A Homogeneous Polysaccharide from the Medicinal Plant Curcuma wenyujin and Its Application in Immunomodulation

Abstract

The present invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a homogeneous polysaccharide from the medicinal plant Curcuma wenyujin and its application in immunomodulation. The homogeneous polysaccharide from Curcuma wenyujin of the present invention is a newly discovered homogeneous polysaccharide compound for the first time. This homogeneous polysaccharide from Curcuma wenyujin is composed of mannose (Man) and glucose (Glu), with molar ratios of 20.51% and 79.49% respectively, and a molecular weight of 28.1KDa. The present invention provides a preparation method and process parameters of the homogeneous polysaccharide from Curcuma wenyujin. This preparation method is suitable for industrial production, provides a basis for the quality control and standardized production of the homogeneous polysaccharide from Curcuma wenyujin, and broadens the application scope of Curcuma wenyujin. The homogeneous polysaccharide from Curcuma wenyujin (WP-1G) prepared by the present invention has immunomodulatory activity, providing a basis for the development of potential new drugs for enhancing immunity.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a homogeneous polysaccharide of the medicinal plant Curcuma wenyujin and its application in immunomodulation. Background Art

[0002] Polysaccharide, also known as polyglycan, is a high-molecular-weight carbohydrate polymerized by at least 10 monosaccharides through α- or β-glycosidic bonds. Its structure usually consists of 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, and are an important class of biological macromolecules with a wide range of biological effects. Polysaccharides not only provide energy for life activities, and some polysaccharides can form the cytoskeleton such as cellulose and chitin. With the development of analytical and detection techniques, polysaccharide compounds derived from various plants, animals, or microorganisms have been found to have a series of biological activities, such as anti-tumor, antioxidant, immune enhancement, blood pressure lowering, and blood lipid regulation.

[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 activities to attack invaders such as bacteria or viruses. Plant polysaccharides can promote the proliferation of macrophages, enhance the phagocytosis of macrophages, and stimulate the release of cytokines such as NO, TNF-α, and IL-6.

[0004] Curcuma wenyujin Curcuma wenyujin Y. H. chert et C. Ling has a long medicinal history and is one of the famous "Eight Flavors of Zhejiang". It is a perennial herb of the genus Curcuma in the family Zingiberaceae. It was first recorded in "Newly Revised Materia Medica". Its medicinal use is mainly through cultivation, and wild distribution is very rare. Curcuma wenyujin is a medicinal plant that can be used in three ways. Its dried tuberous roots are called "Yujin", which have the effects of promoting blood circulation to relieve pain, regulating qi and relieving depression, clearing the heart and cooling blood, and promoting bile secretion to reduce jaundice. Currently, hundreds of compounds have been 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. The structure of the polysaccharide components in Curcuma wenyujin is not yet clear. In the case of few research reports on the isolation, structure, and pharmacological activities of the active homogeneous polysaccharides of Curcuma wenyujin, the research results of the present invention are particularly important. Summary of the Invention

[0005] The present invention aims to provide a homogeneous polysaccharide of the medicinal plant Curcuma wenyujin and apply it to immunomodulation, so as to overcome the defects in the prior art that the polysaccharide components contained in Curcuma wenyujin are difficult to separate, the polysaccharide components are complex, and the structure is not clear, thereby providing a basis for the development of potential new drugs for immunomodulation and promoting the industrialization process of Curcuma wenyujin at the same time.

[0006] To achieve the above-mentioned invention object, the present invention is realized through the following technical solutions:

[0007] A homogeneous polysaccharide from the medicinal plant Curcuma wenyujin, the Curcuma wenyujin homogeneous polysaccharide includes mannose and glucose;

[0008] The molar ratio of mannose and glucose contained in the Curcuma wenyujin homogeneous polysaccharide is 1:3.87.

[0009] Preferably, the structure of the Curcuma wenyujin homogeneous polysaccharide is shown in the following formula (1):

[0010]

[0011] Formula (1).

[0012] Carbohydrates are important active components of Curcuma wenyujin. Many studies have shown that the enhancement of the effects of Curcuma wenyujin in promoting blood circulation to relieve pain and clearing the heart and cooling blood is closely related to them. The Curcuma wenyujin homogeneous polysaccharide (WP-1G) in the present invention is derived from the dried tuberous roots (radix curcumae) of the perennial herb Curcuma wenyujin ( Curcuma wenyujin Y. H. chert et C. Ling) of the genus Curcuma in the family Zingiberaceae.

[0013] The Curcuma wenyujin homogeneous polysaccharide (WP-1G) in the present invention is a newly discovered homogeneous polysaccharide compound for the first time. Its content is 0.8% - 1.2% of the original medicinal material Curcuma wenyujin. The HPGPC chart shows that WP-1G presents a symmetrical elution peak, indicating that it is a homogeneous polysaccharide; the gas chromatogram shows that the Curcuma wenyujin homogeneous polysaccharide (WP-1G) is composed of mannose (Man) and glucose (Glu) in a molar ratio of 1:3.87, and the molecular weight is 28.1 kDa.

