Scrophularia ningpoensis polysaccharide decolorization method

Abstract

The invention relates to the technical field of medicine component extraction, in particular to a scrophularia ningpoensis polysaccharide decoloring method. The problems that in the prior art, activated carbon adsorption selectivity is poor, a chemical oxidation method damages a polysaccharide structure, and an organic solvent method has potential safety hazards are solved. The method comprises the following steps: preparing radix scrophulariae powder; preparing defatted radix scrophulariae powder, preparing defatted and deproteinized radix scrophulariae powder, preparing radix scrophulariae polysaccharide by using the defatted and deproteinized radix scrophulariae powder, and decolorizing the radix scrophulariae polysaccharide by using macroporous resin. According to the method, by accurately controlling physical and chemical parameters and process conditions of the resin, efficient and selective removal of pigments is achieved, the decolorization rate exceeds 95%, the polysaccharide recovery rate is remarkably increased, the whole process is mild and non-toxic, the structural integrity and biological activity of the polysaccharide are guaranteed, the product safety meets the medical standard, the resin can be regenerated and recycled, and the process is green and economical.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug component extraction, and particularly relates to a scrophularia ningpoensis polysaccharide decolorization method. BACKGROUND

[0002] Scrophularia ningpoensis polysaccharide is an important active ingredient extracted from dried roots of scrophularia ningpoensis, has multiple pharmacological activities such as improving muscle glycogen and liver glycogen reserves, scavenging free radicals, relieving exercise-induced fatigue, and has important application value in functional food and modernization of traditional Chinese medicine. However, the crude extract of scrophularia ningpoensis polysaccharide often contains a large amount of pigment impurities, which not only affects the appearance color of the polysaccharide, but also seriously interferes with the subsequent purification and classification, structure analysis and biological activity evaluation, and becomes a key bottleneck restricting the high-value development of the polysaccharide.

[0003] Among them, the decolorization and purification of scrophularia ningpoensis polysaccharide is the core link to improve its quality. The commonly used decolorization methods in industry and laboratory at present mainly include activated carbon adsorption method, hydrogen peroxide oxidation method and organic solvent extraction method. Although activated carbon has strong adsorption capacity, its non-specific adsorption characteristics can easily lead to co-adsorption of polysaccharide, which significantly reduces the product yield; although the oxidation method can effectively degrade pigments, the strong oxidation environment can easily damage the glycosidic bond or active group of polysaccharide, causing structure damage and loss of physiological function; and the organic solvent extraction method has the risk of solvent residue, which is difficult to meet the safety standards of drug or food grade products. The existing technology has multiple contradictions in the process of decolorization of scrophularia ningpoensis polysaccharide, such as difficult to balance the decolorization efficiency and polysaccharide retention rate, insufficient process safety and insufficient protection of the structural integrity of polysaccharide. Especially in the application scene of pursuing high purity and high activity, the traditional method is difficult to realize efficient removal of pigments while maintaining the natural configuration and functional characteristics of polysaccharide, and a new decolorization technology scheme with high selectivity, high safety and effective retention of polysaccharide activity is urgently needed.

[0004] Therefore, the application provides a method for decolorizing scrophularia ningpoensis polysaccharide by using macroporous resin to solve the problems in the prior art. SUMMARY

[0005] Therefore, the main purpose of the application is to provide a scrophularia ningpoensis polysaccharide decolorization method, which solves the problems of low polysaccharide yield and safety hazards in the existing scrophularia ningpoensis polysaccharide decolorization method.

[0006] The technical scheme of the application is as follows:

[0007] A scrophularia ningpoensis polysaccharide decolorization method, comprising the steps of:

[0008] Step S1: preparing scrophularia ningpoensis powder;

[0009] Step S2: preparing defatted scrophularia ningpoensis powder;

[0010] Weigh 200 g of dried Scrophularia powder, add 1000 mL of petroleum ether, heat under reflux for 1 h and then filter. Dry the Scrophularia powder in a fume hood for 2 h. After drying, transfer the Scrophularia powder to a 50℃ oven and dry to constant weight to obtain defatted Scrophularia powder.

[0011] Step S3: Prepare defatted and deproteinized Scrophularia powder;

[0012] Step S4: Scan the Scrophularia powder preservation solution obtained in step S3 at 280 nm using a UV-Vis spectrophotometer to detect whether there is a protein absorption peak. If so, repeat step S3.

[0013] Step S5: Prepare Scrophularia polysaccharide using defatted and deproteinized Scrophularia powder;

[0014] Step S6: Decolorize the Scrophularia polysaccharide using macroporous resin;

[0015] The macroporous resin is ADS-7, and when ADS-7 is used to decolorize Scrophularia polysaccharide, the liquid-to-solid ratio is 5:1, the decolorization time is 50 min, the decolorization temperature is 55℃, and the pH is 7.

[0016] In a preferred embodiment of the present invention, the preparation process of the defatted and deproteinized Scrophularia powder in step S3 includes:

[0017] Weigh 150 g of defatted Scrophularia powder, add 1500 mL of distilled water, add 2% enzyme solution (by volume), mix well, seal, and then enzymatically hydrolyze in a 40°C water bath for 60 min.

[0018] In the enzyme solution, the ratio of papain to cellulase is 1:5;

[0019] After enzymatic hydrolysis, the enzyme was inactivated at 100℃ for 5 min, filtered, and the filtrate was collected. 1500 mL of distilled water was added to the filter cake.

