Amomum villosum polysaccharide, its preparation process and use in preparing anti-complement drugs

Abstract

The invention discloses tsaoko polysaccharide and a preparation process thereof and use thereof in preparing an anti-complement drug, belonging to the technical field of polysaccharide drugs. The invention separates three tsaoko polysaccharide components ATP-1-1, ATP-4-1 and ATP-5-1 with anti-complement activity from tsaoko, a medicinal material having both medicinal and edible properties. Experiments have confirmed that the tsaoko polysaccharides have significant inhibitory effects on complement activation and can act on C2, C4 and Factor B targets at the same time, and can be further used as active ingredients to prepare novel anti-complement drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide drugs, and specifically relates to three kinds of tsaoko polysaccharides with anti-complement activity and preparation processes thereof and uses thereof in preparing anti-complement drugs. Background Art

[0002] The prior art discloses that the complement system is an important part of the body's natural immune defense. While ensuring accurate removal of foreign pathogens invading with the participation of different regulatory proteins, in order to prevent damage to host tissues, complement is strictly controlled by cell surface regulators to maintain homeostasis. However, disorders of the complement system can lead to excessive inflammation and a variety of diseases, such as complement-mediated immune complex diseases, neurodegenerative diseases, cancers, and metabolic diseases. Recent clinical studies have shown that overactivation of the complement system also plays a key role in the pathogenesis and severity of new coronary infection. Although immunosuppressants such as glucocorticoids, cyclophosphamide, and methotrexate, which are widely used in clinical practice, have a certain therapeutic effect on certain diseases related to excessive complement activation, since such drugs are not specific complement inhibitors, long-term use has the disadvantages of reducing the body's defense function, resulting in decreased anti-infection ability, easy secondary infection, and the spread of potential lesions, and producing a variety of complications and side effects. Therefore, there is an urgent need for efficient, low-toxic, and specific new complement inhibitors in clinical practice.

[0003] Active ingredients with anti-complement effects are widely present in Chinese medicinal materials. my country is rich in Chinese medicinal resources, and many Chinese medicinal materials have obvious regulatory effects on the immune system, which are valuable resources for finding anti-complement drug precursors. Polysaccharides, as the main active ingredients of Chinese medicine, are safe, low-toxic, and have good biocompatibility. They have broad application prospects in disease prevention and clinical new drug research and development. In addition, Chinese medicinal polysaccharide anti-complement active substances usually have advantages such as multiple targets, multiple pathways, and long-term effects. Therefore, it is of great significance to find new complement inhibitors from Chinese medicinal polysaccharides. In particular, the polysaccharide components in Chinese medicinal materials with medicinal and edible properties are safe, reliable, non-toxic, and have unique immune regulatory mechanisms and potential anti-complement activity.

[0004] Tsaoko is the dried mature fruit of the perennial herbaceous plant Amomum tsao-ko Crevost et Lemarie of the Zingiberaceae family. It is also known as grass fruit kernel and grass fruit. It is distributed in tropical and subtropical regions of Asia, and is mainly produced in Guangxi and Yunnan in my country. It is warm in nature, pungent in taste, and belongs to the lung and stomach meridians. It has the effects of drying dampness and warming the middle, stopping malaria and removing phlegm. It can be used to treat internal obstruction of cold and dampness, abdominal distension and pain, fullness and vomiting, malaria fever and fever, and plague fever. Because of its spicy aroma, fishy smell removal, and appetite enhancement, Tsaoko has been included in the list of "medicine and food of the same origin" by the National Health Commission as one of the "five spices" in food seasoning. In addition, according to the diagnosis and treatment plan for novel coronavirus infection publicly released by the National Health Commission and the State Administration of Traditional Chinese Medicine, Amomum villosum has played an important role in the TCM treatment of mild, common, and severe COVID-19 patients. Its use in the treatment of COVID-19 is based on the book "On Plague" written by Wu Youke, a famous plague doctor in the Ming Dynasty. In the book, the TCM prescription "Dayuanyin" recorded in the book, Amomum villosum plays the role of ministerial medicine in dispelling evil spirits. However, there has been no report on the extraction, purification, and separation process of uniform polysaccharides with anti-complement activity in Amomum villosum. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide three kinds of Amomum villosum polysaccharides with anti-complement activity and their preparation process and application in the preparation of anti-complement drugs.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a preparation process of amomum villosum polysaccharide, which mainly comprises the following steps:

[0008] (1) Take dried Amomum villosum, crush it, add water to extract it at a liquid-to-solid ratio of 20:1 to 40:1 mL / g, the extraction temperature is 80 to 90° C., the extraction time is 80 to 120 min, the extraction is repeated 1 to 3 times, the filtrate is filtered, concentrated, 95% ethanol is added to precipitate polysaccharides, centrifuged, the precipitate is collected, evaporated to dryness, and ground into powder to obtain Amomum villosum crude polysaccharides;

[0009] (2) taking the crude amomum villosum polysaccharide obtained in step (1), dissolving it in distilled water, loading it into an S-8 macroporous adsorption resin chromatography column, eluting it with distilled water, collecting the eluate, concentrating it, dialyzing it, drying it, and grinding it into powder to obtain purified amomum villosum polysaccharide;

