) buffer-acetonitrile (v / v 83:17); detection wavelength: 250nm; flow rate: 0.8mL / min; injection volume: 20μL.

[0092] 2. Infrared spectroscopy detection

[0093] The dried polysaccharide RTP2-3 of Yanzhizi test material 2.0mg was ground with KBr, pressed into a tablet, and scanned with IR Affinity-1 in the range of 4000-400cm -1

[0094] 3. Methylation / GC-MS analysis

[0095] ​Weigh 8.0 mg of dried *Solanum nigrum* polysaccharide RTP2-3 into a reaction flask, add 8 mL of anhydrous DMSO, then add 800 mg of dried sodium hydroxide, sonicate for 30 min, and under ice bath conditions, add 3.0 mL of iodomethane in three portions, sonicating for 30 min each time in an ice bath. After the reaction is complete, add 2 mL of distilled water to decompose the residual iodomethane, and add 1 mL of chloroform for extraction. Centrifuge and collect the chloroform layer to obtain the methylated *Solanum nigrum* polysaccharide RTP2-3.

[0096] The methylated *Solanum lyratum* polysaccharide RTP2-3 sample was placed in a stoppered test tube and hydrolyzed in a constant temperature oil bath at 120℃ for 4 h with 2 mol / L TFA. The solution was then evaporated to dryness under reduced pressure. The process was repeated several times with the addition of methanol until the pH reached neutral. The hydrolysate was then reduced with 20 mg of NaBH4 at 40℃ for 30 min. The reaction was terminated with 100 μL of glacial acetic acid. The sample was evaporated to dryness under low pressure, and then 2 mL of acetic anhydride and pyridine were added for acetylation to obtain the acetylated product.

[0097] The acetylated product was stirred magnetically at 95°C for 2 hours. Then methanol was added three times, the mixture was evaporated to dryness, dissolved in 1 mL of chloroform, washed three times with an equal volume of distilled water to remove the aqueous layer, and finally placed in a fume hood to evaporate the chloroform layer for GC-MS analysis.

[0098] (III) Test Results:

[0099] 1. Structural analysis of RTP2-3 polysaccharide from *Solanum nigrum*

[0100] (1) Monosaccharide composition analysis

[0101] like Figure 1 As shown in the HPLC chromatogram, the RTP2-3 polysaccharide from *Solanum nigrum* contains mannose, glucose, galactose, and arabinose. (Chromatographic peak order: 1: rhamnose; 2: galactose; 3: arabinose)

[0102] (2) Infrared spectroscopy analysis

[0103] like Figure 2 As shown in the infrared spectrum of RTP2-3 polysaccharide from *Solanum nigrum*, it can be seen that RTP2-3 contains the characteristic infrared absorption peaks of polysaccharides.

[0104] (3) Methylation / GC-MS analysis

[0105] Methylation analysis of polysaccharide RTP2-3 from S. involucrata, after hydrolysis, reduction and acetylation, GC-MS detection, from the GC-MS spectrum, polysaccharide RTP2-3 from S. involucrata contains t-α-L-Araf, t-α-L-Rhap, →1)-α-L-Araf-(3→, →1)-α-L-Araf-(5→, t-α-D-Galp, →1)-α-L-Araf-(3,5→, →1)-β-D-Galp(4→, →1)-α-L-Araf-(2,3,5→, →1)-β-D-Galp-(3→, →1)-α-D-Galp-(3,6→and →1)-β-D-Galp-(3,4,6→and other sugar residues.

[0106] In summary: RTP2-3 is a heteropolysaccharide mainly composed of mannose, glucose, galactose and arabinose, from methylation analysis, it contains t-α-L-Araf, t-α-L-Rhap, →1)-α-L-Araf-(3→, →1)-α-L-Araf-(5→, t-α-D-Galp, →1)-α-L-Araf-(3,5→, →1)-β-D-Galp(4→, →1)-α-L-Araf-(2,3,5→, →1)-β-D-Galp-(3→, →1)-α-D-Galp-(3,6→and →1)-β-D-Galp-(3,4,6→and other sugar residues.

