Method for extracting acid-soluble lignin from sample, acid-soluble lignin, pharmaceutical composition and application

Abstract

The invention belongs to the field of tobacco chemistry, and particularly relates to a method for extracting acid-soluble lignin from a sample, which comprises the following steps: pretreating the sample by using an organic solvent, carrying out solid-liquid separation, and collecting a solid-phase substance; wherein the sample is a tobacco raw material and / or a tobacco product; mixing the solid-phase substance with 1-10% by mass of an acid solution, carrying out an acidolysis reaction at 60-95 DEG C, and carrying out solid-liquid separation on the obtained acidolysis product to obtain an acidolysis liquid-phase substance and residues; adjusting the acidolysis liquid phase substance to be neutral, performing ultrafiltration treatment, and collecting components with the molecular weight of 1kDa-10kDa; and purifying the components to obtain the acid-soluble lignin. The invention also relates to the acid-soluble lignin, a pharmaceutical composition and application. The active structure of the acid-soluble lignin is basically reserved, and the acid-soluble lignin is high in purity, concentrated in molecular weight and high in antioxidant activity.

Description

Technical Field

[0001] This invention belongs to the field of tobacco chemistry, and specifically relates to a method for extracting acid-soluble lignin from a sample, acid-soluble lignin, pharmaceutical compositions, and uses. Background Technology

[0002] In the health and consumer products sector, the market demand for safe, natural, and highly effective antioxidants is growing. Chemically synthesized additives pose potential safety risks and raise consumer trust issues, making the development of natural alternatives from renewable resources a crucial trend. Lignin is a naturally abundant aromatic polymer found in plant cell walls. In traditional industry, acid-soluble lignin is considered a byproduct, and its research and utilization are severely insufficient. However, recent studies have discovered that lignin possesses a complex phenolic structure, theoretically exhibiting excellent free radical scavenging capabilities. Currently, lignin is extracted from the acid hydrolysis residue of wood or gramineous plants (also known as acid-soluble lignin), but this process suffers from the following problems: the extracted lignin's functional groups and active structures are altered, and its molecular weight distribution is uneven, leading to reduced and unstable antioxidant activity, failing to meet application requirements.

[0003] The tobacco industry generates a large amount of tobacco waste such as tobacco stems and stalks every year, which is usually discarded or disposed of at a low value, resulting in resource waste.

[0004] Therefore, there is an urgent need to develop a method for extracting acid-soluble lignin with intact structure, high antioxidant activity, and concentrated molecular weight from tobacco raw materials. This is of great significance for realizing the resource utilization of tobacco raw materials, especially tobacco waste, and for developing new natural additives. Summary of the Invention

[0005] One objective of this invention is to provide a method for extracting acid-soluble lignin from a sample. The acid-soluble lignin extracted by this method retains its active structure, has high purity, concentrated molecular weight, and high antioxidant activity. Based on this, the invention also provides the obtained acid-soluble lignin, pharmaceutical compositions, and pharmaceutical uses.

[0006] To achieve the above objectives, the first aspect of the present invention provides a method for extracting acid-soluble lignin from a sample, comprising the following steps:

[0007] The sample is pretreated with an organic solvent, followed by solid-liquid separation, and the solid phase is collected; wherein the sample is tobacco raw material and / or tobacco product;

[0008] The solid phase is mixed with an acid solution of 1%-10% by mass (e.g., 2%, 3%, 4%, 5%, 7%, 9%) and subjected to acid hydrolysis at 60°C-95°C (e.g., 65°C, 70°C, 75°C, 80°C, 85°C, 90°C). The resulting acid hydrolysis product is separated into solid and liquid phases to obtain the acid hydrolysis liquid phase and residue.

[0009] The acid hydrolysate was adjusted to neutral and then subjected to ultrafiltration to collect the components with molecular weights of 1kDa-10kDa.

[0010] The component was purified to obtain acid-soluble lignin.