[0020] Repeat the above steps, finally combine the filtrates, concentrate under reduced pressure to 100 mL, add anhydrous ethanol to the concentrate to a volume ratio of 90%, refrigerate at 4℃ for more than 12 hours, centrifuge, discard the supernatant, dissolve the precipitate in a small amount of distilled water, and then freeze dry to obtain defatted and deproteinized Scrophularia powder.

[0021] In a preferred embodiment of the present invention, the process of preparing Scrophularia polysaccharide using defatted and deproteinized Scrophularia powder in step S5 includes:

[0022] Weigh 100g of defatted and deproteinized Scrophularia powder and add it to 1000mL of deionized water. Extract the powder in a hot water bath at 50℃ for 1 hour and then filter.

[0023] Add 1000 mL of deionized water to the filter residue and extract at 50 °C for 1 h. Combine the two extracts and centrifuge. Concentrate the supernatant obtained after centrifugation under reduced pressure to 500 mL, and add anhydrous ethanol to make the final concentration 90%.

[0024] After shaking evenly, the mixture was refrigerated at 4°C for more than 12 hours. The next day, it was centrifuged, the supernatant was discarded, and the precipitate was freeze-dried to obtain Scrophularia polysaccharide.

[0025] In a preferred embodiment of the present invention, step S6, which involves decolorizing the Scrophularia polysaccharide with macroporous resin, includes:

[0026] Step S6.1: Accurately weigh an appropriate amount of macroporous resin ADS-7, and soak it in 95% ethanol solution at 25°C for 24 hours to allow the resin to fully swell and completely remove residual organic matter inside. After soaking, wash the resin repeatedly with deionized water until there is no alcohol odor, ensuring that the ethanol is completely removed. Then, dynamically soak it in 5% HCl solution for 3 hours, followed by rinsing with deionized water until the effluent is neutral. Finally, soak it in 2% NaOH solution under the same conditions for 3 hours, and continue to wash it with deionized water until neutral. The pretreated resin is then sealed and stored at 4°C for later use.

[0027] Step S6.2: Accurately weigh 20 mg of Scrophularia polysaccharide powder that has been defatted and deproteinized, add it to ultrapure water, and prepare a polysaccharide solution with a concentration strictly controlled at 1.0 mg / mL; pre-filter the solution using a 0.45 μm microporous membrane to remove any possible suspended particles and ensure that the subsequent decolorization process is not disturbed;

[0028] Step S6.3: Weigh the resin precisely according to the mass-volume ratio of polysaccharide solution to pretreated resin of 10:1 mL / g, and add it to the above Scrophularia polysaccharide solution; place the mixture in a constant temperature shaker and shake at 150 r / min for 60 min at 45 ± 0.5°C. After shaking, use a Buchner funnel with medium-speed qualitative filter paper to filter and collect the filtrate.

[0029] In a preferred embodiment of the present invention, step S6.4 further includes taking the filtrate and determining the decolorization rate and component content of the ADS-7 resin. The calculation process for the decolorization rate of the ADS-7 resin includes:

[0030] Decolorization rate D r = (Absorbance before decolorization - Absorbance after decolorization) ÷ Absorbance before decolorization * 100%;

[0031] Wherein, the absorbance before decolorization is: the absorbance of a sample solution prepared with defatted and deproteinized Scrophularia polysaccharide powder and ultrapure water at a concentration of 1 mg / mL before decolorization detection, or the absorbance of a sample solution prepared with polysaccharide preservation solution and ultrapure water at a concentration of 1 mg / mL before decolorization detection.

[0032] The absorbance after decolorization is: the absorbance of a decolorized sample solution prepared with polysaccharide preservation solution and ultrapure water at a concentration of 1 mg / mL, or the absorbance of a decolorized sample solution prepared with polysaccharide concentrate and ultrapure water at a concentration of 1 mg / mL.

[0033] In a preferred embodiment of the present invention, the absorbance of all solutions is measured using a UV-Vis spectrophotometer at a wavelength of 238 nm; the selection of the 238 nm wavelength is determined by obtaining the maximum absorption wavelength after a full wavelength scan of the UV spectrophotometer, as follows:

[0034] A 1 mg / mL polysaccharide solution of Scrophularia ningpoensis polysaccharide was prepared. A full-wavelength scan using a UV-Vis spectrophotometer revealed a maximum absorption peak at 238 nm. Therefore, 238 nm was selected as the detection wavelength. The absorbance of the crude polysaccharide before and after decolorization was measured, and the decolorization rate D was calculated. r .

[0035] In a preferred embodiment of the present invention, the component content mentioned in step S6.4 includes the determination of polysaccharide content and polysaccharide retention rate.

[0036] In a preferred embodiment of the present invention, the polysaccharide content is determined by the following steps:

[0037] 1) Prepare a 1 mg / mL sample solution of defatted Scrophularia polysaccharide powder before decolorization, and prepare a 1 mg / mL sample solution of defatted Scrophularia polysaccharide powder after decolorization. Use ultrapure water as the solvent for both.

[0038] 2) Weigh 5 g of phenol, dissolve it in ultrapure water and make up to 100 mL to obtain a 5% phenol aqueous solution;

[0039] 3) Take 0.5 mL of the sample solution before decolorization and the sample solution after decolorization into test tubes, add 1 mL of 5% phenol solution to the test tubes, then add 5 mL of concentrated sulfuric acid, shake to mix, and then water bath for 20 min, and then cool to room temperature to obtain a control group with the sample solution before decolorization as the main component and an experimental group with the sample solution after decolorization as the main component. Measure the absorbance of both the control group and the experimental group at a wavelength of 490 nm using a UV spectrophotometer, and use the standard curve plotted with glucose as the standard as a reference to calculate the polysaccharide content of the control group and the experimental group to obtain the polysaccharide content.

