Extraction process and application of American ginseng polysaccharide

Abstract

The invention discloses an extraction process and application of panax quinquefolius polysaccharide, and relates to the technical field of medicines.The panax quinquefolius is pulverized and dried, obtained panax quinquefolius powder is placed in a three-phase distribution system to be subjected to ultrasonic extraction, a water phase is taken to be concentrated and dried, and the panax quinquefolius polysaccharide is obtained, the salting-out solution is an ammonium sulfate solution. The optimized process for extracting the panax quinquefolius polysaccharide by adopting a three-phase distribution method of an ultrasonic-assisted deep-eutectic solvent has the advantages of high yield, greenness, safety and no toxic residue. And meanwhile, the green and safe American ginseng polysaccharide product also opens up a wide space for application of the American ginseng polysaccharide in the field of medicines, and the American ginseng polysaccharide prepared by the method disclosed by the invention has the activity of resisting ulcerative colitis, so that the development of American ginseng related industries is greatly promoted.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to an extraction process and application of American ginseng polysaccharide. Background Art

[0002] American ginseng (Panax quinquefolius L.) is a perennial herbaceous plant of the genus Panax in the Araliaceae family. Native to the northern United States and southern Canada, American ginseng was successfully introduced to China in the 1980s and is now widely cultivated in Liaoning, Heilongjiang and Jilin provinces in the northeast. American ginseng has significant pharmacological activities such as anti-tumor, cardiovascular protection and antioxidant activities, so it has been widely used in biomedicine and health products. These activities are closely related to its active compounds such as saponins, peptides, essential oils and polysaccharides. Ginsenosides have long been considered to be the main contributors to its pharmacological effects. Recently, an increasing number of studies have shown that polysaccharides contribute to the medicinal value of American ginseng, which can effectively enhance immunity, inhibit tumor growth, lower blood sugar and repair intestinal damage. However, in the past few decades, due to the complex structural characteristics of American ginseng polysaccharides and the erroneous belief that they are not easily absorbed by the human body, they are often discarded as ineffective ingredients, resulting in a large amount of resource waste. Therefore, developing efficient methods to extract American ginseng polysaccharides and studying their biological activities is of great significance for optimizing the utilization of American ginseng resources.

[0003] Extraction technology has a significant impact on the development, progress and application of natural polysaccharides. Hot water extraction is a green, economical and effective method for polysaccharide extraction. However, the disadvantage of low extraction efficiency has seriously limited the development of hot water extraction. With the development of technology, the extraction methods of polysaccharides have been continuously innovated and improved, and more and more auxiliary extraction methods have been applied to improve the extraction efficiency of bioactive polysaccharides, such as ultrasound, microwave, enzyme, etc. Dilute acid / alkali or organic solvents are also used for polysaccharide extraction to increase yield, but this method changes the structure and activity of the polymer, and the extraction process may cause harm to personnel and the environment. Enzyme-assisted extraction is milder and more environmentally friendly than acid / alkali or organic solvent extraction, but the problems of enzyme residue and instability still exist. Therefore, it is still crucial to find an efficient and environmentally friendly method for extracting natural polysaccharides.

[0004] Three-phase partitioning (TPP) refers to the formation of three different phases under various effects by adding inorganic salts and organic solvents to the original extract. The top phase is the organic phase, which is mainly composed of lipids and other substances with low polarity, while most proteins are deposited in the middle layer. The bottom phase is the aqueous phase, which mainly contains water-soluble substances such as polysaccharides. Due to its high efficiency and convenience, TPP is often used for the extraction and purification of polysaccharides and proteins. Although TPP is superior to traditional extraction techniques in many aspects, there are also some problems in practical applications. The commonly used volatile, flammable and toxic tert-butyl alcohol is a major challenge in TPP operations. Therefore, it is important to find alternative, less toxic solvents.

[0005] Deep eutectic solvents (DESs) are ionic complex solutions formed by hydrogen bond acceptors and hydrogen bond donors, with a freezing point significantly lower than that of any of their components. DESs have many advantages, such as simple preparation, environmental friendliness, low or non-toxicity, and good recyclability. Due to their environmentally friendly properties, DESs have become a potential alternative to traditional organic solvents in extraction and separation processes. They have been effectively used to extract a variety of bioactive substances, such as flavonoids, alkalis, and polysaccharides. TPP is an effective and convenient method for polysaccharide extraction and separation, but the volatility, toxicity, and difficulty in recycling of organic solvents such as tert-butyl alcohol limit its practical application. The green, non-toxic, and recyclable properties of DESs make it possible to further apply TPP in the extraction and separation of natural components. Therefore, the strategic combination of TPP and DESs is predicted to be an innovative approach for the separation and purification of natural bioactive polysaccharides.

[0006] Model organisms are crucial in studying the pathogenesis of diseases and evaluating and screening drug activity. Traditional high-throughput drug screening methods rely on in vitro cell models, biochemical assays, or receptor binding assays. Although in vitro cell models are often used for drug screening, their toxicity results do not represent tissue-specific responses in the human body. In addition to cell models, mammals are also often used as drug screening models. However, their use is often plagued by long experiments, high costs, and reproductive and ethical restrictions. Recently, Drosophila melanogaster has been shown to be an excellent organism for studying the pathogenesis of various diseases and performing drug screening. Its advantages include small size, rapid reproduction, short life cycle, simple physiological structure, low culture cost, clear genetic background, and ease of genetic manipulation. In addition, Drosophila shows significant homology with human organs and genes. These characteristics make Drosophila a valuable tool in modern research, including studies on the prevention and treatment mechanisms of colitis (UC), as well as drug screening. Summary of the invention

[0007] The first technical problem to be solved by the present invention is: in view of the deficiencies in the prior art, a process for extracting American ginseng polysaccharides is provided, which is environmentally friendly and has a good extraction effect of American ginseng polysaccharides.