[0010] (3) taking the purified Amomum villosum polysaccharide obtained in step (2), dissolving it in deionized water, loading it onto a DEAE-52 cellulose chromatography column, and eluting it with a gradient of 0, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L and 0.5 mol / L NaCl solution in sequence, combining the same polysaccharide components according to the phenol-sulfuric acid color reaction, collecting the secondary polysaccharide components eluted under 0, 0.3 mol / L and 0.4 mol / L NaCl solution, respectively, concentrating, dialyzing and freeze-drying them to obtain Amomum villosum polysaccharide ATP-1, Amomum villosum polysaccharide ATP-4 and Amomum villosum polysaccharide ATP-5, respectively;

[0011] (4) Using deionized water, the Amomum villosum polysaccharide ATP-1, Amomum villosum polysaccharide ATP-4 and Amomum villosum polysaccharide ATP-5 obtained in step (3) were respectively re-dissolved and loaded onto a Sephadex G-100 chromatography column, and distilled water was used as the elution solvent. The elution was performed at a flow rate of 0.5 mL / min for 500 to 600 min. The same polysaccharide components were combined according to the phenol-sulfuric acid color reaction, and the polysaccharide components were concentrated, dialyzed and freeze-dried to obtain Amomum villosum polysaccharide ATP-1-1, ATP-4-1 and ATP-5-1.

[0012] Based on the above technical solution, further, in step (1), the volume is concentrated to 1 / 10 to 1 / 3 of the original volume, and the specific process of precipitating polysaccharides is to add 95% ethanol until the final ethanol concentration of the extract is 75 to 85%, and let it stand for 24 to 48 hours.

[0013] Based on the above technical solution, further, in step (1), the extraction rate of crude polysaccharides from Amomum villosum is 11.02±0.49%, and the polysaccharide content is 26.73±1.52%.

[0014] Based on the above technical solution, further, in step (2), the sample concentration is 1-3 mg / mL, the sample volume is 0.3-0.5 BV; the elution flow rate is 1-3 BV / h, and the elution volume is 1-3 BV.

[0015] Based on the above technical scheme, further, in step (2), the deproteinization rate, decolorization rate and polysaccharide recovery rate were 69.4±0.92%, 78.21±1.12% and 81.6±1.71% respectively, the comprehensive score of the purification effect was 73.45±0.32%, and the yield of the purified Amomum villosum polysaccharide was 26.31±0.41%.

[0016] Based on the above technical solution, further, in step (3), the loading concentration is 50-100 mg / mL, the loading volume is 0.2-0.4 BV, the elution flow rate is 1 mL / min, and each gradient elution is 125-150 min.

[0017] Another aspect of the present invention provides the Amomum villosum polysaccharide obtained by the above-mentioned preparation process, wherein the Amomum villosum polysaccharide includes Amomum villosum polysaccharide ATP-1-1, ATP-4-1 and ATP-5-1.

[0018] Based on the above technical solution, further, the Amomum villosum polysaccharide ATP-1-1 is a neutral polysaccharide with a molecular weight of about 9.15×10 2 kDa; the total sugar content was 91.44±1.13%; it did not contain uronic acid, and the monosaccharide composition showed that it was composed of mannose, rhamnose, glucose, galactose and arabinose, with a molar ratio of 1.0:0.7:5.6:3.4:0.9.

[0019] Based on the above technical solution, further, the Amomum villosum polysaccharide ATP-4-1 is an acidic polysaccharide with a molecular weight of about 2.46×10 4 kDa; the total sugar content was 94.54±1.87%, the uronic acid content was 39.59±0.22%, and the monosaccharide composition showed that it was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, with a molar ratio of 1.0:4.8:2.4:20.7:2.0:10.6:5.3:5.3.

[0020] Based on the above technical solution, further, the Amomum villosum polysaccharide ATP-5-1 is an acidic polysaccharide with a molecular weight of about 2.69×10 5 kDa; the total sugar content was 95.81±1.33%; the uronic acid content was 33.23±0.19%, and the monosaccharide composition showed that it was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, with a molar ratio of 1.0:6.6:2.7:22.0:3.3:12.8:9.2:8.4.

[0021] The present invention provides the use of the Amomum villosum polysaccharide in preparing anti-complement drugs.

[0022] Based on the above technical scheme, further, the Amomum villosum polysaccharide ATP-1-1, Amomum villosum polysaccharide ATP-4-1 and Amomum villosum polysaccharide ATP-5-1 have obvious inhibition on cell hemolysis caused by activation of classical and alternative complement pathways, that is, have significant anti-complement effect.

[0023] Based on the above technical solution, further, the anti-complement activity of Amomum villosum polysaccharide ATP-1-1 is CH 50 =6.28±0.23μg / mL, AP 50 =45.33±0.16μg / mL, can interact with targets C2, C4 and Factor B to inhibit complement activation;

[0024] Based on the above technical solution, further, the anti-complement activity of the Amomum villosum polysaccharide ATP-4-1 is CH 50 =7.39±0.36μg / mL, AP 50 =8.62±0.46μg / mL, can interact with targets C2, C4 and Factor B to inhibit complement activation.