[0014] A preparation method of the homogeneous polysaccharide of the medicinal plant Curcuma wenyujin as described above includes the following steps:

[0015] (S.1) Use ethanol reflux to degrease Curcuma wenyujin, then obtain the Curcuma wenyujin polysaccharide extract by water decoction, and perform alcohol precipitation on the Curcuma wenyujin polysaccharide extract to obtain the alcohol-precipitated crude polysaccharide;

[0016] (S.2) Use the Sevag method to remove proteins from the alcohol-precipitated crude polysaccharide obtained in step (S.1) to obtain the total Curcuma wenyujin polysaccharide;

[0017] (S.3) Separate and purify the total Curcuma wenyujin polysaccharide obtained in step (S.2) through an ion exchange column and gel column chromatography in sequence, then concentrate and freeze-dry to obtain the Curcuma wenyujin homogeneous polysaccharide.

[0018] Preferably, in step (S.1), the extraction temperature of water decoction is 80 - 100 °C, and the extraction time is 0.5 - 2 h.

[0019] As a further preference, in step (S.1), the extraction temperature for decocting with water is 95 °C and the extraction time is 1 h.

[0020] As a preference, in step (S.1), when performing alcohol precipitation, the volume concentration of alcohol in the solution is 80 - 95%.

[0021] As a further preference, in step (S.1), when performing alcohol precipitation, the volume concentration of alcohol in the solution is 90%.

[0022] As a preference, in step (S.2), when removing proteins by the Sevag method, an extraction is performed using a mixed solution of chloroform and n-butanol; the volume ratio of chloroform to n-butanol in the mixed solution is 3 - 5:1.

[0023] As a further preference, in step (S.2), when removing proteins by the Sevag method, an extraction is performed using a mixed solution of chloroform and n-butanol; the volume ratio of chloroform to n-butanol in the mixed solution is 4:1.

[0024] As a preference, the packing material of the ion exchange column is DEAE-52, and the packing material of the gel column is Sephadex G-100.

[0025] As a preference, the elution gradient for ion exchange column chromatography is 0 mol / L and 0.1 mol / L NaCl solution.

[0026] As a preference, the eluate with an elution concentration of 0 - 0.1 mol / L NaCl from the ion exchange column is purified by a Sephadex G-100 gel column, and the solution corresponding to the peak at 55 - 75 min is collected.

[0027] Use of the homogeneous polysaccharide of the medicinal plant Curcuma wenyujin described above in the preparation of immunomodulatory drugs.

[0028] Therefore, the present invention has the following beneficial effects:

[0029] (1) The homogeneous polysaccharide of the medicinal plant Curcuma wenyujin of the present invention is a newly discovered novel homogeneous polysaccharide compound. This Curcuma wenyujin homogeneous 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;

[0030] (2) The present invention provides a preparation method and process parameters for the homogeneous polysaccharide of the medicinal plant Curcuma wenyujin. This preparation method is suitable for industrial production, provides a basis for the quality control and standardized production of the Curcuma wenyujin homogeneous polysaccharide, and broadens the application scope of Curcuma wenyujin;

[0031] (3) The homogeneous polysaccharide of the medicinal plant Curcuma wenyujin (WP-1G) prepared by the present invention has immunomodulatory activity, providing a basis for the development of potential new drugs for enhancing immunity. Description of the Drawings

[0032] Figure 1 It is the elution curve of Curcuma wenyujin polysaccharide (WP-1) in Example 1.

[0033] Figure 2 It is the process diagram of gel filtration chromatography of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 1.

[0034] Figure 3 It is the HPGPC chart of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 4.

[0035] Figure 4 It is the gas chromatogram of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 4.

[0036] Figure 5 It is the UV spectrum of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 4.

[0037] Figure 6 It is the infrared spectrum of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 4.

[0038] Figure 7 It is the 13 C NMR spectrum of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 4.

[0039] Figure 8 It is the 1 H NMR spectrum of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 4.

[0040] Figure 9 It is the COSY spectrum of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 4.

[0041] Figure 10 It is the HSQC spectrum of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 4.

[0042] Figure 11 It is the fitting curve of the activity of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in stimulating macrophages in Example 5.

[0043] Figure 12 It is the phagocytic activity of macrophages stimulated by Curcuma wenyujin homogeneous polysaccharide (WP-1G) at different concentrations in Example 5 for neutral red.

[0044] Figure 13 It is the NO concentration secreted by macrophages stimulated by Curcuma wenyujin homogeneous polysaccharide (WP-1G) at different concentrations in Example 5.

