A polysaccharide CWP-3 extracted from cuperus rotundus and its preparation method and use

Abstract

The application belongs to the technical field of traditional Chinese medicine polysaccharide development, and particularly relates to an active polysaccharide CWP-3 extracted from medicinal materials of Coptis deltoidea or residues after extraction of volatile oil, and a preparation method and application thereof. The homogeneous polysaccharide CWP-3 obtained by the application is a heteropolysaccharide, monosaccharide composition includes rhamnose (Rha), galacturonic acid (GalA), galactose (Gal) and arabinose (Ara), the molecular weight is 400-1000 kDa, the types and connection modes of sugar residues are determined by using HPLC, FTIR, GC-MS and NMR and other analysis technologies, and the atomic force microscope (AFM) analysis shows that the CWP-3 has a clear three-strand helical structure. In addition, the pharmacological experiments show that the CWP or CWP-3 has good antioxidant and treatment effects on blood stasis syndrome, cardiovascular diseases, dysmenorrhea and functional constipation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine polysaccharide development, and particularly relates to an active polysaccharide CWP-3 extracted from medicinal materials of Curcuma wenyujin or residues after extraction of volatile oil, and a preparation method and application thereof. BACKGROUND

[0002] Traditional Chinese medicine Curcuma wenyujin is the dried rhizome of Curcuma wenyujin Y.H.Chen et C.Ling, a perennial herbaceous plant of the genus Curcuma, which is one of the base origins of 'Zedoary' and is recorded in the 2020 edition of Chinese Pharmacopoeia (Volume I) and has the effects of 'promoting blood flow and resolving blood stasis, eliminating accumulation and relieving pain', and is used for treating masses and blood stasis, chest pain, and food accumulation and abdominal pain. The volatile oil extracted from Zedoary by steam distillation is the source of 'Zedoary Oil' in Chinese Pharmacopoeia (Volume I).

[0003] It is currently believed that the main bioactive components in Zedoary are volatile oils, including zedoary alcohol, zedoarondione, germacrone, beta-elemene, and neo-zedoarondione. Among them, germacrone, zedoarondione, zedoary alcohol, and beta-elemene have obvious physiological activities such as anti-tumor, anti-virus, and anti-bacterial activities, and beta-elemene has been widely used in clinical practice as a non-cell virus anticancer drug. In addition, Zedoary contains curcumin compounds, polysaccharides, and various trace elements. In particular, there is still a lack of reports on the structure and activity of polysaccharide components in Curcuma wenyujin. The present inventors have found that Curcuma wenyujin and its oil-extracted residues both contain a large amount of polysaccharide components, and have obvious regulatory effects on blood stasis-related diseases.

[0004] The production of Curcuma wenyujin is currently mainly used for industrial extraction of volatile oil, and the extraction rate is about 2%. The annual demand for Zedoary oil in China is about 40 tons. After extraction of volatile oil from Curcuma wenyujin, the residues are discarded. The polysaccharide components (CWP) in Curcuma wenyujin are not destroyed during the extraction of Zedoary oil, so the polysaccharide components can be obtained.

[0005] Chinese patent application CN202410218040.7 discloses a uniform polysaccharide SEP from Rhizoma Polygoni and Curcuma zedoary, a preparation method and application thereof. The polysaccharide SEP is obtained by mixing Rhizoma Polygoni and Curcuma zedoary at a ratio of 1:1, water decocting, and then separating and purifying. The polysaccharide SEP is composed of glucose, has a weight average molecular weight of 1.236 KDa, and has an anti-colorectal cancer effect. Compared with the polysaccharide SEP, the polysaccharide obtained by the present application is derived from the traditional Chinese medicine Curcuma zedoary and does not involve the traditional Chinese medicine Rhizoma Polygoni. The obtained homogeneous polysaccharide CWP-3 is a heteropolysaccharide, the monosaccharide composition of which includes Rha, GalA, Gal and Ara, and the molecular weight of the polysaccharide is greater than 400 KDa. The types and connection modes of the sugar residues are determined by using FTIR, GC-MS, NMR and other analysis techniques. The atomic force microscope (AFM) analysis shows that the CWP-3 has a clear triple helix structure. In addition, the in vitro culture of HUVEC cells and the replication of the gas stagnation and blood stasis type primary dysmenorrhea mouse model show that the CWP-3 has good antioxidant and anti-blood stasis effects.

[0006] In addition, Chinese patent application CN202210126413.9 discloses a polysaccharide nCKCP-2 obtained by water extraction, alcohol precipitation and separation and purification from Curcuma kwangsiensis S.G.Lee et C.F.Liang. The molecular weight of the polysaccharide is 76.4-214 kDa, and the monosaccharide composition thereof contains a large amount of Ara, Xyl, Gal, and a small amount of Man, Rha, Glc, GalA and GlcA. The polysaccharide can induce MSC2 cells to undergo apoptosis in the G0 / G1 phase, thereby reducing the effect of T cell proliferation inhibition, and may have an anti-tumor effect. Compared with the polysaccharide nCKCP-2, the present application relates to the reuse of Curcuma zedoary and the residue after the extraction of volatile oil from Curcuma zedoary. The molecular weight of the obtained product CWP-3 is greater than 400 kDa. The monosaccharide composition of the product CWP-3 is mainly composed of Rha, GalA, Gal and Ara. The product CWP-3 has different pharmacological effects and has antioxidant and anti-blood stasis effects. SUMMARY

[0007] In order to overcome the defects and deficiencies in the prior art and fill the gaps in the prior art, the present application provides an active polysaccharide CWP-3 extracted from Curcuma zedoary medicinal materials or residue after the extraction of volatile oil, a preparation method and use thereof.

[0008] Specifically, the present application is realized through the following technical solutions:

[0009] In a first aspect, the present application provides a polysaccharide CWP-3 separated from Curcuma zedoary medicinal materials or residue after the extraction of volatile oil. The polysaccharide is derived from Curcuma zedoary medicinal materials or residue after the extraction of volatile oil from the medicinal materials. The separated CWP-3 is an acidic polysaccharide, the total sugar content of which is greater than 65%, the uronic acid content of which is about 20%, the protein content of which is less than 5%, and the CWP-3 has good homogeneity.

[0010] As an alternative, in the above-mentioned Curcuma zedoaria polysaccharide CWP-3, the molecular weight of CWP-3 is 400-1000kDa; the monosaccharide composition contains a large amount of Rha, GalA and Gal, wherein Rha is mainly composed of →2)-α-L-Rhap-(1→ sugar residues; GalA is mainly composed of →4)-α-D-GalpA-(1→ sugar residues; Gal is mainly composed of →4)-β-D-Galp-(1→ and →6)-β-D-Galp-(1→ sugar residues; CWP-3 has a distinct triple helix structure.

[0011] Preferably, in the above-mentioned Curcuma zedoaria polysaccharide CWP-3, the molecular weight of CWP-3 is about 600 kDa.