[0107] Example 3

[0108] Effect of polysaccharide RTP2-3 from S. involucrata on treating ulcerative colitis in zebrafish

[0109] (I) Test material: polysaccharide RTP2-3 from S. involucrata in Example 1.

[0110] (II) Test object: zebrafish (purchased from Nanjing Yeshuli Flower Technology Co., Ltd.).

[0111] (III) Test method:

[0112] 1. Zebrafish culture:

[0113] Adult zebrafish were raised in an automatic system at 28±0.5℃, with 14h light and 10h dark alternation. Ova were induced and collected, then transferred to E3 medium (0.33mmol / L CaCl2, 5.00mmol / L NaCl, 0.18mmol / L KCl, 0.34mmol / L MgSO4, pH 7.40) and placed in a 28.5℃ incubator.

[0114] 2. Drug administration

[0115] AB and Tg(lyz:DsRed) zebrafish larvae fertilized to 3dpf were randomly transferred to each well of 24-well plate, 20 larvae were put into each well. Blank control group, model group, positive control group and different concentrations of polysaccharide administration group were set up, and cultured in a constant temperature incubator at 28.5℃ to 6dpf (14h light / 10h dark).

[0116] Blank group: add E3 medium 2mL

[0117] Model group: add 0.25% DSS solution 2mL

[0118] Positive control group: add 0.25% DSS solution and 10 μg / ml SASP solution 2mL

[0119] Polysaccharide group: add RTP2-3 to get 0.25% DSS solution and different concentrations of RTP2-3 (100, 200, 400 μg / mL) 2mL solution.

[0120] 3. Zebrafish intestinal length experiment

[0121] AB wild type after 6dpf experiment, wash off the drug, anesthetize with MS222, observe the length of zebrafish under a body microscope, and quantify with image J.

[0122] 4. Granulocyte fluorescence intensity experiment in the intestine

[0123] Tg(lyz:DsRed) after 6dpf experiment, wash off the drug, anesthetize with MS222, observe the number of neutrophils under a fluorescence microscope, and quantify with image J.

[0124] 5. Zebrafish intestinal ROS and NO experiment

[0125] AB wild type after 6dpf experiment, wash off the drug, add NO probe (DAF-FMDA, 20 μg / mL) dark incubation for 2h or ROS probe (DCFH-DA, 5 μM) dark incubation for 1h, wash off the probe, anesthetize, observe the fluorescence intensity under a fluorescence microscope, and quantify with image J.

[0126] (IV) Experimental results

[0127] As shown in Figures 3-5 , Shanlanzi polysaccharide RTP2-3 can restore the length of DSS damaged intestine, inhibit the massive aggregation of neutrophils, and reduce the levels of intestinal ROS and NO to exert the activity of treating ulcerative colitis.

[0128] In summary, the prepared Shanlanzi polysaccharide pure product RTP2-3 can play a role in treating ulcerative colitis by restoring the length of the intestinal tract, inhibiting neutrophil aggregation, and reducing the levels of ROS and NO in the intestinal tract.

[0129] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. A polysaccharide RTP2-3 from the roots of Silybum marianum, characterized by: The polysaccharide RTP2-3 of the mountain twist mainly consists of arabinose, galactose and rhamnose, and the molecular weight ranges from 1000 to 100000 Da.

2. The kind of mountain twist sub-polysaccharide RTP2-3 according to claim 1, characterized in that: The polysaccharide RTP2-3 of the mountain twist mainly consists of t-alpha-L-Araf, t-alpha-L-Rhap, 1)-alpha-L-Araf-(3 1)-alpha-L-Araf-(5 t-alpha-D-Galp, 1)-alpha-L-Araf-(3,5 1)-beta-D-Galp(4 1)-alpha-L-Araf-(2,3,5 1)-beta-D-Galp-(3 1)-alpha-D-Galp-(3,6 and 1)-beta-D-Galp-(3,4,6.