[0025] Based on the above technical solution, further, the anti-complement activity of Amomum villosum polysaccharide ATP-5-1 is CH 50 =4.47±0.44μg / mL, AP 50 =9.41±0.83μg / mL, can interact with targets C2, C4 and Factor B to inhibit complement activation.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The invention extracts, purifies and separates the amomum villosum to obtain three amomum villosum polysaccharides ATP-1-1, ATP-4-1 and ATP-5-1. In vitro experiments confirm that the three amomum villosum polysaccharide components have significant complement inhibition activity and can act on C2, C4 and Factor B targets. They can be developed and manufactured as anti-complement drugs, providing a material basis for the treatment of complement-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0029] Figure 1 This is the single factor experimental result of optimizing the extraction process of Amomum villosum polysaccharide, where a: solid-liquid ratio, b: extraction temperature, and c: extraction time.

[0030] Figure 2 This is the single-factor experimental result for optimizing the purification process of Amomum villosum polysaccharide, where a: elution volume, b: flow rate, c: sample concentration, and d: sample volume.

[0031] Figure 3 This is the separation elution curve of Amomum villosum polysaccharide on DEAE-52 chromatographic column.

[0032] Figure 4 The elution curves of ATP-1 (a), ATP-4 (b) and ATP-5 (c) on Sephadex G-100 are shown.

[0033] Figure 5 The following are the gel permeation chromatograms of ATP-1-1 (a), ATP-4-1 (b), and ATP-5-1 (c).

[0034] Figure 6These are the liquid chromatograms of the monosaccharide compositions of standard monosaccharide (a), ATP-1-1 (b), ATP-4-1 (c) and ATP-5-1 (d).

[0035] Figure 7 It is the target of ATP-1-1(a), ATP-4-1(b) and ATP-5-1(c) in the classical pathway.

[0036] Figure 8 It is the target of ATP-1-1(a), ATP-4-1(b) and ATP-5-1(c) in the alternative pathway. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Unless otherwise specified, the experimental methods adopted in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be purchased from chemical companies.

[0038] The Amomum tsao-ko Crevost et Lemarie used in the examples were purchased from the Anhui Bozhou Chinese herbal medicine wholesale market.

[0039] Example 1 Extraction of Amomum villosum polysaccharide and its process optimization

[0040] Take the dried Amomum villosum, crush it through a 10-mesh sieve, extract it twice with hot water reflux, and filter to obtain the Amomum villosum polysaccharide extract. Concentrate the water extract to 1 / 5 of the original volume, add 95% ethanol to a final ethanol concentration of 85%, let it stand for 24 hours, centrifuge, collect the precipitate, evaporate it in a water bath, grind it into powder, and obtain the Amomum villosum crude polysaccharide. Measure the polysaccharide concentration by the phenol-sulfuric acid method, and calculate the polysaccharide extraction rate. The effects of liquid-to-solid ratio (10:1~50:1mL / g), extraction temperature (50~95℃) and extraction time (30~210min) on the Amomum villosum extraction rate were investigated. The results of the single-factor experiment on the extraction of Amomum villosum polysaccharides are shown in the figure. Figure 1 As shown in the figure, the optimal extraction conditions of Amomum villosum polysaccharide under the single factor experiment were: liquid-to-solid ratio 30:1 mL / g, extraction temperature 85°C and extraction time 90 min.

[0041] According to the results of the single factor experiment, the optimal condition in the single factor experiment was set as the 0 level, and the two conditions before and after the optimal condition were set at -1 and 1 levels respectively. Using Design-Expert.V8.0.6.1 software, 17 groups of response surface optimization experiments were designed using Box-Behnken (BBD) in the response surface, with liquid-to-solid ratio X1, extraction time X2 and extraction temperature X3 as independent variables, and polysaccharide extraction rate as the response value Y. The 17 groups of running experimental schemes and experimental results are shown in Table 1.

[0042] Table 1 17 experimental schemes and results of response surface design for extracting amomum villosum polysaccharides

[0043]

[0044]

[0045] Based on the multiple regression analysis of 17 groups of response surface experimental data, the second-order polynomial equation of the optimization model is: Y = 10.22 + 0.39 × X1 + 0.055 × X2 + 0.11 × X3 + 0.040 × X1 × X2 - 0.12 × X1 × X3 - 0.060 × X2 × X3 - 2.28 × X1 2

[0046] -0.77×X2 2 -0.90×X3 2 .

[0047] The results of variance analysis of the response surface quadratic model for the optimization of the extraction process of Amomum villosum polysaccharides are shown in Table 2. The F value of the model is 382.38 and the P value is <0.0001, indicating that the model has a highly significant response in terms of extraction rate. The F value of the Lack of Fit is 0.30 and the P value is 0.8233>0.05, indicating that the lack of fit is not related to pure error, which fully proves the reliability of the model. In addition, the coefficient of determination (R 2 ) is 0.9980, and the adjusted coefficient of determination (Adj-R 2 ) was 0.9954, and the coefficient of variation (CV%) was 1.14, which indicated that the experimental value and the predicted value of the extraction rate of cardamom polysaccharides were highly correlated. The linear term coefficient (X1) and the quadratic term coefficient (X1 2 , X2 2 and X3 2 ) had a very significant effect on the extraction rate of cardamom polysaccharides (P < 0.0001), the linear term coefficient (X3) and the interaction term coefficient (X1X3) had a significant effect (P < 0.05), while the effects of other coefficients (X2, X1X2 and X2X3) were not significant (P > 0.05).