[0040] The polysaccharide retention rate was determined by the following steps:

[0041] Retention rate = (Absorbance after decolorization ÷ Absorbance before decolorization R) r ) × 100%;

[0042] Wherein, the absorbance before decolorization is: the absorbance of a sample solution prepared with defatted and deproteinized Scrophularia polysaccharide powder and ultrapure water at a concentration of 1 mg / mL before decolorization detection, or the absorbance of a sample solution prepared with polysaccharide preservation solution and ultrapure water at a concentration of 1 mg / mL before decolorization detection.

[0043] The absorbance after decolorization is defined as: the absorbance of a decolorized sample solution prepared with polysaccharide preservation solution and ultrapure water at a concentration of 1 mg / mL, or the absorbance R of a decolorized sample solution prepared with polysaccharide concentrate and ultrapure water at a concentration of 1 mg / mL. r .

[0044] In a preferred embodiment of the present invention, step S6.5: online detection of polysaccharide components using gel permeation chromatography, and calculation of a comprehensive score based on decolorization rate and component content; wherein, the process of online detection of polysaccharide components using gel permeation chromatography includes:

[0045] 1) Take 10-20 mg of Scrophularia polysaccharide sample before and after decolorization into a 10 mL hydrolysis tube, add 5 mL of 2 mol / L TFA, seal the tube with N2, and hydrolyze in a 110 ℃ oven for 2 h; after cooling, open the cap, take out 1 mL, add 1 mL of methanol, and dry with N2 in a 70 ℃ water bath. Repeat the process of adding methanol and drying with N2 twice to remove TFA; add 1 mL of 0.3 mol / L NaOH solution to fully dissolve the residue to obtain the polysaccharide hydrolysate, and perform derivatization analysis after certain dilution.

[0046] 2) Take 400 μL of the mixed monosaccharide standard solution or the hydrolysate of Scrophularia polysaccharide into 5 mL stoppered test tubes, add 400 μL of PMP methanol solution, and vortex to mix; react in a 70℃ water bath for 2 h; remove and let cool to room temperature; add 400 μL of 0.3 mol / L HCl to neutralize; add 1200 μL of water, then add an equal volume of chloroform, vortex to mix, shake, let stand, discard the chloroform phase, and filter the aqueous phase through a 0.45 μm microporous membrane (for HPLC analysis).

[0047] 3) Determine changes in monosaccharide composition based on the monosaccharide composition diagram;

[0048] The change in monosaccharide composition was determined by using a PMP pre-column derivatization reaction to measure the monosaccharide composition of the sample solution before and after decolorization, and the change in monosaccharide composition was determined based on the monosaccharide composition spectra of the two solutions.

[0049] In a preferred embodiment of the present invention, the process of calculating the comprehensive score of the component content includes:

[0050] The comprehensive score Cs was calculated by weighting the polysaccharide decolorization rate and polysaccharide retention rate using the analytic hierarchy process (AHP), as shown in the following formula:

[0051] Cs = 0.6667 × D r +0.3333×R r .

[0052] Compared with the prior art, the present invention provides a method for decolorizing Scrophularia polysaccharides, which has the following beneficial effects:

[0053] 1. Highly selective decolorization, reducing polysaccharide loss;

[0054] This method compares the decolorization effects of macroporous resins with different polarities to screen out the optimal resin model that achieves the highest decolorization rate and polysaccharide retention rate for Scrophularia ningpoensis polysaccharides, thus solving the problems of poor selectivity and severe polysaccharide adsorption in traditional activated carbon decolorization. Experiments show that the optimized resin achieves a decolorization rate of over 89% and a polysaccharide retention rate of over 68%, significantly superior to conventional methods.

[0055] 2. The process parameters were optimized, improving the decolorization stability;

[0056] This method optimizes decolorization conditions (resin dosage, temperature, time, pH, etc.) through single-factor experiments and response surface methodology, determining the optimal decolorization process. This makes the decolorization process more controllable and avoids the problem of unstable decolorization effects caused by parameter fluctuations in traditional methods. The optimized process has high decolorization efficiency, good repeatability, and is suitable for industrial production.

[0057] 3. It avoids organic solvent residue, making it green and environmentally friendly;

[0058] Compared to organic solvent extraction, this method uses macroporous resin for adsorption and decolorization, eliminating the need for toxic solvents and avoiding the risk of solvent residue, thus meeting green production requirements. Furthermore, the resin can be regenerated and reused multiple times, reducing production costs.

[0059] 4. It maintains the biological activity of polysaccharides;

[0060] This method employs gentle physical adsorption decolorization, avoiding the damage to polysaccharide structures caused by strong oxidation or extreme pH conditions. The decolorized Scrophularia polysaccharides retain high bioactivity, making them suitable for pharmaceutical and health product development. This method solves the problems of low polysaccharide yield and potential safety hazards associated with existing Scrophularia polysaccharide decolorization methods. Attached Figure Description

[0061] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0062] Figure 1 This is a flowchart of the method for decolorizing Scrophularia polysaccharides according to the present invention;

[0063] Figure 2 This is a graph showing the decolorization rate of Scrophularia polysaccharides treated with different resins according to the present invention;

[0064] Figure 3 This is a graph showing the retention rates of Scrophularia polysaccharides treated with different resins according to the present invention.

[0065] Figure 4 This is a comprehensive scoring chart of Scrophularia polysaccharides treated with different resins according to the present invention;

[0066] Figure 5 Figures showing different resin treatments of Scrophularia polysaccharide solutions according to the present invention;

[0067] Figure 6 This is a graph showing the effect of the liquid-to-solid ratio on the decolorization effect of Scrophularia polysaccharide in this invention.

