Efficient extraction process of SOD (superoxide dismutase) in silphium perfoliatum

Abstract

The invention relates to the technical field of biology, and discloses an efficient extraction process of SOD in silphium perfoliatum, which comprises the following steps: mixing a silphium perfoliatum superoxide dismutase crude extract, polyethylene glycol and a first salt, and adjusting the pH value to form an aqueous two-phase system to enrich SOD; adding polysaccharide hydrolase and protease into the system to carry out in-situ enzymolysis so as to degrade macromolecular impurities; after the enzymolysis is finished, adding a second salt for competitive distribution, and selectively removing the added functional enzyme to a salt phase; and finally, separating and collecting a supernatant phase rich in superoxide dismutase. By integrating aqueous two-phase extraction, in-situ enzymolysis and competitive distribution into a single reaction system, a tedious intermediate separation unit in a traditional process is replaced, and by continuous and targeted removal of endogenous macromolecular impurities and exogenous functional enzymes, the purity and specific activity of a final product are remarkably improved, so that the method is suitable for industrial production. And the biological activity of SOD is effectively protected by the mild separation conditions in the whole process.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a highly efficient extraction process for SOD from *Heliotropium indicum*. Background Technology

[0002] Silphium perfoliatum is a plant resource rich in various bioactive substances, among which superoxide dismutase (SOD) is particularly abundant, making it one of the ideal plant sources for preparing natural SOD. Superoxide dismutase is a type of metalloenzyme widely found in organisms. It catalyzes the dismutation reaction of superoxide anion free radicals to generate oxygen and hydrogen peroxide, thereby eliminating harmful substances produced during metabolism and forming a core component of the biological antioxidant defense system. Due to its outstanding antioxidant function, SOD shows great application potential and commercial value in the fields of medicine, functional foods, and daily chemical products.

[0003] Silphium perfoliatum, a plant resource rich in various bioactive substances, has a high SOD content, making it an ideal source for the preparation of natural SOD. Currently, the extraction and purification of target enzyme proteins such as SOD from plant raw materials typically follows a relatively conventional technical route. This route usually begins with homogenizing and crushing plant tissues to prepare a crude extract, followed by preliminary concentration and purification using methods such as ammonium sulfate precipitation or organic solvent precipitation. However, these preliminary purification methods have poor selectivity, and a large amount of impurities and other macromolecules are also precipitated along with the extract.

[0004] To obtain high-purity SOD products, more refined separation techniques are required, with multi-step column chromatography being the most common. While this series of chromatographic processes, such as ion exchange chromatography, molecular sieve chromatography, and hydrophobic interaction chromatography, can progressively improve product purity, it also introduces several insurmountable technical bottlenecks. First, the entire process is lengthy and complex due to the inclusion of multiple separation units, with each step resulting in the loss of the target product and a significant reduction in the overall SOD recovery rate. Second, the high cost of chromatographic media and the time-consuming and labor-intensive operation directly increase the production cost of the final product, limiting its widespread application.

[0005] More importantly, the high content of plant polysaccharides, pectin, and pigments in plant extracts not only significantly increases the viscosity of the solution, severely affecting the separation efficiency and throughput of the chromatography process, but also easily leads to contamination and clogging of the chromatography media, shortening its lifespan. As an improved technology, aqueous two-phase extraction (APE) is used for protein separation due to its advantages such as mild operation, environmental friendliness, and suitability for handling complex solutions. However, conventional APE processes still have insufficient separation capabilities when processing plant extracts, making it difficult to achieve effective separation of the target product from structurally similar proteins and various coexisting macromolecular impurities in one step. It often requires subsequent purification steps, failing to address the problem of complex process flows. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a highly efficient extraction process for SOD from *Heliotropium indicum*, which solves the problems of lengthy traditional processes, cumbersome separation steps, and incomplete removal of large molecular impurities such as polysaccharides, pigments, and proteins in plant extracts, resulting in difficulty in achieving both final product purity and total recovery rate.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a highly efficient extraction process for SOD from *Silphium perfoliatum*, comprising the following steps: S1: Mix the crude extract of superoxide dismutase from *Heliotropium indicum*, polyethylene glycol, and the first salt used to form the aqueous two-phase system; S2: Adjust the pH of the system to form a stable aqueous two-phase system, in which superoxide dismutase preferentially partitions to the supernatant phase rich in polyethylene glycol; S3: Add polysaccharide hydrolase and protease to the stable aqueous two-phase system to carry out in-situ enzymatic hydrolysis to degrade macromolecular impurities in the crude extract. S4: After the in-situ enzymatic hydrolysis reaction is completed, a second salt for competitive partitioning is added to the system to selectively partition the polysaccharide hydrolase and protease to the salt phase; S5: Separate and collect the supernatant phase rich in superoxide dismutase.

[0008] By employing the above technical solution, this invention organically integrates three unit operations—aqueous two-phase extraction, in-situ enzymatic hydrolysis, and competitive partitioning—into a single reaction system, achieving highly efficient purification of SOD from *Heterophyllum sylvestris*. Its core technical principle lies in: First, an initial aqueous two-phase partitioning system is constructed. Steps S1 and S2 involve mixing polyethylene glycol (PEG) with a first salt (such as phosphate) to form two immiscible phases, upper and lower, in an aqueous environment. SOD, being a protein, preferentially partitions to the relatively hydrophobic PEG phase (supernatant) based on its surface physicochemical properties. Most water-soluble small molecule impurities, such as plant pigments and free sugars, are partitioned to the salt phase (lower phase), thus achieving preliminary and gentle solid-liquid separation and impurity removal.

[0009] Second, in-situ enzymatic hydrolysis is performed to degrade macromolecular impurities. Step S3 is one of the key innovations of this invention. Complex polysaccharide hydrolases (cellulase and pectinase) and proteases are directly introduced into the formed aqueous two-phase system. These enzymes, along with the target product SOD, are distributed in the supernatant phase and react here with the goal of degrading impurities. Cellulase and pectinase specifically hydrolyze the macromolecular plant polysaccharides in the crude extract that cause excessive viscosity, while the neutral protease hydrolyzes some of the impurity proteins. This "in-situ" enzymatic hydrolysis process significantly reduces the system viscosity, releases SOD molecules encapsulated in the polysaccharide network, improves subsequent separation efficiency, and the entire process is completed in a mild, single reactor, simplifying the process flow.

