Method for optimizing extraction of oligosaccharides from mesona aculeata using water extraction and alcohol precipitation by response surface methodology and application

Abstract

The application belongs to the field of mesona oligosaccharide extraction, and particularly relates to a method for optimizing water extraction and alcohol precipitation of mesona oligosaccharide by a response surface method and application. The mesona is used as raw material, and on the basis of single factor experiment, the ratio of material to liquid, reflux time, ethanol amount and ethanol volume fraction are determined as the main factors influencing the total sugar content of oligosaccharide. The process parameters of water extraction and alcohol precipitation of mesona oligosaccharide are optimized by the response surface method. The optimal process parameters are as follows: the ratio of material to liquid (g / mL) is 1:10, the reflux time is 1.5 h, the ethanol amount is 4 times of the volume of filtrate, and the ethanol volume fraction is 80%, and the total sugar content is 33.22%. Liquid chromatography-tandem mass spectrometry analysis shows that the obtained oligosaccharide is composed of hexose units with a polymerization degree of 2-6. The oligosaccharide hydrolysate contains mannose (Man), glucose (Glc) and galactose (Gal) three monosaccharides with a molar ratio of 1:23.53:10.89, and the monosaccharide composition result is consistent with the conclusion of liquid chromatography-tandem mass spectrometry analysis.

Description

Technical Field

[0001] This invention belongs to the field of oligosaccharide extraction from Mesona chinensis, specifically involving the method and application of response surface methodology for optimizing the water extraction and alcohol precipitation extraction of oligosaccharides from Mesona chinensis. Background Technology

[0002] Mesona chinensis ( Mesona chinensis Benth. is a perennial herb belonging to the genus *Benth.* of the Lamiaceae family. It is mainly distributed in Guangdong, Guangxi, Taiwan, and Zhejiang provinces of my country. As a typical plant used for both food and medicine, it has a long history of application and is also known as "Immortal Herb." This plant contains abundant active ingredients such as sugars, flavonoids, and phenols. Oligosaccharides, as a class of small molecule compounds composed of 2-10 monosaccharides linked by glycosidic bonds, not only have the advantages of good water solubility and easy absorption by the human body, but also exhibit various biological activities such as anti-oxidation, regulation of intestinal flora, anti-inflammation, and hypoglycemia. They have broad application prospects in functional food additives and natural health product raw materials. Currently, there are three main methods for preparing oligosaccharides: direct extraction, degradation, and synthesis. Direct extraction, also known as solvent extraction, extracts natural free oligosaccharides based on the principle of "like dissolves like," and is simple to operate. Degradation is obtained by hydrolyzing polysaccharides, but this method is complex. Synthesis is extremely difficult.

[0003] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease with an unclear etiology, primarily affecting the colonic mucosa and submucosa. Its clinical manifestations are mainly recurrent diarrhea, bloody and mucous stools, abdominal pain, and tenesmus. The pathogenesis of UC is complex, currently believed to be closely related to genetic susceptibility, environmental factors, gut microbiota dysbiosis, and abnormal intestinal mucosal immune responses. Conventional medications for treating UC include aminosalicylic acid derivatives, glucocorticoids, immunosuppressants, and biologics. However, these traditional drugs generally suffer from significant side effects, high dependence rates, high relapse rates after discontinuation, high cost, and poor primary or secondary responses in some patients. Therefore, finding safe, effective, multi-targeted novel anti-UC drugs that can fundamentally regulate gut microbiota and immune homeostasis has become an urgent need in the pharmaceutical field.

[0004] In recent years, natural plant polysaccharides have shown great potential in the adjuvant treatment and drug development of ulcerative colitis due to their excellent immunomodulatory, anti-inflammatory, antioxidant, and intestinal microecological regulation functions, as well as their extremely low toxicity. Existing literature also records some studies using *Mesona chinensis* polysaccharides to treat ulcerative colitis, such as the Chinese patent: Colon-targeting nanoparticles and their preparation method and application; publication number: CN117297088A; which includes *Mesona chinensis* polysaccharides with a uronic acid content of 56.82±1.02%. Another published paper: Mechanism of action of *Mesona chinensis* polysaccharides in alleviating Caco-2 cell damage induced by sodium dextran sulfate and ulcerative colitis in mice; author: Lu Hanyu; Master's thesis, published on May 26, 2023. However, no literature records the preparation of *Mesona chinensis* oligosaccharides and their application in drugs for treating ulcerative colitis. Due to their high viscosity, high degree of polymerization, large molecular weight, and complex structure, the pharmacological research and application of polysaccharides remain limited. Summary of the Invention

[0005] This invention aims to obtain natural, free oligosaccharides from *Mesona chinensis*. Using *Mesona chinensis* as raw material, a water extraction and alcohol precipitation method is employed. Large molecular impurities are precipitated using differences in solubility to obtain an oligosaccharide solution. The extraction process is optimized through single-factor experiments combined with response surface methodology, with the goal of increasing the total sugar content of *Mesona chinensis* oligosaccharides. The monosaccharide composition is also discussed to provide a reference for the industrial utilization of *Mesona chinensis*. Furthermore, compared to polysaccharides, oligosaccharides have good water solubility, are more easily absorbed by the body after oral administration, and have high bioavailability with fewer adverse reactions. Simultaneously, due to their low degree of polymerization and minimal steric hindrance, oligosaccharides can easily interact directly with receptors on the surface of intestinal and immune cells, and can also act as prebiotics to regulate the balance of intestinal flora and protect intestinal health. Therefore, this study explores the effect of *Mesona chinensis* oligosaccharides in the treatment of ulcerative colitis, providing a basis for the further development and utilization of *Mesona chinensis* oligosaccharides and demonstrating their application in the treatment of ulcerative colitis.