[0068] Figure 7 This is a graph showing the effect of decolorization time on the decolorization effect of Scrophularia polysaccharide in this invention.

[0069] Figure 8 This is a graph showing the effect of decolorization temperature on the decolorization effect of Scrophularia polysaccharide in this invention.

[0070] Figure 9 This is a curve showing the effect of pH on the decolorization effect of Scrophularia polysaccharides in this invention;

[0071] Figure 10 This is a response diagram showing the monosaccharide composition of Scrophularia polysaccharide before decolorization in this invention;

[0072] Figure 11 This is a response diagram showing the monosaccharide composition of Scrophularia polysaccharide after decolorization according to the present invention. Detailed Implementation

[0073] The principle and process of this Scrophularia polysaccharide decolorization method will be further explained in detail below with reference to the accompanying drawings and embodiments of the present invention.

[0074] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0075] A method for decolorizing Scrophularia polysaccharides, comprising the following steps:

[0076] Step S1: Prepare Scrophularia powder;

[0077] After harvesting, the above-ground parts of the Scrophularia plants are removed, the root parts are retained, the fibrous roots are removed, the mud and sand are washed away, and the plants are dried at 40℃ to constant weight. The dried Scrophularia powder is then pulverized, sieved through a 60-mesh sieve, and the sieved Scrophularia powder is collected and stored in a dry place for later use.

[0078] Step S2: Prepare defatted Scrophularia powder;

[0079] Accurately weigh 200 g of dried Scrophularia powder, add 1000 mL of petroleum ether, heat under reflux for 1 h and then filter. Dry the Scrophularia powder in a fume hood for 2 h. After drying, transfer the Scrophularia powder to a 50℃ oven and dry to constant weight to obtain defatted Scrophularia powder.

[0080] Step S3: Prepare defatted and deproteinized Scrophularia powder;

[0081] Accurately weigh 150 g of defatted Scrophularia powder, add 1500 mL of distilled water, add 2% enzyme solution (papain:cellulase = 1:5 by volume), mix well, seal, and incubate in a 40℃ water bath for 60 min for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate at 100℃ for 5 min, filter, collect the filtrate, add 1500 mL of distilled water to the filter cake, repeat the above steps, finally combine the filtrates, concentrate under reduced pressure to 100 mL, add anhydrous ethanol to the concentrate to 90% (by volume), refrigerate at 4℃ for more than 12 h, centrifuge, discard the supernatant, dissolve the precipitate in a small amount of distilled water, and then freeze dry to obtain defatted and deproteinized Scrophularia powder.

[0082] Step S4: Scan the preservation solution obtained in step S3 at 280 nm using a UV-Vis spectrophotometer to detect whether there is a protein absorption peak. If so, repeat step S3.

[0083] Step S5: Prepare Scrophularia polysaccharide using defatted and deproteinized Scrophularia powder;

[0084] Weigh 100g of defatted and deproteinized Scrophularia powder and add it to 1000mL of deionized water. Extract in a hot water bath at 50℃ for 1h, filter, add another 1000mL of deionized water to the residue, and extract at 50℃ for 1h. Combine the two extracts and centrifuge. Concentrate the supernatant obtained after centrifugation under reduced pressure to 500mL, add anhydrous ethanol to make the final concentration 90% (v / v), shake well, and refrigerate at 4℃ for more than 12h. Centrifuge the next day, discard the supernatant, and freeze-dry the obtained precipitate to obtain Scrophularia polysaccharide.

[0085] Step S6: Decolorize the Scrophularia polysaccharide using macroporous resin;

[0086] This step, to obtain higher purity Scrophularia polysaccharides, involves selecting a suitable macroporous resin for decolorization. Specifically, this includes:

[0087] Step S6.1: Select macroporous resins of different polarities. Non-polar resins include X-5 (polystyrene-type non-polar macroporous adsorption resin) and HPD826 (modified polystyrene-type non-polar macroporous adsorption resin). Weakly polar resins include AB-8 (polystyrene-divinylbenzene-type weakly polar macroporous adsorption resin). Medium polar resins include CAD-40 (medium polar macroporous adsorption resin containing amide groups). Polar resins include ADS-7 (polar macroporous adsorption resin containing sulfonic acid groups).

[0088] Step S6.2: Weigh appropriate amounts of macroporous resin and soak them in 95% ethanol for 24 hours to allow the resin to fully swell and remove organic residues. After soaking, wash away the 95% ethanol with deionized water until there is no alcohol odor (no white precipitate is detected by silver nitrate solution) to ensure complete removal of ethanol. Then, dynamically soak the resin in 5% HCl solution for 3 hours, followed by rinsing with deionized water until the effluent is neutral (pH ≈ 7.0). Finally, soak the resin in 2% NaOH solution under the same conditions for 3 hours, and continue to wash with deionized water until neutral. The pretreated resin is then sealed and stored at 4°C for later use.

[0089] Step S6.3: Accurately weigh 20 mg of Scrophularia polysaccharide powder that has been defatted and deproteinized, add it to ultrapure water, and prepare a polysaccharide solution with a concentration strictly controlled at 1.0 mg / mL; pre-filter the solution using a 0.45 μm microporous membrane to remove any possible suspended particles and ensure that the subsequent decolorization process is not disturbed;

[0090] Step S6.4: Using the static adsorption method, weigh 2g of each macroporous resin pretreated in step S6.1, and accurately weigh the resin according to the mass-volume ratio of polysaccharide solution to pretreated resin of 10:1 (mL / g). Add the resin to the Scrophularia polysaccharide solution prepared in step S6.3. Place the mixture in a constant temperature shaker and shake at 150 r / min for 60 min at 45 ± 0.5°C. After shaking, use a Buchner funnel with medium-speed qualitative filter paper to filter the solution and collect the filtrate.