[0010] Third, functional enzymes are removed through competitive partitioning. Step S4 is another key innovation of this invention. After the enzymatic hydrolysis reaction is complete, the added tool enzymes (polysaccharide hydrolases and proteases) themselves constitute new impurities in the system. At this point, a second salt (such as ammonium sulfate or sodium citrate) is introduced into the system, utilizing the salting-out effect and the competitive mechanism of phase partitioning. The second salt has stronger phase-forming ability and hydrophilicity; its addition reconstructs the phase equilibrium, causing the relatively more hydrophilic tool enzyme protein to be "displaced" from the PEG phase and selectively partitioned into the lower phase where the salt concentration increases sharply. Meanwhile, the target product SOD, due to its unique surface properties, maintains a high affinity for the PEG phase and is retained in the supernatant. This step cleverly utilizes the principle of phase partitioning to achieve effective removal of the tool enzymes, avoiding the introduction of additional chromatographic separation steps.

[0011] In summary, through the above-described dynamically controlled tandem steps, this invention achieves the enrichment of SOD, the degradation of endogenous macromolecular impurities, and the removal of exogenous tool enzymes within a single system, ultimately obtaining a high-purity, high-activity SOD product and significantly improving the overall recovery rate.

[0012] Preferably, the amounts of each raw material used in the process are as follows: relative to 100 parts by weight of the crude extract of superoxide dismutase from *Pteris vittata*, the amount of polyethylene glycol is 20-27 parts by weight, the amount of the first salt is 15-20 parts by weight, and the amount of the second salt is 13-27 parts by weight.

[0013] By adopting the above technical solution, the ratio range is the optimized result of the best synergistic effect of each component, which can ensure that the system can quickly form a clear phase interface and provide the optimal reaction microenvironment for the initial distribution of SOD, in-situ enzymatic hydrolysis reaction and subsequent competitive distribution, thereby achieving a balance in multiple indicators such as purity, activity and recovery rate.

[0014] Preferably, prior to step S1, the superoxide dismutase crude extract is prepared by crushing, pressing, and filtering the leaves of *Heliotropium indicum* through a 200-mesh filter.

[0015] By adopting the above technical solution, this pretreatment step ensures the full release of SOD from plant cells and effectively removes insoluble plant tissue fragments through filtration with a specific mesh size, providing a homogeneous and clear starting material for the subsequent construction of the aqueous two-phase system.

[0016] Preferably, the in-situ enzymatic hydrolysis reaction in step S3 is carried out at a temperature of 40-45°C, a reaction time of 60-80 minutes, and a stirring speed of 80 rpm.

[0017] By adopting the above technical solution, the process parameters comprehensively consider the optimal activity temperature range of the enzyme system and the optimized selection of the thermal stability of the target product SOD. A gentle stirring rate ensures the stability of the phase interface and the exchange of matter between the two phases, promoting the full progress of the enzymatic hydrolysis reaction while avoiding the destruction of SOD activity by severe shear forces.

[0018] Preferably, the number-average molecular weight of the polyethylene glycol in step S1 is 3600 g / mol to 8800 g / mol, and its molecular weight distribution index is 1.05 to 1.20.

[0019] By employing the above technical solution, PEG within a specific molecular weight range is key to forming a stable aqueous two-phase system and achieving effective enrichment of SOD. A lower molecular weight distribution index indicates better homogeneity of PEG, which contributes to the formation of a clear phase interface and reproducible partitioning behavior.

[0020] Preferably, the first salt in step S1 is a phosphate composed of dipotassium hydrogen phosphate and potassium dihydrogen phosphate.

[0021] By adopting the above technical solution, the phosphate system is not only a highly efficient phase-forming salt, but also an excellent biological buffer system. In step S2, the pH value of the system can be precisely controlled by adjusting the ratio of the two, providing a stable acid-base environment for subsequent enzyme reactions.

[0022] Preferably, the polysaccharide hydrolase in step S3 comprises cellulase derived from Trichoderma reesei and pectinase derived from Aspergillus niger, and the protease is a neutral protease derived from Bacillus subtilis.

[0023] By adopting the above technical solution, the biological source of the enzyme is clarified. These enzyme preparations from specific sources have a basis for industrial production, and their enzymatic properties (such as optimal pH and optimal temperature) are highly matched with the process conditions of this invention, ensuring the efficiency and reliability of the enzymatic hydrolysis step.

[0024] Preferably, the cellulase and the pectinase are pre-mixed in a buffer solution to form an enzyme stock solution before being added to step S3, and the ratio of activity units is (1-2):1.

[0025] By adopting the above technical solution, the pre-mixing operation simplifies the production feeding steps, and the specific vitality unit ratio is an optimized ratio of polysaccharide components in the cell wall of *Heteropogon schreberi*, which can achieve synergistic and efficient degradation of cellulose and pectin.

[0026] Preferably, in step S4, the second salt is ammonium sulfate or sodium citrate, and the second salt is added in the form of an aqueous solution with a concentration of 3.6-4.0M.

[0027] By employing the above technical solution, ammonium sulfate and sodium citrate are commonly used salts in the separation of bioproducts, exhibiting a strong salting-out effect, which can effectively achieve competitive partitioning of the enzyme. Adding them in the form of high-concentration solutions can rapidly increase the salt concentration in the system, instantly disrupting the original phase equilibrium and promoting efficient partitioning.

[0028] Preferably, after step S5, the method further includes performing tangential flow ultrafiltration on the supernatant phase with a molecular weight cutoff of 10 kDa and freeze-drying under a vacuum of less than 10 Pa to obtain superoxide dismutase lyophilized powder.

[0029] By employing the above technical solution, tangential flow ultrafiltration is a gentle yet highly efficient concentration and desalination technology. The selection of a molecular weight cutoff of 10 kDa effectively removes PEG, residual salts, and small molecule impurities, while simultaneously recovering the target product, SOD. Subsequent freeze-drying removes moisture under low temperature and high vacuum, maximizing the preservation of SOD's bioactivity and yielding a stable, easily stored powdered product.

[0030] This invention provides a highly efficient extraction process for SOD from *Silphium perfoliatum*. It offers the following advantages: 1. This invention integrates multiple unit operations, such as aqueous two-phase extraction, in-situ enzymatic hydrolysis, and competitive partitioning, into a single reaction system, replacing the sequential precipitation, centrifugation, and multi-step chromatographic separation typically involved in traditional processes. This integrated design not only eliminates the dependence on a large number of intermediate separation and purification equipment (such as chromatography columns and high-pressure pump systems), but also significantly reduces product loss by avoiding material transfer between different operating units.