[0008] To solve the above-mentioned first technical problem, the technical solution of the present invention is:

[0009] A process for extracting American ginseng polysaccharides comprises the following steps:

[0010] A: After the American ginseng is ground and dried, take the American ginseng powder;

[0011] B: American ginseng powder is placed in a three-phase distribution system for ultrasonic extraction, and the aqueous phase is concentrated and dried to obtain American ginseng polysaccharide, wherein the organic solvent in the three-phase distribution system is a low eutectic solvent, and the salting-out solution is an ammonium sulfate solution.

[0012] Preferably, in the low eutectic solvent used, the hydrogen bond acceptor is selected from any one of decanoic acid, nonanoic acid and octanoic acid, and the hydrogen bond donor is lauric acid.

[0013] Preferably, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:1 to 1:2.

[0014] Preferably, the hydrogen bond donor is lauric acid, the hydrogen bond acceptor is nonanoic acid, and the molar ratio of lauric acid to nonanoic acid in the system is 1:1.

[0015] Preferably, in step B, the ultrasonic extraction temperature is 30-70° C., and the extraction time is 10-50 min.

[0016] Preferably, in step B, the solid-liquid ratio of the mass of American ginseng powder to the volume of the organic solvent is 1:10 to 1:50.

[0017] Preferably, the volume ratio of the upper phase to the lower phase in the three-phase distribution system in step B is 1:0.25 to 1:2.

[0018] Preferably, the concentration of the ammonium sulfate solution in step B is 5 to 25%wt.

[0019] The second technical problem to be solved by the present invention is: in view of the deficiencies in the prior art, to provide an application of American ginseng polysaccharide in the treatment of ulcerative colitis-related diseases.

[0020] The third technical problem to be solved by the present invention is: in view of the deficiencies in the prior art, a medicine for treating ulcerative colitis-related diseases is provided, comprising American ginseng polysaccharide as an active ingredient.

[0021] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0022] 1. The present invention provides an optimized process for extracting American ginseng polysaccharides by a three-phase distribution method based on a low eutectic solvent. The American ginseng polysaccharides extracted by the process of the present invention have the advantages of high yield, green safety and non-toxic residue.

[0023] 2. The green and safe American ginseng polysaccharide product obtained by the present invention also opens up a broad space for its application in the medical field. The American ginseng polysaccharide prepared by the method of the present invention can be used for the treatment of ulcerative colitis-related diseases, which will greatly promote the development of American ginseng-related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a DES screening and selection diagram for AGP extraction;

[0025] Figure 2 It is the response surface diagram and contour diagram of the interaction of various experimental factors;

[0026] Figure 3 It is the Fourier transform infrared spectrum, monosaccharide composition and nuclear magnetic resonance spectrum of AGP-DES-4;

[0027] Figure 4 This is a diagram showing the effects of AGP-DES-4 on the survival rate, intestinal morphology, and intestinal barrier function of ulcerative colitis flies;

[0028] Figure 5 This is a diagram showing the effect of AGP-EDS-4 on the fluorescence intensity of intestinal stem cells in esg-Gal4-UAS-GFP / Cyo ulcerative colitis Drosophila;

[0029] Figure 6 This is a diagram showing the effect of AGP-EDS-4 on the number of intestinal epithelial cells in esg-Gal4-UAS-GFP / Cyo ulcerative colitis flies. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the embodiments.

[0031] In order to further develop the resources of American ginseng polysaccharides and explore a more efficient TPP system, the present invention aims to establish an ultrasound-assisted three-phase distribution system of low eutectic solvents for the extraction and purification of polysaccharides from American ginseng roots. The extraction process of American ginseng polysaccharides was optimized by single factor experiments and Box-Behnken design (BBD). The structural characteristics of the obtained American ginseng polysaccharide AGP-DES-4 were further analyzed by molecular weight determination, monosaccharide composition analysis, Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. Finally, the protective effect of American ginseng polysaccharide AGP-DES-4 on DSS-induced intestinal damage in vivo was evaluated using a Drosophila model.

[0032] Example 1

[0033] Optimization of Extraction Technology of American Ginseng Polysaccharide

[0034] 1. Experimental Materials

[0035] The roots of American ginseng were purchased from Guangdong Kangzhou Pharmaceutical Co., Ltd. (Guangdong, China). The dried American ginseng was crushed with a grinder and stored in a sealed bag in the Guangdong Provincial Key Laboratory of Natural Medicine Research and Development, Guangdong Medical University, with the number 20231024.

[0036] 2. Experimental instruments

[0037] High-power CNC ultrasonic cleaner (model KQ-400KDB, Kunshan Ultrasonic Instrument Co., Ltd.); high-speed desktop centrifuge (model H1650, Changsha Xiangyi Centrifuge Instrument Co., Ltd.); rotary evaporator (model RE52-99, Shanghai Yarong Biochemical Instrument Factory); circulating water vacuum pump (model SHB-III, Hebei Songdao Instrument Equipment Co., Ltd.); freeze dryer (model FD-1A-50, Shanghai Yuming Instrument Co., Ltd.); microplate reader (model ST-360, Shanghai Kehua Experimental System Co., Ltd.); one-tenth balance (model AL 204-IC, Mettler-Toledo Instrument Co., Ltd.).