[0091] Step S6.5: Use gel permeation chromatography with a differential detector to detect polysaccharide components online, and calculate a comprehensive score based on the decolorization rate and component content to screen the most suitable resin.

[0092] The most suitable resin type is ADS-7 (a polar macroporous adsorption resin with different functional groups).

[0093] Step S7: After obtaining the resin type, in order to further optimize the decolorization process of Scrophularia polysaccharide, the effects of resin addition amount, temperature, time, and pH on the decolorization effect were investigated. ADS-7 resin was used for single-factor experiments, and the experimental steps are as follows:

[0094] Step S7.1: Effect of liquid-to-solid ratio on the decolorization effect of Scrophularia polysaccharide;

[0095] The solvent was fixed as ultrapure water with pH 7, the decolorization temperature was 45℃, the decolorization time was 60 min, and the shaking speed was 150 r / min. The decolorization effect of Scrophularia polysaccharide was investigated when the liquid-to-solid ratio was 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, and 30:1, and the optimal liquid-to-solid ratio was selected.

[0096] The optimal liquid-to-solid ratio is 5:1.

[0097] Step S7.2: Effect of decolorization time on the decolorization effect of Scrophularia polysaccharide;

[0098] The solvent was fixed as ultrapure water with pH 7, the decolorization temperature was 45℃, the liquid-to-solid ratio was 5:1, and the shaking speed was 150 r / min. The decolorization effect of Scrophularia polysaccharide was investigated at decolorization times of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 min, and the optimal decolorization time was selected.

[0099] The optimal decolorization time is 50 minutes.

[0100] Step S7.3: Effect of decolorization temperature on the decolorization effect of Scrophularia polysaccharide;

[0101] The solvent was fixed as ultrapure water with pH 7, the decolorization time was 60 min, the liquid-to-solid ratio was 5:1, and the shaking speed was 150 r / min. The decolorization effect of Scrophularia polysaccharide was investigated at decolorization temperatures of 25, 35, 45, 55, and 65℃, and the optimal decolorization temperature was selected.

[0102] The optimal decolorization temperature is 55℃.

[0103] Step S7.4: Effect of pH on the decolorization effect of Scrophularia polysaccharides;

[0104] With a fixed liquid-to-solid ratio of 5:1, a decolorization temperature of 45℃, a decolorization time of 60 min, and a shaking speed of 150 r / min, the decolorization effect of Scrophularia polysaccharide was investigated at pH values ​​of 3, 5, 7, 9, and 11, and the optimal pH was selected.

[0105] The optimal pH is 7.

[0106] In one specific implementation, the calculation process for the decolorization rate of the ADS-7 resin in step S6.4 includes:

[0107] Decolorization rate D r = (Absorbance before decolorization - Absorbance after decolorization) ÷ Absorbance before decolorization * 100%;

[0108] The absorbance before decolorization is: the absorbance of a sample solution prepared with defatted and deproteinized Scrophularia polysaccharide powder and ultrapure water at a concentration of 1 mg / mL before decolorization detection, or the absorbance of a sample solution prepared with polysaccharide preservation solution and ultrapure water at a concentration of 1 mg / mL before decolorization detection.

[0109] The absorbance after decolorization is: the absorbance of the decolorized sample solution prepared with polysaccharide preservation solution and ultrapure water at a concentration of 1 mg / mL, or the absorbance of the decolorized sample solution prepared with polysaccharide concentrate and ultrapure water at a concentration of 1 mg / mL.

[0110] The absorbance of all solutions was measured using a UV-Vis spectrophotometer at a wavelength of 238 nm. The selection of the 238 nm wavelength was determined by obtaining the maximum absorption wavelength after a full-wavelength scan of the UV spectrophotometer, as follows:

[0111] Accurately weigh Scrophularia polysaccharide to prepare a 1 mg / mL polysaccharide solution. A full-wavelength scan using a UV-Vis spectrophotometer revealed a maximum absorption peak at 238 nm. Therefore, 238 nm was selected as the detection wavelength. The absorbance of the crude polysaccharide before and after decolorization was measured, and the decolorization rate D was calculated. r .

[0112] In one specific implementation, the component content mentioned in step S6.4 includes the determination of polysaccharide content and polysaccharide retention rate, wherein:

[0113] The polysaccharide content was determined by the following steps:

[0114] 1) Prepare a 1 mg / mL sample solution of defatted Scrophularia polysaccharide powder before decolorization, and prepare a 1 mg / mL sample solution of defatted Scrophularia polysaccharide powder after decolorization. Use ultrapure water as the solvent for both.

[0115] 2) Weigh 5 g of phenol, dissolve it in ultrapure water and make up to 100 mL to obtain a 5% phenol aqueous solution;

[0116] 3) Take 0.5 mL of the sample solution before and after decolorization into test tubes, add 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid to the test tubes, shake to mix, and then incubate in a water bath for 20 min. Then cool to room temperature to obtain a control group with the sample solution before decolorization as the main component and an experimental group with the sample solution after decolorization as the main component. Measure the absorbance of both the control group and the experimental group at a wavelength of 490 nm using a UV spectrophotometer. Using a standard curve plotted with glucose as the standard, calculate the polysaccharide content of the control group and the experimental group to obtain the polysaccharide content.