[0031] 2. This invention achieves a significant improvement in product purity and specific activity through a dual, tandem impurity removal strategy. First, through an in-situ enzymatic hydrolysis step, large-molecule endogenous impurities (such as polysaccharides and extraneous proteins) in the crude plant extract that affect separation efficiency and product purity are specifically degraded into smaller molecules that are easily partitioned into the salt phase. Subsequently, through a competitive partitioning step, functional enzymes (exogenous impurities) introduced as processing aids are selectively removed from the product phase. This continuous and targeted removal mechanism of endogenous and exogenous impurities ensures that the final SOD product has higher purity.

[0032] 3. This invention utilizes a mild aqueous system and neutral pH conditions throughout the separation process, avoiding harsh treatments that could cause protein denaturation, such as extreme pH, organic solvents, or high temperatures. These mild process conditions effectively protect the fine three-dimensional structure of SOD, thereby maximizing its inherent biological activity. The retention of high activity, combined with the reduced physical losses resulting from the integrated process, ensures an excellent overall recovery rate of the target product SOD, thereby increasing the total amount of effective active product obtained from a unit weight of raw material. Attached Figure Description

[0033] Figure 1 This is a process flow diagram of the present invention; Figure 2 This is a comparative curve showing the change of apparent viscosity of the reaction system of the present invention over time. Figure 3 This is a bar chart comparing the specific activity of SOD in the final product of this invention. Figure 4 This is a bar chart comparing the total SOD recovery rate of the present invention; Figure 5 This is a comparative column chart showing the SEC-HPLC purity of the final product of this invention. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0036] Polyethylene glycol (PEG), CAS number 25322-68-3, is a linear polyether homopolymer with the chemical formula H-(O-CH2-CH2)n-OH. The PEG used in the examples is a commercially available product with a number-average molecular weight ranging from 3600 g / mol to 8800 g / mol, and a molecular weight distribution index between 1.05 and 1.20. It is a white, waxy solid at room temperature.

[0037] Dipotassium hydrogen phosphate, CAS number 7758-11-4, molecular formula K₂HPO₄. Potassium dihydrogen phosphate, CAS number 7778-77-0, molecular formula KH₂PO₄.

[0038] Ammonium sulfate, CAS number 7783-20-2, molecular formula (NH4)2SO4.

[0039] Trisodium citrate, in its actual form as trisodium citrate dihydrate, has the CAS number 6132-04-3 and the molecular formula C6H5Na3O7·2H2O.

[0040] Cellulase, CAS No. 9012-54-8, is a complex enzyme preparation produced by fermentation of Trichoderma reesei. Its main active ingredient is cellulose-1,4-β-glucanase, and its enzyme activity, measured in filter paper units, is 10,000 U / g to 50,000 U / g. It is a commercially available powder product.

[0041] Pectinase, CAS No. 9032-75-1, is a complex enzyme preparation derived from Aspergillus niger fermentation. Its main active ingredient is polygalacturonase. Enzyme activity, expressed in International Units (SI), is defined as the amount of enzyme required to catalyze the production of 1 μmol of galacturonic acid per minute from a substrate under specific conditions. It is a commercially available powder product ranging from 30,000 U / g to 100,000 U / g.

[0042] Neutral protease, CAS No. 9014-01-1, is an endopeptide produced by fermentation of Bacillus subtilis. Its optimal pH range is 6.0 to 8.0. The enzyme activity is determined by the Folin-Ciocalteu method and is defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute under specific conditions. It is a commercially available powder product with an activity of 50,000 U / g to 200,000 U / g.

[0043] Preparation Example 1: Preparation of Complex Polysaccharide Hydrolytic Enzyme Solution Preparation Example 1-1: Weigh commercially available powders of cellulase (10,000 U / g activity) and pectinase (30,000 U / g activity) and mix them at a 1:1 ratio. Dissolve the mixed enzyme powder in 20 mM phosphate buffer (pH 7.0) to prepare an enzyme stock solution with a total activity of 1,000 U / mL, and store it at 4°C protected from light for later use.

[0044] Preparation Examples 1-2: Weigh commercially available powders of cellulase (10,000 U / g activity) and pectinase (30,000 U / g activity) and mix them at a 2:1 ratio. Dissolve the mixed enzyme powder in 20 mM phosphate buffer (pH 7.0) to prepare an enzyme stock solution with a total activity of 1,000 U / mL, and store it at 4°C protected from light for later use.

[0045] Preparation Example 2: Preparation of "Displacement Buffer" Preparation Example 2-1: Weigh 132.14 g of analytical grade ammonium sulfate, dissolve it in deionized water, and bring the volume to 250 mL. After thorough mixing, a 4.0 M ammonium sulfate displacement buffer solution is obtained and stored in a sealed container at 4 °C for later use.

[0046] Preparation Example 2-2: Weigh 264.65 g of trisodium citrate dihydrate, dissolve it in deionized water, and bring the volume to 250 mL. After thorough mixing, a 3.6 M sodium citrate displacement buffer solution is obtained and stored in a sealed container at 4 °C for later use.

[0047] Example 1: This embodiment provides a method for efficient purification of SOD from *Heterophyllum ferox* based on dynamic phase equilibrium regulation, including the following steps: Weigh 1 kg of cleaned fresh leaves of *Silphium perfoliatum* and mix with 1.5 L of deionized water. Process the mixture in a high-speed tissue homogenizer at 10,000 rpm for 3 minutes to obtain a green slurry. Press the slurry using a screw press, collect the filtrate, and filter it through a 200-mesh stainless steel filter to obtain crude SOD extract.

[0048] In a 5L reaction vessel equipped with a temperature-controlled jacket, add 3kg of the above-mentioned crude SOD extract, 600g of polyethylene glycol (number-average molecular weight 6000g / mol), and 450g of phosphate (a mixture of K2HPO4 and KH2PO4), stir until dissolved, and adjust the pH of the system to 7.0. At this point, the mass percentages of each component in the system are as follows: crude SOD extract 74.1%, polyethylene glycol 14.8%, and phosphate 11.1%.

[0049] After the system forms a stable two-phase system, 15 mL of the complex polysaccharide hydrolase solution prepared in Preparation Example 1-1 and 10 mL of commercially available neutral protease solution (activity unit 100,000 U / g) are added.

[0050] The temperature of the reaction vessel was controlled at 40℃, and the mixture was stirred at a low speed of 80 rpm to carry out the in-situ enzymatic hydrolysis reaction for 80 minutes.