[0006] A response surface methodology-optimized method for water extraction and alcohol precipitation of oligosaccharides from *Mesona chinensis* is presented. Based on the Box-Behnken experimental design principle, Design-Expert software was used to design and optimize the process conditions for extracting oligosaccharides from *Mesona chinensis*, including the solid-liquid ratio, reflux time, ethanol dosage, and ethanol concentration. The steps are as follows: (1) Take the whole grass of Mesona chinensis, wash it, dry it in the sun, dry it in an oven at 50 ℃, crush it and pass it through a 40-mesh sieve, and store it in a desiccator at room temperature; (2) Weigh the grass powder, use the water extraction and alcohol precipitation method, add distilled water to a round bottom flask, heat and reflux twice, filter under reduced pressure, combine the filtrates, add ethanol solution, and let stand for 24 h to settle. (3) Take the supernatant and centrifuge at 12000 rpm and 4 ℃ for 10 min; (4) Take the supernatant, concentrate under reduced pressure, and freeze dry to obtain the grass jelly oligosaccharide sample; (5) Based on steps (1) to (4), design several groups of single-factor experiments with four single factors: material-liquid ratio, reflux time, ethanol dosage and ethanol concentration. (6) Using the total sugar content (Y) of grass jelly oligosaccharides as the response value, based on the experimental results of step (4), a multiple regression analysis was performed on the material-to-liquid ratio (A), reflux time (B), ethanol dosage (C), and ethanol volume fraction (D) in the reaction system to obtain the regression equation: .

[0007] In step (2), the ratio of the herb powder to the distilled water is 1:8 to 1:12; the reflux time is 1 to 2 hours; in step (2), the amount of ethanol used is 3.5 to 4.5 times the volume of the liquid in the flask; and the volume fraction of ethanol used is 75 to 85%.

[0008] The optimal extraction process parameters obtained from the regression equation calculation in step (6) above are: a material-to-liquid ratio of 1:10.11, a reflux time of 1.44 h, an ethanol dosage of 4.11 times, and an ethanol volume fraction of 80.12%. Under these process parameters, the theoretical total sugar content of the grass jelly oligosaccharide is 33.41%. The preferred process parameters are: a material-to-liquid ratio of 1:10, a reflux time of 1.5 h, an ethanol dosage of 4 times, and an ethanol volume fraction of 80%. Under these process parameters, the total sugar content of the grass jelly oligosaccharide is 33.22%.

[0009] The grass jelly oligosaccharide prepared by the above process is composed of three monosaccharides: Man, Glc, and Gal. The molar ratio of the three monosaccharides in the grass jelly oligosaccharide is Man:Glc:Gal = 1:23.53:10.89.

[0010] Application of the grass oligosaccharide obtained by the above extraction method in the treatment of ulcerative colitis.

[0011] Extraction principle: Free oligosaccharides have excellent water solubility, so hot water extraction is used; ethanol is added to the extract to adjust the polarity of the system, and the difference in solubility of the components causes impurities such as proteins and macromolecular polysaccharides to precipitate, thereby obtaining a supernatant rich in oligosaccharides, and subsequent high-speed centrifugation can separate oligosaccharides and polysaccharides.

[0012] The advantages of this invention are: 1. This invention optimizes the water extraction and alcohol precipitation process using response surface methodology, overcoming the limitation of traditional single-factor experiments in examining the interactions between factors. The established multiple regression model exhibits high goodness of fit and accurate prediction, yielding stable and reliable optimal process parameters that result in a total sugar content of up to 33.22% for the oligosaccharides from *Gynostemma pentaphyllum*. This process is simple to operate, operates under mild conditions, and is low in cost, without using toxic or harmful organic solvents, filling a gap in existing technologies and demonstrating strong feasibility for large-scale industrial production.

[0013] 2. The oligosaccharides extracted from *Mesona chinensis* using this invention have a low degree of polymerization (2-6), exhibiting excellent water solubility and extremely low solution viscosity compared to macromolecular *Mesona chinensis* polysaccharides. This characteristic completely solves the pharmaceutical challenges of formulating high-concentration oral solutions and performing microporous membrane sterilization filtration on macromolecular polysaccharides, greatly expanding their dosage form options in the pharmaceutical and food fields (such as oral solutions, granules, injections, etc.) and improving patient compliance.

[0014] 3. This invention discovers the application of this specific composition of *Mesona chinensis* oligosaccharide in the preparation of drugs for treating ulcerative colitis. Compared to macromolecular *Mesona chinensis* polysaccharides, the small-molecule oligosaccharides of this invention do not rely on the slow degradation by the severely imbalanced gut microbiota of ulcerative colitis patients. Instead, they can be directly and rapidly utilized by probiotics such as *Bifidobacterium* in the proximal colon, quickly producing a large amount of short-chain fatty acids to lower intestinal pH, repair the intestinal mucosal barrier, and inhibit inflammatory responses. This provides advantages such as faster onset of action, higher bioavailability, and multi-target intervention, offering a safe and efficient novel natural drug candidate for the clinical treatment of ulcerative colitis. Attached Figure Description