[0012] Preferably, in the above-mentioned Curcuma zedoaria polysaccharide CWP-3, CWP-3 mainly contains rhamnose, galacturonic acid and galactose, and also contains a small amount of arabinose. The monosaccharide composition percentage is Rha:GalA:Gal:Ara = 24.59:29.63:36.77:9.01.

[0013] In a second aspect, the present invention provides a method for preparing the Curcuma zedoaria polysaccharide CWP-3 described in the first aspect above, the preparation method comprising the following steps:

[0014] The residue of Curcuma zedoaria or its extracted volatile oil was defatted by reflux with ethanol to obtain pretreated residue. A certain amount of amylase was added to the aqueous extract of the residue after reflux extraction, and the mixture was concentrated by rotary evaporation while incubating to remove starch. Sevage test solution was added to the concentrate, and the mixture was centrifuged after vigorous shaking to remove protein precipitate. The supernatant was collected, and Sevage test solution was added again to repeat the above steps until no protein rings appeared. 95% ethanol was added to the supernatant while stirring, and the mixture was allowed to stand overnight at low temperature. After filtration, precipitate 1 was collected. The filtrate was concentrated, and 95% ethanol was added again to repeat the above steps to collect precipitate 2. The two precipitates were combined and freeze-dried to obtain crude Curcuma zedoaria polysaccharide CWP. Homogeneous Curcuma zedoaria polysaccharide CWP-3 was obtained after separation and purification by column chromatography.

[0015] Alternatively, in the above-mentioned method for preparing Curcuma zedoaria polysaccharide CWP-3, the amylase is α-amylase, the incubation temperature range is 50-60℃, the pH value is 5.5-7.5, the rotary evaporation concentration temperature is 50-60℃, and the concentration volume is equivalent to 2-3 g / mL of the residue.

[0016] As an optional mode, in the preparation method of the above-mentioned CWP-3, the volume ratio of the concentrated solution to the Sevage reagent is 2:1-5:1; the shaking time is 10-20 min; the centrifugal speed is 6000-10000 r / min; the repeating number is 5-15 times, and the Sevage reagent is dichloromethane:n-butanol=4:1 in volume ratio.

[0017] As an optional mode, in the preparation method of the above-mentioned CWP-3, the amount of 95% ethanol added to the concentrated solution during the alcohol precipitation is 2-5 times; the standing time is 12-48 h; and the drying method of the crude polysaccharide precipitate after suction filtration is freeze drying.

[0018] As an optional mode, in the preparation method of the above-mentioned CWP-3, a DEAEcellucose-52 anion exchange column is used for separation and purification, the polysaccharide component having anthrone sulfuric acid coloration effect in the 0.4 mol / L NaCl elution component is collected, concentrated and dialyzed by using a dialysis bag with a molecular weight cut-off of 8000-14000 Da to remove NaCl and other small molecular substances, and vacuum freeze drying is adopted.

[0019] In a third aspect, the present application provides a use of the above-mentioned CWP-3 or the crude CWP or CWP-3 prepared by the above-mentioned preparation method in the preparation of a drug for treating blood stasis syndrome.

[0020] As an optional mode, in the above-mentioned use, the drug has antioxidant and in vivo anticoagulant activity.

[0021] As an optional mode, in the above-mentioned use, the drug is used for treating cardiovascular diseases or dysmenorrhea of the type of blood stasis due to stagnation of QI.

[0022] Preferably, the drug is used for treating primary dysmenorrhea of the type of blood stasis due to stagnation of QI.

[0023] Preferably, the drug is used for treating thrombus.

[0024] In a fourth aspect, the present application provides a use of the above-mentioned CWP-3 or the crude CWP or CWP-3 prepared by the above-mentioned preparation method in the preparation of a drug for treating functional constipation.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] (1) The product CWP-3 of the present application belongs to the re-development of the active part and active ingredient of traditional Chinese medicine Wenyin Zhi, and has certain innovation. In addition, the production raw material of the product of the present application relates to the reuse of waste residues after Wenyin Zhi oil extraction, and has the advantages of low cost, green safety, no toxic and side effects, reduces the damage of traditional Chinese medicine extraction to the environment, improves the utilization rate of traditional Chinese medicine Wenyin Zhi resources, and meets the sustainable development strategy.

[0027] (2) The Wenyin Zhi purified polysaccharide CWP-3 extracted by the method of the present application has good homogeneity, simple production process, high yield, good color and luster, and the physicochemical properties and structure are clearly characterized.

[0028] (3) The CWP or CWP-3 provided by the present application has the potential to be applied to the treatment of blood stasis syndrome, cardiovascular diseases, dysmenorrhea and functional constipation. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 : UV scanning diagram of CWP-3.

[0030] Figure 2 : FT-IR scanning diagram of CWP-3.

[0031] Figure 3 : HPGPC chromatogram of CWP-3.

[0032] Figure 4 : Chromatogram of monosaccharide composition of CWP-3 determined by PMP-HPLC method.

[0033] Figure 5 : AFM diagram of CWP-3.

[0034] Figure 6 : Scavenging ability of CWP-3 on DPPH, ABTS+· and ·OH.

[0035] Figure 7 : Influence of TMAO and CWP-3 on cell viability of HUVEC.

[0036] Figure 8 : Inhibitory effect of CWP-3 on ROS level increase of HUVEC cells caused by TMAO.

[0037] Figure 9 : Flow cytometry detection result of protective effect of CWP-3 on apoptosis of HUVEC cells caused by TMAO.

[0038] Figure 10 : Influence of CWP-3 on the latency period (A), the number of writhing (B) and the coagulation time (C) of mice with qi stagnation and blood stasis type primary dysmenorrhea.

[0039] Figure 11Effect of CWP-3 on the liver and brain index and uterus and brain index of the mice with primary dysmenorrhea of qi stagnation and blood stasis.

[0040] Figure 12 Effect of CWP-3 on the PGF2α and PGE2 contents in the uterus of the mice with primary dysmenorrhea of qi stagnation and blood stasis.

[0041] Figure 13 Effect of CWP-3 on the uterus morphology of the mice with primary dysmenorrhea of qi stagnation and blood stasis.

[0042] Figure 14 Effect of CWP on the tail thrombus model of mice.

[0043] Figure 15 Effect of CWP on the tail thrombus model of mice (left: tail length; right: coagulation time).

[0044] Figure 16 Effect of CWP on the serum SOD (left) and MDA (right) levels of the tail thrombus model of mice.

[0045] Figure 17 Effect of CWP on the stool morphology of the functional constipation model of rats (14th day).

[0046] Figure 18 Effect of CWP on the water content of the stool of the functional constipation model of rats.

[0047] Figure 19 Effect of CWP on the small intestine propulsion rate of the functional constipation model of rats (14th day).

[0048] Figure 20 Effect of CWP on the motilin (MTL) (left) and gastrin (GAS) (right) contents of the functional constipation model of rats.

[0049] Figure 21 Effect of CWP on the colon morphology of the functional constipation model of rats. DETAILED DESCRIPTION

[0050] The present application will be further described below with reference to the specific examples. It should be understood that the specific examples described herein are intended to serve only for the purpose of explanation and are not intended to limit the scope of the present application.