[0051] After the reaction was complete, 750 mL of the 4.0 M ammonium sulfate displacement buffer prepared in Preparation Example 2-1 was rapidly pumped into the reaction vessel to bring the final ammonium sulfate saturation in the system to approximately 60%. The reaction was then stirred at 40 °C and 80 rpm for 20 minutes.

[0052] The mixture was subjected to phase separation in a continuous flow tube centrifuge at a centrifugal force of 4000xg, and the upper clear phase rich in PEG was collected.

[0053] The collected supernatant was pumped into a tangential flow ultrafiltration system with a molecular weight cutoff of 10 kDa and concentrated to 1 / 8 of the initial volume under an operating pressure of 0.2 MPa. Then, constant-volume dialysis was performed using 20 mM phosphate buffer (pH 7.2) at a volume six times that of the concentrate.

[0054] The dialyzed SOD solution was pre-frozen at -80°C for 4 hours, and then freeze-dried under a vacuum of less than 10 Pa to obtain freeze-dried SOD powder.

[0055] Example 2: This embodiment provides a method for efficient purification of SOD from *Heterophyllum ferox* based on dynamic phase equilibrium regulation, including the following steps: Weigh 1 kg of cleaned fresh leaves of *Silphium perfoliatum* and mix with 1.5 L of deionized water. Process the mixture in a high-speed tissue homogenizer at 10,000 rpm for 3 minutes to obtain a green slurry. Press the slurry using a screw press, collect the filtrate, and filter it through a 200-mesh stainless steel filter to obtain crude SOD extract.

[0056] In a 5L reaction vessel equipped with a temperature-controlled jacket, add 3kg of the above-mentioned crude SOD extract, 800g of polyethylene glycol (number-average molecular weight 4000g / mol), and 600g of phosphate (a mixture of K2HPO4 and KH2PO4), and stir until dissolved. Adjust the pH of the system to 6.5. At this point, the mass percentages of each component in the system are as follows: crude SOD extract 68.2%, polyethylene glycol 18.2%, and phosphate 13.6%.

[0057] After the system forms a stable two-phase system, 15 mL of the complex polysaccharide hydrolase solution prepared in Preparation Example 1-1 and 10 mL of commercially available neutral protease solution (activity unit 100,000 U / g) are added.

[0058] The temperature of the reaction vessel was controlled at 40℃, and the mixture was stirred at a low speed of 80 rpm to carry out the in-situ enzymatic hydrolysis reaction for 80 minutes.

[0059] After the reaction was complete, 750 mL of the 4.0 M ammonium sulfate displacement buffer prepared in Preparation Example 2-1 was rapidly pumped into the reaction vessel to bring the final ammonium sulfate saturation in the system to approximately 60%. The reaction was then stirred at 40 °C and 80 rpm for 20 minutes.

[0060] The mixture was subjected to phase separation in a continuous flow tube centrifuge at a centrifugal force of 4000xg, and the upper clear phase rich in PEG was collected.

[0061] The collected supernatant was pumped into a tangential flow ultrafiltration system with a molecular weight cutoff of 10 kDa and concentrated to 1 / 8 of the initial volume under an operating pressure of 0.2 MPa. Then, constant-volume dialysis was performed using 20 mM phosphate buffer (pH 7.2) at a volume six times that of the concentrate.

[0062] The dialyzed SOD solution was pre-frozen at -80°C for 4 hours, and then freeze-dried under a vacuum of less than 10 Pa to obtain freeze-dried SOD powder.

[0063] Example 3: This embodiment provides a method for efficient purification of SOD from *Heterophyllum ferox* based on dynamic phase equilibrium regulation, including the following steps: Weigh 1 kg of cleaned fresh leaves of *Silphium perfoliatum* and mix with 1.5 L of deionized water. Process the mixture in a high-speed tissue homogenizer at 10,000 rpm for 3 minutes to obtain a green slurry. Press the slurry using a screw press, collect the filtrate, and filter it through a 200-mesh stainless steel filter to obtain crude SOD extract.

[0064] In a 5L reaction vessel equipped with a temperature-controlled jacket, add 3kg of the above-mentioned crude SOD extract, 600g of polyethylene glycol (number-average molecular weight 6000g / mol), and 450g of phosphate (a mixture of K2HPO4 and KH2PO4), stir until dissolved, and adjust the pH of the system to 7.0. At this point, the mass percentages of each component in the system are as follows: crude SOD extract 74.1%, polyethylene glycol 14.8%, and phosphate 11.1%.

[0065] After the system forms a stable two-phase system, 15 mL of the complex polysaccharide hydrolase solution prepared in Preparation Examples 1-2 and 10 mL of commercially available neutral protease solution (activity unit 100,000 U / g) are added.

[0066] The temperature of the reaction vessel was controlled at 45℃, and the mixture was stirred at a low speed of 80 rpm for 60 minutes to carry out the in-situ enzymatic hydrolysis reaction.

[0067] After the reaction was complete, 750 mL of the 4.0 M ammonium sulfate displacement buffer prepared in Preparation Example 2-1 was rapidly pumped into the reaction vessel to bring the final ammonium sulfate saturation in the system to approximately 60%. The reaction was then stirred at 45 °C and 80 rpm for 20 minutes.

[0068] The mixture was subjected to phase separation in a continuous flow tube centrifuge at a centrifugal force of 4000xg, and the upper clear phase rich in PEG was collected.

[0069] The collected supernatant was pumped into a tangential flow ultrafiltration system with a molecular weight cutoff of 10 kDa and concentrated to 1 / 8 of the initial volume under an operating pressure of 0.2 MPa. Then, constant-volume dialysis was performed using 20 mM phosphate buffer (pH 7.2) at a volume six times that of the concentrate.

[0070] The dialyzed SOD solution was pre-frozen at -80°C for 4 hours, and then freeze-dried under a vacuum of less than 10 Pa to obtain freeze-dried SOD powder.

[0071] Example 4: This embodiment provides a method for efficient purification of SOD from *Heterophyllum ferox* based on dynamic phase equilibrium regulation, including the following steps: Weigh 1 kg of cleaned fresh leaves of *Silphium perfoliatum* and mix with 1.5 L of deionized water. Process the mixture in a high-speed tissue homogenizer at 10,000 rpm for 3 minutes to obtain a green slurry. Press the slurry using a screw press, collect the filtrate, and filter it through a 200-mesh stainless steel filter to obtain crude SOD extract.

