Medicinal plant curcuma wenyujin homogeneous polysaccharide and application of medicinal plant curcuma wenyujin homogeneous polysaccharide in immunoregulation

Abstract

The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a medicinal plant curcuma wenyujin homogeneous polysaccharide and application thereof in immunoregulation. The curcuma wenyujin homogeneous polysaccharide is a new homogeneous polysaccharide compound found for the first time, the curcuma wenyujin homogeneous polysaccharide is composed of mannose (Man) and glucose (Glu), the molar ratios are 20.51% and 79.49% respectively, and the molecular weight is 28.1 KDa. The preparation method and process parameters of the curcuma wenyujin homogeneous polysaccharide are provided, the preparation method is suitable for industrial production, a basis is provided for quality control and standardized production of the curcuma wenyujin homogeneous polysaccharide, and the application range of the curcuma wenyujin homogeneous polysaccharide is widened. The curcuma wenyujin homogeneous polysaccharide (WP-1G) prepared by the invention has immunomodulatory activity, and provides a basis for developing a potential new drug for enhancing immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a uniform polysaccharide of a medicinal plant Curcuma zedoariae and an application thereof in immunoregulation. Background Art

[0002] Polysaccharide, also known as polysaccharide, is a high molecular carbohydrate composed of at least 10 monosaccharides polymerized through α- or β-glycosidic bonds. It usually has a long straight chain as the main chain with a large number of branches on the main chain. Polysaccharides are widely present in animals, plants and microorganisms. They are an important class of biological macromolecules with a wide range of biological effects. Polysaccharides not only provide energy for life activities, but some polysaccharides can also form cell skeletons such as cellulose and chitin. With the development of analytical detection technology, it has been found that polysaccharide compounds from various plants, animals or microorganisms have a series of biological activities, such as anti-tumor, anti-oxidation, immune enhancement, lowering blood pressure and regulating blood lipids.

[0003] Studies have found that polysaccharides from traditional Chinese medicine / natural medicines have the effect of regulating immunity. Macrophages play an important role in the host defense system, which produces various inflammatory mediators, cytokines and phagocytic activity to attack invaders such as bacteria or viruses. Plant polysaccharides can promote the proliferation of macrophages, enhance the phagocytic function of macrophages, and stimulate the release of cytokines such as NO, TNF-α and IL-6.

[0004] Wen Yujin ( Curcuma wenyujin YH chert et C. Ling) has a long history of medicinal use and is one of the famous "Eight Zhejiang Flavors". It is a perennial herb of the genus Curcuma in the Zingiberaceae family. It was first recorded in the "New Compendium of Materia Medica". It is mainly cultivated for medicinal purposes, and its wild distribution is rare. Curcuma wenyujin is a "three-purpose" medicinal plant. Its dried root is called "curcuma", which has the effects of promoting blood circulation and relieving pain, promoting qi and relieving depression, clearing the heart and cooling blood, and promoting gallbladder and relieving jaundice. At present, there are hundreds of compounds isolated from Curcuma wenyujin, mainly including volatile oil (2.05%), curcumin (1.46%), polysaccharides (8.90%), etc. Polysaccharides are one of the important components of Curcuma wenyujin, and the structure of the polysaccharide components of Curcuma wenyujin is still unclear. In the case of few research reports on the homogeneity of the activity of Curcuma wenyujin, the separation and structure of polysaccharides, and the pharmacological activity, the research results of the present invention are particularly important. Summary of the invention

[0005] The present invention aims to provide a uniform polysaccharide of the medicinal plant Curcuma aromatica and apply it to immunoregulation, so as to overcome the defects of the prior art that the polysaccharide components contained in Curcuma aromatica are difficult to separate, the polysaccharide components are complex, and the structure is unclear, thereby providing a basis for the development of potential new drugs for immunoregulation and promoting the industrialization process of Curcuma aromatica.

[0006] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions: A medicinal plant Curcuma wenyujin uniform polysaccharide, wherein the Curcuma wenyujin uniform polysaccharide comprises mannose and glucose; The molar ratio of mannose to glucose contained in the Curcuma aromatica uniform polysaccharide is 1:3.87.

[0007] Preferably, the structure of the Curcuma zedoariae homogeneous polysaccharide is shown in the following formula (1): Formula (1).

[0008] Sugar is an important active ingredient of Curcuma wenyujin. Many studies have shown that the effects of Curcuma wenyujin on promoting blood circulation and relieving pain, clearing the heart and cooling blood are closely related to it. The Curcuma wenyujin homogeneous polysaccharide (WP-1G) in the present invention comes from the perennial herb Curcuma wenyujin ( Curcuma wenyujin YH chert et C. Ling) and its dried root tubers (Curcuma zingiberensis).

[0009] The Curcuma aromatica uniform polysaccharide (WP-1G) in the present invention is a new uniform polysaccharide compound discovered for the first time, and its content is 0.8%~1.2% of the original medicinal material Curcuma aromatica. The HPGPC graph shows that WP-1G presents a symmetrical elution peak, indicating that it is a uniform polysaccharide; the gas chromatogram shows that the Curcuma aromatica uniform polysaccharide (WP-1G) is composed of mannose (Man) and glucose (Glu) in a molar ratio of 1:3.87, and has a molecular weight of 28.1kDa.