[0051] The specific techniques or conditions not specified in the examples were performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not specified by the manufacturer were all conventional products that can be purchased through regular channels.

[0052] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available products unless otherwise specified.

[0053] Preparation of Examples:

[0054] 1. Preparation of crude polysaccharide from Curcuma wenyujin Y.H.Chen et C.L.Shi

[0055] Preparation of CWP: A certain amount of Curcuma wenyujin Y.H.Chen et C.L.Shi residue powder was accurately weighed, crushed through a No.3 sieve, and added with 80% ethanol solution according to a solid-liquid ratio of 1:10 (g / mL) for reflux extraction for 5 h. The residue was collected by filtration and dried at 50°C. After drying, the residue was added with distilled water according to a solid-liquid ratio of 1:20 (g / mL) for extraction at 90°C for 1 h. The filtrate was collected by centrifugation and combined after repeated extraction for three times. A certain amount of α-amylase was added, and the mixture was incubated at 60°C in a water bath to remove starch while the extract was concentrated to a proper volume by rotary evaporation. Then, free proteins in the polysaccharide were removed by the Sevage method (dichloromethane:n-butanol = 4:1, v / v). The Sevage reagent and the extract were added into a sealed container according to a ratio of 1:4, and shaken vigorously for 20 min. The mixture was centrifuged at 4000 r / min for 10 min to remove the middle layer of milky protein and the lower layer of organic phase. The upper layer of water phase was collected and repeated until the middle layer of milky protein disappeared. Finally, 95% ethanol was added to the treated extract to a final concentration of 80% (v / v) ethanol, and the mixture was allowed to stand at 4°C for 24 h. The precipitate was obtained by centrifugation and freeze-dried to obtain crude polysaccharide CWP from Curcuma wenyujin Y.H.Chen et C.L.Shi.

[0056] 2. Separation and purification of CWP

[0057] DEAE-52 cellulose was mixed with a certain amount of water and packed into a column. CWP from Curcuma wenyujin Y.H.Chen et C.L.Shi was weighed and dissolved in distilled water by ultrasonic treatment. The polysaccharide solution was uniformly added to the column bed surface. After loading, the sample was allowed to adsorb on the column bed surface. Then, the column was eluted with distilled water, 0.1, 0.2, 0.3, 0.4 and 0.5 mol / L NaCl solutions at a flow rate of 0.8 mL / min. Each tube was collected for about 10 mL, and the absorbance was measured by the anthrone sulfuric acid method. Fractions with an absorbance value greater than 1.0 were combined, concentrated, and dialyzed in a dialysis bag with a molecular weight cutoff of 8000-14000 Da for 48 h. The freeze-dried product was used for further experiments. The results showed that the acidic polysaccharide CWP-3 was obtained from the 0.4 mol / L NaCl elution fraction after purification of CWP.

[0058] 3. UV and FTIR analysis of CWP-3

[0059] A certain amount of dried CWP-3 was prepared into a sample solution of 1.0 mg / mL, and water was used as a blank solvent. UV scanning was performed in the range of 190-400 nm. The results are shown inFigure 1 As shown in Figure 3, CWAP-3 does not show obvious absorption at 260 nm and 280 nm, so it can be judged that the CWP-3 obtained after preliminary separation and purification contains almost no protein and nucleic acid impurities.

[0060] Dry CWP-3 1 mg was weighed, ground with 10 times the amount of spectral pure KBr to make transparent pressed tablets, and infrared spectrum scanning analysis was performed in the range of 400-4000 cm -1 by using a Nicolet 5700 Fourier infrared spectrometer. The results are shown in Figure 4. Figure 2 As shown in Figure 4, the strong absorption peak at 3410 cm -1 is O-H stretching vibration; the absorption peak at 2920 cm -1 is C-H stretching vibration; the absorption peak at 1430 cm -1 is C-H bending vibration; the presence of the above three absorption peaks indicates that this substance is a polysaccharide component, and CWP-3 has this characteristic. The regions at 1735 cm -1 and 1616 cm -1 are respectively attributed to the presence of -COOR and -COOH, which is related to the content of uronic acid, and CWP-3 is an acidic polysaccharide, which has this absorption peak. 1200-1000 cm -1 is caused by two C-O stretching vibrations, and the absorption peak at 1098 cm -1 is attributed to the stretching vibration of C-OH and C-O-C bond, which can be inferred that the polysaccharide CWP-3 may exist in pyran ring. 1000-800 cm -1 is the fingerprint region of infrared spectrum, in which the configuration of polysaccharide can be observed. There are absorptions at 930 cm -1 and 890 cm -1 , indicating that both β configuration and α configuration exist in the polysaccharide CWP-3.

[0061] 4. Determination of total sugar, uronic acid and protein content of CWP-3

[0062] The anthrone sulfuric acid method was used to determine the total sugar content of polysaccharide CWP-3, with anhydrous glucose as the standard to prepare a standard curve; the m-phenolsulfonic acid method was used to determine the uronic acid content of CWP-3, with galacturonic acid as the standard to prepare a standard curve; and the BCA kit was used to determine the protein content of CWP-3 according to the instructions. The results showed that the total sugar content of CWP-3 was 65.3%; the uronic acid content was 23.8%, which is consistent with the result that it is an acidic polysaccharide; and the protein content was 2.6%.

[0063] 5. Determination of molecular weight and homogeneity

[0064] The average molecular weight and homogeneity of the polysaccharide were determined by high performance gel permeation chromatography (HPGPC). 2 mg of polysaccharide sample was weighed and dissolved in 2 mL of ultrapure water. After complete dissolution, the sample was filtered with an inorganic filter membrane (pore size of 0.22 μm) and the filtrate was loaded into a sample bottle for use. The detection system was an Agilent 1260 system equipped with an evaporative light scattering detector (ELSD). The chromatographic column was a TSK-GEL PW type column TSKgel G4000PWxL column. The mobile phase was ultrapure water, the flow rate was 0.5 mL / min, the column temperature was 40°C, and the injection volume was 10 μL. The molecular weight standard curve was plotted using the GBW(E) series of glucose standards. The results are shown in Figure 3 CWP-3 peak is single and symmetrical, and is a homogeneous polysaccharide with a molecular weight of about 600 kDa.

[0065] 6. Determination of monosaccharide composition of CWP-3

[0066] The chromatographic conditions were as follows: chromatographic column Inertsil ODS-3 (5 μm, 4.6 μm x 250 mm), mobile phase A acetonitrile, mobile phase B 0.05 mol / L phosphate buffer solution, pH 6.6, detection wavelength 250 nm, flow rate 0.8 mL / min, column temperature 30°C, injection volume 5 μL, elution conditions water: phosphate buffer solution = 17:83, elution time 70 min.