[0038] 3. Experimental reagents

[0039] Choline chloride, 1,6-hexanediol, capric acid, lauric acid, terpineol, nonanoic acid, and octanoic acid were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China). Monosaccharide standards (glucose, galactose, xylose, arabinose, rhamnose, mannose, galacturonic acid, and glucuronic acid) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). 1-Phenyl-3-methyl-5-pyrazolone (PMP) and sodium dextran sulfate (DSS) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Isoflurane was purchased from Suzhou Ruigu Medical Technology Co., Ltd. (Suzhou, China). Paraformaldehyde (PFA) was purchased from Langer Technology Co., Ltd. (Beijing, China), while DAPI and 7-AAD staining reagents were purchased from Sevier Biotechnology Co., Ltd. (Wuhan, China) and Abcam (Shanghai) Trading Co., Ltd. (Shanghai, China), respectively.

[0040] 4. Drawing of glucose standard curve

[0041] D-anhydrous glucose was prepared into 6 groups of glucose standard solutions with concentrations of 0.75, 0.375, 0.1875, 0.09375, 0.046875, and 0 mg / mL, respectively. Then 30 μL was pipetted into a 96-well plate in sequence, and 30 μL of 5% phenol solution and 160 μL of concentrated sulfuric acid were added. After reacting for 15 minutes, the absorbance was measured at 490 nm, and the glucose standard curve was drawn based on the absorbance.

[0042] 5. Extraction of American ginseng polysaccharide AGP-TPP by ultrasound-assisted three-phase partitioning (UA-TPP)

[0043] 0.1g of American ginseng dry powder was extracted in 25%wt (NH2)2SO4 solution at a solid-liquid ratio of 1:20. Then, 2mL of tert-butanol was added to the mixture and extracted for 30min at 60°C and 100W ultrasonic conditions. After extraction, the sample was centrifuged at 400rpm for 10min to obtain the lower layer liquid. The lower layer liquid was then concentrated and further treated with dialyzed water (48h) to obtain the American ginseng polysaccharide AGP-TPP by freeze drying. The yield of A GP-TPP was determined by the phenol-sulfuric acid method and the glucose standard curve.

[0044] 6. Ultrasound-assisted three-phase distribution method of low eutectic solvent (UA-TPP-DES) was used to extract American ginseng polysaccharides AGP-D ES-1~5.

[0045] 6.1 Preparation of DESs

[0046] DESs for polysaccharide extraction were prepared as follows. Briefly, hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) were thoroughly mixed in a round-bottom flask and sonicated at 70 °C until a transparent and homogeneous solution was obtained. The detailed DES compositions are shown in Table 1.

[0047] Table 1

[0048]

[0049] 6.2 DES solvent optimization

[0050] 0.1 g of American ginseng powder was mixed with different DESs (DES-1, DES-2, DES-3, DES-4 and DES-5) at a solid-liquid ratio of 1:2. Then, 2 mL of 25% wt ammonium sulfate was added to the solution to generate a three-phase distribution system, and the extraction was carried out under the following conditions: temperature 60°C, moisture content 30%, ultrasonic power 100 W, and ultrasonic time 30 min. After extraction, the sample was centrifuged at 400 rpm for 10 min. The lower layer of liquid was collected, concentrated, and dialyzed in running tap water for 48 h. After that, the dialysate was frozen. The polysaccharides obtained from DES-1 to DES-5 were labeled as AGP-DES-1 to AGP-DES-5, respectively. The yields of AGP-DES-1 to 5 were determined by the phenol-sulfuric acid method and the glucose standard curve.

[0051] 7. Investigation of single factors

[0052] In order to establish the optimal conditions for ultrasonic-assisted DES extraction of AGP, single factor experiments were conducted to investigate the effects of extraction time (10min, 20min, 30min, 40min and 50min), extraction temperature (30℃, 40℃, 50℃, 60℃ and 70℃), ammonium sulfate mass fraction (5%, 10%, 15%, 20% and 25%wt), solid-liquid ratio of American ginseng powder mass to organic solvent volume (1:10, 1:20, 1:30, 1:40 and 1:50) and volume ratio of upper and lower phases (1:0.25, 1:0.5, 1:1, 1:1.5 and 1:2) on the yield of AGP-DES-4. Based on the results of the single factor experiments, three main influencing factors, namely solid-liquid ratio (A), volume ratio of upper and lower phases (B) and ammonium sulfate mass fraction (C), were selected as independent variables, and a three-level (1,0,-1) Box-Behnken design (BBD) was adopted. A total of 17 groups of experiments were conducted, with the yield of AGP-DES-4 as the evaluation index. The three-factor three-level experimental design is shown in Table 2, and each group of experiments was randomly conducted during the experiment. In addition, the optimal extraction process was determined by regression analysis, and a verification experiment was carried out under the obtained conditions to verify the accuracy of the experiment.

[0053] Table 2

[0054]

[0055] 8. Determination and calculation of American ginseng polysaccharides

[0056] The content of American ginseng polysaccharide was determined according to the polysaccharide determination method in the glucose standard curve, and the calculation formula is:

[0057]

[0058] Wherein: W: polysaccharide extraction rate (%); C: polysaccharide concentration (mg / mL); V: volume of polysaccharide solution (mL); N: dilution multiple; M: mass of American ginseng medicinal material (g).

[0059] 9. Structural characterization of American ginseng polysaccharide AGP-DES-4

[0060] 9.1. Relative molecular mass distribution

[0061] The relative molecular mass distribution of AGP-DES-4 was analyzed by Agilent 1200 high performance liquid chromatography system (HPGPC) equipped with a differential refractive index detector and a TSK-Gel G3000 PWXL column (Tosoh Biosep, To okyo, Japan). 0.2 M KH2PO4 was used as the mobile phase with a flow rate of 0.5 ml / min. The column temperature and the RID detector temperature were set at 35 °C, respectively. The relative molecular mass distribution of AGP-DES-4 was estimated by the calibration curve of pullulan molecular weight standards (201000, 107000, 4940022000, 9800, 2000 and 1000 Da).