[0010] The preparation method of the uniform polysaccharide of Curcuma aromatica from the medicinal plant as described above comprises the following steps: (S.1) defatting the Curcuma aromatica by ethanol reflux, then decocting the Curcuma aromatica polysaccharide extract by water, and subjecting the Curcuma aromatica polysaccharide extract to alcohol precipitation to obtain alcohol-precipitated crude polysaccharide; (S.2) removing protein from the crude polysaccharide precipitated by alcohol obtained in step (S.1) by Sevag method to obtain total polysaccharide of Curcuma aromatica; (S.3) The total polysaccharide of Curcuma aromatica obtained in step (S.2) is separated and purified by ion exchange column and gel column chromatography, and then concentrated and freeze-dried to obtain uniform polysaccharide of Curcuma aromatica.

[0011] Preferably, the water decoction extraction temperature in step (S.1) is 80-100°C, and the extraction time is 0.5-2h.

[0012] As a further preference, the water decoction extraction temperature in step (S.1) is 95°C and the extraction time is 1 hour.

[0013] Preferably, the volume concentration of alcohol in the solution during alcohol precipitation in step (S.1) is 80-95%.

[0014] As a further preference, the volume concentration of alcohol in the solution during alcohol precipitation in step (S.1) is 90%.

[0015] Preferably, when the Sevag method is used to remove the protein in step (S.2), a mixture of chloroform and n-butanol is used for extraction; the volume ratio of chloroform to n-butanol in the mixture is 3-5:1.

[0016] As a further preference, when the Sevag method is used to remove the protein in step (S.2), a mixture of chloroform and n-butanol is used for extraction; the volume ratio of chloroform to n-butanol in the mixture is 4:1.

[0017] Preferably, the filler of the ion exchange column is DEAE-52, and the filler of the gel column is Sephadex G-100.

[0018] Preferably, the elution gradient of the ion exchange column chromatography is 0 mol / L and 0.1 mol / L NaCl solution.

[0019] Preferably, the eluate with a NaCl concentration of 0-0.1 mol / L from the ion exchange column is purified by a Sephadex G-100 gel column, and the solution corresponding to the 55-75 min peak is collected.

[0020] The use of the homogeneous polysaccharide of the medicinal plant Curcuma aromatica as described above in the preparation of immunomodulatory drugs.

[0021] Therefore, the present invention has the following beneficial effects: (1) The medicinal plant Curcuma australis uniform polysaccharide of the present invention is a new uniform polysaccharide compound discovered for the first time. The Curcuma australis uniform polysaccharide is composed of mannose (Man) and glucose (Glu), with molar ratios of 20.51% and 79.49%, respectively, and a molecular weight of 28.1 kDa; (2) The present invention provides a preparation method and process parameters for a uniform polysaccharide of Curcuma aromatica, a medicinal plant. The preparation method is suitable for industrial production, provides a basis for quality control and standardized production of the uniform polysaccharide of Curcuma aromatica, and broadens the application scope of Curcuma aromatica. (3) The uniform polysaccharide of Curcuma wenyujin (WP-1G) prepared by the present invention has immunomodulatory activity, which provides a basis for the development of potential new drugs for enhancing immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the elution curve of Curcuma aromatica polysaccharide (WP-1) in Example 1.

[0023] Figure 2 This is a diagram of the gel filtration chromatography process of the Curcuma aromatica uniform polysaccharide (WP-1G) in Example 1.

[0024] Figure 3 This is the HPGPC diagram of the Wenyujin uniform polysaccharide (WP-1G) in Example 4.

[0025] Figure 4 This is the gas chromatogram of the Curcuma aromatica uniform polysaccharide (WP-1G) in Example 4.

[0026] Figure 5 This is the UV spectrum of the Curcuma aromatica uniform polysaccharide (WP-1G) in Example 4.

[0027] Figure 6 This is the infrared spectrum of the Curcuma aromatica uniform polysaccharide (WP-1G) in Example 4.

[0028] Figure 7 The uniform polysaccharide of Curcuma aromatica (WP-1G) in Example 4 13 C NMR spectrum.

[0029] Figure 8 The uniform polysaccharide of Curcuma aromatica (WP-1G) in Example 4 1 H NMR spectrum.

[0030] Fig. 9 This is the COSY spectrum of the Wenyujin uniform polysaccharide (WP-1G) in Example 4.

[0031] Fig.10 This is the HSQC spectrum of the Curcuma zedoaria uniform polysaccharide (WP-1G) in Example 4.

[0032] Fig.11 This is the fitting curve of the activity of macrophages stimulated by the uniform polysaccharide of Curcuma aromatica (WP-1G) in Example 5.

[0033] Fig.12 The phagocytic activity of macrophages towards neutral red stimulated by different concentrations of Curcuma aromatica uniform polysaccharide (WP-1G) in Example 5.