[0067] The elution results are shown in Figure 4 The monosaccharide composition ratio of the polysaccharide was calculated by peak area normalization method. CWP-3 mainly contains rhamnose, galacturonic acid and galactose, in addition to a small amount of arabinose. The percentage of monosaccharide composition is Rha: GalA: Gal: Ara = 24.59: 29.63: 36.77: 9.01.

[0068] 7. Methylation analysis of CWP-3

[0069] 50 mg of CWP-3 polysaccharide sample was precisely weighed, and after the steps of uronic acid reduction, methylation, acid hydrolysis, reduction and acetylation, partial methylation alditol acetate derivative (PMAA) was obtained. It was dissolved in 1.0 mL of chromatographic grade CH2Cl2, filtered through a 0.22 μm filter membrane, and then subjected to GC-MS analysis.

[0070] Detection condition: GC-MS analysis was used, and the chromatographic system used was a Shimadzu gas chromatograph system equipped with an HP-5MS chromatographic column (25 m x 0.22 mm x 0.25 μm), with high-purity helium as the carrier gas, 1 μL of sample injection, and a split ratio of 10:1. The initial temperature of the column oven was 60°C, which was increased to 160°C at 10°C / min, and then increased to 260°C at 4°C / min, and maintained for 5 min. A high-precision all-metal molybdenum quadrupole detection system was used, with an electron impact ion source (EI) temperature of 170°C, an electron energy of 70 eV, and a quadrupole rod temperature of 240°C. The spectrum was recorded in full scan mode.

[0071] The methylation results showed that the homogeneous polysaccharide CWP-3 contained 10 sugar residues: T-Araf, 2-linked Rhap, T-Galp, 2,4-linked Rhap, 4-linked Galp, 4-linked GalAp, 3-linked Galp, 6-linked Galp, 4,6-linked Galp, and 3,6-linked Galp, with a relative molar ratio of 1.9:21.3:4.3:3.2:9.1:25.6:4.2:6.7:5.2:5.6.

[0072] 8. NMR analysis of CWP-3

[0073] The structure of CWP-3 was characterized by nuclear magnetic resonance spectroscopy. The hydrogen signals and carbon signals of CWP-3 mainly appeared in the 3.0-5.5 ppm and 60-110 ppm regions of the 1 H and 13 C NMR spectra, respectively. Combined with the HSQC spectrum, 9 correlation peak signals were observed, including 5.16 / 109.16 ppm, 5.18 / 98.49 ppm, 5.32 / 99.55 ppm, 5.01 / 98.66 ppm, 4.91 / 97.47 ppm, 4.64 / 100.67 ppm, 4.41 / 102.67 ppm, 4.35 / 103.28 ppm, and 4.61 / 103.72 ppm, indicating that CWP-3 contains 9 sugar residues (named A, B, D, E, F, G, I, J, and K). Due to the characteristic high chemical shift of the anomeric signal of the α-arabinofuranosyl unit in the 13 C NMR spectrum, the signal at 109.16 ppm was assigned to the anomeric carbon of arabinose. Then, through HSQC, HMBC, and 1 H- 1H COSY spectra assigned signals of H2 / C2, H3 / C3, H4 / C4 and H5 / C5, respectively. Based on HSQC, HMBC spectra, monosaccharide composition analysis and methylation result analysis, two low field signals at 175.21 and 175.26 ppm were assigned to C-6 of uronic acid. The cross peaks in HSQC (1.88 / 22.12 ppm) and HMBC (1.88 / 173.41 ppm) spectra proved the presence of acetyl group. The combination of anomeric signal at 5.01 / 98.66 ppm in HMQC spectrum, low field shift of C-4 (78.86 ppm) and the correlation peak at 4.67 / 175.21 ppm confirmed that residue E was (1→4)-linked glucuronic acid. Similarly, the anomeric signal at 4.91 / 97.47 ppm in HMQC spectrum, low field shift of C-3 (80.08, 81.70 ppm) and the correlation peak at 4.73 / 175.26 ppm in HMBC spectrum confirmed that residue F was →4)-3-OAc-α-D-GalpA-(1→. In addition, the correlation peak around 1.2 / 16.5 ppm was obviously assigned to the methyl group of α-L-Rhap (H6 / C6), which was further assigned to →2)-α-L-Rhap-(1→ and →2,4)-α-L-Rhap-(1→, based on the data of HSQC and HMBC spectra, and corresponding to the methylation result. Based on the data of HSQC and HMBC spectra, and the result of methylation data analysis, the complete description of residues A, B, D, E, F, G, I, J and K was shown in Table 1. The comprehensive results showed that CWP-3 was composed of →2)-α-L-Rhap-(1→ and →4)-α-D-GalpA-(1→ linked alternately, in which the 2 or 3 position of galacturonic acid was connected with acetyl group; on the 4th position of rhamnose, there was a neutral side chain, mainly composed of polygalactose of →4)-β-D-Galp-(1→ and →6)-β-D-Galp-(1→, in addition, there was a small amount of arabinose.

[0074] Table 1 The assignment of signals of CWP-3 1 H and 13 C signals

[0075]

[0076] 9. Atomic force microscopy analysis of CWP-3

[0077] CWP-3 (1 mg) was dissolved in deionized water and diluted to 10 μg / mL. 10 μL of the diluted solution was dropped on the surface of freshly cleaved mica and dried at room temperature. The sample was imaged using a Bruker Dimension Icon AFM instrument, with a scanning range of 5 μm*5 μm. Image analysis was performed using Nano Scope Analysis. The analysis results are shown in Figure 9.Figure 5 As shown, CWP-3 presents a clear triple helix structure.

[0078] Effect implementation examples:

[0079] 1. In vitro antioxidant activity of CWP-3

[0080] The antioxidant activity of CWP-3 was evaluated by investigating the scavenging capacity of polysaccharides on 1,1-diphenyl-2-trinitrophenylhydrazine free radical (DPPH), 2,2-diamine-bis (3-ethyl-benzothiazoline-6-sulfonic acid) diamine salt free radical (ABTS+·) and hydroxyl radical (·OH).

[0081] The results are shown in Figure 6 As shown, it was found that the scavenging rate of CWP-3 on DPPH was 43.95% at a concentration of 2.5 mg / mL, the scavenging rate on ABTS+· was 47.88%, and the scavenging rate on ·OH was as high as 61.61%, indicating that CWP-3 had certain antioxidant activity.

[0082] 2. Protective effect of CWP-3 on HUVEC cells

[0083] 2.1 CCK-8 experiment

[0084] Logarithmic growth phase human umbilical vein endothelial cells (HUVEC) were seeded in a 96-well plate at a density of 5000 cells / well, and incubated with 5% serum medium containing 0, 25, 50, 100 and 200 μg / mL of oxidized trimethylamine (TMAO) for 24 h, respectively. The effect of different concentrations of TMAO on the survival of HUVEC cells was detected by CCK-8 method to obtain a suitable modeling concentration.

[0085] The cell viability of cells incubated with serum-free medium containing 0, 25, 50, 100 and 200 μg / mL of CWP-3 for 36 h was detected by the same method to determine whether CWP-3 had cytotoxic or cell proliferation effect on HUVEC cells.