[0062] 9.2. FT-IR analysis

[0063] An infrared spectrometer (Shimadzu) was used at 4000-400 cm -1 The FT-IR spectrum of AGP-DES-4 was measured in a wide range of wavenumbers.

[0064] 9.3 Monosaccharide composition

[0065] The monosaccharide composition of AGP-DES-4 was analyzed by 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization method. The specific steps are as follows: First, 4 mg of AGP-DES-4 was dissolved in 2 M trifluoroacetic acid (TFA) and reacted at 120 ° C for 6 h. After the reaction, the reaction product was co-concentrated with anhydrous methanol 4 times to remove excess TFA. Subsequently, the hydrolyzate was redissolved in 1 mL of distilled water. Next, 500 μL of PMP-methanol solution (0.5 M) and 500 μL of NaOH solution (0.3 M) were added to the above hydrolyzate, and further reacted at 75 ° C for 30 min. The reaction product was neutralized with 0.3 M HCl solution, and then extracted with an equal volume of chloroform to remove unreacted substances. The reaction products (PMP derivatives) were further analyzed using an Agilent 1200 high performance liquid chromatography (HPLC) system equipped with a UV detector and an Agilent ZORBAX Eclipse XDB-C18 column (4.6×250 mm, 5 μm). The mobile phase was acetonitrile and phosphate buffer solution (17:83, v / v, pH=6.9) at a flow rate of 1.0 mL / min. The column temperature was set at 35°C and the detection wavelength was 250 nm.

[0066] 9.4 Nuclear Magnetic Resonance Analysis

[0067] AGP-DES-4 (50 mg) was dissolved in 500 μl of heavy water for NMR analysis. The 1H spectrum of AGP-DES-4 was obtained on a Bruker AV-500 instrument ( 1H NMR) and carbon spectroscopy ( 13 C NMR) spectra were analyzed by MestReNova software.

[0068] 10. Results and Analysis

[0069] 10.1 Screening and selection of deep eutectic solvent (DES) for extracting American ginseng polysaccharide AGP.

[0070] The composition of deep eutectic solvent (DES) has a significant effect on the extraction efficiency of plant polysaccharides. Therefore, in order to find a suitable DES solvent to form a three-phase partition method (TPP) system to extract American ginseng polysaccharide AGP, the tert-butyl alcohol-based TPP system was used as a control to evaluate the effects of five DES-based TPP systems (DES-1, DES-2, DES-3, DES-4 and DES-5) on the yield of American ginseng polysaccharide AGP. Figure 1 The standard curve of glucose (y = 4.4811x + 0.2439, R 2 =0.9985), and found that the production of AGP in the UA-TPP-DESs group was mainly distributed in the range of 20%-25% ( Figure 1 A), which was much higher than that of the UA-TPP group (14.38%). These results indicate that DES is a more effective solvent that can replace tert-butyl alcohol to form the TPP system for the extraction of AGP. In addition, by comparing the TPP extraction systems based on DES, we found that the TPP system formed by lauric acid and nonanoic acid (molar ratio = 1:1; DES-4) was more suitable for the extraction of AGP (yield of 24.95%). Therefore, the TPP system formed by DES-4 (UA-TPP-DES-4) was further used in subsequent experiments.

[0071] 10.2 Effect of ultrasound time

[0072] Effect of ultrasonic time on the extraction rate of American ginseng polysaccharides Figure 1 B.

[0073] The experimental results show that when the ultrasonic time is 20 minutes, the yield of AGP-DES-4 reaches the maximum value (16.68%). Therefore, in the subsequent research, the extraction time is set to 20 minutes to ensure the best extraction effect.

[0074] 10.3 Effect of Ultrasonic Temperature

[0075] Effect of ultrasonic temperature on the extraction rate of American ginseng polysaccharides Figure 1 C.

[0076] As can be seen from the figure, with the increase of ultrasonic temperature, the yield of AGP-DES-4 first increased and then decreased. In addition, when the temperature was 60°C, the yield of AGP-DES-4 reached the highest (13.54%), and this temperature was selected for subsequent experiments.

[0077] 10.4 Effect of Ammonium Sulfate Mass Fraction

[0078] Effect of ammonium sulfate mass fraction on the extraction rate of American ginseng polysaccharides Figure 1 D.

[0079] The experimental results show that with the increase of (NH4)2SO4 mass fraction, the extraction yield of AGP-DES-4 first increases and then decreases. When the (NH4)2SO4 mass fraction is 10%, the yield of AGP-DES-4 reaches the maximum value (12.27%), and this mass fraction is selected as the optimal content for the next experiment.

[0080] 10.5 Effect of material-liquid ratio

[0081] Effect of solid-liquid ratio on the extraction rate of American ginseng polysaccharides Figure 1 E.

[0082] The experimental results show that when the solid-liquid ratio changes from 1:10 to 1:50, the yield of AGP-DES-4 first increases and then decreases. When the solid-liquid ratio is 1:20, the extraction rate of AGP-DES-4 reaches the maximum value (7.81%). Therefore, the optimal solid-liquid ratio for extracting American ginseng polysaccharides is 1:20.

[0083] 10.6 Impact of DES-4 Ratio

[0084] The effect of DES-4 ratio on the extraction rate of American ginseng polysaccharides is shown in Figure 1 F.

[0085] As can be seen from the figure, when the volume ratio of the top phase to the bottom phase increases from 1:0.25 to 1:2, we found that the extraction yield of AGP-DES-4 first increased and then decreased. When the volume ratio of the top phase to the bottom phase was 1:0.5, the extraction yield of AGP-DES-4 reached the maximum value of 14.76%.