[0034] Fig.13 The NO concentration secreted by macrophages stimulated by different concentrations of Wenyujin uniform polysaccharide (WP-1G) in Example 5.

[0035] Fig.14It is a concentration graph of the cellular inflammatory factors IL-1β, IL-6, and TNF-α secreted by macrophages stimulated by different concentrations of Wenyujin uniform polysaccharide (WP-1G) in Example 5, wherein A is the concentration change of the cellular inflammatory factor IL-1β secreted by macrophages stimulated by different concentrations of Wenyujin uniform polysaccharide (WP-1G) in Example 5, B is the concentration change of the cellular inflammatory factor IL-6 secreted by macrophages stimulated by different concentrations of Wenyujin uniform polysaccharide (WP-1G) in Example 5, and C is the concentration change of the cellular inflammatory factor TNF-α secreted by macrophages stimulated by different concentrations of Wenyujin uniform polysaccharide (WP-1G) in Example 5.

[0036] Fig.15 The figure is a graph of the relative mRNA expression of different cellular inflammatory factors secreted by macrophages stimulated by different concentrations of Wenyujin uniform polysaccharide (WP-1G) in Example 5, wherein A is the relative mRNA expression of the cellular inflammatory factor IL-1β secreted by macrophages stimulated by different concentrations of Wenyujin uniform polysaccharide (WP-1G) in Example 5, B is the relative mRNA expression of the cellular inflammatory factor IL-6 secreted by macrophages stimulated by different concentrations of Wenyujin uniform polysaccharide (WP-1G) in Example 5, and C is the relative mRNA expression of the cellular inflammatory factor TNF-α secreted by macrophages stimulated by different concentrations of Wenyujin uniform polysaccharide (WP-1G) in Example 5. In addition, Fig.13 , 14 ns in , 15 means no significant difference compared with the negative control group; * means p < 0.05, significant difference compared with the negative control group; ** means p < 0.01, extremely significant difference compared with the negative control group; *** means p < 0.001, more significant difference compared with the negative control group; **** means p < 0.0001, extremely significant difference compared with the negative control group. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0038] Example 1 This embodiment provides a medicinal plant Curcuma australis uniform polysaccharide (WP-1G) and a preparation method thereof.

[0039] A uniform polysaccharide of a medicinal plant Curcuma wenyujin, the uniform polysaccharide of Curcuma wenyujin comprising mannose and glucose. The molar ratio of mannose to glucose in the uniform polysaccharide of Curcuma wenyujin is 1:3.87. The structure of the uniform polysaccharide of Curcuma wenyujin is shown in the following formula (1): Formula (1).

[0040] The preparation method of the uniform polysaccharide of Curcuma aromatica from the medicinal plant as described above comprises the following steps: (S.1) Crush 25g of Curcuma aromatica into powder, add 100 times the amount of 80% ethanol and soak for 0.5h. After soaking, reflux and extract at 100℃ for 4h, take the residue, evaporate the ethanol, and complete defatting. Take the defatted sample and add 100 times the amount of distilled water for water decoction, continue to decoct at 100℃ for 1h, cool, filter, and take the filtrate to obtain the Curcuma aromatica polysaccharide extract. Concentrate the Curcuma aromatica polysaccharide extract using a rotary thin film evaporator to obtain a fluid extract. Add industrial ethanol to a final concentration of 90% to the fluid extract, let it stand for 24h, centrifuge, take the precipitate, evaporate the ethanol, and obtain alcohol-precipitated crude polysaccharide; (S.2) Add 1 volume of distilled water to the crude polysaccharide precipitated from alcohol obtained in step (S.1), and add a mixture of chloroform and n-butanol for extraction according to the Sevag method (wherein the volume ratio of the total volume of the mixture to distilled water is 5:1, and the volume ratio of chloroform to n-butanol in the mixture is 4:1), shake on a shaker for 20 minutes, transfer to a separatory funnel, let stand, take the upper layer of liquid, centrifuge for 1 minute, and remove residual protein precipitate. Repeat the operation 3 to 5 times to obtain a crude polysaccharide solution of Curcuma aromatica after protein removal. Concentrate under reduced pressure and freeze-dry to obtain 2.94 g of total polysaccharides of Curcuma aromatica; (S.3) A DEAE-52 column is used and connected to a fraction collector and a peristaltic pump. The total polysaccharides of Curcuma aromatica obtained in step (S.2) are eluted with distilled water at a flow rate of 1 mL / min. The eluate is collected in 10 mL test tubes and labeled. The absorbance value of each tube of eluate at 490 nm is measured by the phenol-sulfuric acid method, and a scatter plot is drawn. The eluates from 11 to 17 tubes corresponding to the peak are combined (distilled water eluate), concentrated, dialyzed with a 3500Da dialysis bag, and freeze-dried to obtain an eluted component named Curcuma aromatica polysaccharide (WP-1), with a mass of 0.24 g. The elution curve of Curcuma aromatica polysaccharide (WP-1) in this embodiment is shown in FIG. Figure 1As shown. Take 100 mg of WP-1 polysaccharide component, add 3 mL of distilled water to dissolve, centrifuge at 12000 rpm for 10 min, and take the supernatant. The supernatant is further separated and purified by a dextran gel Sephadex G-100 column, and the absorbance value of each tube of eluate at 490 nm is measured by the phenol-sulfuric acid method. A scatter plot is drawn, and the eluate of the 7th tube at the corresponding peak is collected, concentrated by a rotary evaporator, and freeze-dried to obtain an eluted component and named it WP-1G, which is a uniform polysaccharide of Curcuma aromatica, with a mass of 30 mg. The gel filtration chromatography process diagram of the uniform polysaccharide of Curcuma aromatica (WP-1G) in this embodiment is shown in Figure 2 As shown. Figure 1~2 Analysis showed that a non-adsorbed component (WP-1) was obtained by eluting with ultrapure water. After collecting it (tubes 11 to 17), it was eluted using Sephadex G-100, and the peak was in tube 7. The eluate in tube 7 was collected to obtain a homogenized polysaccharide (WP-1G).