[0011] The inventors of this invention have creatively discovered that the pretreatment process is beneficial for removing small molecules such as hemicellulose, pigments, and lipids, while lignin concentrates in the solid phase, creating favorable conditions for subsequent processing. The acid hydrolysis process selectively cleaves the cross-linked network structure formed by lignin with substances such as cellulose and pectin, facilitating the dissolution of acid-soluble lignin in the acid solution while preserving its active structure. Furthermore, cellulose and pectin do not decompose during acid hydrolysis and remain insoluble in the acid solution, which is beneficial for their removal. Although starch and protein decompose in the acid hydrolysis environment, most of the starch decomposition product glucose and protein decomposition product amino acids are excluded during subsequent ultrafiltration to collect components with molecular weights of 1kDa-10kDa, and undecomposed proteins are also removed. Subsequently, due to the strong water solubility and polarity of protein decomposition products amino acids and starch decomposition products glucose, the remaining protein decomposition products amino acids and starch decomposition products glucose are completely removed after ethyl acetate extraction, thereby improving the purity and antioxidant activity of acid-soluble lignin.

[0012] In any embodiment of the first aspect, the acidolysis reaction takes 0.5-4 hours, for example, 1 hour, 2 hours, 3 hours, or 4 hours.

[0013] In any embodiment of the first aspect, the acidolysis reaction is carried out under reflux and stirring conditions.

[0014] In any embodiment of the first aspect, the tobacco raw material includes tobacco waste, such as tobacco stems and stalks.

[0015] In any embodiment of the first aspect, the ratio of the solid to the acid solution is 1:50 g / mL to 1:10 g / mL, for example, 1:50 g / mL, 1:45 g / mL, 1:40 g / mL, 1:35 g / mL, 1:30 g / mL, 1:25 g / mL, 1:20 g / mL, or 1:15 g / mL.

[0016] In any embodiment of the first aspect, the acid solution includes one or more of sulfuric acid solution, hydrochloric acid solution, and acetic acid solution.

[0017] In any embodiment of the first aspect, the organic solvent is an eutectic solvent.

[0018] In this application, the eutectic solvent is a mixture of multiple components that are bonded together by intermolecular forces such as hydrogen bonds, and whose melting point is significantly lower than that of each component.

[0019] Unconstrained by theoretical limitations, in existing technologies using eutectic solvents formed from choline chloride and lactic acid, the proportion of choline chloride is typically much lower than that of lactic acid. This solvent ratio results in a highly acidic solution that promotes the dissolution of hemicellulose and catalyzes the acid-induced breakage of bonds in lignin, causing the lignin macromolecules to degrade into smaller fragments. This invention employs a eutectic solvent composed of a specific ratio of choline chloride and lactic acid. This solvent possesses relatively weak acidity and generates different hydrogen bonding environments. It tends to dissolve hydroxyl-rich, highly polar hemicellulose, as well as some small pigments and lipids, through hydrogen bonding and ionic interactions. However, it is essentially insoluble in lignin macromolecules with more complex structures, rich in aromatic rings and ether bonds. This achieves both the "retention" of lignin and the "selective dissolution" of hemicellulose and other macromolecules.

[0020] In any embodiment of the first aspect, the eutectic solvent comprises choline chloride and lactic acid, wherein the volume ratio of choline chloride to lactic acid is 2:1 to 5:1, for example 3:1 or 4:1.

[0021] In any embodiment of the first aspect, the pretreatment temperature is 20°C-35°C, for example 25°C or 30°C.

[0022] In any embodiment of the first aspect, the preprocessing time is 1-5 hours, for example 2, 3, 4, or 5 hours.

[0023] In any embodiment of the first aspect, the ratio of the sample to the organic solvent is 1:10 g / mL to 1:50 g / mL.

[0024] In any embodiment of the first aspect, the purification process comprises: extracting the component at least once (e.g., 2 or 3 times) with ethyl acetate, concentrating the resulting extract, mixing the obtained concentrate with n-hexane, collecting the precipitate, and optionally washing and drying it to obtain acid-soluble lignin.

[0025] In any embodiment of the first aspect, the volume of the ethyl acetate is equal to that of the component during each extraction.

[0026] In any embodiment of the first aspect, concentration is carried out by vacuum rotary evaporation, for example, vacuum rotary evaporation at 40-50°C.