[0086] 10.7 Statistical Analysis and Model Fitting

[0087] Based on the experimental results of signal factors, a BBD experiment was further conducted to optimize the extraction conditions of AGP-DES-4. In this BBD experiment, the solid-liquid ratio (A), the volume ratio of the upper phase to the lower phase (B), and the mass fraction of (NH4)2SO4 (C) were set as independent variables at three different levels (1, 0, -1), and the extraction rate of AGP-DE S-4 was used as the response value for multivariate regression fitting analysis. The results of the multivariate regression fitting analysis are shown in Table 3, and the corresponding second-order polynomial regression equation is shown below.

[0088] Y=34.58-4.63×A-1.75×B-0.1603×C-0.3328×AB+0.1525×AC+0.1784×BC-13.03×A 2 -3.32×B 2 -2.03×C 2 .

[0089] Table 3

[0090]

[0091] As shown in Table 4, the P value of the regression model is less than 0.0001, and the lack of fit value is 0.0997, which is higher than 0.05, indicating that the model fits the experimental data well, has low error, and is less affected by unknown factors. According to the F value, it can be concluded that the order of influence of each factor on the extraction rate of AGP-DES-4 is solid-liquid ratio (A)>upper phase to lower phase volume ratio (B)>(NH4)2SO4 mass fraction (C).

[0092] Table 4

[0093]

[0094] ***referred to highly significant (P<0.001); nsreferred to no significant.

[0095] 10.8 Analysis of the interaction of various factors in the model

[0096] A three-dimensional response surface was used to further predict the relationship between factor interactions and AGP-DES-4 extraction efficiency. Figure 2 As shown in AD, the interaction term between the solid-liquid ratio and the upper and lower phases (AB) and the interaction term between the solid-liquid ratio and the mass fraction of (NH4)2SO4 (AC) showed a higher inclination angle, a steeper slope and an elliptical boundary, indicating that the interaction term between the solid-liquid ratio and the upper and lower phases (AB) and the interaction term between the solid-liquid ratio and the mass fraction of (NH4)2SO4 (AC) had a significant effect on the extraction rate of AGP-DES-4. On the contrary, Figure 2 As shown in EF, the interaction term (BC) between the upper and lower phase ratios and the (NH4)2SO4 mass fraction showed a relatively flat response surface and an elliptical contour line, indicating that the interaction term (BC) between the upper and lower phase ratios and the (NH4)2SO4 mass fraction had the least effect on the extraction rate of AGP-DES-4.

[0097] The software further predicted the optimal conditions for extracting AGP-DES-4 as follows. Ultrasonic time: 20min, ultrasonic temperature: 60℃, ultrasonic power: 100W, molar ratio of lauric acid to nonanoic acid = 1:1, solid-liquid ratio = 1:18.1836, volume ratio of upper phase to lower phase = 1:0.492131, mass fraction of (NH4)2SO4 = 9.33953%wt. The predicted AGP-DES-4 extraction rate is 35.1735%. The actual experimental conditions are set as follows: ultrasonic time: 20min, ultrasonic temperature: 60℃, ultrasonic power: 100W, molar ratio of lauric acid to nonanoic acid = 1:1, solid-liquid ratio = 1:20, volume ratio of upper phase to lower phase = 1:0.5, mass fraction of (NH4)2SO4 = 10%wt. After calculation, the actual extraction rate of AGP-DES-4 is 35.28%, which is almost the same as the predicted value, indicating that the proposed model is accurate and reliable.

[0098] 10.9 Relative Molecular Mass Distribution

[0099] According to the regression equation (y = -0.3382x + 9.2806, R 2 =0.9997), the relative molecular weight of A GP-DES-4 is between 2.48453-174.64405 kDa.

[0100] 10.10 Infrared spectroscopy analysis

[0101] Figure 3 A shows the Fourier transform infrared spectrum of AGP-DES-4. 3425cm -1 The prominent peak at 2924cm is caused by the OH stretching of the hydroxyl group, indicating the existence of hydrogen bonding within the polysaccharide. -1 The relatively weak band at 1645 cm corresponds to CH bending. -1 The band at 1424 cm -1 The band at 1000-1200 cm -1 The three peaks in the region represent the asymmetric COC and COH stretching of the pyranose ring. -1 The characteristic absorption near 569cm indicates the coexistence of α and β configurations. -1 The peak at indicates the presence of a pyranose ring.

[0102] 10.11 Analysis of Monosaccharide Composition

[0103] The monosaccharide composition of AGP-DES-4 was determined by PMP pre-column derivatization. The analysis showed that it mainly contained glucose, galactose, and arabinose (87.7%, 7.3%, and 4.0%, respectively) ( Figure 3 B).

[0104] 10.12 Nuclear Magnetic Resonance Analysis

[0105] AGP-DES-4 1 H and 13 C NMR spectroscopy was further used to characterize the structural features of AGP-DES-4. Figure 3 As shown in CD, 1 A group of anomeric proton signals with chemical shifts ranging from 4.50 to 5.35 ppm were observed in the H NMR spectrum, while 13 A group of anomeric carbon signals with chemical shifts ranging from 91 to 110 ppm were observed in the C NMR spectrum, indicating that AGP-DES-4 is a heteropolysaccharide. These results are consistent with the results of monosaccharide analysis. The strongest signals at 5.32 ppm and 99.6 ppm were assigned to the anomeric proton and anomeric carbon signals of →4)-α-G lcp-(1→. In addition, the signals at 5.28 / 99.9 ppm are the anomeric proton and anomeric carbon signals of →4,6)-α-Glcp-(1→. The signals at 4.89 / 98.5 ppm are →6)-α-Gl The signals at 5.25 / 99.7 ppm are caused by the anomeric proton and anomeric carbon signals of α-Glcp-(1→. In addition, the signals at 5.15 ppm and 110.2 ppm can be assigned to the anomeric proton and anomeric carbon signals of α-Araf sugar residues, whose anomeric proton and anomeric carbon signals are mainly distributed in the range of 5.10-5.19 ppm and 106-110 ppm, respectively. The signals at 4.56 (4.58) / 103.6 ppm are caused by the anomeric proton and anomeric carbon signals of β-Ga The anomeric proton and anomeric carbon signals of the lp sugar residue are reported to be mainly distributed in the range of 4.40-4.70 ppm and 102-104.0 ppm, respectively. In addition, the signal at 91.9 ppm is the anomeric carbon signal of the α-Glcp(4→ residue. The signal at 95.7 ppm is the anomeric carbon signal of the β-Glcp(4→ residue. In addition, the signals at 3.34 / 54.3 ppm are typical signals caused by -OCH3 groups, indicating that AGP-DES-4 contains -OCH3 groups.