[0117] The polysaccharide retention rate was determined by the following steps:

[0118] Retention rate (%) = (Absorbance after decolorization ÷ Absorbance before decolorization R) r ) × 100%;

[0119] The absorbance before decolorization is: the absorbance of a sample solution prepared with defatted and deproteinized Scrophularia polysaccharide powder and ultrapure water at a concentration of 1 mg / mL before decolorization detection, or the absorbance of a sample solution prepared with polysaccharide preservation solution and ultrapure water at a concentration of 1 mg / mL before decolorization detection.

[0120] The absorbance after decolorization is defined as: the absorbance of a decolorized sample solution prepared with polysaccharide preservation solution and ultrapure water at a concentration of 1 mg / mL, or the absorbance R of a decolorized sample solution prepared with polysaccharide concentrate and ultrapure water at a concentration of 1 mg / mL. r Based on the above data, the retention rates of Scrophularia polysaccharides treated with different resins were obtained as follows: Figure 3 As shown.

[0121] In one specific implementation, step S6.5, which involves online detection of polysaccharide components using gel permeation chromatography equipped with a differential detector and calculation of a comprehensive score based on the decolorization rate and component content, includes:

[0122] The comprehensive score Cs was calculated by weighting the polysaccharide decolorization rate and polysaccharide retention rate using the analytic hierarchy process (AHP), as shown in the following formula:

[0123] Cs = 0.6667 × D r +0.3333×R r ;

[0124] The comprehensive scores of Scrophularia polysaccharides treated with different resins were obtained as follows: Figure 4 As shown.

[0125] In one specific embodiment, the polysaccharide component described in step S6.5 is determined by the following steps:

[0126] 1) For solid samples, take 10-20 mg of Scrophularia polysaccharide before and after decolorization into a 10 mL hydrolysis tube, add 5 mL of 2 mol / L TFA (for liquid samples, mix an equal volume of 4 mol / L TFA with the sample solution at a 1:1 ratio), seal the tube with N2 (10 L / min, 1 min), and hydrolyze in a 110 ℃ oven for 2 h; after cooling, open the cap, take out 1 mL, add 1 mL of methanol, and dry with N2 in a 70 ℃ water bath. Repeat this process of adding methanol and drying with N2 twice to remove TFA; add 1 mL of 0.3 mol / L NaOH solution to fully dissolve the residue, resulting in a polysaccharide hydrolysate. After dilution, perform derivatization analysis to obtain Scrophularia polysaccharide solutions treated with different resins, as shown below. Figure 5 As shown;

[0127] 2) Take 400 μL of the mixed monosaccharide standard solution or the hydrolysate of Scrophularia ningpoensis polysaccharide into 5 mL stoppered test tubes, add 400 μL of PMP methanol solution, and vortex to mix. React in a 70℃ water bath for 2 h. Remove and allow to cool to room temperature. Neutralize with 400 μL of 0.3 mol / L HCl (pH 6-7). Add 1200 μL of water, then add an equal volume of chloroform, vortex to mix, shake, and let stand. Discard the chloroform phase. Repeat this extraction process twice. Filter the aqueous phase through a 0.45 μm microporous membrane (water-based) for HPLC analysis.

[0128] 3) Determine changes in monosaccharide composition based on the monosaccharide composition graph.

[0129] The monosaccharide composition change was determined by using PMP pre-column derivatization reaction to measure the monosaccharide composition of the sample solution before and after decolorization. The monosaccharide composition change was determined based on the monosaccharide composition spectrum of the two solutions. The monosaccharide composition is shown in Table 1, and there is no significant difference in the monosaccharide composition.

[0130] The effect of liquid-to-solid ratio on the decolorization effect of Scrophularia polysaccharide was obtained through the above method, as follows: Figure 6 As shown, the effect of decolorization time on the decolorization effect of Scrophularia polysaccharide is as follows: Figure 7 As shown, the effect of decolorization temperature on the decolorization effect of Scrophularia polysaccharide is as follows: Figure 8 As shown, the effect of pH on the decolorization effect of Scrophularia polysaccharides is as follows: Figure 9 As shown, the monosaccharide composition of Scrophularia polysaccharide before decolorization is as follows: Figure 10 As shown, the monosaccharide composition of Scrophularia polysaccharide after decolorization is as follows: Figure 11 As shown in Table 1, the changes in the monosaccharide composition of Scrophularia polysaccharides are as follows;

[0131] Table 1: Changes in the monosaccharide composition of Scrophularia polysaccharides (molar percentage %)

[0132]

[0133] Based on the above results, it can be seen that:

[0134] 1. Using the above-mentioned ADS-7 at a liquid-to-solid ratio of 5:1, a decolorization time of 50 min, a decolorization temperature of 55℃, and a pH of 7, the decolorization rate can reach over 92%, and the polysaccharide retention rate exceeds 95%, providing a reliable process basis for the subsequent large-scale preparation of high-purity Scrophularia polysaccharides.

[0135] 2. This method fully measures the changes in polysaccharide content, polysaccharide retention rate, and polysaccharide structural components before and after decolorization of Scrophularia ningpoensis polysaccharide by measuring indicators such as polysaccharide content, polysaccharide retention rate, and polysaccharide components. The changes are then displayed using data to show the level of change before and after decolorization.