3. A method of preparing the RTP2-3 of claim 1 or 2, characterized by: The method comprises the following steps: (1) crushing the dried fruits of the mountain twist into powder, adding water and heating to extract to obtain an extract; (2) performing multiple alcohol precipitation on the extract of step (1), and after each alcohol precipitation, performing alcohol precipitation again on the supernatant obtained after the alcohol precipitation; and obtaining crude polysaccharide RT1, crude polysaccharide RT2 and crude polysaccharide RT3 respectively after each alcohol precipitation; (3) purifying the crude polysaccharide RT2 to obtain the polysaccharide RT2 of the mountain twist, and performing ion exchange column chromatography on the polysaccharide RT2 of the mountain twist, gradient elution with 0-2M NaCl solution, and according to the elution curve, collecting the sugar part, concentrating, and freeze-drying to obtain the polysaccharide RT2-3 of the mountain twist; (4) adding an aqueous solution to the polysaccharide RT2-3 of the mountain twist, centrifuging, taking the supernatant, performing gel column chromatography with a molecular sieve, eluting with water, and according to the elution curve, collecting the sugar part, concentrating, and freeze-drying to obtain the polysaccharide RTP2-3 of the mountain twist.

4. The method of claim 3, wherein: In step (1), the amount of water added is 6-10 times the weight of the mountain twist powder, the heating temperature is 60-80 DEG C, the heating extraction time is 1-3h, and the heating extraction times is 2-4 times.

5. The method of claim 3, wherein: In step (2), the multiple alcohol precipitation process is as follows: the extract of step (1) is concentrated under reduced pressure to obtain concentrated solution 1; ethanol is added to the concentrated solution 1 until the volume concentration of ethanol is a%, and then it is left to stand, the precipitate and the supernatant are collected, and the crude polysaccharide RT1 and the supernatant 1 are obtained; the supernatant 1 is concentrated under reduced pressure to obtain concentrated solution 2; ethanol is added to the concentrated solution 2 until the volume concentration of ethanol is b%, and then it is left to stand, the precipitate and the supernatant are collected, and the crude polysaccharide RT2 and the supernatant 2 are obtained; the supernatant 2 is concentrated under reduced pressure to obtain concentrated solution 3; ethanol is added to the concentrated solution 3 until the volume concentration of ethanol is c%, and then it is left to stand, and the precipitate is collected to obtain the crude polysaccharide RT3; wherein 10≤a 6. The method of claim 5, wherein: The temperature for the concentration under reduced pressure is 40-70 DEG C, and the standing time is 10-15h.

7. A method of identifying the spliced polysaccharide RTP2-3 of claim 1 or 2, characterized by: The method comprises the following steps: (1) taking the polysaccharide RTP2-3 of the mountain twist, hydrolyzing with 1.8mL of 0.2M TFA, and detecting the hydrolyzate by high performance liquid chromatography; (2) taking the polysaccharide RTP2-3 of the mountain twist, drying, tabletting, and detecting by infrared spectrum; (3) taking the polysaccharide RTP2-3 of the mountain twist, methylating, hydrolyzing, reducing, acetylating, and detecting by GC-MS.

8. The method of claim 7, wherein: In step (3), the methylation process is as follows: the polysaccharide RTP2-3 is added into a reaction bottle with anhydrous DMSO, dry sodium hydroxide is added, and ultrasonic treatment is performed for 20-40 min; under ice bath condition, iodomethane is added in three times under dark condition, and ultrasonic treatment is performed for 20-40 min each time; after the reaction is completed, distilled water is added to decompose the residual iodomethane, chloroform is added for extraction, centrifugation is performed to obtain the chloroform layer, and the methylated polysaccharide RTP2-3 is obtained.

9. The method of claim 8, wherein: In step (3), the hydrolysis and reduction process is as follows: the methylated polysaccharide RTP2-3 is taken, TFA is added, and hydrolysis is performed at 110-120 ℃ constant temperature oil bath for 4-6 h; the product is evaporated to dryness under reduced pressure, and then methanol is repeatedly added and dried to neutral pH; then, NaBH4 is added to react at 35-45 ℃ for 25-35 min to reduce the hydrolysis product. The acetylation process is as follows: the reduced hydrolysis product is taken, glacial acetic acid is added to terminate the reaction and dry the product, acetic anhydride and pyridine are added, and the acetylated product is obtained.

10. Use of the polysaccharide RTP2-3 of claim 1 or 2 in the preparation of a drug or health care product for treating colitis.

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