[0106] Example 2

[0107] In vivo anti-colitis activity of American ginseng polysaccharide

[0108] 1. Experimental Materials

[0109] Freeze-dried powder of crude polysaccharide from American ginseng, W1118 wild type and esg-Gal4, UAS-GFP / Cyo transgenic Drosophila melanogaster.

[0110] 2. Experimental instruments

[0111] Small high-speed refrigerated centrifuge (model Bioc-ir, Shanghai Dove Industrial Co., Ltd.), embedding machine (model JB-P5, Wuhan Junjie Electronics Co., Ltd.), staining machine (model Giotto, DIAPATH), tissue slide machine (model KD-P, Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd.), pathological slicer (model RM2016, Shanghai Leica Instrument Co., Ltd.), upright optical microscope (model Nikon Eclipse E100, Nikon, Japan), high-speed tissue grinder (model KZ-II, Wuhan Sewell Biotechnology Co., Ltd.).

[0112] 3. Experimental reagents

[0113] Dextran sulfate sodium (DSS) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Isoflurane was purchased from Suzhou Ruigu Medical Technology Co., Ltd. (Suzhou, China). Paraformaldehyde (PFA) was purchased from Langer Technology Co., Ltd. (Beijing, China), while DAPI and 7-AAD staining reagents were purchased from Sevier Biotechnology Co., Ltd. (Wuhan, China) and Abcam (Shanghai) Trading Co., Ltd. (Shanghai, China), respectively.

[0114] 4. Preparation of American ginseng polysaccharide

[0115] The polysaccharide was extracted according to the optimal conditions of the American ginseng polysaccharide extraction process optimization in Example 1, and the bottom phase was collected for dialysis, concentration, and freeze-drying to obtain the crude American ginseng polysaccharide.

[0116] 5. Establishment of Drosophila ulcerative colitis model and survival analysis

[0117] The fruit fly strain W1118 3-5 days after eclosion was selected and randomly divided into a control group (NC group), a model group (DSS group) and an AGP-DES-4 administration group, with 30 fruit flies in each group (half male and half female). Before the experiment, the fruit flies were starved for 2 hours. Afterwards, each group of fruit flies was divided into an empty culture tube containing three filter papers, and the filter papers were soaked in 5% sucrose, 3% dextran sulfate sodium (DSS) and different concentrations of AGP-DE S-4. The entire experimental period was 7 days, and the filter paper was replaced every 24 hours. At the same time, the survival rate of each group of fruit flies was recorded and calculated. The calculation formula for the survival rate is as follows.

[0118]

[0119] 6. Intestinal morphology

[0120] Female fruit flies (W1118 type) at 4-5 days of eclosion were randomly divided into three groups, namely the normal control group (NC group, containing 5% sucrose), the dextran sulfate sodium group (DSS group, containing 5% sucrose and 3% dextran sulfate sodium), and the 5% AGP-DES-4 treatment group (containing 5% sucrose, 3% dextran sulfate sodium, and 5% AGP-DES-4). The fruit flies were starved for 2 hours before being distributed into empty culture tubes containing 3 circular filter papers. The total duration of the experiment was 4 days, and the filter paper was changed every day. At the end of the experiment, each group of fruit flies was anesthetized with isoflurane, and the intestines of each group of fruit flies were collected. The collected intestines were sealed with 78% glycerol and then imaged under a microscope. The length of the intestine was measured and quantified using Image J software.

[0121] 7. Blue light dye feeding test

[0122] The blue dye feeding test was used to assess the integrity of the intestine. Edible blue dye was diluted to a concentration of 2.5% (w / v) using a 5% sucrose solution. Three stock solutions were further prepared using 2.5% edible blue dye (diluted with a 5% sucrose solution). NC solution: 2.5% edible blue dye (diluted with a 5% sucrose solution). DSS solution: DSS was diluted to a concentration of 4% (w / v) using a NC solution. AGP-DES-4 solution: AGP-DES-4 was diluted to a concentration of 4% (w / v) using a DSS solution. Female fruit flies (W1118 type) at 3-4 days of eclosion were randomly divided into three groups, namely, the NC group (treated with 200 μL NC solution), the DSS group (treated with 200 μL DSS solution), and the 5% AGP-DES-4 treatment group (treated with 200 μL AGP-DES-4 solution). The total experimental time was 7 days, and the filter paper was replaced every day. After the experiment, each group of flies was anesthetized with isoflurane and imaged using a stereomicroscope. After imaging, the flies were crushed and homogenized using phosphate buffered saline, and the absorbance was measured at 632 nm to assess the incorporation of the blue dye into the flies.

[0123] 8. Effect of AGP-DES-4 on Drosophila intestinal precursor cells.