[0027] In any embodiment of the first aspect, during the purification process, the volume of the concentrate is 1 / 20 to 1 / 5 of the volume of the extract, for example, 1 / 10.

[0028] In any embodiment of the first aspect, the volume ratio of the concentrate to the n-hexane is 1:30 to 1:10, for example, 1:20.

[0029] In any embodiment of the first aspect, during the purification process, the precipitate is allowed to stand before washing.

[0030] In any embodiment of the first aspect, the precipitate is washed with n-hexane.

[0031] In any embodiment of the first aspect, during the purification process, the drying is performed in a vacuum environment at room temperature, for example, drying at 35°C for 12 hours in a vacuum dryer.

[0032] In any embodiment of the first aspect, prior to the pretreatment, the sample is dried, crushed, and sieved (e.g., through a 30-50 mesh sieve or a 40 mesh sieve).

[0033] In any embodiment of the first aspect, after the pretreatment, the solid phase is dried and then mixed with a 1%-6% sulfuric acid solution.

[0034] In any embodiment of the first aspect, the drying is freeze drying or vacuum drying at room temperature.

[0035] In any embodiment of the first aspect, after the pretreatment, solid-liquid separation is performed by filtration.

[0036] In any embodiment of the first aspect, the acid hydrolysis product is subjected to solid-liquid separation by centrifugation. Optionally, centrifugation is performed at 7000-9000 rpm (e.g., 8000 rpm) for 5-15 minutes (e.g., 10 minutes).

[0037] In any embodiment of the first aspect, the method further includes: washing the residue with water, and incorporating the resulting washing liquid into the acid hydrolysate.

[0038] In any embodiment of the first aspect, the acid hydrolysate is adjusted to neutral using a sodium hydroxide solution (e.g., 1M).

[0039] In any embodiment of the first aspect, the ultrafiltration process is performed using a tangential flow ultrafiltration system.

[0040] A second aspect of the present invention provides an acid-soluble lignin, which is prepared by the method of the first aspect of the present invention.

[0041] In any embodiment of the second aspect, the purity of the acid-soluble lignin is ≥90% by mass, for example ≥91% by mass, ≥92% by mass, ≥93% by mass, or ≥94% by mass.

[0042] In any embodiment of the second aspect, the weight-average molecular weight of the acid-soluble lignin is 1800-4500, for example 2000, 2300, 2600, 2800, 3000, 3300, 3500, 3700, 4000, 4200, 4500.

[0043] In any embodiment of the second aspect, the number average molecular weight of the acid-soluble lignin is 1500-4000, for example 1800, 2000, 2300, 2600, 2800, 3000, 3300, 3500, 3700, 4000.

[0044] In any embodiment of the second aspect, the molecular weight polydispersity index of the acid-soluble lignin is 1.01-1.2, for example 1.01, 1.03, 1.05, 1.07, 1.1, 1.15, 1.2.

[0045] A third aspect of the present invention provides a pharmaceutical composition comprising acid-soluble lignin prepared by the method of the first aspect of the present invention or the acid-soluble lignin of the second aspect of the present invention, and pharmaceutically acceptable excipients.

[0046] In any embodiment of the third aspect, the pharmaceutical composition is an antioxidant.

[0047] The fourth aspect of the present invention provides the use of the acid-soluble lignin of the second aspect of the present invention or the pharmaceutical composition of the third aspect of the present invention in the preparation of antioxidant drugs.

[0048] The present invention achieves at least one of the following technical effects:

[0049] 1. The acid-soluble lignin extracted by the method of the present invention has a basically preserved active structure, high purity, and narrow molecular weight distribution.

[0050] 2. The acid-soluble lignin extracted by the method of this invention has high antioxidant activity, and its performance is even higher than that of the natural reference standard. It is a safe and natural antioxidant that can be widely used in food preservation, functional cosmetics and other fields, and has significant economic and social benefits.

[0051] 3. The method of the present invention uses tobacco raw materials, especially tobacco waste, as extraction raw materials, which is low in cost and sustainable in source.