[0041] Example 2 The difference between this embodiment and embodiment 1 is that: A method for preparing a uniform polysaccharide of Curcuma wenyujin, a medicinal plant, wherein the decocting is continued at 80°C for 2h in step (S.1); industrial ethanol is added to the fluid extract to a final concentration of 80%; and a mixed solution of chloroform and n-butanol is added for extraction in step (S.2) (wherein the volume ratio of the total volume of the mixed solution to distilled water is 5:1, and the volume ratio of chloroform to n-butanol in the mixed solution is 3:1). The rest is the same as in Example 1. 2.68g of total polysaccharide of Curcuma wenyujin is obtained; finally, a uniform polysaccharide of Curcuma wenyujin (WP-1G) is obtained, with a mass of 24.7mg.

[0042] Example 3 The difference between this embodiment and embodiment 1 is that: A method for preparing a uniform polysaccharide of Curcuma australis, a medicinal plant, wherein the decocting is continued at 90°C for 0.5h in step (S.1); industrial ethanol is added to the fluid extract to a final concentration of 95%; and a mixed solution of chloroform and n-butanol is added for extraction in step (S.2) (wherein the volume ratio of the total volume of the mixed solution to distilled water is 5:1, and the volume ratio of chloroform to n-butanol in the mixed solution is 5:1). The rest is the same as in Example 1. 2.54g of total polysaccharide of Curcuma australis is obtained; finally, a uniform polysaccharide of Curcuma australis (WP-1G) is obtained, with a mass of 22.3mg.

[0043] Example 4: Structural Identification of Curcuma wenyujin Uniform Polysaccharide (WP-1G) (1) The uniform polysaccharide of Curcuma wenyujin (WP-1G) was prepared according to the method in Example 1. The molecular weight of the polysaccharide was determined by HPGPC, and the optimized conditions were: Shimadzu LC 10A high performance liquid chromatograph, differential detector RID-20A, tandem gel chromatography column (8×300mm); mobile phase was 0.05M NaCl solution, flow rate: 0.8mL / min, column temperature: 40℃; injection volume: 25μL. A regression curve was established with different molecular weight dextran standards to calculate the molecular weight. Different molecular weight dextran standards (1152, 5000, 11600, 23800, 48600, 80900, 148000, 273000) were prepared into 5 mg / mL solutions with mobile phase before use, and the lg value of the number average molecular weight (Mn) was used as the horizontal coordinate (i.e., lgMn was used as the horizontal coordinate) and the retention time RT was used as the vertical coordinate to draw a standard curve (lgMn-RT). The equation of the standard curve was: y=-0.209x+10.95 (R²=0.999). WP-1G was prepared in the same way, and the number average molecular weight of WP-1G was calculated by the standard curve equation. The HPGPC diagram of the Curcuma zedoaria uniform polysaccharide (WP-1G) in this example is shown in FIG. Figure 3 shown. Figure 3 The results showed that the retention time of WP-1G was 31.103 min. Substituting y=31.103 into the standard curve equation, the solution was x=lgMn. Further calculation showed that the number average molecular weight of WP-1G was 28.1 kDa.

[0044] (2) Analysis of monosaccharide composition of WP-1G by gas chromatography: a. Use mixed monosaccharide standards of rhamnose (label 1), arabinose (label 2), xylose (label 3), mannose (label 4), glucose (label 5), and galactose (label 6) as the control group; b. Weigh 10.00 mg of polysaccharide sample (WP-1G) and place it in a hydrolysis tube. Add 0.4 mL of water to dissolve it, then add 0.4 mL of 4 mol / L trifluoroacetic acid solution, seal the tube, and hydrolyze it in a 100°C water bath for 5 hours. After evaporating the hydrolyzate under reduced pressure, add 1.5 mL of methanol to dissolve it, continue to evaporate it under reduced pressure, and repeat 3 times to remove the residual trifluoroacetic acid. Add 10 mg of hydroxylamine hydrochloride and 0.5 mL of pyridine, heat and react in a 90°C water bath for 30 minutes and shake. After taking it out, cool it to room temperature, add 0.5 mL of acetic anhydride, acetylate it at 90°C for 30 minutes, and dilute it to 10 mL with chloroform. HP-5 capillary column (30m×0.32mm, 0.25μm), injection port temperature 250℃, injection volume 1μL, split ratio 10:1, carrier gas is high-purity nitrogen, flow rate 50mL / min; detector temperature 270℃; hydrogen flow rate 40mL / min, air flow rate 400mL / min. Column heating program: initial temperature 150℃, hold for 0.5min; heat up to 250℃ at 8℃ / min, hold for 8min.