[0072] In a 5L reaction vessel equipped with a temperature-controlled jacket, add 3kg of the above-mentioned crude SOD extract, 600g of polyethylene glycol (number-average molecular weight 6000g / mol), and 450g of phosphate (a mixture of K2HPO4 and KH2PO4), stir until dissolved, and adjust the pH of the system to 7.0. At this point, the mass percentages of each component in the system are as follows: crude SOD extract 74.1%, polyethylene glycol 14.8%, and phosphate 11.1%.

[0073] After the system forms a stable two-phase system, 15 mL of the complex polysaccharide hydrolase solution prepared in Preparation Example 1-1 and 10 mL of commercially available neutral protease solution (activity unit 100,000 U / g) are added.

[0074] The temperature of the reaction vessel was controlled at 40℃, and the mixture was stirred at a low speed of 80 rpm to carry out the in-situ enzymatic hydrolysis reaction for 80 minutes.

[0075] After the reaction was complete, 750 mL of the 3.6 M sodium citrate displacement buffer prepared in Preparation Example 2-2 was rapidly pumped into the reaction vessel. The reaction was then continued with stirring at 40 °C and 80 rpm for 20 minutes.

[0076] The mixture was subjected to phase separation in a continuous flow tube centrifuge at a centrifugal force of 4000xg, and the upper clear phase rich in PEG was collected.

[0077] The collected supernatant was pumped into a tangential flow ultrafiltration system with a molecular weight cutoff of 10 kDa and concentrated to 1 / 8 of the initial volume under an operating pressure of 0.2 MPa. Then, constant-volume dialysis was performed using 20 mM phosphate buffer (pH 7.2) at a volume six times that of the concentrate.

[0078] The dialyzed SOD solution was pre-frozen at -80°C for 4 hours, and then freeze-dried under a vacuum of less than 10 Pa to obtain freeze-dried SOD powder.

[0079] Comparative Example 1: Compared to Example 1, the difference lies in that an aqueous two-phase system is not used for extraction, nor are in-situ enzymatic hydrolysis and competitive partitioning steps performed. Instead, a conventional acid-thermal precipitation method is used to purify SOD. Specifically, the crude SOD extract, identical to that in Example 1, is heated to 80°C, its pH is adjusted to 4.5 with lactic acid, kept at this temperature for 30 minutes, cooled to room temperature, and the precipitate is removed by centrifugation. The supernatant is then collected. Subsequent processing of this supernatant, including ultrafiltration and freeze-drying, is the same as in Example 1.

[0080] Comparative Example 2: Compared to Example 1, the difference lies in that after constructing the aqueous two-phase system, no complex polysaccharide hydrolase or neutral protease was added, and subsequent phase equilibration, phase separation, and purification steps were performed directly. All other steps and parameters were the same as in Example 1.

[0081] Comparative Example 3: Compared to Example 1, the difference lies in that the enzymatic hydrolysis and aqueous two-phase extraction are performed in separate steps. Specifically, the crude SOD extract is first subjected to enzymatic hydrolysis for 80 minutes at 40°C and pH 7.0 with an enzyme preparation of the same volume as in Example 1. After the enzymatic hydrolysis is completed, polyethylene glycol and phosphate are added to the hydrolysate to construct an aqueous two-phase system, and then subsequent steps are performed. All other steps and parameters are the same as in Example 1.

[0082] Comparative Example 4: Compared to Example 1, the difference lies in omitting the step of adding replacement buffer. Specifically, after the in-situ enzymatic digestion reaction is completed, ammonium sulfate-based replacement buffer is not added, and the reaction mixture is directly centrifuged for phase separation. All other steps and parameters are the same as in Example 1.

[0083] Test Example 1: Validation of the effectiveness of the in-situ enzymatic hydrolysis process Experimental description: This test case aims to characterize the impact of in-situ enzymatic hydrolysis on the macroscopic physical properties of an aqueous two-phase system by monitoring changes in apparent viscosity. The test subjects were the reaction systems of Example 1 and Comparative Example 2. In aqueous two-phase extraction systems, the macromolecular polysaccharides, pectin, and some proteins introduced by the crude plant extract are the main factors leading to excessively high system viscosity and even the formation of a stable emulsion layer. This severely hinders phase separation efficiency and the mass transfer process of the target product. Therefore, apparent viscosity is a direct and crucial macroscopic indicator for evaluating the effectiveness of in-situ enzymatic hydrolysis.

[0084] Experimental steps: Aqueous two-phase systems were prepared according to the procedures of Example 1 and Comparative Example 2, respectively, with enzyme preparation added to the system of Example 1 and not added to the system of Comparative Example 2. At the start of the reaction (denoted as 0 min), approximately 50 mL samples were immediately taken from each reaction vessel. Using a rotational viscometer at a constant temperature of 40°C, a suitable SC4-27 rotor was selected, and the rotation speed was set to 60 rpm to measure the apparent viscosity (unit: mPa·s). Subsequently, the sampling and measurement operations were repeated at 15 min, 30 min, 45 min, 60 min, and 80 min of the reaction, and the apparent viscosity data measured at all time points were recorded.

[0085] Experimental data: Table 1. Apparent viscosity changes of the reaction systems in Example 1 and Comparative Example 2.

[0086] in conclusion: The test results show that the apparent viscosity of the reaction system in Example 1 decreased significantly with increasing reaction time. In contrast, the viscosity of the Comparative Example 2 system, which did not contain any enzyme, remained essentially stable throughout the process, without any significant change.

[0087] The decrease in system viscosity is attributed to the in-situ hydrolysis of macromolecular polysaccharides, proteins, and other impurities in the crude SOD extract by the compound enzyme preparation. This degradation of macromolecules disrupts their tendency to form a dense emulsion layer at the aqueous two-phase interface or a network structure within the phase, thereby reducing the overall flow resistance of the system. This result confirms the effectiveness of the in-situ enzymatic hydrolysis step in the method of this invention, creating crucial physical conditions for subsequent efficient phase separation and the release of the target product SOD from impurity binding and its migration to the target phase. These data collectively demonstrate that in-situ enzymatic hydrolysis, as the initial key step in the method of this invention, solves the technical challenge of handling high-viscosity plant crude extracts in aqueous two-phase systems and is a prerequisite for achieving subsequent high-purity, high-recovery purification.