[0052] 4. The process conditions of the method of the present invention are mild, avoiding the use of large amounts of strong acids, strong alkalis and toxic reagents, which is in line with the concept of green chemistry and sustainable development. Attached Figure Description

[0053] Figure 1 This is a flowchart of the extraction method of acid-soluble lignin in Examples 1-2 of the present invention.

[0054] Figure 2 This is the carbon NMR spectrum of the final product of Example 1 of the present invention.

[0055] Figure 3 This is the infrared spectrum of the final product of Example 1 of the present invention.

[0056] Figure 4 This is a high-performance liquid chromatogram of the final product of Example 1 of the present invention.

[0057] Figure 5 This is a high-performance liquid chromatogram of the final product for comparison.

[0058] Figure 6 This is a high-performance gel permeation chromatogram of the final product of Example 1 of the present invention.

[0059] Figure 7 This is a high-performance gel permeation chromatogram of the final product for comparison. Detailed Implementation

[0060] The embodiments of the present invention will now be clearly and completely described in conjunction with examples. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0061] Example 1: Extraction of acid-soluble lignin

[0062] The acid-soluble lignin extraction method in this embodiment is as follows: Figure 1 As shown.

[0063] Take 100g of dried tobacco stems, crush them and pass them through a 40-mesh sieve. Extract them at 25℃ for 2 hours with 1000 mL of eutectic solvent of choline chloride and lactic acid (volume ratio 3:1). Filter the extract, collect the filter residue, freeze-dry the filter residue to obtain pretreated tobacco stem powder.

[0064] Weigh 10g of pretreated tobacco stem powder and place it in a 500mL round-bottom flask. Add 200mL of 3% (w / w) dilute sulfuric acid solution (solid-to-liquid ratio 1:20g / mL) and mix. Reflux the mixture in an 85℃ water bath for 2 hours with stirring. After the reaction is complete, cool the reaction product and centrifuge at 8000rpm for 10 minutes. Collect the supernatant (acid hydrolysis solution). Wash the residue with a small amount of deionized water and centrifuge (under the same conditions as above). Combine the supernatants. Slowly adjust the pH of the combined supernatant to 7.0 with 1mol / L NaOH solution. Pass the neutral solution through a tangential flow ultrafiltration system to remove macromolecules such as cellulose and pectin, as well as lignin with non-target molecular weights. Collect the 1kDa~10kDa molecular weight fraction. Transfer the obtained filtrate to a separatory funnel and extract three times with an equal volume of ethyl acetate. Combine the ethyl acetate phases. Evaporate the ethyl acetate phase under reduced pressure at 40℃ to concentrate it to 1 / 20 of the original volume (approximately 10mL). Under vigorous stirring, the concentrated solution was added dropwise to 200 mL of n-hexane, and a brown precipitate immediately formed. After standing, the supernatant was discarded, and the precipitate was washed twice with a small amount of n-hexane. The precipitate was placed in a vacuum drying oven and dried at 35°C for 12 hours to obtain 0.35 g of brown powdery acid-soluble lignin.

[0065] Example 2: Extraction of acid-soluble lignin

[0066] The acid-soluble lignin extraction method in this embodiment is as follows: Figure 1 As shown.

[0067] Take 100g of tobacco waste and pretreat it in the same way as in Example 1.

[0068] Weigh 10g of pretreated tobacco powder, add 300mL of 5% hydrochloric acid solution (material-to-liquid ratio 1:30g / mL) and mix. Stir and reflux the mixture in a 70℃ water bath for 3 hours.

[0069] The remaining operations were the same as in Example 1; 0.28 g of dark brown powdery acid-soluble lignin was obtained.

[0070] Comparative analysis of traditional methods for extracting acid-soluble lignin from tobacco waste

[0071] Take 100g of tobacco waste and pretreat it in the same way as in Example 1.

[0072] Weigh 10g of pretreated tobacco powder, add 200mL of 72% by mass concentrated sulfuric acid solution and mix. Sulfonate the mixture at 120℃ for 3 hours. Dilute the reaction product to a sulfuric acid concentration of 20% by mass and stir and reflux at 100℃ for 4 hours.