[0045] The gas chromatogram of the Curcuma zedoariae uniform polysaccharide (WP-1G) in this example is as follows: Figure 4 As shown. Figure 4 Comparative analysis of the retention times of the control group and WP-1G showed that the Curcuma zedoariae uniform polysaccharide (WP-1G) was composed of mannose (Man) and glucose (Glu). The corresponding peaks of mannose and glucose in the gas phase peak of Curcuma zedoariae uniform polysaccharide (WP-1G) were integrated to obtain the peak areas of mannose and glucose. The ratio of the peak areas of mannose and glucose was the molar ratio of mannose and glucose in Curcuma zedoariae uniform polysaccharide (WP-1G). Therefore, the molar ratios of mannose and glucose in Curcuma zedoariae uniform polysaccharide (WP-1G) were 20.51% and 79.49%, respectively.

[0046] (3) Determination of the purity of the uniform polysaccharide of Curcuma aromatica by ultraviolet spectroscopy: The UV spectrum of the Curcuma zedoariae uniform polysaccharide (WP-1G) in this example is as follows: Figure 5 As shown. Since nucleic acid has a strong absorption peak at a wavelength of 260nm, protein has a strong absorption peak at a wavelength of 280nm. Figure 5 Display: No absorption peaks were observed at wavelengths of 260nm and 280nm (only obvious descending peaks), indicating that the content of protein or nucleic acid contained in the Curcuma zedoariae uniform polysaccharide (WP-1G) was extremely low.

[0047] (4) Through methylation and uronic acid reduction analysis, as well as infrared spectroscopy, 1 H NMR spectrum, 13C NMR spectrum, one-dimensional spectrum and two-dimensional spectrum are used to assign the glycosidic bond signals of polysaccharides. It is speculated that →6)-α-D-Glc p -(1→,→3,6)-α-D-Glc p -(1→glycosidic bond connection mode and β-D-Man p -(1→The analysis results are shown in Table 1~2: Table 1: Analysis of methylated sugar alcohol acetyl ester (PMAA) of WP-1G Retention time Methylated sugar Fragment ions (m / z) Connection 12.58 2,3,4,6-Me4-Man 43,59,71,87,99,101,113,117,129,145,161,205 β-D-Man-(1→ 14.20 2,3,4-Me3-Glc 43,59,87,99,101,117,129,145,159,189 →6)-α-D-Glc-(1→ 17.54 2,4-Me2-Glc 43,59,74,87,101,118,129,139,160,189 →3,6)-α-D-Glc-(1→ Table 2: Hydrogen and carbon signal assignments of WP-1G The present invention also carries out a series of structural characterizations, including: using infrared spectra to analyze the glycosidic bond configuration and functional group information of the uniform polysaccharide of Curcuma australis, using methylation test to analyze its sugar residues, and performing nuclear magnetic spectrum analysis to determine the glycosidic bond connection mode. The infrared spectrum of the uniform polysaccharide of Curcuma australis (WP-1G) in this embodiment is as follows: Figure 6 The uniform polysaccharide of Curcuma wenyujin (WP-1G) in this example 13 C NMR spectrum Figure 7 The uniform polysaccharide of Curcuma wenyujin (WP-1G) in this example 1 H NMR spectrum Figure 8 The COSY spectrum of the Curcuma wenyujin uniform polysaccharide (WP-1G) in this example is shown in Fig. 9 The HSQC spectrum of the Curcuma zedoariae homogeneous polysaccharide (WP-1G) in this example is shown in Fig.10 shown.

[0048] Depend on Figures 6 to 10 As shown in the data analysis of Tables 1 and 2, the uniform polysaccharide of Curcuma wenyujin (WP-1G) exists in the form of →6)-α-D-Glc p -(1→,→3,6)-α-D-Glc p -(1→main chain and β-D-Man p -(1→ is the glycosidic bond connection mode of the branched chain, and the branched chain β-D-Man p -(1→through the main chain unit→3,6)-α-D-Glc p -(1→ carbon 3 is connected to the main chain, and according to the monosaccharide composition ratio, one →3,6)-α-D-Glc appears in every 3 glucose structural units p -(1→unit, from which the structural formula of Wenyujin uniform polysaccharide (WP-1G) can be drawn. The structural formula of Wenyujin uniform polysaccharide (WP-1G) is shown in the following formula (1): Formula (1).