[0045] Figure 14Concentration diagrams of cytokines IL-1β, IL-6, and TNF-α secreted by macrophages stimulated with different concentrations of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 5. Among them, A shows the concentration change of cytokine IL-1β secreted by macrophages stimulated with different concentrations of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 5, B shows the concentration change of cytokine IL-6 secreted by macrophages stimulated with different concentrations of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 5, and C shows the concentration change of cytokine TNF-α secreted by macrophages stimulated with different concentrations of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 5.

[0046] Figure 15 Diagrams of relative mRNA expression levels of different cytokines secreted by macrophages stimulated with different concentrations of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 5. Among them, A shows the relative mRNA expression level of cytokine IL-1β secreted by macrophages stimulated with different concentrations of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 5, B shows the relative mRNA expression level of cytokine IL-6 secreted by macrophages stimulated with different concentrations of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 5, and C shows the relative mRNA expression level of cytokine TNF-α secreted by macrophages stimulated with different concentrations of Curcuma wenyujin homogeneous polysaccharide (WP-1G) in Example 5.

[0047] In addition, Figure 13 、 14 In Figures 15, ns indicates no significant difference compared with the negative control group; * indicates p < 0.05, significant difference compared with the negative control group; ** indicates p < 0.01, extremely significant difference compared with the negative control group; *** indicates p < 0.001, more significant difference compared with the negative control group; **** indicates p < 0.0001, particularly significant difference compared with the negative control group. Detailed implementation manners

[0048] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. 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 usually only a part of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention should fall within the scope of protection of the present invention.

[0049] Example 1

[0050] This example provides a Curcuma wenyujin homogeneous polysaccharide (WP-1G) for medicinal use and its preparation method.

[0051] A homogeneous polysaccharide from the medicinal plant Curcuma wenyujin, the homogeneous polysaccharide of Curcuma wenyujin includes mannose and glucose. Among them, the molar ratio of mannose to glucose contained in the homogeneous polysaccharide of Curcuma wenyujin is 1:3.87. The structure of the homogeneous polysaccharide of Curcuma wenyujin is shown in the following formula (1):

[0052]

[0053] Formula (1).

[0054] A preparation method of the homogeneous polysaccharide from the medicinal plant Curcuma wenyujin as described above, comprising the following steps:

[0055] (S.1) Break 25 g of Curcuma wenyujin into powder, add 100 times the amount of 80% ethanol and soak for 0.5 h. After the soaking is completed, reflux and extract at 100 °C for 4 h. Take the medicinal residue, evaporate the ethanol to dryness to complete defatting. Add 100 times the amount of distilled water to the defatted sample for water decoction, continue to decoct at 100 °C for 1 h, cool, filter, take the filtrate to obtain the Curcuma wenyujin polysaccharide extract. Concentrate the Curcuma wenyujin polysaccharide extract using a rotary thin-film evaporator to obtain a flow extract. Add industrial ethanol with a final concentration of 90% to the flow extract, let it stand for 24 h, centrifuge, take the precipitate, evaporate the ethanol to dryness to obtain the alcohol-precipitated crude polysaccharide;

[0056] (S.2) Add 1 volume of distilled water to the alcohol-precipitated crude polysaccharide obtained in step (S.1), and perform extraction according to the Sevag method by adding a mixture of chloroform and n-butanol (wherein the total volume of the mixture is 5:1 with the volume of distilled water, and the volume ratio of chloroform to n-butanol in the mixture is 4:1). Place it on a shaker and shake for 20 min, transfer it to a separatory funnel, let it stand, take the upper layer liquid, centrifuge for 1 min to remove the residual protein precipitate. Repeat the operation 3 - 5 times to obtain the Curcuma wenyujin crude polysaccharide solution after removing proteins. Concentrate under reduced pressure and freeze-dry to obtain 2.94 g of the total Curcuma wenyujin polysaccharide;

[0057] (S.3) Use a DEAE-52 column, connected to a fraction collector and a peristaltic pump. Elute the total Curcuma wenyujin polysaccharide obtained in step (S.2) with distilled water at a flow rate of 1 mL / min, collect the eluate in 10 mL test tubes and label them. Measure the absorbance value of each tube of eluate at 490 nm by the phenol-sulfuric acid method, draw a scatter plot, combine the eluates in tubes 11 - 17 corresponding to the peak (distilled water eluate), concentrate, dialyze with a 3500 Da dialysis bag, and freeze-dry to obtain the elution fraction and name it Curcuma wenyujin polysaccharide (WP-1), with a mass of 0.24 g. The elution curve of the Curcuma wenyujin polysaccharide (WP-1) in this example is as Figure 1As shown in the figure. Take 100 mg of the WP-1 polysaccharide component, add 3 mL of distilled water to dissolve it, centrifuge at 12,000 rpm for 10 min using a centrifuge, and take the supernatant. The supernatant is further separated and purified through a Sephadex G-100 column. Use the phenol-sulfuric acid method to measure the absorbance value of each tube of the eluate at 490 nm, draw a scatter plot, collect the eluate of the 7th tube corresponding to the peak value, concentrate it using a rotary evaporator, and freeze-dry it to obtain the eluted component, which is named WP-1G, that is, the homogeneous polysaccharide of Curcuma wenyujin, with a mass of 30 mg. The gel filtration chromatography process diagram of the homogeneous polysaccharide (WP-1G) of Curcuma wenyujin in this example is as Figure 2 shown. From Figures 1 - 2 analysis, it can be seen that when ultra-pure water is used for elution, an unadsorbed component (WP-1) is obtained. After collecting it (tubes 11 - 17), Sephadex G-100 is used for elution, and a peak appears in the 7th tube. Collect the eluate of the 7th tube to obtain the homogenized polysaccharide (WP-1G).