[0073] The remaining operations are the same as in Example 1; the final product is dark black.

[0074] Experimental Example 1: Structural Confirmation of Acid-Soluble Lignin

[0075] (1) Nuclear magnetic resonance analysis

[0076] The final product of Example 1 was subjected to nuclear magnetic resonance analysis, and the specific method is as follows:

[0077] 50 mg of sample was dissolved in 0.5 mL of heavy water, and one-dimensional carbon NMR spectroscopy was performed at 25 °C using a nuclear magnetic resonance spectrometer. The analytical results of Example 1 are as follows: Figure 2 As shown.

[0078] Lignin is an amorphous polymer composed of three phenylpropane units linked by ether bonds and / or carbon-carbon bonds, including p-hydroxyphenyl, guaiacol, and syringyl structures. Figure 2 The presence of a methoxy group at 50 ppm indicates the presence of lignin in the sample. The signal at 104-106 ppm is characteristic of the syringyl structure, the signal near 128 ppm belongs to the p-hydroxyphenyl structure, the signal near 90 ppm is a typical β-O-4 linkage signal, and the chemical shifts in the 100-130 ppm range correspond to the CH bonds in the aromatic ring. Based on the above characterization information, the sample contains lignin.

[0079] (2) Infrared spectroscopy analysis

[0080] The final product of Example 1 was subjected to infrared spectroscopy analysis, and the specific method is as follows:

[0081] Take approximately 1 mg of the dried sample, mix it with KBr, grind it evenly in an agate mortar, and compress it into a tablet; using KBr as a blank control, perform Fourier transform infrared spectroscopy at 4000-400 cm⁻¹. -1 Spectral scanning was performed within the specified range. The analytical results of Example 1 are as follows: Figure 3 As shown.

[0082] Depend on Figure 3 It can be known that 3442 cm -1 It is the stretching vibration of phenolic hydroxyl and aliphatic hydroxyl groups, 2800 cm. -1 It is the stretching of CH on the aromatic ring and side chain, 1617 cm -1 It is the skeletal vibration of the aromatic ring, a typical characteristic of lignin, 1114 cm -1 The signal is an in-plane bending absorption signal of the aromatic ring CH, a characteristic peak of the guaiac lignin unit. The above characterization information indicates that the sample contains lignin.

[0083] Experimental Example 2: Purity Test of Acid-Soluble Lignin

[0084] (1) Determination of the purity of acid-soluble lignin by ultraviolet spectrophotometry

[0085] Lignin includes p-hydroxyphenyl, guaiacol, and syringyl structures, and has strong absorption peaks near 210 nm, 260 nm, 267 nm, and 280 nm. However, since the byproduct furfural also has strong absorption near 260 nm, 267 nm, and 280 nm, the 210 nm wavelength was chosen for the determination of acid-soluble lignin content.

[0086] Acid-soluble lignin standard (Aladdin, purity >98%) was dissolved in water and diluted to a series of standard solutions of 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, and 12.0 mg / L. The series of standard solutions were analyzed by ultraviolet spectrophotometry, and regression analysis was performed on the absorbance (y) at 210 nm and the concentration (x) to obtain the working curve with the regression equation: y = 0.0723x - 0.0003, R0. 2 = 0.9995, detection limit 0.8 mg / L (equivalent to 0.4% mass fraction of sample).

[0087] The final product of Example 1 was diluted with water to prepare a 10 mg / L test solution. Ultraviolet spectrophotometry analysis was performed on the test solution, and the absorbance at 210 nm was 0.702. Substituting this into the regression equation y = 0.0723x - 0.0003, the concentration x of acid-soluble lignin in the test solution was calculated to be 9.71 mg / L. Therefore, the purity of the acid-soluble lignin in the final product of Example 1 was calculated to be 97.1% by mass, indicating that the acid-soluble lignin extracted by the method of this invention has high purity.

[0088] Following the above method, the purity of acid-soluble lignin in the final product of Example 2 was measured to be 94.8% by mass.

[0089] (2) High performance liquid chromatography (HPLC) was used to analyze the impurities in the product.