[0086] The results are shown in Figure 7 As shown, compared with the normal group, the cell viability of HUVEC cells incubated with 200 μg / mL of TMAO was significantly decreased, and 200 μg / mL concentration was finally selected as the modeling concentration for subsequent experiments.

[0087] In addition, compared with the normal group, the cell viability of HUVEC cells incubated with serum-free medium containing different concentrations of CWP-3 increased in a dose-dependent manner compared with the blank group, indicating that CWP-3 had significant activity in promoting cell proliferation. Finally, 25, 50 and 100 μg / mL drug concentrations were selected as low (CWP-3L), medium (CWP-3M) and high dose groups (CWP-3H) for subsequent experiments.

[0088] 2.2 Detection of reactive oxygen species

[0089] First, HUVEC cells 8 x 10 4 were seeded in a 6-well plate and cultured with medium containing 10% fetal bovine serum for 12 h. The original culture medium was discarded, and the normal and model groups were added with serum-free medium, and the low, medium and high drug groups were added with serum-free medium containing 25, 50 and 100 μg / mL CWP-3, respectively, and cultured for 24 h. Then 5% serum medium containing TMAO was added to each well to make the final concentration of TMAO 200 μg / mL and cultured for 12-24 h, with a cell confluence of 70%. Then the DCFH-DA probe was loaded in situ according to the ROS kit instructions, and the data were analyzed and optimized by FlowJo_V10 software. The ROS level of the normal group was close to 0, and the other groups were batched out according to the normal group treatment.

[0090] The results are shown in Figure 8 Compared with the blank group, the ROS content of the model group (TMAO) increased significantly; compared with the TMAO group, the ROS content of each drug group decreased significantly and showed a dose-dependent manner. It is proved that CWP-3 can significantly inhibit the production of reactive oxygen species in HUVEC cells caused by TMAO.

[0091] 2.3 Annexin V-FITC / PI double staining

[0092] The cells were treated by the same method as in the active oxygen detection, and then the Annexin V-FITC / PI double staining kit was used to load the probe after digestion and flow cytometry detection according to the instructions.

[0093] The results are shown in Figure 9 Compared with the normal control group, the proportion of double positive in the first quadrant of the TMAO group increased significantly, while the early apoptotic cells in the fourth quadrant were almost none, indicating that TMAO caused pyroptosis in HUVEC cells. After drug administration, the proportion of double positive decreased in a dose-dependent manner compared with the TMAO group, indicating that CWP-3 indeed had a significant effect on inhibiting cell pyroptosis.

[0094] 3. Improvement of CWP-3 on mice with dysmenorrhea of stagnation of Qi and blood stasis

[0095] 3.1 Animal source

[0096] 30 SPF female non-pregnant and non-mated KM mice (body weight 20±2g), 6-8 weeks old, provided by Changsheng Biotechnology Co., Ltd. (SCXK(Liaoning)-2020-0001). Adapted to feed for 1 week before the experiment, free access to food and water, laboratory temperature 25±1℃, relative humidity 60±5%.

[0097] 3.2 Experimental method

[0098] The experimental animals were randomly divided into blank control group (Control), model group (Model), ibuprofen group (ibuprofen), CWP-3 low dose group (CWP-3L) and high dose group (CWP-3H), 6 in each group. According to the dosage of Zedoary Turmeric in Chinese Pharmacopoeia 2020 (Part I) is 6-9g, take the average value 7.5g, the low dose group is converted into the dosage of mice according to the clinical equivalent dose, which is equivalent to the crude drug amount of 0.79g / kg, and then converted to about 0.4g / kg according to the extraction rate of CWP-3 of 0.5%; the high dose group is converted by 4 times of the clinical equivalent dose, with a dosage of 1.2g / kg. The ibuprofen group is converted into 0.06g / kg according to the same method of clinical equivalent dose.

[0099] The method of "comprehensive environmental stimulation + hydrochloric acid adrenaline" was used to establish the blood stasis model of mice, and the method of "benzoic acid estradiol + oxytocin" was used to establish the primary dysmenorrhea model of mice.

[0100] Except for the blank control group, each group of mice was injected with 0.1% hydrochloric acid adrenaline (0.9mg / kg / d) for 4h, and then the random comprehensive stimulation was started (A: sound stimulation (60dB, (10±5)kHz, intensity level 3), 10min / time, 1 time / day; B: light stimulation (flickering light, frequency (2±1)Hz), 10min / time, 1 time / day; C: restraint cylinder restraint, 10min / time, 1 time / day; D: book clip tail, 10min / time, 1 time / day; E: 0-4℃ ice water bath, 5min / time, 1 time / day). On the 1st and 10th day, each mouse was given benzoic acid estradiol 2.5mg / kg / d subcutaneously, and on the 2nd-9th day, the dosage of benzoic acid estradiol was 1.25mg / kg / d. From the 4th day, each group was given gavage, once a day, for 7 consecutive days. On the 10th day of modeling, except for the blank control group, each group of mice was given oxytocin (1U / each) intraperitoneally after injection of benzoic acid estradiol, and the blank control group was given intraperitoneal injection of the same volume of normal saline.

[0101] In addition, the body weight and hair color of the mice were observed and recorded daily to ensure the success of the establishment of the primary dysmenorrhea model of mice with qi stagnation and blood stasis.

[0102] Pharmacodynamic index determination

[0103] (1) Observation of mouse writhing reaction

[0104] On the 10th day of modeling, after intraperitoneal injection of oxytocin in mice of each group, the writhing times and writhing latency of mice within 0-30 min were recorded and counted by camera.

[0105] (2) Determination of mouse blood coagulation time and viscera-brain index

[0106] After recording the mouse writhing reaction for 30 min, the eyeball was enucleated to collect blood, and the glass plate method was immediately used to determine the blood coagulation time. The mouse liver, uterus and brain were dissected and weighed accurately with a one-hundredth balance. The blood coagulation time and viscera-brain ratio of mice in each group were compared.

[0107] (3) Detection of mouse dysmenorrhea-related factor content

[0108] After the mouse blood collection was completed, the mouse uterus was taken, the connective tissue was removed, and the ice physiological saline was rinsed, the blood was removed, and the uterus tissue was dried. About 100 mg of uterus tissue was accurately weighed, placed in an ice water bath beaker, 1 mL of ice physiological saline was added per 100 mg of uterus tissue, the tissue was cut as soon as possible, placed in a homogenizer tube, and placed in an ultrasonic wave crusher to homogenize the tissue. Then, it was transferred to a 4℃ refrigerated centrifuge, centrifuged at 3000 r / min for 15 min, and the supernatant was taken. The PGF2α and PGE2 levels of each sample group were determined by ELISA method. The determination was strictly performed according to the kit instructions.

[0109] (4) Histopathological observation of mouse uterus tissue

[0110] The remaining part of the mouse uterus was fixed in 4% paraformaldehyde solution, frozen sectioned, HE stained, and observed under an optical microscope for pathological changes in the uterus of mice in each group.