[0048] Table 2 Response surface quadratic model variance analysis for optimization of amomum villosum polysaccharide extraction process

[0049]

[0050]

[0051] Note: * indicates P < 0.05, the effect is significant; ** indicates P < 0.01, the effect is very significant; *** indicates P < 0.001, the effect is extremely significant.

[0052] The optimal values ​​of the selected variables were obtained by solving the regression equation using Design-Expert Software. The theoretical optimal extraction process of Amomum villosum polysaccharide was a liquid-to-solid ratio of 30.85:1 mL / g, an extraction temperature of 85.18°C, and an extraction time of 91.62 min, and the predicted extraction rate was 10.23%. Considering the actual industrial extraction situation, the optimal extraction process parameters were a liquid-to-solid ratio of 30:1 mL / g, an extraction temperature of 85°C, and an extraction time of 100 min. The actual Amomum villosum extraction rate was measured to be 11.02±0.49% (n=3), which was not significantly different from the predicted extraction rate (P>0.05), indicating that the optimal extraction process of Amomum villosum polysaccharide was stable and reliable.

[0053] Example 2 Purification of Amomum villosum polysaccharide and its process optimization

[0054] (1) Preliminary screening of the best purification resin

[0055] The best purification resin for Amomum villosum polysaccharide was selected from five different types of macroporous adsorption resins (S-8, AB-8, NKA-II, D101 and X-5) by static adsorption method. 20 mL of 2 mg / mL Amomum villosum crude polysaccharide solution was added to 3 g of resin, shaken at 35 ° C in a constant temperature shaker for 30 min, filtered, and the adsorption solution was collected. Distilled water was added to the resin again for desorption and shaken for 30 min, and the desorption solution was collected. The polysaccharide concentrations in the crude polysaccharide solution, adsorption solution and desorption solution were determined by phenol-sulfuric acid method, and the adsorption capacity (Ac, mg / g), adsorption rate (Ae, %) and desorption rate (De, %) were calculated. The results are shown in Table 3. The Ac and Ae of S-8 resin for Amomum villosum polysaccharide were significantly higher than those of other resins (P<0.05). The De of S-8 for Amomum villosum polysaccharide was only lower than that of NKA-II. Therefore, S-8 is the best macroporous adsorption resin for purifying Amomum villosum polysaccharide.

[0056] Table 3 Adsorption capacity, adsorption rate and desorption rate of Amomum villosum polysaccharide in different types of resins

[0057] Resin Ac(mg / g) Ae(%) De(%) S-8 2.14±0.13 42.18±2.12 35.74±1.12 AB-8 1.68±0.15 30.89±1.87 25.52±1.45 NKA-II 0.83±0.11 15.28±1.24 48.33±1.34 D101 1.64±0.12 30.30±1.50 27.57±1.82 X-5 1.95±0.12 35.90±1.23 27.70±1.64

[0058] (2) Determination of purification index weight values

[0059] The weight value of each purification index was calculated by analytic hierarchy process, and the comprehensive score of purification effect was used as the decision target, and polysaccharide recovery rate (Pr), deproteinization rate (Dp) and decolorization rate (Dc) were used as decision criteria to establish a hierarchical structure. Based on the nine-level evaluation rule, a pairwise matrix structure N was constructed (as shown in Formula 1 below), and the priority weights of Pr, Dp and Dc were set to w1, w2 and w3 respectively. According to the following comparison matrix calculation, the priority weight values ​​of decision criteria Pr, Dp and Dc were obtained.

[0060]

[0061] The maximum eigenvalue of the matrix N is 3.0059, and the consistency ratio CR = 0.0086 < 0.1, which indicates that the consistency test has passed. The calculated initial weight coefficients are normalized to obtain values ​​of w1, w2, and w3 of 0.59, 0.28, and 0.13, respectively. Finally, the comprehensive score of the purification effect of Amomum villosum polysaccharide can be calculated based on the weights of Pr, Dp, and Dc (as shown in Formula 2 below).

[0062] Comprehensive score (%) = 0.59 × Pr (%) + 0.28 × Dp (%) + 0.13 × Dc (%) (Formula 2)

[0063] (2) Optimization of purification process parameters by single factor and response surface experiments

[0064] Dynamic adsorption method was used to purify Amomum villosum polysaccharides. The crude polysaccharides were wet loaded and gradually dripped into the pretreated macroporous adsorption resin chromatography column (2.6cm×20cm), eluted with distilled water, and the eluted solution was collected, evaporated to dryness in a water bath, and ground to obtain the purified polysaccharide for later use. The phenol-sulfuric acid method and Coomassie brilliant blue method were used to determine the polysaccharide and protein content before and after purification, respectively, and the determination of Pr and Dp pigments was calculated by calculating the absorbance value at 420nm to obtain Dc. The effects of elution volume (1~5BV), elution flow rate (1~5BV / h), loading concentration (1~5mg / mL) and volume (0.2~0.6BV) on the purification of crude Amomum villosum polysaccharides were investigated. The results of the single factor experiment are shown in the figure. Figure 2 As shown, the optimal resin dynamic adsorption purification conditions for Amomum villosum polysaccharide under single factor experiment are: elution volume 2BV, elution flow rate 2BV / h, loading concentration 2mg / mL and loading volume 0.4BV.