[0090] The final products of Example 1 and the comparative example were detected according to the method in the reference (Zhao Yan, Huang Yanjun, Liu Xiucai, et al. Analysis of monosaccharide composition of water-soluble polysaccharides in tobacco leaves by pre-column derivatization HPLC [J]. China Tobacco Science, 2025, 46, 02). The detection spectrum of the final product of Example 1 is shown below. Figure 4 As shown, the detection spectrum of the final product in the comparative example is as follows. Figure 5 As shown.

[0091] Depend on Figure 4It can be seen that galacturonic acid, glucose, arabinose, xylose, galactose and mannose were not detected in the acid hydrolysate of the final product of Example 1. Since the acid hydrolysis product of pectin is galacturonic acid, the acid hydrolysis product of cellulose is glucose, and the acid hydrolysis products of hemicellulose are arabinose, xylose, galactose and mannose, the final product of Example 1 does not contain macromolecular substances such as pectin, cellulose and hemicellulose.

[0092] Depend on Figure 5 It can be seen that galacturonic acid and glucose were clearly detected in the acid hydrolysate of the final product of the comparative example, indicating that the final product of the comparative example contained a lot of pectin and cellulose impurities, and the impurity content was calculated to be as high as 18.5%, which significantly affected the purity of acid-soluble lignin.

[0093] As can be seen from the above analysis, the method of the present invention can effectively remove impurities such as pectin, cellulose, and hemicellulose, significantly improve the purity of acid-soluble lignin, and form a sharp contrast with the acid-soluble lignin obtained by the comparative method, thereby ensuring the purity and bioactivity of acid-soluble lignin.

[0094] Experimental Example 3: Determination of Molecular Weight of Acid-Soluble Lignin

[0095] The molecular weight distribution of acid-soluble lignin in the final products of Example 1 and the comparative example was determined by high-performance gel permeation chromatography (HPGPC). The specific method is as follows:

[0096] Dextran standards of different molecular weights were prepared into 5 mg / mL aqueous solutions and determined by high-performance gel permeation chromatography (HPGPC). A standard curve was plotted using the retention time (x) and the logarithm (y) of the corresponding molecular weights of the dextran standards.

[0097] The final product was prepared into a 5 mg / mL aqueous solution, filtered through a 0.22 μm filter membrane, and the sample to be tested was determined by high-performance gel permeation chromatography (HPGPC).

[0098] Operating conditions for high-performance gel permeation chromatography: Waters high-performance liquid chromatography is equipped with a TSK-gel G3000PWXL column; the mobile phase is ultrapure water, the flow rate is 0.5 mL / min; the detector is a differential detector.

[0099] The weight-average molecular weight and number-average molecular weight of acid-soluble lignin were calculated by substituting the retention time determined from the final product into the standard curve.

[0100] Figure 6The figure shows the high-performance gel permeation chromatogram of the final product of Example 1. As can be seen, the retention time of the final product of Example 1 is 19.98 min, and the chromatographic peaks are relatively sharp and single, indicating that the method of the present invention can obtain acid-soluble lignin with uniform molecular weight. Based on the chromatogram, the weight-average molecular weight of the acid-soluble lignin is calculated to be 2900 Da, the number-average molecular weight is 2685 Da, and the polydispersity index (PDI) is 1.08, indicating an extremely narrow molecular weight distribution. This is superior to acid-soluble lignin obtained by traditional wood extraction methods (PDI is usually greater than 1.5), and this method can extract acid-soluble lignin with uniform molecular weight.

[0101] Following the above method, the weight-average molecular weight of the acid-soluble lignin in the final product of Example 2 was measured to be 3140 Da, the number-average molecular weight was 3050 Da, and the polydispersity index (PDI) was 1.03.

[0102] Figure 7 The figure shows the high-performance gel permeation chromatogram of the final product of the comparative example. As can be seen from the figure, the retention times determined by the final product of the comparative example are 19.71 min and 18.60 min. The chromatographic peaks are extremely broad and non-uniform. The corresponding molecular weights calculated are 3727 Da and 9948 Da, respectively. The polydispersity index (PDI) value is greater than 1.5, indicating that the comparative method cannot obtain acid-soluble lignin with uniform molecular weight.