[0124] Female esg-Gal4, UAS-GFP / Cyo transgenic fruit flies were randomly divided into 3 groups (30 fruit flies in each group), including a control group (NC group), a dextran sulfate sodium (DSS) group, and a 5% AGP-DES-4 treatment group. Each group of fruit flies was treated with DSS or 5% AGP-DES-4. At the end of the experiment, the fruit fly intestines were collected, rinsed twice with PBS solution, and then fixed in 4% paraformaldehyde for 20 minutes. Subsequently, the fixed intestines were rinsed with PBS for three cycles to remove residual fixatives and impurities. After that, the fixed intestines were further stained with DAPI solution (50 microliters) for 10 minutes under dark conditions. After staining, the samples were rinsed twice with PBS to remove unreacted DAPI. Finally, the obtained samples were sealed with 78% glycerol and examined using a fluorescence microscope. The fluorescence intensity was evaluated using ImageJ software.

[0125] 9. Effects of AGP-DES-4 on Drosophila intestinal epithelial cells

[0126] Female W1118 fruit flies were randomly divided into 3 groups (30 fruit flies in each group), including a control group (NC group), a dextran sulfate sodium treatment group (DSS group), and a 5% AGP-DES-4 treatment group. After drug treatment, the fruit fly intestines were collected, washed twice with PBS solution, and then incubated with 50 μL of 7-AAD solution (5 μg / mL) for 30 min under dark conditions. After staining, the samples were further washed three times with PBS to remove excess 7-AAD solution. Subsequently, the samples were fixed with 200 μL of paraformaldehyde solution (4%). After fixation, the samples were centrifuged at 500 rpm for 2 min to remove excess paraformaldehyde solution. Subsequently, the samples were washed twice with PBS, and then 50 μL DAPI was added and incubated in the dark for 10 min. After incubation, the samples were washed twice with PBS solution to remove unreacted DAPI solution. Finally, the samples were sealed with 78% glycerol and examined using a fluorescence microscope. The fluorescence intensity was evaluated using ImageJ software.

[0127] 10. Results and Analysis

[0128] 10.1 Effect of AGP-DES-4 on the survival rate of Drosophila with ulcerative colitis

[0129] The effects of AGP-DES-4 on the survival of Drosophila with ulcerative colitis were evaluated. Figure 4 As shown in A. Figure 4A shows that, compared with the standard control group, the survival rate of DSS group fruit flies on the 7th day was significantly reduced (P<0.001), indicating that the ulcerative colitis fruit fly model was successfully established. After 7 days of treatment with different concentrations of AGP-DES-4, we found that 1% AGP-DES-4 and 10% AGP-DES-4 could slightly increase the survival rate of ulcerative colitis fruit flies, while 5% AGP-DES-4 could significantly increase the survival rate of ulcerative colitis fruit flies (P<0.001), indicating that AGP-DES-4 has significant anti-ulcerative colitis activity. In addition, considering that the promoting effect of 5% AGP-DES-4 is better than that of 1% AGP-DES-4 and 10% AGP-DES-4, 5% AGP-DES-4 was selected as the optimal experimental concentration for subsequent fruit fly anti-colitis experiments.

[0130] 10.2 Effect of AGP-DES-4 on intestinal morphology of Drosophila with ulcerative colitis.

[0131] In mammals, ulcerative colitis lesions involve the rectum and sigmoid colon, which can damage the intestinal barrier, cause intestinal atrophy and change intestinal morphology. In this study, to explore whether AGP-DES-4 can protect ulcerative colitis lesions caused by dextran sulfate sodium (DSS), the intestines of ulcerative colitis fruit flies were collected and compared to evaluate the rescue effect of AGP-DES-4 on the intestinal atrophy phenotype of fruit flies. Figure 4 As shown in BC, the intestinal length of DSS-treated fruit flies was significantly shortened compared with that of the normal control group, indicating that the ulcerative colitis Drosophila model was successfully established. However, the addition of 5% AGP-DES-4 significantly rescued the shorter intestinal length of Drosophila under DSS stimulation (P<0.05), which was 32.1% longer than that of the DSS group. This result further confirmed the intestinal protective effect of AGP-DES-4.

[0132] 10.3 Effect of AGP-DES-4 on intestinal barrier function in Drosophila with ulcerative colitis.

[0133] The intestinal barrier function was evaluated by blue dye feeding test. Impaired intestinal barrier function will cause blue dye to spread all over the fruit fly. In order to explore the protective effect of AGP-DES-4 on the intestinal barrier function of fruit flies with ulcerative colitis, this study conducted a blue dye feeding test. Figure 4As shown in D, compared with the normal control group, the abdomen of the fruit flies induced by dextran sulfate sodium (DSS) showed a large area of ​​blue, indicating that DSS destroyed the intestinal permeability and intestinal barrier function of ulcerative colitis fruit flies. After treatment with AGP-DES-4, the blue infiltration in the abdomen of ulcerative colitis fruit flies was significantly improved. In addition, the absorbance of the fruit fly pulverized homogenate at 632nm was further quantified, and we found that the absorbance of the DSS group was 111.03% higher than that of the normal control group (P<0.001), while the absorbance of the AGP-DES-4-treated group was 34.10% lower than that of the DSS group (P<0.001). These results indicate that AGP-DES-4 can effectively alleviate the intestinal barrier damage caused by DSS.

[0134] 10.4 Effect of AGP-DES-4 on intestinal stem cells (ISCs) in Drosophila with ulcerative colitis (UC).

[0135] The intestine can defend against harmful substances ingested through food and is a major barrier. Intestinal stem cells (ISCs) are one of the most active cell populations. Under normal physiological conditions, ISCs proliferate and differentiate into intestinal epithelial cells (IECs) in an orderly manner to maintain the integrity of the intestinal barrier. When the intestinal barrier is damaged, ISCs will continue to proliferate and differentiate to replace lost or damaged IECs. Therefore, excessive proliferation and differentiation of ISCs can be used as an indicator to observe intestinal barrier damage. In order to further study the effect of AGP-DES-4 on UC fruit fly ISCs, this experiment used transgenic esg-Gal4 UAS-GFP / Cyo fruit flies, whose ISCs were marked with GFP. Figure 5 As shown in AB, the relative fluorescence intensity increased significantly after induction with dextran sulfate sodium (DSS) (P<0.001), reaching more than ten times that of the normal control group (NC group). This indicates that the DSS group destroyed the intestinal barrier, leading to significant proliferation of ISCs, thus successfully establishing a UC model. Compared with the DSS group, the relative fluorescence intensity was significantly reduced under AGP-DES-4 treatment (P<0.01), indicating that AGP-DES-4 can reverse DSS-induced excessive proliferation of ISCs and restore intestinal homeostasis.