[0049] Example 5: Experiment on immunomodulatory activity of Curcuma wenyujin uniform polysaccharide (WP-1G) Instruments and Materials: Mouse macrophage cell line RAW264.7, 1% double antibody (penicillin and streptomycin) and 10% fetal bovine serum (FBS) DMEM complete medium, placed in a 37 ° C, 5% CO2 incubator. Wenyujin uniform polysaccharide (WP-1G) was prepared according to the method in Example 1. Among them, the immunomodulatory experiment statistical analysis of Wenyujin uniform polysaccharide (WP-1G) was performed by one-way ANOVA using software GraphpadPrism 10.4, and P<0.05 was considered statistically significant.

[0050] (1) Cell viability assay Take RAW264.7 cells in the logarithmic growth phase and adjust the cell concentration to 1×10 5 / mL, 100μL per well was inoculated in a 96-well plate and placed in a 37°C, 5% CO2 incubator for 24h. WP-1G was dissolved in serum-free medium DMEM to a concentration of 2mg / mL, and WP-1G was added in the form of liquid replacement to a concentration of 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0mg / mL. After continuing to culture for 12h, the cell activity was determined using a CCK-8 kit, and the absorption of 450nm wavelength was measured on an ELISA reader. The results are shown in Table 3.

[0051] Table 3: Cell viability results Group Mass concentration (mg / mL) <![CDATA[OD 450 nm ]]> 1 Blank control 1.634±0.083 2 0.025 1.775±0.052* 3 0.05 1.708±0.277* 4 0.10 1.774±0.129* 5 0.20 1.574±0.119* 6 0.40 1.545±0.130 7 0.60 1.557±0.134* 8 0.80 1.550±0.095* 9 1.00 1.465±0.090* Note: *P<0.05, there is a significant difference compared with the blank control.

[0052] From Table 3, we can see the absorbance of RAW264.7 cells treated with different concentrations of Wenyujin polysaccharide at a wavelength of 450nm measured by CCK-8 kit, and the size of the absorbance is positively correlated with cell activity. The absorbance data in Table 3 were imported into the software Graphpad Prism 10.4, and the absorbance data in groups (2-9) and group (1) (i.e., blank control group) in Table 3 were subjected to one-way analysis of variance by software Graphpad Prism 10.4 to determine whether the absorbance data in groups (2-9) had significant differences. The analysis results showed that Wenyujin uniform polysaccharide (WP-1G) significantly promoted cell proliferation at low concentrations (0.025-0.1mg / mL) (P < 0.05), and inhibited cell proliferation at high concentrations (0.6-1.0mg / mL) (P < 0.05).

[0053] The cell survival rate corresponding to the absorbance data of the blank control group was set as 100%, and then the absorbance data in groups (2-9) in Table 3 were converted into the corresponding cell survival rate, and the concentration unit of the corresponding Curcuma polysaccharide was converted into μg / mL, and the corresponding logarithmic conversion was performed to obtain the corresponding log C The value is obtained by performing nonlinear fitting between the cell survival rate and the drug concentration (i.e., the concentration of Curcuma zedoaria polysaccharide), and then obtaining the fitting curve of the activity of macrophages stimulated by Curcuma zedoaria uniform polysaccharide (WP-1G). The fitting curve of the activity of macrophages stimulated by Curcuma zedoaria uniform polysaccharide (WP-1G) in this embodiment is shown in FIG. Fig.11 shown.

[0054] from Fig.11 The analysis showed that RAW264.7 affected by WP-1G had a good cell activity fitting curve, and its R 2 =0.9329, with the log value of the concentration of Curcuma zedoariae homogeneous polysaccharide (WP-1G) as the horizontal axis (i.e., log C The horizontal axis is the survival rate of RAW264.7 cells, and the vertical axis is the survival rate of RAW264.7 cells. C When log is 0-2, the survival rate of RAW264.7 cells is greater than 100%, which effectively promotes cell proliferation. C >2, the cell survival rate drops to less than 100%, which inhibits cell proliferation. When the survival rate of RAW264.7 cells drops to half of the initial survival rate, the corresponding log C =2.488. Since IC50 half-inhibitory concentration is a key indicator for evaluating drug potency, the survival rate curve of RAW264.7 cells is in log C =2.488 and then dropped to 50%, indicating that the IC50 half inhibition concentration of WP-1G was 0.308mg / mL. When the IC50 half inhibition concentration value was higher than 0.1mg / mL, it indicated that the drug safety was high and WP-1G was effective within the safe concentration range.