[0058] Example 2

[0059] The difference between this example and Example 1 is that:

[0060] A preparation method of a homogeneous polysaccharide of the medicinal plant Curcuma wenyujin, wherein, in step (S.1), continue to decoct at 80 °C for 2 h; add industrial ethanol with a final concentration of 80% to the flow extract; in step (S.2), add a mixed solution of chloroform and n-butanol for extraction (wherein, the total volume of the mixed solution and the volume of distilled water are in a ratio of 5:1, and the volume ratio of chloroform to n-butanol in the mixed solution is 3:1). Others are the same as in Example 1. Obtain 2.68 g of the total polysaccharide of Curcuma wenyujin; finally obtain the homogeneous polysaccharide (WP-1G) of Curcuma wenyujin, with a mass of 24.7 mg.

[0061] Example 3

[0062] The difference between this example and Example 1 is that:

[0063] A preparation method of a homogeneous polysaccharide of the medicinal plant Curcuma wenyujin, wherein, in step (S.1), continue to decoct at 90 °C for 0.5 h; add industrial ethanol with a final concentration of 95% to the flow extract; in step (S.2), add a mixed solution of chloroform and n-butanol for extraction (wherein, the total volume of the mixed solution and the volume of distilled water are in a ratio of 5:1, and the volume ratio of chloroform to n-butanol in the mixed solution is 5:1). Others are the same as in Example 1. Obtain 2.54 g of the total polysaccharide of Curcuma wenyujin; finally obtain the homogeneous polysaccharide (WP-1G) of Curcuma wenyujin, with a mass of 22.3 mg.

[0064] Example 4: Structural identification of the homogeneous polysaccharide (WP-1G) of Curcuma wenyujin

[0065] (1)The Curcuma wenyujin homogeneous polysaccharide (WP-1G) was prepared according to the method in Example 1. The molecular weight of the polysaccharide was determined by HPGPC. The optimized conditions were as follows: Shimadzu LC 10A high performance liquid chromatograph, differential refractive index detector RID-20A, tandem gel chromatographic column (8×300 mm); the mobile phase was 0.05 M NaCl solution, flow rate: 0.8 mL / min, column temperature: 40 °C; injection volume: 25 μL. Regression curves were established with dextran standards of different molecular weights to calculate the molecular weight. Dextran standards of different molecular weights (1152, 5000, 11600, 23800, 48600, 80900, 148000, 273000) were respectively prepared into solutions with a concentration of 5 mg / mL using the mobile phase immediately before use. With the lg value of the number average molecular weight (Mn) as the abscissa (i.e., with lgMn as the abscissa) and the retention time RT as the ordinate, a standard curve (lgMn-RT) was plotted, and the standard curve equation was obtained as: 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 from the standard curve equation. The HPGPC diagram of the Curcuma wenyujin homogeneous polysaccharide (WP-1G) in this example is as 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 to solve for x = lgMn, and further calculating, the number average molecular weight of WP-1G was obtained as 28.1 kDa.

[0066] (2)The monosaccharide composition of WP-1G was analyzed by gas chromatography:

[0067] a. A mixed monosaccharide standard of rhamnose (label 1), arabinose (label 2), xylose (label 3), mannose (label 4), glucose (label 5), and galactose (label 6) was used as the control group;

[0068] b. Weigh 10.00 mg of the 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 water bath at 100 °C for 5 h. After evaporating the hydrolysis solution under reduced pressure, add 1.5 mL of methanol to dissolve it, and continue to evaporate it under reduced pressure, repeating 3 times to completely remove the residual trifluoroacetic acid. Add 10 mg of hydroxylamine hydrochloride and 0.5 mL of pyridine, heat and react in a water bath at 90 °C for 30 min with shaking. After taking it out and cooling to room temperature, add 0.5 mL of acetic anhydride, carry out acetylation reaction at 90 °C for 30 min, and make up the volume to 10 mL with chloroform. HP-5 capillary chromatographic column (30 m × 0.32 mm, 0.25 μm), inlet temperature 250 °C, injection volume 1 μL, split ratio 10:1, carrier gas is high-purity nitrogen, flow rate 50 mL / min; detector temperature 270 °C; hydrogen flow rate 40 mL / min, air flow rate 400 mL / min. Column temperature programming: initial temperature 150 °C, hold for 0.5 min; increase the temperature to 250 °C at a rate of 8 °C / min and hold for 8 min.