[0065] On the basis of the single factor experiment, the response surface Box-Behnken (BBD) method was used to design 29 groups of response surface optimization purification experiments. The elution volume A, elution flow rate B, loading concentration C and loading volume D were used as independent variables, and the comprehensive score of the purification effect Y0 was used as the response value. The 29 groups of experimental schemes and experimental results are shown in Table 4.

[0066] Table 4 Experimental schemes and results of 29 groups of response surface design for purification of Amomum villosum polysaccharides

[0067]

[0068]

[0069] Based on the 29 groups of response surface experimental data, the polynomial regression equation was obtained: Y0 = +74.71 + 0.31 × A + 0.46 × B - 0.18 × C + 0.29 × D - 0.61 × A × B + 0.005 × A × C - 1.23 × A × D + 0.67 × B × C

[0070] -0.13×B×D-1.63×C×D-4.98×A 2 -3.67×B 2 -5.11×C 2 -9.57×D 2

[0071] The variance analysis and statistical significance results of the multivariate regression model of Amomum villosum polysaccharides are shown in Table 5. The P values ​​of the "Model" of the regression model are all <0.0001, which proves that the model is significant. The P value of the lack of fit term is 0.7480>0.05, indicating that the error is negligible. At the same time, R 2 , Adj R 2 、Pred R 2 The P values ​​of the CV% values ​​were 0.9961, 0.9921, 0.9838, and 0.70, respectively. The Pred R 2 and Adj R 2 The difference between them was 0.0083<0.1, which revealed a good correlation between the predicted purification effect and the actual purification effect of Amomum villosum polysaccharide. 2 , B 2 , C 2 and D 2 ) had an extremely significant effect on the purification of Amomum villosum (P<0.0001), the linear coefficient (B) had a very significant effect (P<0.01), the A, D, AB and BC variables had a significant effect (P<0.05), while the C, AC and BD variables had no significant effect (P>0.05).

[0072] Table 5 Response surface quadratic model variance analysis for optimization of amomum villosum polysaccharide purification

[0073]

[0074] Note: * indicates significant impact; ** indicates very significant impact; *** indicates extremely significant impact.

[0075] The optimal purification process of Amomum villosum polysaccharide was calculated by polynomial regression equation as follows: S-8 resin, elution volume 2.03BV, flow rate 2.06BV / h, sample concentration 1.98mg / mL, sample volume 0.4BV, and predicted comprehensive score 74.72%. Considering the actual industrial production, the parameters were adjusted to: elution volume 2BV, elution flow rate 2BV / h, sample concentration 2mg / mL and sample volume 0.4BV. Under these conditions, Pr, Dp and Dc were 69.4±0.92%, 78.21±1.12% and 81.6±1.71% respectively, the actual comprehensive score of purification effect was 73.45±0.32% (n=3), the content of cardamom polysaccharide increased from 26.73±1.52% to 70.48±2.36%, and the yield was 26.31±0.41%, indicating that the purity of cardamom polysaccharide purified by macroporous adsorption resin was significantly improved, the purification process was stable and reliable, and the purification effect was excellent.

[0076] Example 3 Separation of Amomum villosum polysaccharide

[0077] Take the purified Amomum villosum polysaccharide, add deionized water to dissolve it again, and separate it with a DEAE-52 cellulose chromatography column. Elute it with deionized water, 0.1mol / L, 0.2mol / L, 0.3mol / L, 0.4mol / L and 0.5mol / L NaCl solution at a flow rate of 1mL / min, 5mL / bottle, collect the eluate, and number it according to the collection order. Each gradient elution lasts 125min, and each fraction is collected. According to the phenol-sulfuric acid color reaction, the bottle number is used as the horizontal axis and the absorbance value is used as the vertical axis. The following is the attached figure. Figure 3 The elution curves shown were shown. The same polysaccharide components were combined, and the secondary polysaccharide components eluted with distilled water, 0.3 mol / L and 0.4 mol / L NaCl solutions were collected, concentrated, dialyzed and freeze-dried to obtain three secondary Amomum villosum polysaccharide components ATP-1, ATP-4 and ATP-5. Deionized water was then added to dissolve the three secondary polysaccharide components, and the components were separated by Sephadex G-100 chromatography. Distilled water was used as the eluent, the flow rate was 0.5 mL / min, and the eluent was collected at 5 mL / bottle. The elution lasted for about 500 minutes. According to the phenol-sulfuric acid method, the attached Figure 4 The ATP-1-1, ATP-4-1 and ATP-5-1 polysaccharide components shown were concentrated, dialyzed and freeze-dried to obtain uniform Amomum villosum polysaccharides ATP-1-1, ATP-4-1 and ATP-5-1.

[0078] Example 4 Structural Characterization of Amomum villosum Polysaccharides (ATP-1-1, ATP-4-1 and ATP-5-1)

[0079] (1) Molecular weight determination

[0080] The molecular weights of the Amomum villosum polysaccharides ATP-1-1, ATP-4-1 and ATP-5-1 were determined by high performance gel permeation chromatography combined with a differential refractive index detector (HPGPC-RID). A 2.0 mg / mL Amomum villosum polysaccharide solution was passed through a 0.45 μm microporous filter membrane and injected into a Shodexsugar KS-804 sugar column (8.0 mm×300 mm). The chromatographic conditions were: injection volume 20 μL, ultrapure water as the mobile phase, flow rate 1.0 mL / min, column temperature 50°C, and differential refractive index detector temperature 35°C. Data were recorded and processed on the N2000 GPC chromatography workstation. The third-order calibration curve drawn based on dextran standards of different molecular weights is Log Mw=35.47-26.01x+7.642x 2 -0.78x 3 , the retention time of the polysaccharide on the HPGPC chromatogram was substituted into the calibration curve to calculate the molecular weight.