[0055] (2) Cell phagocytic ability test RAW264.7 cells in the logarithmic growth phase were inoculated into 96-well plates for culture, with 1×10 cells per well. 5After 24 hours of adherent culture, the excess culture medium was discarded, and a positive control (only lipopolysaccharide (LPS) was added without any preparation) group and a negative control (no LPS and any preparation) group were set up at the same time. Different concentrations of Wenyujin Uniform Polysaccharide (WP-1G) groups (62.5, 125, 250, 500 mg / mL) were set up, and 6 replicate wells were set up in each group. 100 μL of sample was injected into each well. After 24 hours of intervention with Wenyujin Uniform Polysaccharide (WP-1G), the supernatant was discarded, and the cells were washed twice with PBS. 0.09% neutral red solution was injected into each well, incubated at 37°C for 3 hours, the upper layer of neutral red was discarded, and PBS was rinsed three times. 100 μL of cell lysate was injected, and the OD was measured. 550nm The value was calculated based on the absorbance value, and the macrophage phagocytosis rate was calculated based on the absorbance value. In this example, different concentrations of Wenyujin uniform polysaccharide (WP-1G) stimulated the macrophage phagocytosis activity of neutral red. Fig.12 As shown. Fig.12 The analysis showed that WP-1G can promote the phagocytic ability of RAW264.7 cells for neutral red. When WP-1G was at a low concentration (<125μg / mL), the phagocytic rate of RAW264.7 cells for neutral red was low, indicating that low concentrations of WP-1G had no significant effect on the phagocytic ability of RAW264.7 cells; when the concentration of WP-1G was increased (>250μg / mL), the phagocytic rate of RAW264.7 cells for neutral red was significantly increased, and reached more than twice the phagocytic rate of the negative control group (P<0.01), indicating that high concentrations of WP-1G can significantly enhance the phagocytic ability of RAW264.7 cells for neutral red.

[0056] (3) Immunomodulatory effect experiment RAW264.7 cells in the logarithmic growth phase were inoculated into 96-well plates for culture, with 1×10 cells per well. 5 After 24 hours of adherence culture, the excess culture medium was discarded, and a positive control (only LPS was added without any preparation) group and a negative control (without LPS and any preparation) group were set up. Different concentrations of Curcuma aromatica uniform polysaccharide (WP-1G) groups (62.5, 125, 250, 500 mg / mL) were set up, and 6 replicates were set up in each group. After the culture was completed, the cell culture medium was collected and centrifuged at 4°C and 12000 r / min for 10 min. The NO content was determined by a NO kit, and the expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 were determined by ELISA kits. In this example, the NO concentration secreted by macrophages stimulated by different concentrations of Curcuma aromatica uniform polysaccharide (WP-1G) is as follows. Fig.13In this example, the concentrations of inflammatory factors IL-1β, IL-6, and TNF-α secreted by macrophages stimulated by different concentrations of Wenyujin uniform polysaccharide (WP-1G) are shown in Fig.14 As shown. Among them, Fig.14 A in the figure is the concentration change of IL-1β, a cellular inflammatory factor secreted by macrophages stimulated by different concentrations of Curcuma aromatica uniform polysaccharide (WP-1G) in this example. Fig.14 B in the figure is the concentration change of IL-6, a cellular inflammatory factor secreted by macrophages stimulated by different concentrations of Curcuma zedoariae uniform polysaccharide (WP-1G) in this example. Fig.14 C in this example is the concentration change of TNF-α, a cell inflammatory factor secreted by macrophages stimulated by different concentrations of Wenyujin uniform polysaccharide (WP-1G). Figures 13 and 14 Analysis showed that different concentrations of Wenyujin uniform polysaccharide (WP-1G) can activate the mouse macrophage cell line RAW264.7 to increase the concentration of NO secreted by it, and can also increase the concentration of inflammatory factors TNF-α, IL-1β, and IL-6 secreted by it, thereby enhancing the immune activity of the cells.

[0057] (4) Immune factor mRNA experiment RAW264.7 cells in the logarithmic growth phase were inoculated into 6-well plates for culture, with 1×10 cells per well. 6 The cells were cultured for 24 hours. A positive control group (LPS only, without any preparation) and a negative control group (without LPS and any preparation) were set up at the same time. Different concentrations of WP-1G groups (62.5, 125, 250, 500 mg / mL) were set up, and 6 replicates were set up in each group. 1000 μL of sample was injected into each well according to the setting. After 24 hours of intervention with WP-1G, the supernatant was removed, the cells were washed twice with PBS at 4°C, and 1 mL of Trizol RNA extraction reagent was added.

[0058] Extract RNA: inject 200 μL of chloroform into each well, shake and mix, centrifuge at 4°C and 10,000 r / min to obtain the supernatant, add 500 μL of isopropanol, mix evenly, and centrifuge to obtain the precipitate; add 1 mL of 75% ethanol, centrifuge to obtain the precipitate, and dry the RNA; add DEPC water and store for later use.

[0059] RNA purity assessment: Comparison of OD 260nm / OD 280nm Is the value between 1.8 and 2.2?

[0060] RNA reverse transcription: performed according to the Transcriptor cDNA Synth Kit reverse transcription kit.

[0061] PCR amplification reaction: The specific system and amplification procedure are shown in Tables 4 to 6. -ΔΔCT The relative expression was calculated using the formula.