[0015] Figure 1 For glucose standard curve; Figure 2 The graph shows the effect of the material-to-liquid ratio on the total sugar content of *Gynostemma pentaphyllum* oligosaccharides. Figure 3 Figure showing the effect of reflux time on the total sugar content of mesona chinensis oligosaccharides; Figure 4 The graph shows the effect of ethanol dosage on the total sugar content of mesona chinensis oligosaccharides. Figure 5 The graph shows the effect of ethanol volume fraction on the total sugar content of mesona chinensis oligosaccharides. Figure 6 The graph shows the effect of the interaction of various factors on the total sugar content of oligosaccharides in *Mesona chinensis*. Figure 6 (a) Response surface plot of reflux time-to-liquid ratio to total sugar content; Figure 6 (b) Contour plot of reflux time-to-liquid ratio versus total sugar content; Figure 6 (c) Response surface plot of ethanol dosage-liquid-to-solid ratio to total sugar content; Figure 6(d) Contour plot of the effect of ethanol dosage-liquid-to-solid ratio on total sugar content; Figure 6 (e) Response surface plot of ethanol volume fraction - liquid-to-solid ratio to total sugar content; Figure 6 (f) Contour plot of ethanol volume fraction - liquid-to-solid ratio versus total sugar content; Figure 6 (g) Response surface plot of ethanol dosage-reflux time to total sugar content; Figure 6 (h) Contour plot of ethanol dosage - reflux time versus total sugar content Figure 7 The total ion chromatogram of the grass jelly oligosaccharide sample; Figure 8 HPLC chromatograms of monosaccharide standards and grass jelly oligosaccharide samples ( Figure 8 (a) is the HPLC chromatogram of the monosaccharide standard; Figure 8 (b) is the HPLC chromatogram of the grass jelly oligosaccharide sample; 1: PMP. 2: Glc. 3: Rib. 4: Rha. 5: Glc. 6: Gal. 7: Xyl. 8: Ara. Figure 9 Effect of material-to-liquid ratio on the yield of mesona chinensis oligosaccharides; Figure 10 The effect of reflux time on the yield of mesona chinensis oligosaccharides; Figure 11 Effect of ethanol dosage on the yield of mesona chinensis oligosaccharides; Figure 12 Effect of ethanol volume fraction on the yield of mesona chinensis oligosaccharides; Figure 13 The effect of solid-liquid ratio on the extraction quality of grass jelly oligosaccharide samples; Figure 14 The effect of reflux time on the extraction quality of mesona chinensis oligosaccharide samples; Figure 15 Effect of ethanol dosage on the extraction quality of mesona chinensis oligosaccharide samples; Figure 16 Effect of ethanol volume fraction on the extraction quality of mesona chinensis oligosaccharide samples; Figure 17 This is a graph showing the change in mouse body weight, compared with the Model group (*). p <0.05,** p <0.01, *** p <0.001, **** p< 0.0001); Figure 18 This is a graph showing the changes in DAI scores in mice, compared with the Model group (*). p <0.05,** p <0.01, *** p <0.001, **** p<0.0001); Figure 19 is the colon morphology diagram of mice; Figure 20 is the statistical diagram of mouse colon length ( p<0.001, compared with the Control group, * p <0.05, ** p <0.01, compared with the Model group); Figure 21 is the ELISA analysis of the expression of TNF-α, IL-1β and IL-10 in mouse colon tissue ( p <0.001, compared with the Control group, * p <0.05, ** p <0.01, *** p <0.001, compared with the Model group); Figure 22 H&E staining of mouse colon tissue;( Figure 22 (a) is the H&E staining diagram of colon tissue of mice in the Control group (scale bar 500μm); Figure 22 (b) is the H&E staining diagram of colon tissue of mice in the Control group (scale bar 100μm); Figure 22 (c) is the H&E staining diagram of colon tissue of mice in the Model group (scale bar 500μm); Figure 22 (d) is the H&E staining diagram of colon tissue of mice in the Model group (scale bar 100μm); Figure 22 (e) is the H&E staining diagram of colon tissue of mice in the MCO-H group (scale bar 500μm); Figure 22 (f) is the H&E staining diagram of colon tissue of mice in the MCO-H group (scale bar 100μm); Figure 22 (g) is the H&E staining diagram of colon tissue of mice in the MCO-L group (scale bar 500μm); Figure 22 (h) is the H&E staining diagram of colon tissue of mice in the MCO-L group (scale bar 100μm); Figure 22 (i) is the H&E staining diagram of colon tissue of mice in the 5-ASA group (scale bar 500μm); Figure 22 (j) is the H&E staining diagram of colon tissue of mice in the 5-ASA group (scale bar 100μm)); Figure 23 is the AB-PAS staining of mouse colon tissue ( Figure 23 (a) is the AB-PAS staining diagram of colon tissue of mice in the Control group (scale bar 500μm); Figure 23 (b) is the AB-PAS staining diagram of colon tissue of mice in the Control group (scale bar 100μm); Figure 23(c) AB-PAS staining image of colon tissue from Model group mice (scale bar 500 μm); Figure 23 (d) AB-PAS staining image of colon tissue from Model group mice (scale bar 100 μm); Figure 23 (e) AB-PAS staining image of colon tissue from MCO-H group mice (scale bar 500 μm); Figure 23 (f) is an AB-PAS staining image of colon tissue from mice in the MCO-H group (scale bar 100 μm). Figure 23 (g) is an AB-PAS staining image of colon tissue from MCO-L group mice (scale bar 500 μm); Figure 23 (h) is an AB-PAS staining image of the colon tissue of mice in the MCO-L group (scale bar 100μm). Figure 23 (i) AB-PAS staining image of colon tissue from mice in the 5-ASA group (scale bar 500 μm); Figure 23 (j) is an AB-PAS staining image of colon tissue from mice in the 5-ASA group (scale bar 100 μm). Detailed Implementation

[0016] Source of raw materials The grass used in the experiment was provided by Researcher Tang Danfeng of the Guangxi Zhuang Autonomous Region Medicinal Botanical Garden, and was identified as *Mesona chinensis*, belonging to the genus *Mesona* of the family Lamiaceae. Mesona chinensis Benth. Dried whole herb.

[0017] Main instruments and reagents RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); 1-2 Ldplus freeze dryer (Christ GmbH, Germany); T700 UV-Vis UV-Vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.); XMTD-205C digital display constant temperature water bath (Jiangsu Kexi Instrument Co., Ltd.); XS205DU 0.0001 g balance (Mettler-Toledo International Trading Co., Ltd.), ATY224 0.0001 g balance, CBM-20A high performance liquid chromatograph (Shimadzu Instrument Co., Ltd.); Alpha Thermo Fisher Scientific MICRO17R centrifuge, Thermo Fisher Scientific TSQ Quantum ultra-high performance liquid chromatography-mass spectrometer with triple quadrupole (Thermo Fisher Scientific, USA).