[0088] Test Example 2: Verification of the Competitive Allocation Effect Experimental description: This test case aims to quantify the distribution of the target product, superoxide dismutase (SOD), and total protein (representing most impurity proteins, including the added enzyme) in the polyethylene glycol upper and salt phases after the introduction of a replacement buffer and the attainment of a new phase equilibrium. The test subjects were the upper and lower phase samples separated after the competitive partitioning step in Example 1. Before the introduction of the high-concentration salt, both the target product SOD and the enzyme added as a processing aid tended to accumulate in the upper phase of the aqueous two-phase system. The core objective of this test step is to verify whether the introduction of a high-concentration replacement salt can disrupt the original partitioning equilibrium, achieving selective retention of SOD and selective removal of impurity proteins (especially the enzyme).

[0089] Experimental steps: The upper and lower phase samples obtained from Example 1 after ammonium sulfate replacement and centrifugation were subjected to the following determinations: First, accurately measure the volumes of the upper and lower phases. Use a pipette to precisely aspirate samples from both phases and perform serial dilutions with 20 mM phosphate buffer (pH 7.2) for subsequent activity and concentration determinations.

[0090] Secondly, the SOD activity was determined using the pyrogallol autoxidation method. The diluted sample was added to a reaction buffer containing pyrogallol, and the rate of change in absorbance over time was monitored using a spectrophotometer at 325 nm. One unit of SOD activity (U) was defined as the amount of sample required to inhibit 50% of the pyrogallol autoxidation rate. The SOD activity concentration (U / mL) in the original phase was calculated based on the dilution factor.

[0091] Next, the total protein concentration was determined using the BCA method. The diluted sample was mixed with the BCA working solution and incubated at 60°C for 30 minutes. After cooling to room temperature, the absorbance was measured at 562 nm. A standard curve was prepared using bovine serum albumin as a standard, and the total protein concentration (mg / mL) in the original phase was calculated based on the standard curve. The total protein concentration measured here includes the target product SOD, endogenous plant proteins, and the protein from the enzyme preparation added during the enzymatic digestion step.

[0092] Finally, based on the measured concentration and volume data, the partition coefficient (K) and the distribution rate in the upper phase were calculated. The partition coefficient K is defined as the ratio of the concentration of the substance in the upper phase to the concentration in the lower phase (K = Cupper phase / Clower phase). The distribution rate in the upper phase is defined as the percentage of the total amount of the substance in the upper phase to the sum of the total amounts in both phases. All activity and concentration measurements were performed three times, and the average value was used for the final calculation to ensure the accuracy of the data.

[0093] Experimental data: Table 2. Phase distribution data of SOD and total protein after competitive partitioning in Example 1.

[0094] in conclusion: Table 2 shows the experimental data, which quantitatively reveal the effectiveness of the competitive partitioning step. The partition coefficient K of SOD is as high as 17.06, indicating that SOD exhibits a very strong affinity for the PEG upper phase under high ionic strength conditions. This phenomenon stems from the fact that high concentrations of ammonium sulfate significantly enhance the hydrophobic effect of the system. For SOD molecules with relatively strong surface hydrophobicity, their migration from the highly polar saline phase to the more hydrophobic PEG phase gains a more favorable driving force. Over 90% of the total SOD activity is enriched in the upper phase, demonstrating the effectiveness of this step in the efficient recovery of the target product.

[0095] Conversely, the partition coefficient K value for total protein was only 0.17, indicating that the vast majority of protein impurities were partitioned into the lower salt phase. These included cellulase, pectinase, and protease added during the enzymatic hydrolysis step, as well as some hydrophilic impurities from the crude extract of *Silphium perfoliatum*. The high-salt environment produced a strong salting-out effect on these proteins, significantly reducing their solubility and thus repelling them from the PEG phase, concentrating them in the lower phase. Less than 9% of the total protein remained in the upper phase, demonstrating that this step effectively removed a large amount of protein impurities. This significant difference in the partitioning behavior of SOD and total protein directly led to a substantial increase in the specific activity of SOD in the upper phase.

[0096] Comprehensive analysis shows that this competitive partitioning step achieves the two core purification goals—targeted enrichment of SOD and separation and removal of contaminating proteins—in a single operation. Data confirms that by altering the ionic environment of the system, the partitioning behavior of different proteins in the two phases can be selectively regulated, thereby effectively separating the target product from the enzyme introduced as a processing aid and other endogenous contaminating proteins. These results provide direct experimental evidence that the method of this invention simplifies the purification process and avoids introducing additional enzyme removal steps. This single-step operation integrates the separation effect that traditional purification processes require additional steps such as hydrophobic chromatography, demonstrating significant advantages in process integration and enhancement.

[0097] Test Example 3: Determination of SOD specific activity in the final product Experimental description: This test case aims to evaluate the impact of different purification processes on product purity by measuring and comparing the specific activity of the superoxide dismutase (SOD) lyophilized powders obtained in each example and the comparative example. Specific activity, which is the number of enzyme activity units per milligram of protein, is a core indicator for measuring enzyme purity. Therefore, specific activity not only reflects the purity of the target protein relative to other impurities, but also comprehensively reflects the degree of removal of non-protein impurities in the final product.

[0098] Experimental steps: Accurately weigh approximately 10.0 mg of the SOD lyophilized powder obtained in each example and comparative example, dissolve it in 20 mM, pH 7.2 phosphate buffer, and bring the volume to 10.0 mL to obtain the sample stock solution.

[0099] The sample stock solution was serially diluted, and its SOD activity was determined using the pyrogallol autoxidation method. The sample dilution factor required to inhibit the pyrogallol autoxidation rate by 50% was recorded, and the SOD activity of the original lyophilized powder was calculated accordingly, in U / mg lyophilized powder.

[0100] Simultaneously, the total protein concentration in the sample stock solution was determined using the BCA method. A standard curve was constructed using bovine serum albumin as a standard, and the protein content in the original lyophilized powder was calculated based on the sample absorbance value, expressed in mg protein / mg lyophilized powder. This protein content value (mg protein / mg lyophilized powder) directly reflects the proportion of other components (such as residual salts, buffers, etc.) present in the final powder besides protein, and is an important indicator of product specifications.

[0101] Finally, the specific activity of SOD for each sample was calculated using the following formula: Specific activity (U / mg protein) = SOD activity (U / mg lyophilized powder) / Protein content (mg protein / mg lyophilized powder) Experimental data Table 3. Comparative activity of the final SOD product in each embodiment and comparative example.

[0102] in conclusion: Test results show that the specific activity of the SOD products obtained in all examples (1-4) is at a high level and is significantly higher than that of the products in all comparative examples (1-4).