[0111] (5) Statistical analysis

[0112] The data were statistically plotted and analyzed by using GraphPad Prsim 8.0 statistical plotting software. The experimental data were expressed as mean ± standard deviation (Mean ± SD), and P<0.05 was statistically significant.

[0113] 3.3 Experimental results

[0114] As Figure 10 A and Figure 10As shown in B, normal mice did not appear writhing reaction, while model mice appeared severe writhing reaction, which was manifested in short writhing latency and high writhing frequency. The average writhing latency of mice was 2.13 min, and the average writhing frequency was 37 times (P<0.001). Compared with model mice, the average writhing latency of mice in CWP-3L group was 4.69 min, and the average writhing frequency was 17 times (P<0.05). The average writhing latency of mice in CWP-3H group was 12.5 min, and the average writhing frequency was 11 times, and 2 mice did not appear writhing reaction (P<0.01). The average writhing latency of mice in positive drug ibuprofen group was 19 min, and the average writhing frequency was 5 times, and 3 mice did not appear writhing reaction (P<0.001). However, due to the large difference within each experimental group, the statistical results were not significant.

[0115] As shown in Figure 10 As shown in C, the average blood clotting time of normal mice was 3.5 min. Compared with normal mice, the blood clotting time of model mice showed a decreasing trend, and the average blood clotting time was 1.6 min (P<0.001). Compared with model mice, the blood clotting time of mice in each administration group was prolonged. The average blood clotting time of mice in CWP-3L group was 1.7 min (P<0.05), the average blood clotting time of mice in CWP-3H group was 2.2 min (P<0.01), and the blood clotting time of mice in positive drug ibuprofen group was 3.1 min (P<0.001). However, due to the large difference within each group, the same was not significant.

[0116] The results of mouse viscera-brain index are shown in Figure 11 Compared with normal mice, the liver-brain index and uterus-brain index of model mice were significantly increased (P<0.001), indicating that the liver and uterus of the mice were abnormal, and the model was successfully replicated. Compared with model mice, the liver-brain index and uterus-brain index of mice in each administration group were significantly decreased, indicating that CWP-3 had obvious protective effect on liver and uterus damage caused by dysmenorrhea of blood stasis and blood stasis type. Compared with model mice, CWP-H group was the most significant (P<0.01), and the remaining administration groups showed a decreasing trend, and there was no significant difference between groups.

[0117] The contents of two kinds of prostaglandins related to blood stasis and dysmenorrhea in mouse uterus tissue were detected by ELISA method, and the results are shown in Figure 12The abnormal PGF2a / PGE2 caused uterine smooth muscle spasm is the main pathophysiological mechanism of primary dysmenorrhea, which can be used as one of the main pharmacodynamic indexes of blood stasis and dysmenorrhea. The experimental results show that compared with the normal group, the PGF2a content in the model group is significantly increased (P<0.05), and the PGE2 content in the CWP-3 low and high dose groups has a decreasing trend. Among them, the PGF2a content in the CWP-3 high dose group is significantly reduced (P<0.05). The PGE2 content in the model group is significantly reduced, and the PGE2 content in the CWP-L and CWP-H groups is significantly increased after administration (P<0.01). The ratio of GF2a / PGE2 in the model group is significantly increased (P<0.01), and is significantly reduced after administration of CWP, and has a dose-dependent effect (P<0.01). It shows that CWP-3 can play a therapeutic role in primary dysmenorrhea mice by regulating the levels of PGF2a and PGE2.

[0118] Figure 13 The results of HE staining of uterine tissue sections of mice in each group are shown. The uterine structure of the normal group of mice is clear, and is divided into three layers from inside to outside, which are endometrium, myometrium and perimetrium. The endometrial epithelial cells are complete, the glandular distribution of the lamina propria is normal, and the thickness of the endometrium and myometrium is normal. The uterine structure of the model group of mice is disordered, the uterine cavity is not smooth, the glandular distribution of the lamina propria is abnormal, the glands are hypertrophic, the endometrial hyperplasia is obvious, the myometrial hyperplasia is obvious, and the inflammatory cell infiltration is obvious. Different doses of drug treatment groups show different degrees of improvement, among which the CWP-3H group shows more obvious improvement, which is reflected in clearer structure layering, reduced gland hypertrophy and reduced inflammatory cell infiltration. This shows that CWP-3 has obvious protective effect on the uterine tissue of mice with dysmenorrhea of stagnation of Qi and blood stasis.

[0119] 4. Anti-thrombotic activity of CWP

[0120] 4.1 Animal source

[0121] Healthy adult male KM mice, 60 (SPF level), weighing 20±2g, were provided by Liaoning Changsheng Biotechnology Co., Ltd., with a qualified certificate number of SCXK(Liaoning)-2020-0001.

[0122] 4.2 Experimental method

[0123] KM mice were randomly divided into 6 groups, 10 in each group, namely, blank group (Control), aspirin group (Aspirin), model group (Model), CWP low-dose group (CWP-L), CWP medium-dose group (CWP-M), and CWP high-dose group (CWP-H). The aspirin group was given gavage at a dose of 20 mg / kg, the CWP low-, medium-, and high-dose groups were given gavage at a dose of 200 mg·kg-1, 400 mg·kg-1, and 600 mg·kg-1, respectively, the blank control group was given gavage with the same volume of normal saline, once a day, for 7 consecutive days.

[0124] At 1 h after the 7th administration, the mice in each group were intraperitoneally injected with 20 mg / kg of carrageenan, and the mortality rate, black tail length, and black tail incidence were observed 24 h later. The capillary tube method was used to measure the changes in blood clotting time, 1 mL of blood was taken by enucleation, and the mice were sacrificed by cervical dislocation, and the ears and tails of the mice were cut off and fixed with 10% neutral formalin solution. The obtained blood was immediately centrifuged at 4000 r / min for 20 min, and the supernatant was collected as serum. The biochemical indicators included SOD and MDA, and all operations were performed according to the instructions of the kit.

[0125] All data in the experiment were expressed as mean ± standard deviation (Mean ± SD), and the data were processed by SPSS 19.1 statistical software. The mean values between groups were compared by one-way analysis of variance, and P<0.05 was considered statistically significant.

[0126] 4.3 Experimental results

[0127] After intraperitoneal injection of carrageenan for 24 h, the mortality rate of the model group was 20%, the mortality rate of the CWP-L group was 20%, and no death occurred in the other groups. As shown in Figure 14 and Figure 15 , the black tail length and black tail incidence determination results showed that compared with the blank group, the mice in each group intraperitoneally injected with carrageenan showed a certain degree of black tail phenomenon, among which the black tail incidence (100%) and black tail length of the model group were the most significant (P<0.001), and the ears of the mice also showed obvious congestion (see Figure 14 ), indicating that the hemorheology of the mice changed and the thrombus model was successfully made. After the positive control group was given aspirin for protection, the black tail incidence and black tail length significantly decreased (P<0.01), and the color of the black tail was lighter, and no obvious congestion occurred in the ears. After being given low, medium, and high concentrations of CWP, the black tail length and black tail incidence of each group were improved (see Figure 14 and Figure 15The CWP-H group showed a more significant effect, with a significant decrease in both black tail length and incidence (P<0.05), demonstrating a protective effect similar to that of aspirin. While the CWP-L group showed a slight decrease in black tail length and incidence, severe black tail phenomena still occurred, with no significant difference compared to the model group. CWP can significantly improve tail thrombosis and ear congestion in thrombotic mice.