[0018] Anhydrous ethanol (analytical grade, Chengdu Kelong Chemical Co., Ltd.); anthrone (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); sulfuric acid (analytical grade, Chongqing Chuandong Chemical Co., Ltd.); D(+)-anhydrous glucose (Glc, purity ≥99%, Hefei Bomei Biotechnology Co., Ltd.); D-mannose (Man, purity ≥98%, Shanghai Yuanye Biotechnology Co., Ltd.); D-(-)-arabinose (Ara, purity ≥98%, Shanghai Aladdin Biochemical Technology Co., Ltd.); D-galactose (Gal, purity 99%), L-rhamnose (Rha, purity 99%), D-(+)-xylose (Xyl, purity ≥99%), D-ribose (Rib, purity 99%), trifluoroacetic acid (purity 99%), 1-phenyl-3-methyl-5-pyrazolone (PMP, purity 99%) (Shanghai Maclean Biochemical Technology Co., Ltd.); acetonitrile (mass spectrometry grade, Thermo Fisher Scientific). All water used was distilled water.

[0019] Example 1

[0020] Process flow for extracting oligosaccharides from grass jelly using water extraction and alcohol precipitation (1) Take the whole grass of Mesona chinensis, wash it, dry it in the sun, dry it in an oven at 50 ℃, crush it and pass it through a 40-mesh sieve, and store it in a desiccator at room temperature; (2) Weigh the grass powder, use the water extraction and alcohol precipitation method, add distilled water to a round bottom flask, heat and reflux twice, filter under reduced pressure, combine the filtrates, add ethanol solution, and let stand for 24 h to settle. (3) Take the supernatant and centrifuge it at 12000 rpm and 4 ℃ for 10 min; (4) Take the supernatant, concentrate under reduced pressure, and freeze dry to obtain the grass jelly oligosaccharide sample.

[0021] Determination of total sugar content of oligosaccharides from grass jelly Total sugar content was determined using the sulfuric acid-anthrone method. Anhydrous glucose standard was weighed and prepared into a 0.1 mg / mL glucose stock solution. 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mL of the glucose standard solution were pipetted into test tubes, and water was added to bring the volume to 1 mL. Then, 4 mL of 0.33% anthrone-sulfuric acid solution (freshly prepared before use) was added to each tube. The tubes were shaken well and heated in a boiling water bath for 10 min, followed by cooling in an ice-water bath for 15 min. The absorbance was measured at 620 nm. A standard curve was plotted with absorbance (A) on the ordinate and the standard solution concentration (c) on the abscissa. Figure 1 As shown, the regression equation is Y = 7.468X - 0.001036, R0 2 =0.9992.

[0022] Weigh an appropriate amount of *Gynostemma pentaphyllum* oligosaccharide sample, add it to distilled water to dissolve, vortex to mix, centrifuge at 4℃ and 12000 rpm for 10 min, take the supernatant and measure the absorbance of the sample. Other operating methods are the same as those for preparing the standard curve. Substitute into the standard curve equation to obtain the oligosaccharide mass concentration. Calculate the total sugar content of the *Gynostemma pentaphyllum* oligosaccharide sample according to formula (1):

[0023] In the formula, r is the total sugar content of *Mesona chinensis* (%), c is the mass concentration of *Mesona chinensis* oligosaccharide sample (mg / mL), D is the dilution factor, v is the volume of *Mesona chinensis* oligosaccharide sample liquid (mL), and m is the mass of *Mesona chinensis* oligosaccharide sample (mg).

[0024] Example 2

[0025] Single-factor experimental design - Effect of different solid-liquid ratios on the total sugar content of oligosaccharide samples Weigh 10 g of *Mesona chinensis* powder and add distilled water. Set the material-to-liquid ratio (g:mL) to 1:4, 1:6, 1:8, 1:10, and 1:12. Maintain a fixed reflux time of 2 h, an ethanol volume fraction of 80%, and a volume four times that of the filtrate. Extract the sample according to the method in Example 1 and measure its absorbance. Perform three parallel experiments and take the average value to investigate the effect of different material-to-liquid ratios on the total sugar content of the oligosaccharide sample. Figure 2 As shown; As shown in the figure, under the experimental conditions, the effect of the solid-liquid ratio first increases and then decreases. The total sugar content is highest when the solid-liquid ratio is 1:10 (g:ml), and then decreases as the solid-liquid ratio decreases (solvent increases). This may be because, within a certain range, as the amount of solvent increases, the contact area between the powder and the solvent increases, resulting in more complete sugar diffusion and improved oligosaccharide dissolution efficiency. However, excessive solvent leads to competitive inhibition of oligosaccharide release by other water-soluble components, resulting in a decrease in its relative content. Therefore, response surface methodology experiments were conducted using solid-liquid ratios of 1:8, 1:10, and 1:12 (g:ml).

[0026] Example 3

[0027] Single-factor experimental design - Effect of reflux time on the total sugar content of mesona chinensis oligosaccharides Weigh 10 g of *Gynostemma pentaphyllum* powder, add distilled water, fix the material-to-liquid ratio at 1:8, the ethanol volume fraction at 80%, and the volume of ethanol used at 4 times the filtrate volume. Set reflux times of 1 h, 1.5 h, 2 h, 2.5 h, and 3 h. Extract samples according to the method in Example 1 and measure their absorbance. Perform the experiment in triplicate and take the average value to investigate the effect of different reflux times on the total sugar content of the oligosaccharide samples. Figure 3 As shown; As shown in the figure, under the experimental conditions, the total sugar content peaked at extraction times of 1.5 h and 2.5 h. However, excessively long reflux times may lead to changes in the oligosaccharide structure and decomposition, affecting the total sugar content. Therefore, from the perspective of energy conservation and environmental protection, extraction times of 1, 1.5, and 2 h were selected for response surface methodology optimization experiments.