[0081] The gel chromatograms of ATP-1-1, ATP-4-1 and ATP-5-1 are shown in the attached figure. Figure 5 Furthermore, the molecular weights of ATP-1-1, ATP-4-1, and ATP-5-1 were calculated to be 9.15×10 2 kDa, 2.46×10 4 kDa and 2.69×10 5 kDa.

[0082] (2) Determination of total sugar and uronic acid content

[0083] The total sugar content was determined by the phenol-sulfuric acid method: the total sugar contents of ATP-1-1, ATP-4-1 and ATP-5-1 were 91.44±1.13%, 94.54±1.87% and 95.81±1.33%, respectively.

[0084] The uronic acid content was detected by the m-hydroxybiphenyl method: no uronic acid was detected in ATP-1-1, and the uronic acid content of ATP-4-1 and ATP-5-1 was 39.59±0.22% and 33.23±0.19%, respectively.

[0085] (3) Determination of monosaccharide components

[0086] The monosaccharide components were determined by 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization method. 2 mg of Amomum villosum polysaccharide was accurately weighed and mixed with 1 mL of 2 mol / L trifluoroacetic acid (TFA), and the reaction was sealed and hydrolyzed for 2 h. The hydrolyzate was added with 200 μL of 0.5 mol / L PMP methanol solution and 0.3 mol / L NaOH solution in turn, and the mixture was placed in a 70 °C water bath for 1 h. 200 μL of 0.3 mol / L HCl was added, and the chloroform was extracted three times, and the aqueous layer was collected. Mannose (Man), glucosamine (GlcN), ribose (Rib), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara) and fucose (Fuc) were used as standard controls, and the derivatization steps were the same as above. The pretreated samples and standards were filtered through a 0.45 μm microporous membrane and injected. Chromatographic conditions: Supersil ODS2 column (5 μm, 4.6 mm×250 mm), column temperature 35° C., detector wavelength 245 nm, flow rate 0.8 mL / min, injection volume 20 μL, mobile phase acetonitrile: 0.2 mol / mL phosphate buffer solution (pH 6.8) = 83:17 (v / v).

[0087] The results showed that Figure 6 As shown, ATP-1-1 is composed of five monosaccharides, namely mannose, rhamnose, glucose, galactose and arabinose, with a molar ratio of 1.0:0.7:5.6:3.4:0.9; ATP-4-1 is composed of eight monosaccharides, namely mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, with a molar ratio of 1.0:4.8:2.4:20.7:2.0:10.6:5.3:5.3; ATP-5-1 is composed of eight monosaccharides, namely mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, with a molar ratio of 1.0:6.6:2.7:22.0:3.3:12.8:9.2:8.4.

[0088] Example 5 Classical Pathway Complement Inhibition Assay

[0089] Take 6% sheep red blood cells and use GVB-Ca 2+ / Mg 2+ (Beijing Coolaibo Technology Co., Ltd., catalog number: DZSL0356-500ML) Buffer solution was adjusted to 2×10 9cells / mL, mixed with hemolysin (Nanjing Senbega Biotechnology Co., Ltd., catalog number: SBJ-RXS10) in a 1:1 ratio, and centrifuged at 37°C for 30 minutes to remove unbound hemolysin, and sensitized sheep erythrocytes (EAs) were obtained for use. Normal healthy adult serum (NHSP) was used as the complement source for this classical pathway. Accurately weigh the Amomum villosum polysaccharide and use GVB-Ca 2+ / Mg 2+ The buffer solution was prepared into different concentration gradients, and 1:80 diluted NHSP was added. After pre-incubation in a 37°C water bath for 30 minutes, sensitized sheep red blood cells were added, and the mixture was incubated in a 37°C water bath for 30 minutes. The mixture was centrifuged, and the supernatant was taken and placed in a 96-well plate. The absorbance value was measured at 540 nm by an ELISA reader. At the same time, a blank group, a full hemolysis group, a NHSP group, a negative drug control group (glucose), and a positive drug control group (heparin) were set up in the experiment. The hemolysis inhibition rate of Amomum villosum polysaccharide under different concentration gradients was calculated, and the concentration of polysaccharide required for 50% inhibition of hemolysis of each component was calculated using GraphPadprism 6.0 (CH 50 value).

[0090] The ingredients and addition amounts in each group are as follows:

[0091] Blank group: 40μL EAs+260μL GVB-Ca 2+ / Mg 2+ ;

[0092] Full hemolysis group: 40 μL EAs + 260 μL DDW;

[0093] NHSP group: 150μL NHSP+40μL EAs+110μL GVB-Ca 2+ / Mg 2+ ;

[0094] Negative control group: 30 μL 1 mmol / L glucose + 150 μL NHSP + 40 μL EAs + 80 μL GVB-Ca 2+ / Mg 2+ ;

[0095] Positive control group: 12 μL heparin of different concentrations + 150 μL NHSP + 40 μL EAs + 98 μL GVB-Ca 2+ / Mg 2+ ;

[0096] Drug administration group: 6 μL samples of different concentrations + 150 μL NHSP + 104 μL GVB-Ca 2+ / Mg 2+ +40 μL EAs.