[0069] The gas chromatogram of the Curcuma wenyujin homogeneous polysaccharide (WP-1G) in this example is as Figure 4 shown. By comparing and analyzing the retention times of the control group and WP-1G in Figure 4 , it can be known that this Curcuma wenyujin homogeneous polysaccharide (WP-1G) is composed of mannose (Man) and glucose (Glu). Integrate the corresponding peaks of mannose and glucose in the gas chromatographic peaks of the Curcuma wenyujin homogeneous polysaccharide (WP-1G) respectively to obtain the peak areas of mannose and glucose. The ratio of the peak areas of mannose and glucose is the molar proportion of mannose and glucose in the Curcuma wenyujin homogeneous polysaccharide (WP-1G). Therefore, the molar ratios of mannose and glucose in the Curcuma wenyujin homogeneous polysaccharide (WP-1G) are 20.51% and 79.49% respectively.

[0070] (3) Determine the purity of the Curcuma wenyujin homogeneous polysaccharide by ultraviolet spectroscopy:

[0071] The UV spectrum of the Curcuma wenyujin homogeneous polysaccharide (WP-1G) in this example is as Figure 5 shown. Since nucleic acids have a strong absorption peak at a wavelength of 260 nm and proteins have a strong absorption peak at a wavelength of 280 nm, while Figure 5 shows that there are no absorption peaks (only obvious descending peaks) at wavelengths of 260 nm and 280 nm, indicating that the content of proteins or nucleic acids in the Curcuma wenyujin homogeneous polysaccharide (WP-1G) is extremely low.

[0072] (4) Through methylation and uronic acid reduction analysis, as well as infrared spectroscopy, 1 H NMR spectrum, 1313C NMR spectra, one-dimensional spectra and two-dimensional spectra were used to assign the glycosidic bond signals of the polysaccharide. It was speculated that there was →6)-α-D-Glc p -(1→, →3,6)-α-D-Glc p -(1→ glycosidic bond linkage and β-D-Man p -(1→ The analysis results are shown in Tables 1 to 2:

[0073] Table 1: Analysis of the results of methylated sugar alcohol acetate (PMAA) of WP-1G

[0074] Retention time Methylated sugar Fragment ion (m / z) Linkage mode 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→

[0075] Table 2: Assignment of hydrogen and carbon signals of WP-1G

[0076]

[0077] A series of structural characterizations were also carried out in the present invention, including: analyzing the glycosidic bond configuration and functional group information of the Curcuma wenyujin homogeneous polysaccharide by using infrared spectra, analyzing its sugar residues by using methylation experiments, and analyzing its nuclear magnetic resonance spectra to determine the glycosidic bond linkage. The infrared spectrum of the Curcuma wenyujin homogeneous polysaccharide (WP-1G) in this example is as Figure 6 shown. The Curcuma wenyujin homogeneous polysaccharide (WP-1G) in this example 13 13C NMR spectrum is as Figure 7 shown. The Curcuma wenyujin homogeneous polysaccharide (WP-1G) in this example 1 1H NMR spectrum is as Figure 8 shown. The COSY spectrum of the Curcuma wenyujin homogeneous polysaccharide (WP-1G) in this example is as Figure 9 shown. The HSQC spectrum of the Curcuma wenyujin homogeneous polysaccharide (WP-1G) in this example is as Figure 10 shown.

[0078] From Figures 6 - 10 and the data analysis in Tables 1 to 2, it can be seen that the Curcuma wenyujin homogeneous polysaccharide (WP-1G) has →6)-α-D-Glc p -(1→, →3,6)-α-D-Glc p -(1→ as the main chain and β-D-Man p -(1→ as the side chain glycosidic bond linkage, and the side chain β-D-Man p -(1→ is connected to the main chain through the 3rd carbon on the main chain unit →3,6)-α-D-Glc p -(1→, and one →3,6)-α-D-Glc appears in every 3 glucose structural units according to the monosaccharide composition ratio p-(1→ unit, from which the structural formula of the homogeneous polysaccharide (WP-1G) of Curcuma wenyujin can be drawn. The structural formula of the homogeneous polysaccharide (WP-1G) of Curcuma wenyujin is shown in the following formula (1):

[0079]

[0080] Formula (1).