[0103] As can be seen from the above results, compared with the comparative method, the method of the present invention can obtain acid-soluble lignin with a significantly narrower molecular weight.

[0104] Experiment Example 4: Evaluation of the antioxidant capacity of acid-soluble lignin

[0105] (1) DPPH free radical scavenging test

[0106] The acid-soluble lignin prepared in Example 1 and the comparative example were subjected to a DPPH free radical scavenging test. The specific method was as follows:

[0107] A series of sample solutions with concentrations of 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL were prepared from the acid-soluble lignin sample (final product). 1 mL of each sample solution was placed in a brown centrifuge tube, and 4 mL of anhydrous methanol DPPH solution (DPPH mass fraction 0.0036%) was added. The mixture was vortexed and reacted at 37℃ for 35 min. The absorbance at 517 nm was measured using a UV spectrophotometer. Distilled water was used to replace the acid-soluble lignin sample as a blank control. The DPPH free radical scavenging rate of the sample was calculated using the following formula.

[0108] DPPH free radical scavenging rate = [1 - (A2 - A1) / A0] × 100%

[0109] A0 represents the absorbance value measured in the blank control;

[0110] A2 represents the absorbance value measured for the sample;

[0111] A1 represents the absorbance measured after replacing the DPPH anhydrous methanol solution with an equal volume of methanol.

[0112] The results are shown in Table 1.

[0113] Table 1 Results of DPPH free radical scavenging rate determination

[0114]

[0115] The table above shows that the IC50 of acid-soluble lignin prepared in the comparative example for scavenging DPPH free radicals... 50 Compared to 61.2 μg / mL, the acid-soluble lignin prepared by the method of this invention exhibits a strong dose-dependent DPPH free radical scavenging ability, with an IC50 concentration of 61.2 μg / mL. 50 The value was 22.4 μg / mL, which was significantly better than the comparative ratio and also better than the antioxidant activity of the previously reported Poria cocos polysaccharide (Lu Huajie, Lu Yan, Liu Yanwen, Study on antioxidant activity of Poria cocos polysaccharide [J]. Food Research and Development, 2014, 35(23):1-3.).

[0116] (2) ABTS + Free radical scavenging test

[0117] The ABTS of acid-soluble lignin prepared in Example 1, acid-soluble lignin prepared in the comparative example, and ascorbic acid (positive control) were calculated according to the method in the literature (Zhang Huihui, Li Can, Liu Huiping, Extraction, Purification and In Vitro Antioxidant and Hypoglycemic Activity Analysis of Cinnamon Polysaccharides [J]. Food Industry Technology, 2024, 45(7):15-24.). + Free radical scavenging rate, the results are shown in Table 2.

[0118] Table 2 ABTS + Free radical scavenging rate measurement results

[0119]

[0120] The table above shows that the acid-soluble lignin prepared in the comparative example scavenges ABTS. + IC of free radicals 50 The value was 290 μg / mL, compared to which the acid-soluble lignin-scavenging ABTS scavenging agent prepared by the method of this invention... + IC of free radicals 50 The value was 17.8 μg / mL, indicating that the antioxidant activity of the acid-soluble lignin prepared by the method of the present invention was significantly improved compared with the control group.

[0121] The acid-soluble lignin-based ABTS scavenger prepared by the method of this invention + IC of free radicals 50 The value is slightly higher than that of ascorbic acid scavenging ABTS. + IC of free radicals 50 The value is 21.5 μg / mL, and the ABTS of acid-soluble lignin prepared by the method of the present invention is... + The free radical scavenging ability is superior to that of cinnamon polysaccharide (Zhang Huihui, Li Can, Liu Huiping, Extraction, Purification and In Vitro Antioxidant and Hypoglycemic Activity Analysis of Cinnamon Polysaccharide [J]. Food Industry Technology, 2024, 45(7):15-24.).

[0122] The above demonstrates that the acid-soluble lignin prepared by this invention possesses excellent antioxidant capacity and exhibits diverse antioxidant mechanisms, capable of donating both hydrogen atoms (DPPH model) and electrons (ABTS). + Model).