[0028] Example 4

[0029] Single-factor experimental design - Effect of ethanol dosage on the total sugar content of mesona chinensis oligosaccharides Weigh 10 g of *Mesona chinensis* powder, add distilled water, fix the material-to-liquid ratio at 1:8, reflux time at 2 h, and ethanol volume fraction at 80%. Set the amount of ethanol added to be 3, 3.5, 4, 4.5, and 5 times the volume of the filtrate. Extract samples according to the method in Example 1 and measure their absorbance. Perform three parallel experiments and take the average value to investigate the effect of different ethanol amounts on the total sugar content of the oligosaccharide samples. Figure 4 As shown.

[0030] As shown in the figure, the total sugar content initially increased and then decreased with increasing ethanol dosage, reaching its highest level when the ethanol dosage was four times the filtrate volume. This may be because insufficient ethanol dosage leads to incomplete precipitation of large molecules such as proteins, while excessive ethanol dosage causes oligosaccharides to co-precipitate with impurities, resulting in a decrease in total sugar content. Therefore, response surface methodology experiments were conducted with ethanol dosages of 3.5, 4, and 4.5 times the filtrate volume.

[0031] Example 5

[0032] Single-factor experimental design - Effect of ethanol volume fraction on the total sugar content of mesona chinensis oligosaccharides Weigh 10 g of *Gynostemma pentaphyllum* powder, add distilled water, fix the material-to-liquid ratio at 1:8, reflux time at 2 h, and use ethanol at 4 times the volume of filtrate. Set the ethanol volume fractions to 70%, 75%, 80%, 85%, and 90%. Extract samples according to the method in Example 1 and measure their absorbance. Perform three parallel experiments and take the average value to investigate the effect of different ethanol volume fractions on the total sugar content of oligosaccharide samples. Figure 5 As shown.

[0033] As shown in the figure, the total sugar content increases with increasing ethanol volume fraction, reaching its highest level at an ethanol volume fraction of 80%. However, excessively high ethanol concentrations lead to a decrease in total sugar content. This may be because increasing ethanol concentration alters the polarity of the solution, reducing the solubility of oligosaccharides and thus decreasing the total sugar content. Therefore, response surface methodology experiments were conducted using ethanol volume fractions of 75%, 80%, and 85%.

[0034] Example 6

[0035] Box-Benhnken Response Surface Optimization Experimental Design and Results Analysis Using Design-Expert 13.0 software, regression analysis and analysis of variance were performed on the optimized influencing factors and levels selected from the single-factor experimental data. Based on the Box-Benhnken central composite design principle, a four-factor, three-level experiment was conducted. The central value (represented by "0"), low level (represented by "-1"), and high level (represented by "+1") determined from the single-factor experiment were used. The total sugar content (Y) of *Gynostemma pentaphyllum* oligosaccharides was used as the response value to optimize the material-to-liquid ratio (A), reflux time (B), ethanol dosage (C), and ethanol volume fraction (D) in the reaction system. Specific influencing factors and levels are shown in Table 1.

[0036] The experimental results are shown in Table 2;

[0037] Statistical results of the surface experiment: Using the total sugar content of grass jelly oligosaccharides as the response value, a multiple regression analysis was performed to obtain the regression equation:

[0038] The results of the analysis of variance for the regression model are shown in Table 3.

[0039]

[0040] From Table 3, we can see that the regression model... P A value < 0.0001 indicates that the regression model is statistically significant and that the lack-of-fit term is not present. P The value of 0.2583 > 0.05 indicates that the impact is not significant, suggesting that the regression model can predict the experimental results well. 2 =0.9679, R 2 Adj =0.9359, indicating that the model's predicted value matches the measured value well, and can be used to analyze the process parameters for extracting oligosaccharides from grass jelly.

[0041] The response surface plot and corresponding contour plot were obtained by using Design-Expert 13.0 software for multivariate fitting. Figure 6 It can be seen that in the response surface plot, the surface changes are steep and the ellipse of the contour lines has a large eccentricity, indicating that the interaction between reflux time and liquid-to-material ratio, ethanol dosage and liquid-to-material ratio, ethanol volume fraction and liquid-to-material ratio, and ethanol dosage and reflux time has a significant effect on the total sugar content of grass jelly oligosaccharides.

[0042] In addition, referring to Table 3, the partial regression coefficient C, the interaction term AB, and the quadratic term A of AC are also included. 2 B 2 C2 D 2 of p The value <0.01 indicates that the effect of time alone and the interaction between the material-liquid ratio and reflux time, and between the material-liquid ratio and ethanol dosage, are highly significant on the total sugar content of *Gynostemma pentaphyllum* oligosaccharides. Furthermore, the partial regression coefficients B, and the interaction terms AD and BC... p A value <0.05 indicates a significant impact, suggesting that the single effect and the interaction between the solid-liquid ratio and ethanol volume fraction, as well as the interaction between reflux time and ethanol dosage, have a significant effect on the total sugar content of *Gynostemma pentaphyllum* oligosaccharides. A larger F-value indicates a more significant effect of the factor. Table 3 shows that the order of influence of each factor on the total sugar content of *Gynostemma pentaphyllum* oligosaccharides is: ethanol dosage (C) > reflux time (B) > solid-liquid ratio (A) > ethanol volume fraction (D).