[0097] The results showed that the CH 50 The values ​​were 6.28±0.23μg / mL, 7.39±0.36μg / mL and 4.47±0.44μg / mL respectively. The three Amomum villosum polysaccharides had significant inhibitory activity on the activation of the classical complement pathway (as shown in Table 6).

[0098] Example 6 Alternative Pathway Complement Inhibition Assay

[0099] Normal healthy human serum was measured by GVB-Mg 2+ / EGTA buffer was incubated on ice for 15 min as the complement source for this alternative pathway. 2+ / Mg 2+ Mix the buffer solution with fresh rabbit blood, centrifuge for 5 min in a low-temperature high-speed centrifuge, discard the supernatant, and prepare 2% rabbit red blood cells (Erab) with Aldrich solution. 2+ / EGTA buffer is diluted to 5×10 8 cells / mL, then add NHSP and different concentrations of diluted Amomum villosum polysaccharides, pre-incubate for 15 minutes, add rabbit red blood cells (Erab) and incubate in a 37°C water bath for 30 minutes, cool on ice, terminate the reaction, and centrifuge. Take the supernatant from each tube and dilute it in a 96-well plate, and measure the absorbance value at 412nm. At the same time, set up a blank group, a full hemolysis group, a NHSP group, a negative drug control group (glucose), and a positive drug control group (heparin) in the experiment. Calculate the hemolysis inhibition rate of Amomum villosum polysaccharides under different concentration gradients. And calculate the concentration of polysaccharides required for 50% inhibition of hemolysis of each polysaccharide component (AP 50 value).

[0100] The ingredients and addition amounts in each group are as follows:

[0101] Blank group: 25μL Erab + 75μL GVB-Mg 2+ / EGTA;

[0102] Full hemolysis group: 25 μL Erab + 75 μL DDW;

[0103] NHSP group: 10μL NHSP+25μL Erab+65μL GVB-Mg 2+ / EGTA;

[0104] Negative control group: 1μL 1mmol / L glucose + 10μL NHSP + 25μL Erab + 64μL GVB-Mg 2+ / EGTA;

[0105] Positive control group: 11μL heparin of different concentrations + 54μL NHSP + 25μL Erab + 54μL GVB-Mg 2+ / EGTA;

[0106] Drug administration group: 1μL samples of different concentrations + 10μL NHSP + 25μL Erab + 64μL GVB-Mg 2+ / EGTA.

[0107] The results showed that the AP of ATP-1-1, ATP-4-1 and ATP-5-1 50 The values ​​were 45.33±0.16μg / mL, 8.62±0.46μg / mL and 9.41±0.83μg / mL respectively. The three Amomum villosum polysaccharides had significant inhibitory activity on the activation of the alternative complement pathway (as shown in Table 6).

[0108] Table 6 Inhibitory effect of Amomum villosum polysaccharide on complement activation

[0109] Amomum villosum polysaccharide <![CDATA[CH 50 (μg / mL)]]> <![CDATA[AP 50 (μg / mL)]]> ATP-1-1 6.28±0.23 45.33±0.16 ATP-4-1 7.39±0.36 8.62±0.46 ATP-5-1 4.47±0.44 9.41±0.83 heparin 107.60±1.34 122.80±2.34

[0110] Note: All data are expressed as mean ± SD (n = 3); CH 50 and AP 50 Represents 50% inhibition concentration of hemolysis through the classical pathway and alternative pathway, respectively; heparin is the positive control.

[0111] Example 7 Complement system target determination

[0112] Classical pathway: The minimum concentration of Amomum villosum polysaccharide required when the hemolysis rate is close to 100% under the classical pathway was selected as the critical concentration of the anti-complement target, and the missing serum (C2, C3, C4, C5, C9) and sensitized sheep red blood cells (2.0×10 9 cells / mL), mixed, incubated in a 37°C water bath for 30 min, and centrifuged at 2000×g for 5 min at 4°C. Each group was set up with a sample group, a serum-deficient group, and a positive control group. 0.2 mL of the supernatant from each tube was taken in a 96-well plate, and the absorbance value was measured at 540 nm.

[0113] Alternative pathway: The lowest concentration of Amomum villosum polysaccharide required to inhibit hemolysis by nearly 100% under the alternative pathway was selected as the critical concentration of the anti-complement target, and deficient serum (C3, C5, C9, Factor B, Factor D, Factor P) and rabbit red blood cells (5.0×10 8cells / mL) for 30 min, incubate on ice for 5 min to terminate the reaction, and centrifuge at 1000×g for 3 min at 4°C. Set up a sample group, serum-deficient group, and positive control group. Transfer 30 μL of supernatant to a 96-well plate, and add 270 μL of GVB-Mg to each well. 2+ / EGTA buffer and measure the absorbance at 412 nm.