[0081] Example 5: Immunomodulatory activity experiment of the homogeneous polysaccharide (WP-1G) of Curcuma wenyujin

[0082] Instruments and materials:

[0083] Mouse macrophage cell line RAW264.7, DMEM complete medium containing 1% double antibody (penicillin and streptomycin) and 10% fetal bovine serum (FBS), placed in an incubator at 37°C and 5% CO 2 and cultured. The homogeneous polysaccharide (WP-1G) of Curcuma wenyujin was prepared according to the method in Example 1. Among them, the statistical analysis of the immunomodulation experiment of the homogeneous polysaccharide (WP-1G) of Curcuma wenyujin was performed by one-way ANOVA using the software Graphpad Prism 10.4, and P < 0.05 was considered statistically significant.

[0084] (1) Cell viability experiment

[0085] RAW264.7 cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 1×10 5 cells / mL, and 100 μL per well was inoculated into a 96-well plate and cultured in an incubator at 37°C and 5% CO 2 for 24 h. WP-1G was dissolved in serum-free medium DMEM to a concentration of 2 mg / mL, and WP-1G was added in the form of medium replacement to make the concentration of WP-1G 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg / mL. After continued culture for 12 h, the CCK-8 kit was used to measure cell viability, and the absorbance at a wavelength of 450 nm was measured on an enzyme-labeled instrument. The results are shown in Table 3.

[0086] Table 3: Cell viability results

[0087] 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*

[0088] Note: *P < 0.05 indicates a significant difference compared with the blank control.

[0089] From Table 3, we can obtain the absorbance of RAW264.7 cells treated with Curcuma wenyujin polysaccharides at different concentrations measured using a CCK-8 kit at a wavelength of 450 nm. The magnitude of the absorbance is positively correlated with cell viability. The absorbance data in Table 3 were imported into the software GraphPad Prism 10.4, and one-way ANOVA was performed on the absorbance data of groups (2 - 9) and group (1) (i.e., the blank control group) in Table 3 using the software GraphPad Prism 10.4 to determine whether there were significant differences in the absorbance data of groups (2 - 9). The analysis results showed that Curcuma wenyujin homogeneous polysaccharide (WP-1G) significantly promoted cell proliferation at low concentrations (0.025 - 0.1 mg / mL) (P < 0.05), and inhibited cell proliferation at high concentrations (0.6 - 1.0 mg / mL) (P < 0.05).

[0090] The cell viability corresponding to the absorbance data of the blank control group was set to 100%. Then, the absorbance data of groups (2 - 9) in Table 3 were converted into the corresponding cell viability, and the concentration unit of the corresponding Curcuma wenyujin polysaccharide was converted to μg / mL, and logarithmic conversion was performed to obtain its corresponding log C value, so as to perform non-linear fitting of cell viability and drug concentration (i.e., the concentration of Curcuma wenyujin polysaccharide), and then obtain the fitting curve of Curcuma wenyujin homogeneous polysaccharide (WP-1G) stimulating macrophage activity. The fitting curve of Curcuma wenyujin homogeneous polysaccharide (WP-1G) stimulating macrophage activity in this example is as Figure 11 shown.

[0091] From Figure 11 the analysis, it can be seen that RAW264.7 affected by Curcuma wenyujin homogeneous polysaccharide (WP-1G) has a good cell viability fitting curve, with an R 2 = 0.9329. Taking the log value of the concentration of Curcuma wenyujin homogeneous polysaccharide (WP-1G) as the abscissa (i.e., taking log C as the abscissa), and the viability of RAW264.7 cells as the ordinate. When log C is 0 - 2, the viability of RAW264.7 cells is greater than 100%, effectively promoting cell proliferation. When log C > 2, its cell viability drops to less than 100%, and at this time, cell proliferation will be inhibited. When the viability of RAW264.7 cells drops to half of the initial viability, the corresponding log C = 2.488. Since the IC50 half-inhibitory concentration is a key indicator for evaluating drug potency, the viability curve of RAW264.7 cells at log CWhen it decreased to 50% at = 2.488, it indicated that the IC50 half-inhibitory concentration of the homogeneous polysaccharide of Curcuma wenyujin (WP-1G) was 0.308 mg / mL. When the IC50 half-inhibitory concentration value was higher than 0.1 mg / mL, it showed that the drug had high safety, and the polysaccharide of Curcuma wenyujin (WP-1G) took effect within the safe concentration range.