[0128] Capillary coagulation time assays showed that, compared with the control group, the coagulation time in the model group was significantly decreased (P<0.001), indicating that the blood in the model group exhibited hypercoagulable characteristics. Compared with the model group, the coagulation time in both the aspirin group and the CWP-H group was prolonged, with significant differences (P<0.01). These results indicate that CWP has a certain protective effect on thrombosis model mice, and the CWP-H group showed better efficacy (see...). Figure 15 ).

[0129] like Figure 16 As shown in the results obtained by the SOD and MDA oxidative stress assay kits, compared with the blank group, the serum SOD level of mice in the model group was significantly decreased (P<0.01), and the MDA level was significantly increased (P<0.01). Carrageenan is a potent inflammatory substance that can lead to thrombosis through local vascular inflammation and damage to vascular endothelial cells, as evidenced by the decreased SOD and MDA levels in the model group. Compared with the model group, the serum SOD level of mice in the aspirin group and the high-dose CWP group was significantly increased (P<0.01), and the MDA level was significantly decreased (P<0.01), demonstrating a significant protective effect against oxidative stress in the serum of thrombotic model mice and alleviating the carrageenan-induced inflammatory response. The effect of the high-dose CWP group was comparable to that of aspirin. The protective effect of CWP on thrombotic mice may exert its effect by regulating their oxidative stress level to a normal level.

[0130] 5. CWP's effect on functional constipation

[0131] 5.1 Animal source

[0132] Forty-eight healthy adult male SD rats (SPF grade), weighing 180±20g, were provided by Liaoning Changsheng Biotechnology Co., Ltd., certificate number SCXK(Liaoning)-2020-0001. They were allowed to acclimatize for 7 days in a free-range food and water environment before being used in the experiments.

[0133] 5.2 Experimental Methods

[0134] Forty-eight SD rats were first adaptively fed for 1 week (temperature 25±2℃, light 12h / d, humidity 40-45%), fed with a standard feed in a fixed amount, and free water. According to the body weight, they were randomly divided into 6 groups, 8 rats in each group, and were set as a blank group (Control), a mosapride group (Mos), a model group (Model), a CWP low-dose group (CWP-L), a CWP medium-dose group (CWP-M) and a CWP high-dose group (CWP-H). An appropriate amount of loperamide hydrochloride was prepared into a suspension with a proportion of 5mg / kg and 0.5mL / 100g body weight of normal saline. Except for the blank group, the rest of the groups were subcutaneously injected every day at 9:00 and 18:00 for 7 days, and the blank group was subcutaneously injected with an equal amount of normal saline.

[0135] When the rats showed poor mental state, reduced food and water intake, weight loss, small and hard stool, and reduced defecation, etc., it was indicated that the modeling was successful. From the 8th day, except for the blank group, the rats in each group were subcutaneously injected with loperamide hydrochloride once a day, and the rats in each treatment group were given the corresponding drugs by gavage, and the blank group was given an equal amount of distilled water. The body weight of the rats was recorded every day, and the drug dosage was adjusted according to the body weight change; the general condition of the animals including appetite, behavior, hair, fecal condition and death was observed every day in the first week, and then once every 1 day.

[0136] The results were analyzed and processed using SPSS17.1 software, one-way ANOVA was used to compare the differences between groups, and variance homogeneity test was performed, P<0.05 was considered statistically significant.

[0137] 5.3 Experimental results

[0138] 5.3.1 Effect of CWP on the body weight of rats

[0139] During the modeling period, the diet and activity of each rat in the blank group remained normal, the food intake of each rat in each modeling group decreased to varying degrees, the body weight also decreased with time, the rat hair was dry and yellow, and the rat was weak and irritable. After the second week of treatment with the drug, the body weight of each treatment group gradually returned to the original level, and the body weight of the model group also increased slightly with time, but the growth was slow compared with the treatment group, such as Figure 13 The experimental results showed that the positive drug mosapride and warm turmeric polysaccharide could improve the trend of weight loss in constipated model rats, and the effect of CWP-H group was better than that of the medium and low dose groups, and slightly inferior to that of the positive drug mosapride.

[0140] 5.3.2 Effect of CWP on the fecal morphology of rats

[0141] During the experiment, the feces of the blank group rats remained stable, and the feces shape remained normal. The feces of the model group rats decreased in number and became smaller in shape, with a dry and hard texture, and the anus was red and swollen, showing obvious symptoms of constipation, indicating that the functional constipation model was successfully established.

[0142] After administration of the therapeutic drugs, the feces shape and texture of the rats in the positive drug mosapride group and the various administration groups of cumin polysaccharide were improved. For example, Figure 17 As shown, the fecal particles of the model group rats became smaller, the surface was dry, and the number and water content were significantly reduced. The feces of the rats in the positive drug mosapride group and the high-dose cumin polysaccharide group basically returned to normal, with complete fecal particles and moisture. The feces of the rats in the low-dose and medium-dose groups still contained a small amount of small particles, but the overall trend improved.

[0143] 5.3.3 Effect of CWP on the 24h fecal water content of rats

[0144] During the experiment, the 24h feces of the blank group rats remained stable, and the feces water content remained stable. The feces of the model group rats decreased in frequency, became smaller and harder, and the feces water content decreased significantly. After administration of the therapeutic drugs from the second week, the 24h feces of each treatment group improved, such as Figure 18 As shown. The experimental results show that the positive drug mosapride and cumin polysaccharide high-dose and medium-dose groups can improve the decreasing trend of fecal water content in constipation model rats, and restore it to near normal levels. The high-dose cumin polysaccharide group is better than the low-dose and medium-dose groups, and slightly inferior to the positive drug mosapride group.

[0145] 5.3.4 Effect of CWP on small intestinal propulsion rate

[0146] After the experiment, the stomach and intestinal tissues of each group were surgically removed, carefully separated, and opened. The results are shown in Figure 19 The small intestinal propulsion rate of the model group was significantly lower than that of the treatment group and the blank group (P<0.01). Since the small intestinal propulsion rate experiment can reflect the strength of the gastrointestinal peristalsis function under the same time, the significant decrease in the small intestinal propulsion rate of the model group reflects the consistency of the model with the expectation. Compared with the model group, the small intestinal propulsion rate of the positive control group, the high-dose, medium-dose, and low-dose cumin polysaccharide groups increased, among which the high-dose cumin polysaccharide group increased significantly (P<0.05) and was not significantly different from the positive drug mosapride group. The medium-dose and low-dose cumin polysaccharide groups were not significantly different due to large errors. This shows that high-dose cumin polysaccharide can effectively increase the small intestinal propulsion rate of rats and promote gastrointestinal peristalsis, thereby resisting the slowing down of gastrointestinal movement caused by loperamide hydrochloride.