[0136] 3. This method involves pulverizing dried Scrophularia root, followed by defatting with petroleum ether under reflux. The petroleum ether is then evaporated, and enzymatic hydrolysis using protease breaks down large proteins into smaller peptides and amino acids. The polysaccharides are then precipitated with ethanol, separating the polysaccharides from the proteins to obtain defatted and deproteinized Scrophularia polysaccharides. By employing macroporous adsorption resins of varying polarities, the method utilizes polarity differences and molecular sieve principles to remove pigments from the Scrophularia polysaccharides, thereby improving their purity. The unique porous structure and physical adsorption principle of macroporous adsorption resins do not damage the polysaccharide structure, preserving its activity while maximizing pigment removal. Furthermore, macroporous adsorption resins are highly efficient, environmentally friendly, recyclable, and leave no chemical residues. This method effectively solves the problems of low polysaccharide yield and potential safety hazards associated with existing Scrophularia polysaccharide decolorization methods.

[0137] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for decolorizing Scrophularia polysaccharides, characterized in that, Including the following steps: Step S1: Prepare Scrophularia powder; Step S2: Prepare defatted Scrophularia powder; Weigh 200 g of dried Scrophularia powder, add 1000 mL of petroleum ether, heat under reflux for 1 h and then filter. Dry the Scrophularia powder in a fume hood for 2 h. After drying, transfer the Scrophularia powder to a 50℃ oven and dry to constant weight to obtain defatted Scrophularia powder. Step S3: Prepare defatted and deproteinized Scrophularia powder; Step S4: Scan the Scrophularia powder preservation solution obtained in step S3 at 280 nm using a UV-Vis spectrophotometer to detect whether there is a protein absorption peak. If so, repeat step S3. Step S5: Prepare Scrophularia polysaccharide using defatted and deproteinized Scrophularia powder; Step S6: Decolorize the Scrophularia polysaccharide using macroporous resin; The macroporous resin is ADS-7, and when ADS-7 is used to decolorize Scrophularia polysaccharide, the liquid-to-solid ratio is 5:1, the decolorization time is 50 min, the decolorization temperature is 55℃, and the pH is 7.

2. The method for decolorizing Scrophularia polysaccharides as described in claim 1, characterized in that, The preparation process of the defatted and deproteinized Scrophularia powder in step S3 includes: Weigh 150 g of defatted Scrophularia powder, add 1500 mL of distilled water, add 2% enzyme solution (by volume), mix well, seal, and then enzymatically hydrolyze in a 40°C water bath for 60 min. In the enzyme solution, the ratio of papain to cellulase is 1:5; After enzymatic hydrolysis, the enzyme was inactivated at 100℃ for 5 min, filtered, and the filtrate was collected. 1500 mL of distilled water was added to the filter cake. Repeat the above steps, finally combine the filtrates, concentrate under reduced pressure to 100 mL, add anhydrous ethanol to the concentrate to a volume ratio of 90%, refrigerate at 4℃ for more than 12 hours, centrifuge, discard the supernatant, dissolve the precipitate in a small amount of distilled water, and then freeze dry to obtain defatted and deproteinized Scrophularia powder.

3. The method for decolorizing Scrophularia polysaccharides as described in claim 1, characterized in that, Step S5 describes the process of preparing Scrophularia polysaccharides from defatted and deproteinized Scrophularia powder, which includes: Weigh 100g of defatted and deproteinized Scrophularia powder and add it to 1000mL of deionized water. Extract the powder in a hot water bath at 50℃ for 1 hour and then filter. Add 1000 mL of deionized water to the filter residue and extract at 50 °C for 1 h. Combine the two extracts and centrifuge. Concentrate the supernatant obtained after centrifugation under reduced pressure to 500 mL, and add anhydrous ethanol to make the final concentration 90%. After shaking evenly, the mixture was refrigerated at 4°C for more than 12 hours. The next day, it was centrifuged, the supernatant was discarded, and the precipitate was freeze-dried to obtain Scrophularia polysaccharide.

4. The method for decolorizing Scrophularia polysaccharides as described in claim 1, characterized in that, Step S6 involves decolorizing the Scrophularia polysaccharide with macroporous resin, including: Step S6.1: Accurately weigh an appropriate amount of macroporous resin ADS-7, and soak it in 95% ethanol solution at 25°C for 24 h to allow the resin to fully swell and completely remove residual organic matter inside. After soaking, repeatedly wash the resin with deionized water until there is no alcohol odor to ensure that the ethanol is completely removed. Then, dynamically soak it in 5% HCl solution for 3 h, followed by rinsing with deionized water until the effluent is neutral. Then, soak it in 2% NaOH solution under the same conditions for 3 h, and continue to wash it with deionized water until neutral. Finally, obtain the pretreated resin, seal it and store it in a 4°C environment for later use. Step S6.2: Accurately weigh 20 mg of Scrophularia polysaccharide powder that has been defatted and deproteinized, add it to ultrapure water, and prepare a polysaccharide solution with a concentration strictly controlled at 1.0 mg / mL; pre-filter the solution using a 0.45 μm microporous membrane to remove any possible suspended particles and ensure that the subsequent decolorization process is not disturbed; Step S6.3: Weigh the resin precisely according to the mass-volume ratio of polysaccharide solution to pretreated resin of 10:1 mL / g, and add it to the above Scrophularia polysaccharide solution; place the mixture in a constant temperature shaker and shake at 150 r / min for 60 min at 45 ± 0.5°C. After shaking, use a Buchner funnel with medium-speed qualitative filter paper to filter and collect the filtrate.