[0092] (2) Cell phagocytosis ability experiment

[0093] RAW264.7 cells in the logarithmic growth phase were inoculated into a 96-well plate for culture, with 1×10 5 cells per well. After adherent culture for 24 h, the excess culture medium was discarded. At the same time, a positive control group (only adding lipopolysaccharide (LPS) without adding any preparation) and a negative control group (not adding LPS and any preparation) were set, and different concentration groups of the homogeneous polysaccharide of Curcuma wenyujin (WP-1G) (62.5, 125, 250, 500 mg / mL) were set, with 6 replicate wells in each group. 100 μL of the sample was injected into each well. After intervention with the homogeneous polysaccharide of Curcuma wenyujin (WP-1G) for 24 h, the supernatant was discarded, and the cells were washed twice with PBS. 0.09% neutral red solution was injected into each well and incubated at 37 °C for 3 h. The upper layer of neutral red was discarded, and the cells were washed three times with PBS. 100 μL of cell lysate was injected, and the OD 550nm value was measured, and the macrophage phagocytosis rate was calculated according to the absorbance value. The phagocytic activity of different concentration groups of the homogeneous polysaccharide of Curcuma wenyujin (WP-1G) on macrophages for neutral red in this example was as Figure 12 shown. It can be analyzed from Figure 12 that the homogeneous polysaccharide of Curcuma wenyujin (WP-1G) can promote the phagocytosis ability of RAW264.7 cells for neutral red. When the homogeneous polysaccharide of Curcuma wenyujin (WP-1G) was at a low concentration (<125 μg / mL), the phagocytosis rate of RAW264.7 cells for neutral red was relatively low, indicating that the low concentration of the homogeneous polysaccharide of Curcuma wenyujin (WP-1G) had no significant promoting effect on the phagocytosis ability of RAW264.7 cells; while when the concentration of the homogeneous polysaccharide of Curcuma wenyujin (WP-1G) increased (>250 μg / mL), the phagocytosis rate of RAW264.7 cells for neutral red increased significantly, and was more than twice the phagocytosis rate of the negative control group (P < 0.01), indicating that the high concentration of the homogeneous polysaccharide of Curcuma wenyujin (WP-1G) could significantly enhance the phagocytosis ability of RAW264.7 cells for neutral red.

[0094] (3) Immunomodulatory effect experiment

[0095] RAW264.7 cells in the logarithmic growth phase were inoculated into a 96-well plate for culture, with 1×10 5cells. After adherent culture for 24 h, the excess culture medium was discarded. At the same time, a positive control group (only adding LPS, without adding any preparations) and a negative control group (without adding LPS and any preparations) were set up, and groups with different concentrations of the homogeneous polysaccharide from Curcuma wenyujin (WP-1G) (62.5, 125, 250, 500 mg / mL) were set up, with 6 replicate wells in each group. After the culture was completed, the cell culture medium was collected, centrifuged at 12,000 r / min at 4 °C for 10 min, the content of NO was measured using a NO kit, and the expression levels of the pro-inflammatory factors TNF-α, IL-1β, and IL-6 were measured using ELISA kits respectively. In this example, the concentrations of NO secreted by macrophages stimulated with different concentrations of the homogeneous polysaccharide from Curcuma wenyujin (WP-1G) are as Figure 13 shown. In this example, the concentrations of the cell inflammatory factors IL-1β, IL-6, and TNF-α secreted by macrophages stimulated with different concentrations of the homogeneous polysaccharide from Curcuma wenyujin (WP-1G) are as Figure 14 shown. Among them, Figure 14 A in Figure 14 is the change in the concentration of the cell inflammatory factor IL-1β secreted by macrophages stimulated with different concentrations of the homogeneous polysaccharide from Curcuma wenyujin (WP-1G) in this example. Figure 14 B in Figures 13 - 14 is the change in the concentration of the cell inflammatory factor IL-6 secreted by macrophages stimulated with different concentrations of the homogeneous polysaccharide from Curcuma wenyujin (WP-1G) in this example.

[0096] (4) Immune factor mRNA experiment

[0097] RAW264.7 cells in the logarithmic growth phase were taken and inoculated in a 6-well plate for culture, with 1×10 6 cells in each well, and adherent culture was carried out for 24 h. At the same time, a positive control group (only adding LPS, without adding any preparations) and a negative control group (without adding LPS and any preparations) were set up, and groups with different concentrations of the homogeneous polysaccharide from Curcuma wenyujin (WP-1G) (62.5, 125, 250, 500 mg / mL) were set up, with 6 replicate wells in each group. According to the setting, 1000 μL of the sample was injected into each well. After 24 h of intervention with the homogeneous polysaccharide from Curcuma wenyujin (WP-1G), the supernatant was removed, washed 2 times with PBS at 4 °C, and 1 mL of Trizol RNA extraction reagent was added.

[0098] RNA extraction: Inject 200 μL of chloroform into each well, mix thoroughly by shaking, centrifuge at 4°C and 10,000 r / min to obtain the supernatant, add another 500 μL of isopropanol, mix well, 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.

[0099] RNA purity identification: Compare whether the value of OD 260nm / OD 280nm is between 1.8 and 2.2.

[0100] RNA reverse transcription: Operate according to the Transcriptor cDNA Synth Kit reverse transcription kit.