[0147] 5.3.5 Effect of CWP on the content of motilin MTL and gastrin GAS in rat serum

[0148] The results of the measurement of biochemical indicators of gastrointestinal hormones related to gastrointestinal motility in the serum of experimental rats are shown in the figure. Figure 20 Compared with the control group, the levels of motilin (MTL) and gastrin (GAS) in the serum of rats in the model group were significantly reduced (P<0.05); indicating that the subcutaneous injection of loperamide hydrochloride to establish the model led to a decrease in the levels of motilin and gastrin in the serum of rats in the model group, which in turn inhibited gastrointestinal motility and produced symptoms of functional constipation.

[0149] Compared with the model group, the levels of GAS and MTL in the mosapride positive control group and the high-dose Curcuma zedoaria polysaccharide group in the treatment group were significantly increased (P<0.05). The medium- and low-dose Curcuma zedoaria polysaccharide groups showed some improvement, but there was no significant difference. This indicates that the high-dose Curcuma zedoaria polysaccharide can effectively increase the levels of motilin GAS and gastrin MTL in rat serum. Curcuma zedoaria polysaccharide counteracts the gastrointestinal motility disorder caused by loperamide hydrochloride by increasing the level of gastrointestinal hormones related to gastrointestinal motility in serum.

[0150] 5.3.6 Pathological section results of rat colon

[0151] The effects of loperamide hydrochloride on colonic pathology in rats of different groups were observed in HE-stained sections. Figure 21 In the control group, the colonic mucosal layer thickness of all rats was normal, the epithelial cells were regularly arranged, the goblet cells were normally morphological, and the colonic muscle layer thickness was normal. Loperamide hydrochloride caused thinning of the colonic mucosal layer, a large accumulation of inflammatory cells, a reduction in goblet cells, and significant thinning of the muscle layer in constipated rats, indicating that loperamide hydrochloride altered the colonic morphology and affected colonic function, leading to constipation. After administration of various concentrations of Curcuma zedoaria polysaccharide, the colonic morphology of rats in all groups showed some improvement. In the positive control group (mosapride), the mucosal epithelial cells were intact and regularly arranged, the goblet cells of the colonic glands in the lamina propria were normally morphological, with a small number of goblet cells missing (black arrows), and the gel layer on the mucosa was thinner. In the low-dose group, occasional damage and loss of mucosal epithelial cells, loss of goblet cells of the colonic glands in the lamina propria (red arrows), scattered inflammatory cell infiltration in the lamina propria and submucosa, and thinning of the gel layer on the mucosa were observed. In the medium-dose group, occasional loss of goblet cells in the colonic glands and thinning of the colonic mucosa were observed in the lamina propria, but no obvious inflammatory cell infiltration was observed, and the gel layer on the mucosa was slightly thinner. In the high-dose group, the mucosal epithelial cells were intact, neatly arranged, and regular, the thickness of the colonic mucosa was normal, the morphology of the colonic gland goblet cells in the lamina propria was normal, the number was not reduced, and the thickness of the gel layer on the mucosa was basically restored to normal.

[0152] 5.3.7 Overall Conclusion

[0153] In summary, by subcutaneously injecting loperamide hydrochloride to SD rats to copy functional constipation model, and giving CWP to observe the disease course changes of rats, the results show that high dose of CWP has good therapeutic effect on functional constipation of rats, significantly improves the defecation and gastrointestinal motility of rats, increases the MTL and GAS levels in the plasma of rats, and reduces the inflammatory response of the colon tissue of rats.

[0154] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. The use of Curcuma zedoaria crude polysaccharide CWP or Curcuma zedoaria polysaccharide CWP-3 in the preparation of drugs for treating thrombosis, dysmenorrhea, or functional constipation, characterized in that: The preparation method of the crude polysaccharide CWP or the polysaccharide CWP-3 of Curcuma Wenyujin includes the following steps: The pre-processed residue is obtained by refluxing and degreasing the Curcuma Wenyujin medicinal material or residue after extracting volatile oil with ethanol; a certain amount of amylase is added to the water extract after reflux extraction, and the concentrated solution is incubated to remove starch while being concentrated by rotary evaporation; Sevage reagent is added to the concentrated solution, and after shaking, the supernatant is collected by centrifugation to remove protein precipitate; the Sevage reagent is repeatedly added to the supernatant until no protein ring appears; 95% ethanol is added to the supernatant, and after standing overnight at low temperature, the precipitate 1 is collected by suction filtration; the filtrate is concentrated and 95% ethanol is added again to repeat the above steps, and the precipitate 2 is collected; the two precipitates are combined, and the crude polysaccharide CWP of Curcuma Wenyujin is obtained by freeze-drying; and the homogeneous polysaccharide CWP-3 of Curcuma Wenyujin is obtained by column chromatography separation and purification.

2. Use according to claim 1, characterized in that: The amylase is α - amylase, incubation temperature range 50-60°C, pH 5.5-7.5; spin concentration temperature 50-60°C.

3. Use according to claim 1, characterized in that: The volume ratio of the concentrated solution to the Sevage reagent is 2:1-5:1; the shaking time is 10-40 min; the centrifugal speed is 5000-10000 r / min; the number of repetitions is 5-15 times, and the Sevage reagent is dichloromethane:n-butanol=4:1 by volume ratio.

4. Use according to claim 1, characterized in that: When the alcohol precipitation is performed, 95% ethanol is added to the concentrated solution in an amount of 2-5 times; the standing time is 12-48 h; and the drying method of the crude polysaccharide precipitate after suction filtration is freeze-drying.

5. Use according to claim 1, characterized in that: The DEAE cellucose-52 anion exchange column is used for separation and purification, the polysaccharide component having anthrone sulfuric acid coloration effect is collected from the 0.4 mol / L NaCl elution component, the concentrated components are combined and dialyzed by using a dialysis bag with a molecular weight cutoff of 8000-14000 Da to remove NaCl and other small molecular substances, and vacuum freeze-drying is performed.

6. Use according to claim 1, characterized in that: The polysaccharide CWP-3 of Curcuma Wenyujin is an acidic polysaccharide, the total sugar content is more than 65%, the uronic acid content is about 20%, the protein content is less than 5%, and the polysaccharide has good homogeneity.

7. Use according to claim 6, characterized in that: The molecular weight of the CWP-3 is 400-1000 kDa; the monosaccharide composition contains a large amount of Rha, GalA and Gal, wherein the Rha is mainly in →2)- α -L-Rha p -(1→ sugar residues; the GalA is mainly in →4)- α -D-Gal p A-(1→ sugar residues; the Gal is mainly in →4)- β -D-Gal p -(1→ and →6)- β -D-Gal p -(1→ sugar residues; the CWP-3 has a clear triple helix structure.

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