[0043] Extracting and optimizing parameters and verifying the results: After optimizing four parameters—the material-to-liquid ratio, reflux time, ethanol dosage, and ethanol volume fraction—the optimal process parameters were obtained: a material-to-liquid ratio of 1:10.11, a reflux time of 1.44 h, an ethanol dosage of 4.11 times the volume fraction, and an ethanol volume fraction of 80.12%. To facilitate practical operation, the predicted results were verified by adjusting the material-to-liquid ratio to 1:10, the reflux time to 1.5 h, the ethanol dosage to 4 times the volume fraction, and the ethanol volume fraction to 80%. The total sugar content of the *Gynostemma pentaphyllum* oligosaccharides was found to be 33.22%, which is close to the predicted value of 33.41% calculated by the regression equation, indicating that the extraction process parameters are reliable.

[0044] Example 7

[0045] The extraction conditions were set as follows: material-to-liquid ratio of 1:8, reflux time of 1 hour, ethanol dosage of 3.5 times, ethanol volume fraction of 75%, and other conditions were the same as in Example 1.

[0046] Example 8

[0047] The extraction conditions were set as follows: material-to-liquid ratio of 1:10, reflux time of 1.5 h, ethanol dosage of 4 times, ethanol volume fraction of 80%, and other conditions were the same as in Example 1.

[0048] Example 9

[0049] The extraction conditions were set as follows: material-to-liquid ratio of 1:12, reflux time of 2 hours, ethanol dosage of 4.5 times, ethanol volume fraction of 85%, and other conditions were the same as in Example 1.

[0050] Example 10

[0051] Identification of Grass Jelly Oligosaccharides using UPLC H-Class PLUS Full Scan Technology Based on Waters Xevo G3 Q-TOF The oligosaccharide sample of *Gynostemma pentaphyllum* was dissolved in 50% acetonitrile water to prepare a concentration of 6 mg / mL. The solution was then centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm membrane to obtain the analyte. LC-MS / MS was performed on a Waters Xevo G3 Q-TOF mass spectrometer and a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatography system equipped with an autosampler. UHPLC separation was performed using a Waters BEH Amide column (2.1 × 100 mm, 1.7 μm). Mobile phase A was 0.1% ammonia-water, and mobile phase B was 0.1% ammonia-acetonitrile. The elution method for the chromatographic system is as follows: 0-2 min, 5%-15% A, 95%-85% B; 2-23 min, 15%-45% A, 85%-55% B; 23-25 ​​min, maintaining phase A at 45% and phase B at 55%; 25-30 min, maintaining phase A at 5% and phase B at 95%. The total run time is 30 min.

[0052] Oligosaccharides from *Gynostemma pentaphyllum* were identified using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The total ion chromatogram is shown below. Figure 7 As shown in the figure, oligosaccharides mainly exist in [Hexn-H] mode under negative ion mode. - and [Hexn+HCOO] - It exists in the form of , where Hex represents a hexose and n is the degree of polymerization. The molecular weight of each oligosaccharide is calculated using formulas (2) and (3):

[0053] In the formula, 162.053 represents the mass of hexose residues, n represents the degree of polymerization, and 1.0078 represents the H+ residue mass. - The monoisotopic mass of 44.9977 is HCOO. - The monoisotopic mass of H₂O is 18.015. Full-scan mass spectrometry revealed that the *Gynostemma pentaphyllum* oligosaccharide sample contained various oligosaccharides composed of hexoses, with a degree of polymerization ranging from 2 to 6.

[0054] Example 11

[0055] Determination of oligosaccharide and monosaccharide composition of grass jelly Hydrolysis of grass jelly oligosaccharides: Weigh 6 mg of *Gynostemma pentaphyllum* oligosaccharide sample and add 1 mL of trifluoroacetic acid (2.0 mol / L). Hydrolyze the sample in a sealed glass tube at 121 °C for 2 h. Concentrate the reaction solution under reduced pressure and evaporate to dryness. Wash the sample residue 2-3 times with 3 mL of methanol, evaporating after each wash to ensure complete removal of trifluoroacetic acid. Finally, dissolve the residue in 1 mL of sterile water for later use.

[0056] PMP Derivatization: Take 300 μL of the test solution, add 300 μL of NaOH (0.5 mol / L) and 750 μL of PMP methanol solution (0.5 mol / L), react at 70℃ for 1 h, remove and cool to room temperature, add 300 μL of 0.5 mol / L HCl to neutralize the reaction solution, then extract three times with chloroform to remove excess PMP. Take the aqueous layer solution, filter it through a 0.22 μm filter, and then use it for high performance liquid chromatography analysis. The same derivatization method is used for Man, Glc, Gal, Rib, Rha, Xyl, and Ara monosaccharide standards.

[0057] HPLC conditions: The monosaccharide composition of oligosaccharides from *Gynostemma pentaphyllum* was analyzed using a Shimadzu CBM-20A HPLC system with a Hypersil BDS C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase was acetonitrile-0.025 mol / L phosphate buffer (pH 6.8) (16:84, V / V); the flow rate was 0.8 mL / min; the column temperature was set at 30 ℃; the PDA detection wavelength was 250 nm; and the injection volume was 10 μL.

[0058] Calculation of the molar ratio of monosaccharides: Mixed monosaccharide standard solutions of different concentrations were prepared, derivatized by PMP, and then injected for determination. A linear regression equation was performed using monosaccharide mass concentration (X) and peak area (Y), and the molar ratio of monosaccharide composition of grass jelly oligosaccharides was calculated according to formula (4):

[0059] In the formula: c is the mass concentration of the grass jelly oligosaccharide sample (mg / mL), V is the constant volume (mL), and M is the relative molecular mass of the monosaccharide.