[0062] Table 4: Gene primer sequences Primer name Gene sequence (5' to 3') TNF-α(upstream primer) CCT CAG GAA CGG GAC TCG AA TNF-α(downstream primer) ATG TAC ACC AAG TCG GTA GCA CCA IL-6 (upstream primer) GGA ATT CGT GGA AAT GAG AA IL-6 (downstream primer) GCA CTA GGA AAG CCG AGT AC IL-1β (upstream primer) TGT GAT GTT CCC ATT AGA C IL-1β (downstream primer) AAT ACC ACT TGT TGG CTT A GADPH (upstream primer) TTT GTC AAG CTC ATT TCC TGG TATG GADPH (downstream primer) TGG GAT AGG GCC TCT CTT GC Table 5: Fluorescence quantitative PCR reaction system Reagent name Reagent volume (μL) 2×SYBR Green PCR Mix 10 DNA template 2 Upstream primer 1 Downstream primer 1 Water (DNase free) 6 total 20 Table 6: Fluorescence quantitative PCR reaction process Reaction process Temperature (℃) Reaction time (min) Number of cycles (times) Pre-denaturation 95 2 1 Heat denaturation 95 0.25 40 annealing 60 0.5 40 extend 60 0.5 40 Stablize 72 0.5 1 In this example, different concentrations of Wenyujin uniform polysaccharide (WP-1G) stimulated macrophages to secrete different inflammatory factors. Fig.15 As shown. Among them, Fig.15 A in the figure is the relative expression level of mRNA of inflammatory factor IL-1β secreted by macrophages stimulated by different concentrations of Curcuma aromatica uniform polysaccharide (WP-1G) in this example. Fig.15 B in the figure is the relative expression of mRNA of inflammatory factor IL-6 secreted by macrophages stimulated by different concentrations of Curcuma aromatica uniform polysaccharide (WP-1G) in this example. Fig.15 C in this example is the relative expression of mRNA of inflammatory factor TNF-α secreted by macrophages stimulated by different concentrations of Curcuma zedoariae uniform polysaccharide (WP-1G). Fig.15 Analysis showed that under the stimulation of different concentrations of Wenyu Jin uniform polysaccharide (WP-1G), the relative mRNA expression levels of related cellular inflammatory factors TNF-α, IL-1β, and IL-6 secreted by macrophages were significantly upregulated, and the results showed the same trend as the protein expression levels of related cellular inflammatory factors TNF-α, IL-1β, and IL-6. The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A medicinal plant Curcuma wenyujin uniform polysaccharide, characterized in that: The Wenyujin uniform polysaccharide includes mannose and glucose; The molar ratio of mannose to glucose contained in the Curcuma aromatica uniform polysaccharide is 1:3.

87.

2. The medicinal plant Curcuma zedoaria homogeneous polysaccharide according to claim 1, characterized in that: The structure of the Curcuma wenyujin uniform polysaccharide is shown in the following formula (1): Formula (1).

3. The method for preparing a uniform polysaccharide of Curcuma aromatica from a medicinal plant as claimed in claim 1 or 2, characterized in that: The following steps are involved: (S.1) defatting the Curcuma aromatica by ethanol reflux, then decocting the Curcuma aromatica polysaccharide extract by water, and subjecting the Curcuma aromatica polysaccharide extract to alcohol precipitation to obtain alcohol-precipitated crude polysaccharide; (S.2) removing protein from the crude polysaccharide precipitated by alcohol obtained in step (S.1) by Sevag method to obtain total polysaccharide of Curcuma aromatica; (S.3) The total polysaccharide of Curcuma aromatica obtained in step (S.2) is separated and purified by ion exchange column and gel column chromatography, and then concentrated and freeze-dried to obtain uniform polysaccharide of Curcuma aromatica.

4. The method for preparing a uniform polysaccharide of Curcuma aromatica from a medicinal plant according to claim 3, characterized in that: In step (S.1), the water decoction extraction temperature is 80-100°C, and the extraction time is 0.5-2h.

5. The method for preparing a uniform polysaccharide of Curcuma aromatica from a medicinal plant according to claim 3, characterized in that: During the alcohol precipitation in step (S.1), the volume concentration of alcohol in the solution is 80-95%.

6. The method for preparing a uniform polysaccharide of Curcuma aromatica from a medicinal plant according to claim 3, characterized in that: When the Sevag method is used to remove proteins in step (S.2), a mixture of chloroform and n-butanol is used for extraction; the volume ratio of chloroform to n-butanol in the mixture is 3-5:

1.

7. The method for preparing a uniform polysaccharide of Curcuma aromatica from a medicinal plant according to claim 3, characterized in that: The filler of the ion exchange column is DEAE-52, and the filler of the gel column is Sephadex G-100.

8. The method for preparing a uniform polysaccharide of Curcuma aromatica from a medicinal plant according to claim 3 or 7, characterized in that: The elution gradient of the ion exchange column chromatography was 0 mol / L and 0.1 mol / L NaCl solution.

9. The method for preparing a uniform polysaccharide of Curcuma aromatica from a medicinal plant according to claim 7, characterized in that: The eluate with a concentration of 0-0.1 mol / L NaCl from the ion exchange column was purified by a Sephadex G-100 gel column, and the solution corresponding to the 55-75 min peak was collected.

10. Use of the medicinal plant Curcuma aromatica uniform polysaccharide as claimed in claim 1 or 2 in the preparation of immunomodulatory drugs.

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