[0101] PCR amplification reaction: The specific system and amplification program are shown in Tables 4 - 6, and calculate the relative expression level through the 2 -ΔΔCT formula.

[0102] Table 4: Gene primer sequences

[0103] Primer name Gene sequence (5'to 3') TNF-α (forward primer) CCT CAG GAA CGG GAC TCG AA TNF-α (reverse primer) ATG TAC ACC AAG TCG GTA GCA CCA IL-6 (forward primer) GGA ATT CGT GGA AAT GAG AA IL-6 (reverse primer) GCA CTA GGA AAG CCG AGT AC IL-1β (forward primer) TGT GAT GTT CCC ATT AGA C IL-1β (reverse primer) AAT ACC ACT TGT TGG CTT A GADPH (forward primer) TTT GTC AAG CTC ATT TCC TGG TATG GADPH (reverse primer) TGG GAT AGG GCC TCT CTT GC

[0104] Table 5: Fluorescent quantitative PCR reaction system

[0105] Reagent name Reagent dosage (μL) 2×SYBR Green PCR Mix 10 DNA template 2 Forward primer 1 Reverse primer 1 Water (DNase free) 6 Total 20

[0106] Table 6: Fluorescent quantitative PCR reaction process

[0107] Reaction procedure Temperature (°C) Reaction time (min) Number of cycles (times) Pre-denaturation 95 2 1 Denaturation 95 0.25 40 Annealing 60 0.5 40 Extension 60 0.5 40 Stabilization 72 0.5 1

[0108] In this example, the relative mRNA expression levels of different cell inflammatory factors secreted by macrophages stimulated with different concentrations of Curcuma wenyujin homogeneous polysaccharide (WP - 1G) are as Figure 15 shown. Among them, Figure 15 A in it is the relative mRNA expression level of cell inflammatory factor IL - 1β secreted by macrophages stimulated with different concentrations of Curcuma wenyujin homogeneous polysaccharide (WP - 1G) in this example. Figure 15 B in it is the relative mRNA expression level of cell inflammatory factor IL - 6 secreted by macrophages stimulated with different concentrations of Curcuma wenyujin homogeneous polysaccharide (WP - 1G) in this example. Figure 15 C in it is the relative mRNA expression level of cell inflammatory factor TNF - α secreted by macrophages stimulated with different concentrations of Curcuma wenyujin homogeneous polysaccharide (WP - 1G) in this example. From Figure 15Analysis shows that under the stimulation of Curcuma wenyujin homogeneous polysaccharide (WP-1G) at different concentrations, the relative mRNA expression levels of related cellular inflammatory factors TNF-α, IL-1β, and IL-6 secreted by macrophages are all significantly up-regulated, and the results show the same trend as the protein expression levels of related cellular inflammatory factors TNF-α, IL-1β, and IL-6.

[0109] The above description only details the preferred embodiments and principles of the present invention. For those of ordinary skill in the art, based on the ideas provided by the present invention, there will be changes in the specific implementation manners, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a uniform polysaccharide of a medicinal plant Curcuma aromatica, characterized in that: The following steps are involved: (S.1) defatting 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 longifolia; (S.3) separating and purifying the total polysaccharide of Curcuma aromatica obtained in step (S.2) by ion exchange column DEAE-52 column and gel column Sephadex G-100 column chromatography, and then concentrating and freeze-drying to obtain uniform polysaccharide of Curcuma aromatica; 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 to 5:1; Step (S.3) comprises: The total polysaccharide of Curcuma aromatica obtained in step (S.2) was eluted with distilled water using a DEAE-52 column at a flow rate of 1 mL / min, and the absorbance of each tube of eluate at 490 nm was measured using a phenol-sulfuric acid method, and a scatter plot was drawn. The eluates corresponding to the peaks were combined, concentrated, dialyzed with a 3500Da dialysis bag, and freeze-dried to obtain an eluted component and named it Curcuma aromatica polysaccharide WP-1; The WP-1 polysaccharide component was taken, distilled water was added to dissolve, and the supernatant was taken by centrifugation. The supernatant was further separated and purified by Sephadex G-100 column. The absorbance value of each tube of eluate at 490 nm was measured by phenol-sulfuric acid method, and a scatter plot was drawn. The eluate at the corresponding peak was collected, concentrated by rotary evaporator, and freeze-dried to obtain the elution component and named it WP-1G, which is the uniform polysaccharide of Curcuma longifolia. The Wenyujin uniform polysaccharide includes mannose and glucose; the molar ratio of mannose to glucose contained in the Wenyujin uniform polysaccharide is 1:3.87; The structure of the Curcuma wenyujin uniform polysaccharide is shown in the following formula (1):

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

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

4. Use of the uniform polysaccharide of Curcuma aromatica L. prepared by the method as claimed in claim 1 in the preparation of immunomodulatory drugs.

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