[0060] Monosaccharide composition analysis of *Mesona chinensis* oligosaccharides: The mixed monosaccharide standard solution and the acid-hydrolyzed *Mesona chinensis* oligosaccharide sample were subjected to derivatization reactions, and the results were analyzed by HPLC as follows: Figure 8 As shown in the chromatogram of the mixed monosaccharide standard solution, peak 1, the solvent peak of the PMP methanol solution, is located before the retention times of each monosaccharide peak and will not interfere with the target monosaccharide chromatographic peaks. The seven target monosaccharide components were essentially separated. Comparison of the chromatograms of the *Mesona chinensis* oligosaccharide sample with the seven monosaccharide standards revealed that the extracted *Mesona chinensis* oligosaccharide was composed of three monosaccharides: Man, Glc, and Gal.

[0061] A linear equation was constructed using monosaccharide concentration (X) and peak area (Y). The detection limit was calculated at the concentration corresponding to a signal-to-noise ratio (SNR) of 3, and the quantitation limit was calculated at the concentration corresponding to a SNR of 10. As shown in Table 4, the linear correlation coefficients of the three monosaccharides were all greater than 0.99, meeting the quantification requirements. The calculated molar ratio of the three monosaccharides in the grass jelly oligosaccharide was Man:Glc:Gal = 1:23.53:10.89.

[0062]

[0063] Example 12

[0064] The yield and extraction quality were calculated for the above embodiments.

[0065] In the formula, r is the total sugar content (%) of the Mesona chinensis sample, m1 is the amount of Mesona chinensis raw material (g), and m2 is the mass (mg) of the Mesona chinensis oligosaccharide sample extracted, i.e., the supernatant freeze-dried product.

[0066] According to formula (5), under the optimal extraction conditions of a solid-liquid ratio (g:mL) of 10:1, a reflux time of 2 h, an ethanol volume of 4 times the filtrate volume, an ethanol volume fraction of 80%, a total sugar content of 33.22%, and a yield of 4.95%, the yield of *Gynostemma pentaphyllum* oligosaccharides was higher than that of the single-factor experimental groups. The effects of each factor on the yield of *Gynostemma pentaphyllum* oligosaccharides are as follows: Figures 9-12 As shown, except for the solid-liquid ratio, the effects of other factors on the yield generally follow the same trend as their effects on the total sugar content. The yield increases as the solid-liquid ratio decreases (solvent volume increases). This is because excess solvent causes other water-soluble components to dissolve, resulting in the supernatant containing many water-soluble components other than oligosaccharides. This increases the mass of the freeze-dried supernatant, thus increasing the yield.

[0067] Extraction quality such as Figures 13-16 As shown.

[0068] Example 13

[0069] Study on the pharmacodynamic mechanism of mesona chinensis oligosaccharide in the treatment of ulcerative colitis - preparation of mouse UC model and drug administration intervention Six-week-old SPF-grade male C57BL / 6 mice (20 ± 2 g) were purchased from the Animal Experiment Center of Guangxi Medical University (Nanning, China). All mice were housed in an IVC mouse system at a temperature of 22 ± 1 ℃ and humidity of 40%–60%, in a stable environment with a regular day-night cycle. All mice had free access to sterile water and a normal diet (20% protein, 70% carbohydrates, and 10% fat). After one week of acclimatization, the mice were randomly divided into five groups: Control group, Model group, MCO-L group, MCO-H group, and 5-ASA group. The control group received normal food and water. The model group received 2.3% DSS (free access to water) from day 1 to 7. The MCO-L group received *Mesona chinensis* oligosaccharide (25 mg / kg / day) via gavage for 11 days, along with 2.3% DSS from day 1 to 7. The MCO-H group received *Mesona chinensis* oligosaccharide (100 mg / kg / day) via gavage for 11 days, along with 2.3% DSS from day 1 to 7. The 5-ASA (mesalazine) group received 5-ASA (100 mg / kg / day) via gavage, along with 2.3% DSS from day 1 to 7. The mice's food and water intake were monitored daily. Animal experiments were conducted in accordance with the requirements of the Animal Ethics Experiment Committee of Guangxi Medical University and the "National Laboratory Animal Use Law".

[0070] Disease Activity Index Score During the experiment, the mice's weight changes, fecal characteristics, and degree of rectal bleeding were observed daily to calculate the Disease Activity Index (DAI). The calculation method was DAI = (weight loss rate score + fecal viscosity score + degree of bleeding score) / 3. The DAI scoring criteria are shown in Table 5.

[0071]

[0072] Colonic histopathology After administration, mice were euthanized by CO2 inhalation. The colon was then dissected from the ileocecal junction to the lower rectum and its length was measured. A portion of the colon was fixed in 4% paraformaldehyde for 48 h. Subsequently, the colon was dehydrated, embedded, cut, stained with hematoxylin and eosin (H&E), and Alsin blue-periodic acid-Schiff stain (AB-PAS). Colon tissue sections were photographed using an optical microscope to observe the inflammatory infiltration and ulceration of the colon tissue.

[0073] Example 14

[0074] The effect of Mesona chinensis oligosaccharide on physiological indicators in UC mice - Mesona chinensis oligosaccharide inhibited the decrease in body weight and the increase in DAI score in UC mice.

[0075] In this study, a UC model was established in C57BL / 6 mice by administering 2.3% DSS. Changes in mouse body weight and fecal characteristics were recorded daily during the experiment.

[0076] Figure 17 The curves showing the weight changes of mice in each group during the process were presented. The results showed that starting from the fourth day after DSS administration, the weight of mice in the UC group began to decrease. Treatment with *Gynostemma pentaphyllum* oligosaccharide alleviated the weight loss in UC mice. On the other hand, the severity of UC was determined by DAI scoring based on mouse weight, fecal viscosity, and degree of bleeding. The results were as follows... Figure 18 As shown, the DAI score of the Model group mice showed a significant upward trend with the increase of modeling time, while the DAI score of the drug-treated group mice was lower than that of the Model group, indicating that Mesona chinensis oligosaccharide can alleviate the course of UC mice.