[0103] The specific activity data of Examples 1 to 4 show that the method of the present invention can stably prepare high-purity SOD products within different ranges of parameters such as polymer molecular weight, system pH, enzymatic hydrolysis ratio and substitution salt type, proving the universality and robustness of the method.

[0104] Compared with Comparative Example 1, the method of the present invention operates under mild conditions, avoiding the damage and irreversible degradation of the three-dimensional structure of SOD caused by acid heat treatment, thereby obtaining a product with higher activity.

[0105] The results, compared with those of Comparative Example 2, demonstrate the necessity of the in-situ enzymatic hydrolysis step. Without enzymatic hydrolysis, a large number of macromolecular impurities enter the upper phase along with SOD, resulting in a final product with low protein content, high impurity content, and extremely low specific activity.

[0106] Compared with the results of Comparative Example 3, it is shown that the method of the present invention performs enzymatic hydrolysis and extraction simultaneously, and its synergistic effect is better than that of conventional combined processes that separate the two steps, resulting in a product with higher purity.

[0107] Compared with the results of Comparative Example 4, one of the core innovations of the present invention is most evident. The lack of a competitive partitioning step, despite enzymatic hydrolysis and extraction, resulted in the ineffective separation of the complex enzymes and some impurities introduced as processing aids into the lower phase. This ultimately led to a product with a significantly lower specific activity than the examples. This directly demonstrates that the competitive partitioning step is a crucial and indispensable step in removing enzymes and proteins introduced as processing aids, thereby achieving high purity in the final product. Through the synergistic effect of enzymatic hydrolysis and extraction, along with subsequent competitive partitioning, highly efficient purification of SOD was achieved. The specific activity data of the obtained product fully supports the advanced nature of this technical solution.

[0108] Test Example 4: Determination of Total SOD Recovery Experimental description: This test case aims to evaluate the impact of different purification processes on the recovery efficiency of the target product by calculating the total activity recovery rate of superoxide dismutase (SOD) from starting materials to the final product. Total recovery rate is a key parameter for evaluating the economics and efficiency of the entire process.

[0109] Experimental steps: Determination of initial total SOD activity: Before the experiments in each example and comparative example, the total volume of the pretreated, homogenized, and fully combined crude SOD extract from the same batch was accurately measured. The SOD activity concentration (U / mL) of the crude extract was determined using the pyrogallol auto-oxidation method. The initial total activity was obtained by multiplying the volume of the crude extract by the activity concentration.

[0110] Determination of the total activity of the final product: Accurately weigh the total mass (mg) of all SOD lyophilized powders obtained in each example and comparative example. The activity (U / mg) of each lyophilized powder sample was determined using the pyrogallol autoxidation method. The total activity of the final product was obtained by multiplying the total mass of the lyophilized powder by the measured activity.

[0111] The total recovery rate of each process is calculated according to the following formula: Total recovery rate (%) = (total vitality of final product / total initial vitality) × 100%. To ensure the reliability of the results, all vitality measurements are performed in triplicate and the average value is used for calculation.

[0112] Experimental data: Table 4. Total SOD recovery rate of each example and comparative example

[0113] Based on the data in Table 4, a bar chart can be drawn, with each embodiment and comparative example as the horizontal axis category and the total recovery rate as the vertical axis, to visually compare the SOD activity recovery capabilities of different process routes.

[0114] in conclusion: Data show that the total SOD recovery rate of Examples 1 to 4 remained above 80%, while the recovery rates of all comparative examples were significantly lower than this level.

[0115] The high recovery rates demonstrated in Examples 1-4 are directly attributed to the inherent mechanism of the method of this invention. First, the in-situ enzymatic hydrolysis under mild conditions effectively degrades the macromolecular matrix binding SOD, achieving full release of the target product, which is the basis for the high recovery rate. Second, the subsequent competitive partitioning step, by introducing a high concentration of salt, not only drives impurities towards the salt phase but also provides a strong thermodynamic driving force for the efficient partitioning of SOD into the PEG phase, ensuring that the released SOD is captured to the maximum extent in the target phase, greatly reducing losses during phase separation. The entire process avoids extreme pH or high temperature conditions, protecting the biological activity of SOD.

[0116] The recovery rate of Comparative Example 1 (acid-heat precipitation method) was extremely low, and its main loss of activity was due to irreversible denaturation of SOD protein caused by high temperature and acidic environment.

[0117] The low recovery rate of Comparative Example 2 (without enzymatic hydrolysis) confirms that in the absence of enzymatic hydrolysis, a large amount of SOD is retained in plant cell wall fragments and macromolecular networks due to physical encapsulation. These impurities are mainly allocated to the lower phase or interface layer and discarded, resulting in a large loss of SOD.

[0118] The recovery rate of Comparative Example 3 (stepwise operation) was lower than that of all other examples, indicating that a synergistic effect exists in the simultaneous enzymatic hydrolysis and extraction. In the in-situ reaction system, the immediate removal or partitioning of enzymatic hydrolysis products may facilitate a positive shift in the hydrolysis equilibrium and reduce product inhibition, thereby achieving a more thorough release and recovery of SOD than the stepwise operation.

[0119] The recovery rate of Comparative Example 4 (without competitive partitioning) was significantly lower than that of the Example, demonstrating the necessity of this step. The lack of a high-concentration salt resulted in a low partition coefficient of SOD between the two phases, leading to insufficient differentiation in its distribution between the upper and lower phases. During phase separation, a considerable portion of SOD was lost with the lower phase, thus reducing the overall recovery rate. By integrating in-situ enzymatic hydrolysis, aqueous two-phase extraction, and competitive partitioning, not only was the high purity of the product ensured, but also efficient recovery of SOD activity was achieved.

[0120] Test Example 5: Determination of the purity of the final product Experimental description: This test example uses size exclusion high-performance liquid chromatography (SEC-HPLC) to analyze the purity of the superoxide dismutase (SOD) lyophilized products obtained in each example and comparative example. This method separates molecules based on their hydrodynamic volume in solution, which can directly reflect the distribution of the main component and impurity proteins (such as aggregates or degradation fragments) in the sample, and quantify the purity of the main peak. This test example complements the specific activity determination in Test Example 3, evaluating purity from different physical dimensions (hydrodynamic volume) to more comprehensively characterize the product quality.

[0121] Experimental steps: The SOD lyophilized powders obtained in each example and comparative example were dissolved in the mobile phase and prepared into a solution with a concentration of approximately 1.0 mg / mL. The solution was then filtered through a 0.22 μm filter membrane and injected.