[0136] Effects of 10.5AGP-DES-4 on Drosophila intestinal epithelial cells (IECs) in ulcerative colitis (UC).

[0137] Intestinal epithelial cells constitute the intestinal barrier and are tightly arranged on the surface of the intestinal epithelium to protect the body from microbial infection. An abnormal increase in the mortality rate of intestinal epithelial cells manifests as epithelial erosion, which is a feature of several intestinal diseases including inflammatory bowel disease and infectious colitis. Therefore, the number of intestinal epithelial cells is an important indicator for assessing the integrity of the intestinal barrier. In order to study the effect of AGP-EDS-4 on UC fruit flies, this experiment used 7-AAD to stain and observe intestinal epithelial cells. 7-AAD is a cell membrane-impermeable fluorescent dye that binds to nucleic acids. It cannot penetrate the membranes of living cells, but it can penetrate the membranes of necrotic cells. Therefore, a higher relative fluorescence intensity after 7-AAD staining indicates a higher cell mortality rate. Figure 6 As shown in AB, DSS treatment resulted in a significant increase in fluorescence intensity compared with the untreated control group (P<0.01), which was approximately twice that of the normal control group (NC group). This indicates that DSS successfully induced the UC model. After AGP-EDS-4 treatment, the relative fluorescence intensity was significantly reduced to the same level as the NC group (P<0.01), indicating that AGP-EDS-4 can restore the damage caused by DSS to intestinal epithelial cells and restore it to normal levels.

[0138] The research object of the present invention is American ginseng polysaccharide with anti-ulcerative colitis activity. At present, no scholar has studied the anti-ulcerative colitis effect of American ginseng polysaccharide, and the present invention is the first to discover that American ginseng polysaccharide has the function of anti-ulcerative colitis, which will greatly expand the application field of American ginseng.

[0139] At present, the extraction of plant polysaccharides at home and abroad mostly adopts water extraction and alcohol precipitation, ultrasound-assisted, microwave-assisted and other methods. The three-phase distribution method is a method widely used in the extraction of various components such as oils and polysaccharides. It has many advantages, but it also has some shortcomings. The main defect is that the three-phase distribution system usually uses volatile, flammable and toxic tert-butanol. The American ginseng polysaccharide prepared by the present invention is obtained by a three-phase distribution method based on a low eutectic solvent, and the extraction method is different from the traditional method. The present invention uses a low eutectic solvent instead of tert-butanol to solve the problems of pollution and toxicity of the original system. In addition, the literature reports that the extraction rate of American ginseng polysaccharides is often 1-11%, and the extraction rate of American ginseng polysaccharides obtained after the process optimization method of the present invention is as high as 35.28%.

[0140] The present invention establishes a Drosophila ulcerative colitis model to evaluate the protective effect of American ginseng polysaccharide AGP-DES-4 on intestinal damage induced by sodium dextran sulfate in vivo. This effect is the first study on American ginseng polysaccharides, which is different from other literature materials. It also provides a new treatment method for the medical field for the treatment of ulcerative colitis-related diseases.

[0141] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0142] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

Claims

1. A process for extracting American ginseng polysaccharides, characterized in that The following steps are involved: A: After the American ginseng is ground and dried, take the American ginseng powder; B: American ginseng powder is placed in a three-phase distribution system for ultrasonic extraction, and the aqueous phase is concentrated and dried to obtain American ginseng polysaccharide, wherein the organic solvent in the three-phase distribution system is a low eutectic solvent, and the salting-out solution is an ammonium sulfate solution.

2. The process for extracting American ginseng polysaccharides according to claim 1, characterized in that: In the low eutectic solvent used, the hydrogen bond acceptor is selected from any one of decanoic acid, nonanoic acid and octanoic acid, and the hydrogen bond donor is lauric acid.

3. The process for extracting American ginseng polysaccharides according to claim 2, characterized in that: The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:1 to 1:

2.

4. The process for extracting American ginseng polysaccharides according to claim 3, characterized in that: The hydrogen bond donor is lauric acid, the hydrogen bond acceptor is nonanoic acid, and the molar ratio of lauric acid to nonanoic acid in the system is 1:

1.

5. The process for extracting American ginseng polysaccharides according to claim 1, characterized in that: In step B, the ultrasonic extraction temperature is 30-70° C., and the extraction time is 10-50 min.

6. The process for extracting American ginseng polysaccharides according to claim 1, characterized in that: In step B, the solid-liquid ratio of the mass of American ginseng powder to the volume of the organic solvent is 1:10 to 1:

50.

7. The process for extracting American ginseng polysaccharides according to claim 1, characterized in that: In step B, the volume ratio of the upper phase to the lower phase in the three-phase distribution system is 1:0.25 to 1:

2.

8. The process for extracting American ginseng polysaccharides according to claim 1, characterized in that: The concentration of the ammonium sulfate solution in step B is 5-25%wt.

9. Use of the American ginseng polysaccharide according to any one of claims 1 to 8 in treating diseases related to ulcerative colitis.

10. A drug for treating ulcerative colitis-related diseases, characterized in that: The medicine contains American ginseng polysaccharide as an active ingredient.

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