[0077] Example 15

[0078] Mesona chinensis oligosaccharides inhibit DSS-induced colonic shortening DSS-induced UC often damages the colon. After DSS administration, shortened colon length and intestinal congestion were observed in mice, with dark red feces in the cecum and colon. Administration of *Gynostemma pentaphyllum* oligosaccharides resulted in a smoother colon surface and reduced cecal and colonic congestion in UC mice compared to the Model group. Figure 19 Statistical analysis of colon length showed that, compared with the Model group, the colon length of mice in the MCO-H group was significantly increased ( ). Figure 20 () p <0.05 or 0.01).

[0079] Example 16

[0080] Mesona chinensis oligosaccharides regulate the expression of colonic inflammatory factors in UC mice. Dysregulation of inflammatory factors is a hallmark of DSS-induced colitis. To further investigate the effects of *Gynostemma pentaphyllum* oligosaccharides on pro-inflammatory and anti-inflammatory factors, this study detected key inflammatory factors. Previous studies have shown that DSS can activate the NLRP3 inflammasome, promote IL-1β maturation, and induce intestinal inflammation. TNF-α is an important target in inflammatory bowel disease, playing a role in leukocyte aggregation, activation of inflammation, and apoptosis; IL-10 is an anti-inflammatory factor that can inhibit local inflammation. Figure 21 As shown, TNF-α and IL-1β levels were significantly increased and IL-10 levels were significantly decreased in the colon tissue of DSS-induced UC mice, indicating that Mesona chinensis oligosaccharide has a significant regulatory effect on cellular inflammatory factors, can significantly reduce the expression of TNF-α and IL-1β, and restore the expression level of IL-10.

[0081] Example 17

[0082] Effects of Mesona chinensis oligosaccharides on colonic structure in UC mice Colonic tissue sections often reflect the degree of colonic inflammation and can also reveal overall colonic pathological features, such as crypt inflammation, structural changes, abscesses, and ulcers. To evaluate the extent of colonic damage, this study performed H&E staining on intestinal sections. The staining results are shown below. Figure 22 The results showed that the colon tissue of the Model group mice was significantly damaged, the colonic mucosal epithelium and crypt structure were destroyed, and there was significant inflammatory cell infiltration. However, after treatment with MCO, the crypts and goblet cells were restored, and the inflammatory infiltration was reduced.

[0083] Example 18

[0084] Effects of Mesona chinensis oligosaccharides on intestinal barrier function in UC mice To investigate the effects of *Gynostemma pentaphyllum* oligosaccharides on intestinal barrier damage, this study performed AB-PAS staining analysis on colon tissues of mice in each group to assess the number of goblet cells and mucus secretion levels in the intestinal epithelium. The results showed ( Figure 23 DSS treatment significantly reduced the number of goblet cells in the colonic tissue of mice, while the number of goblet cells in the colon was restored after treatment with 5-ASA, MCO-L and MCO-H, with MCO-H showing the most significant effect.

Claims

1. A method for optimizing the water extraction and alcohol precipitation extraction of oligosaccharides from *Mesona chinensis* using response surface methodology, characterized in that... Based on the Box-Behnken experimental design principle, the process conditions for extracting oligosaccharides from *Gynostemma pentaphyllum*, including the solid-liquid ratio, reflux time, ethanol dosage, and ethanol concentration, were designed and optimized using Design-Expert software. The steps are as follows: (1) Take the whole herb of Mesona chinensis, wash it, dry it in the sun, dry it in an oven at 50 ℃, pulverize it and pass it through a 40-mesh sieve, and dry it at room temperature for storage; (2) Weigh the grass powder, use the water extraction and alcohol precipitation method, add distilled water to the container, heat and reflux twice, filter under reduced pressure, combine the filtrates, add ethanol solution, and let stand for 24 h to settle; the reflux time is 1~2 h; the volume fraction of the ethanol solution is 75~85%, and the amount used is 3.5~4.5 times the volume of the filtrate; (3) Take the supernatant and centrifuge at 12000 rpm and 4 ℃ for 10 min; (4) Take the supernatant, concentrate under reduced pressure, and freeze dry to obtain the grass jelly oligosaccharide sample; (5) Based on steps (1) to (4), design several groups of single-factor experiments with four single factors: material-liquid ratio, reflux time, ethanol dosage and ethanol concentration. (6) Using the total sugar content (Y) of grass jelly oligosaccharides as the response value, based on the experimental results of step (4), a multiple regression analysis was performed on the material-to-liquid ratio (A), reflux time (B), ethanol dosage (C), and ethanol volume fraction (D) in the reaction system to obtain the regression equation: ; The grass jelly oligosaccharide is composed of three monosaccharides: Man, Glc, and Gal; the molar ratio of the three monosaccharides in the grass jelly oligosaccharide is Man:Glc:Gal = 1:23.53:10.

89.

2. The method according to claim 1, characterized in that, In step (2), the ratio of the herb powder to the distilled water is 1:8~1:12 g / mL.

3. The method according to claim 1, characterized in that, The optimal extraction process parameters obtained from the regression equation calculation in step (6) are: a material-to-liquid ratio of 1:10.11 g / mL, a reflux time of 1.44 h, an ethanol solution volume of 4.11 times the filtrate volume, and an ethanol solution volume fraction of 80.12%.

4. The method according to claim 1, characterized in that, In step (2), if the material-to-liquid ratio is 1:10, the reflux time is 1.5 h, the ethanol solution volume is 4 times the filtrate volume, and the ethanol solution volume fraction is 80%, the oligosaccharide content in the obtained grass jelly oligosaccharide sample is ≥33.22%.

5. The use of the geranium oligosaccharide prepared by the method according to any one of claims 1 to 4 in the preparation of a drug for treating ulcerative colitis.

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