[0123] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for extracting acid-soluble lignin from a sample, comprising the following steps: The sample is pretreated with an organic solvent, solid-liquid separation is performed, and the solid phase is collected; wherein, The sample is a tobacco raw material and / or a tobacco product; The solid phase is mixed with an acid solution with a mass fraction of 1%-10%, and an acidolysis reaction is performed at 60℃-95℃, and the obtained acidolysis product is subjected to solid-liquid separation to obtain an acidolysis liquid phase and a residue; The acidolysis liquid phase is subjected to ultrafiltration treatment after being adjusted to neutral, and a component with a molecular weight of 1 kDa-10 kDa is collected; The component is subjected to purification treatment to obtain acid-soluble lignin.

2. The method of claim 1, wherein, The acidolysis reaction is performed for 0.5-4 hours; and / or, The acidolysis reaction is performed under reflux and stirring; and / or, The ratio of the solid phase to the acid solution is 1:50 g / mL-1:10 g / mL, for example, 1:20 g / mL; and / or, The acid solution comprises one or more of a sulfuric acid solution, a hydrochloric acid solution, and an acetic acid solution.

3. The method of claim 1 or 2, wherein, The organic solvent is a deep eutectic solvent; Optionally, the deep eutectic solvent comprises choline chloride and lactic acid, and the volume ratio of the choline chloride to the lactic acid is 2:1-5:1, for example, 3:

1.

4. The method according to any one of claims 1 to 3, wherein, The temperature of the pretreatment is 20℃-35℃; and / or, The time of the pretreatment is 1-5 hours; and / or, The ratio of the sample to the organic solvent is 1:10 g / mL-1:50 g / mL.

5. The method according to any one of claims 1 to 4, wherein, The purification treatment comprises the following steps: the component is extracted with ethyl acetate at least once, the obtained extract is concentrated, and then the obtained concentrate is mixed with n-hexane, and the precipitate is collected after optional washing and drying to obtain acid-soluble lignin.

6. The method of claim 5, wherein, The purification treatment satisfies one or more of the following conditions: In each extraction, the volume of the ethyl acetate is equal to that of the component; The concentration is performed by rotary evaporation under reduced pressure; The volume of the concentrate is 1 / 20-1 / 5 of that of the extract; The volume ratio of the concentrate to the n-hexane is 1:30-1:10; Before washing, the precipitate is allowed to stand; The precipitate is washed with n-hexane; The drying is performed in a vacuum environment at room temperature.

7. The method according to any one of claims 1 to 6, characterized in that One or more of the following conditions: Before the pretreatment, the sample is dried, crushed, and sieved (for example, through a 30-50 mesh sieve); After the pretreatment, solid-liquid separation is performed by filtration; After the pretreatment, the solid phase is dried and then mixed with a sulfuric acid solution with a mass fraction of 1%-6%; Optionally, the drying is freeze-drying or vacuum drying at room temperature; The acidolysis product is subjected to solid-liquid separation by centrifugation; The method further comprises the following step: the residue is washed with water, and the obtained washing liquid is combined into the acidolysis liquid phase; The acidolysis liquid phase is adjusted to neutral with a sodium hydroxide solution; The ultrafiltration treatment is performed by a tangential flow ultrafiltration system. 8.Acid-soluble lignin obtained by the method according to any one of claims 1-7; Optionally, the acid-soluble lignin satisfies one or more of the following conditions: The purity of the acid-soluble lignin is ≥90% by mass; The weight-average molecular weight of the acid-soluble lignin is 1800-4500; The number-average molecular weight of the acid-soluble lignin is 1500-4000; The molecular weight polydispersity index of the acid-soluble lignin is 1.01-1.

2.

9. A pharmaceutical composition comprising the acid-soluble lignin prepared by the method of any one of claims 1 to 7 or the acid-soluble lignin of claim 8, and a pharmaceutically acceptable excipient. Optionally, the pharmaceutical composition is an antioxidant.

10. Use of the acid-soluble lignin of claim 8 or the pharmaceutical composition of claim 9 in the preparation of an antioxidant medicament.

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