5. The method for decolorizing Scrophularia polysaccharides as described in claim 4, characterized in that, Step S6.4 further includes taking the filtrate and determining the decolorization rate and component content of the ADS-7 resin. The calculation process for the decolorization rate of the ADS-7 resin includes: Decolorization rate D r = (Absorbance before decolorization - Absorbance after decolorization) ÷ Absorbance before decolorization * 100%; Wherein, the absorbance before decolorization is: the absorbance of a sample solution prepared with defatted and deproteinized Scrophularia polysaccharide powder and ultrapure water at a concentration of 1 mg / mL before decolorization detection, or the absorbance of a sample solution prepared with polysaccharide preservation solution and ultrapure water at a concentration of 1 mg / mL before decolorization detection. The absorbance after decolorization is: the absorbance of a decolorized sample solution prepared with polysaccharide preservation solution and ultrapure water at a concentration of 1 mg / mL, or the absorbance of a decolorized sample solution prepared with polysaccharide concentrate and ultrapure water at a concentration of 1 mg / mL.

6. The method for decolorizing Scrophularia polysaccharides as described in claim 5, characterized in that, The absorbance of all solutions was measured using a UV-Vis spectrophotometer at a wavelength of 238 nm. The selection of the 238 nm wavelength was determined by obtaining the maximum absorption wavelength after a full-wavelength scan of the UV spectrophotometer, as follows: A 1 mg / mL polysaccharide solution of Scrophularia ningpoensis polysaccharide was prepared. A full-wavelength scan using a UV-Vis spectrophotometer revealed a maximum absorption peak at 238 nm. Therefore, 238 nm was selected as the detection wavelength. The absorbance of the crude polysaccharide before and after decolorization was measured, and the decolorization rate D was calculated. r .

7. The method for decolorizing Scrophularia polysaccharides as described in claim 5, characterized in that, The component content mentioned in step S6.4 includes the determination of polysaccharide content and polysaccharide retention rate.

8. The method for decolorizing Scrophularia polysaccharides as described in claim 7, characterized in that, The polysaccharide content was determined by the following steps: 1) Prepare a 1 mg / mL sample solution of defatted Scrophularia polysaccharide powder before decolorization, and prepare a 1 mg / mL sample solution of defatted Scrophularia polysaccharide powder after decolorization. Use ultrapure water as the solvent for both. 2) Weigh 5 g of phenol, dissolve it in ultrapure water and make up to 100 mL to obtain a 5% phenol aqueous solution; 3) Take 0.5 mL of the sample solution before decolorization and the sample solution after decolorization into test tubes, add 1 mL of 5% phenol solution to the test tubes, then add 5 mL of concentrated sulfuric acid, shake to mix, and then water bath for 20 min, and then cool to room temperature to obtain a control group with the sample solution before decolorization as the main component and an experimental group with the sample solution after decolorization as the main component. Measure the absorbance of both the control group and the experimental group at a wavelength of 490 nm using a UV spectrophotometer, and use the standard curve plotted with glucose as the standard as a reference to calculate the polysaccharide content of the control group and the experimental group to obtain the polysaccharide content. The polysaccharide retention rate was determined by the following steps: Retention rate = (Absorbance after decolorization ÷ Absorbance before decolorization R) r ) × 100%; Wherein, the absorbance before decolorization is: the absorbance of a sample solution prepared with defatted and deproteinized Scrophularia polysaccharide powder and ultrapure water at a concentration of 1 mg / mL before decolorization detection, or the absorbance of a sample solution prepared with polysaccharide preservation solution and ultrapure water at a concentration of 1 mg / mL before decolorization detection. The absorbance after decolorization is defined as: the absorbance of a decolorized sample solution prepared with polysaccharide preservation solution and ultrapure water at a concentration of 1 mg / mL, or the absorbance R of a decolorized sample solution prepared with polysaccharide concentrate and ultrapure water at a concentration of 1 mg / mL. r .

9. The method for decolorizing Scrophularia polysaccharides as described in claim 7, characterized in that, Step S6 further includes: Step S6.5: Detecting polysaccharide components online using gel permeation chromatography, and calculating a comprehensive score based on the decolorization rate and component content; wherein, the process of detecting polysaccharide components online using gel permeation chromatography includes: 1) Take 10-20 mg of Scrophularia polysaccharide sample before and after decolorization into a 10 mL hydrolysis tube, add 5 mL of 2 mol / L TFA, seal the tube with N2, and hydrolyze in a 110 ℃ oven for 2 h; after cooling, open the cap, take out 1 mL, add 1 mL of methanol, and dry with N2 in a 70 ℃ water bath. Repeat the process of adding methanol and drying with N2 twice to remove TFA; add 1 mL of 0.3 mol / L NaOH solution to fully dissolve the residue to obtain the polysaccharide hydrolysate, and perform derivatization analysis after certain dilution. 2) Take 400 μL of the mixed monosaccharide standard solution or the hydrolysate of Scrophularia polysaccharide into a 5 mL stoppered test tube, add 400 μL of PMP methanol solution, and vortex to mix; react in a 70℃ water bath for 2 h; remove and let cool to room temperature; add 400 μL of 0.3 mol / L HCl to neutralize; add 1200 μL of water, then add an equal volume of chloroform, vortex to mix, shake, let stand, discard the chloroform phase, and filter the aqueous phase through a 0.45 μm microporous membrane (for HPLC analysis). 3) Determine changes in monosaccharide composition based on the monosaccharide composition diagram; The change in monosaccharide composition was determined by using a PMP pre-column derivatization reaction to measure the monosaccharide composition of the sample solution before and after decolorization, and the change in monosaccharide composition was determined based on the monosaccharide composition spectra of the two solutions.

10. The method for decolorizing Scrophularia polysaccharides as described in claim 9, characterized in that, The process of calculating the comprehensive score based on the component content includes: The comprehensive score Cs was calculated by weighting the polysaccharide decolorization rate and polysaccharide retention rate using the analytic hierarchy process (AHP), as shown in the following formula: Cs=0.6667×D r +0.3333×R r 。

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