[0122] The high-performance liquid chromatography (HPLC) conditions are as follows: Instrument: High-performance liquid chromatography system equipped with a UV detector.

[0123] Column: TSKgel-G2000SWxl (7.8mm×300mm, 5μm).

[0124] Mobile phase: 0.1M phosphate buffer containing 0.1M sodium sulfate, pH 6.8. The sodium sulfate added to the mobile phase is intended to shield proteins from any nonspecific electrostatic or hydrophobic interactions that may exist between them and the stationary phase of the column, ensuring that separation is based entirely on molecular size.

[0125] Flow rate: 0.5 mL / min.

[0126] Column temperature: 25℃.

[0127] Injection volume: 20 μL.

[0128] Detection wavelength: 280nm.

[0129] Record the chromatogram and calculate the relative purity of the SOD main peak using the area normalization method.

[0130] Purity (%) = (SOD main peak area / total peak area) × 100% Experimental data: Table 5. SEC-HPLC purity analysis of the final products of each example and comparative example.

[0131] in conclusion: The SEC-HPLC analysis results provide direct evidence for evaluating the purification effect of each process.

[0132] The samples from Examples 1 to 4 all exhibited a purity exceeding 95%, with prominent SOD peaks, stable baselines, and no obvious impurity peaks in their chromatograms. This indicates that the method of the present invention, through multi-step synergistic action, can effectively remove various impurities from the crude extract of *Gnaphalium affine*, including macromolecular polysaccharides, pigments, and impurity proteins with molecular weights similar to or different from SOD, ultimately yielding a high-purity target product.

[0133] Comparative Example 1 (acid-heat method) had one of the lowest purity values, reflecting that while the method precipitates impurities, it also causes the aggregation and degradation of the target protein, resulting in the product containing a large amount of inactive or denatured SOD components.

[0134] Comparative Example 2 (without enzymatic hydrolysis) showed the lowest purity, confirming the crucial role of the in-situ enzymatic hydrolysis step in removing macromolecular impurities. Without this step, a large number of impurities are encapsulated and enter the PEG phase along with SOD, making them ineffective for removal in subsequent steps.

[0135] The purity of Comparative Example 4 (without competitive partitioning) was significantly lower than that of the Examples, and the impurity peaks in its chromatogram mainly corresponded to proteases and polysaccharides added during the enzymatic hydrolysis step that could not be removed. This result directly confirms the effectiveness of the competitive partitioning step: by introducing a high concentration of salt, these enzyme proteins, which act as processing aids, are selectively driven to the salt phase, thereby achieving separation from the target product SOD.

[0136] Comparative Example 3 (stepwise operation) showed better purity than the other comparative examples, but still not as good as the embodiments of the present invention. This indicates that performing enzymatic hydrolysis and extraction simultaneously has advantages in removing impurities compared to traditional stepwise processes. This may be due to the synergistic effect of interfacial reaction and real-time extraction transfer, resulting in more thorough purification. The quantitative data from SEC-HPLC systematically confirmed the necessity of each technical step in the method of the present invention and the superior technical effect produced by their combination from the perspective of component separation, proving that the method can stably prepare high-purity SOD products.

Claims

1. A highly efficient extraction process for SOD from *Silphium perfoliatum*, characterized in that, Includes the following steps: S1: Mix the crude extract of superoxide dismutase from *Heliotropium indicum*, polyethylene glycol, and the first salt used to form the aqueous two-phase system; S2: Adjust the pH of the system to form a stable aqueous two-phase system, in which superoxide dismutase preferentially partitions to the supernatant phase rich in polyethylene glycol; S3: Add polysaccharide hydrolase and protease to the stable aqueous two-phase system to carry out in-situ enzymatic hydrolysis to degrade macromolecular impurities in the crude extract. S4: After the in-situ enzymatic hydrolysis reaction is completed, a second salt for competitive partitioning is added to the system to selectively partition the polysaccharide hydrolase and protease to the salt phase; S5: Separate and collect the supernatant phase rich in superoxide dismutase.

2. The efficient extraction process of SOD from *Silphium perfoliatum* according to claim 1, characterized in that, The amounts of each raw material used in the process are as follows: 100 parts by weight of the crude extract of superoxide dismutase from *Pteris vittata*, 20-27 parts by weight of polyethylene glycol, 15-20 parts by weight of the first salt, and 13-27 parts by weight of the second salt.

3. The efficient extraction process of SOD from *Silphium perfoliatum* according to claim 1, characterized in that, Prior to step S1, the method further includes preparing the crude superoxide dismutase extract by crushing, pressing, and filtering the leaves of *Heliotropium indicum* through a 200-mesh filter.

4. The efficient extraction process of SOD from *Silphium perfoliatum* according to claim 1, characterized in that, The in-situ enzymatic hydrolysis reaction in step S3 is carried out at a temperature of 40-45℃, a reaction time of 60-80 minutes, and a stirring speed of 80 rpm.

5. The efficient extraction process of SOD from *Silphium perfoliatum* according to claim 1, characterized in that, The number-average molecular weight of the polyethylene glycol mentioned in step S1 is 3600 g / mol-8800 g / mol, and its molecular weight distribution index is 1.05-1.

20.

6. The efficient extraction process of SOD from *Silphium perfoliatum* according to claim 1, characterized in that, In step S1, the first salt is a phosphate composed of dipotassium hydrogen phosphate and potassium dihydrogen phosphate.

7. The efficient extraction process of SOD from *Silphium perfoliatum* according to claim 1, characterized in that, The polysaccharide hydrolase in step S3 includes cellulase derived from Trichoderma reesei and pectinase derived from Aspergillus niger, and the protease is a neutral protease derived from Bacillus subtilis.

8. The efficient extraction process of SOD from *Silphium perfoliatum* according to claim 7, characterized in that, The cellulase and the pectinase are pre-mixed in a buffer solution to form an enzyme stock solution before being added to step S3, and the ratio of activity units is (1-2):

1.

9. The efficient extraction process of SOD from *Silphium perfoliatum* according to claim 1, characterized in that, In step S4, the second salt is ammonium sulfate or sodium citrate, and the second salt is added in the form of an aqueous solution with a concentration of 3.6-4.0M.

10. The efficient extraction process of SOD from *Silphium perfoliatum* according to claim 1, characterized in that, After step S5, the process further includes tangential flow ultrafiltration of the supernatant phase with a molecular weight cutoff of 10 kDa and freeze-drying under a vacuum of less than 10 Pa to obtain superoxide dismutase lyophilized powder.

Images