[0114] The experiment also set up a missing serum group, a target detection group and a full hemolysis group, and the hemolysis rate was calculated after subtracting the absorbance value of the relevant control group. A bar graph was drawn with different targets as the horizontal axis and the corresponding hemolysis rate as the vertical axis. By comparing the changes in the hemolysis rates of the missing serum group and the target detection group treated with the test sample, it can be determined whether the sample has an antagonistic effect on complement components C2, C3, C4, C5, C9, Factor B, Factor D, and Factor P. If the hemolytic ability of the target detection group cannot be restored compared to the missing serum group, it is considered that the sample acts on the missing component; if the hemolytic ability of the target detection group is restored, it indicates that the sample does not act on the missing complement component.

[0115] The experimental results are as attached Figure 7 and 8 As shown in the figure, none of the complement-depleted sera was able to lyse red blood cells independently, and the hemolysis percentage did not exceed 20%. After treatment with ATP-1-1, the complement-deficient sera of C2, C4 and Factor B did not restore hemolysis, while the sera deficient in C3, C5, C9, Factor D and Factor P significantly restored hemolytic activity, indicating that ATP-1-1 blocks the activation cascade of the complement system by acting on the C2, C4 and Factor B targets, but does not act on the targets of C3, C5 and C9. Similarly, ATP-4 and ATP-5 can also act on the C2, C4 and Factor B targets.

[0116] The above description of the embodiments is to facilitate the understanding and application of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the embodiments herein, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An application of Amomum villosum polysaccharide in the preparation of an anti-complement drug, characterized in that: The preparation process of the amomum villosum polysaccharide mainly comprises the following steps: (1) Take dried Amomum villosum, crush it, add water to extract it at a liquid-to-solid ratio of 20:1 to 40:1 mL / g, the extraction temperature is 80 to 90° C., the extraction time is 80 to 120 min, filter the filtrate, concentrate it, add 95% ethanol to precipitate polysaccharides, centrifuge it, collect the precipitate, and obtain Amomum villosum crude polysaccharide; (2) taking the crude amomum villosum polysaccharide obtained in step (1), dissolving it in distilled water, loading it into an S-8 macroporous adsorption resin chromatography column, eluting it with distilled water, collecting the eluate, and drying it to obtain purified amomum villosum polysaccharide; (3) taking the purified Amomum villosum polysaccharide obtained in step (2), dissolving it in deionized water, loading it onto a DEAE-52 cellulose chromatography column, and eluting it with a gradient of 0, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L and 0.5 mol / L NaCl solution in sequence, combining the same polysaccharide components according to the phenol-sulfuric acid color reaction, collecting the secondary polysaccharide components eluted under 0, 0.3 mol / L and 0.4 mol / L NaCl solution, respectively, and freeze-drying them to obtain Amomum villosum polysaccharide ATP-1, Amomum villosum polysaccharide ATP-4 and Amomum villosum polysaccharide ATP-5, respectively; (4) Using deionized water to re-dissolve the Amomum villosum polysaccharide ATP-1, Amomum villosum polysaccharide ATP-4 and Amomum villosum polysaccharide ATP-5 obtained in step (3), respectively, and loading them onto Sephadex G-100 chromatography columns, performing constant elution using distilled water as the elution solvent, combining the same polysaccharide components according to the phenol-sulfuric acid color reaction, and freeze-drying to obtain Amomum villosum polysaccharide ATP-1-1, Amomum villosum polysaccharide ATP-4-1 and Amomum villosum polysaccharide ATP-5-1.

2. The use according to claim 1, characterized in that: The extract in step (1) is concentrated to 1 / 10 to 1 / 3 of the original volume, and the specific process of precipitating the polysaccharide is to add 95% ethanol until the final ethanol concentration of the extract is 75 to 85%, and let it stand for 24 to 48 hours.

3. The use according to claim 1, characterized in that: In step (2), the sample concentration is 1-3 mg / mL, and the sample volume is 0.3-0.5 BV; the elution flow rate is 1-3 BV / h, and the elution volume is 1-3 BV.

4. The use according to claim 1, characterized in that: In step (3), the sample concentration is 50-100 mg / mL, the sample volume is 0.2-0.4 BV; the elution flow rate is 1 mL / min, and each gradient elution takes 125-150 min.

5. The use according to claim 1, characterized in that: In step (4), the elution flow rate is 0.5 mL / min and the elution time is 500 to 600 min.

6. The use according to claim 1, characterized in that: Amomum villosum polysaccharide ATP-1-1 is a neutral polysaccharide with a molecular weight of 9.15×10 2 kDa; total sugar content is 91.44±1.13%; composed of mannose, rhamnose, glucose, galactose and arabinose, with a molar ratio of 1.0:0.7:5.6:3.4:0.

9.

7. The use according to claim 1, characterized in that: ATP-4-1 is an acidic polysaccharide with a molecular weight of 2.46×10 4 kDa; the total sugar content is 94.54±1.87%, the uronic acid content is 39.59±0.22%, and it is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, with a molar ratio of 1.0:4.8:2.4:20.7:2.0:10.6:5.3:5.

3.

8. The use according to claim 1, characterized in that: Amomum villosum polysaccharide ATP-5-1 is an acidic polysaccharide with a molecular weight of 2.69×10 5 kDa; the total sugar content is 95.81±1.33%; the uronic acid content is 33.23±0.19%, composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, with a molar ratio of 1.0:6.6:2.7:22.0:3.3:12.8:9.2:8.4.

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