Agmatine and a method for extracting the same

Abstract

The application provides a spermidine and an extraction method thereof, and relates to the technical field of substance extraction. In the application, plant raw materials are first crushed under a low-temperature protective atmosphere, and are mixed with an acid stabilizer to obtain pretreated raw materials. Then, the pretreated raw materials are extracted by using a natural eutectic solvent, and an extract is obtained after solid-liquid separation. Then, the extract is adsorbed by a hydrogen-type cation exchange resin column, and is treated with a washing agent and an eluent in sequence. The eluent containing spermidine is collected. Finally, the eluent is concentrated, anhydrous ethanol is added for crystallization, and spermidine is obtained after solid-liquid separation and drying. The extraction method has the advantages of green safety, mild process, controllable cost, easy industrialization, and high-purity spermidine.

Description

Technical Field

[0001] This invention relates to the field of substance extraction technology, and in particular to spermidine and its extraction method. Background Technology

[0002] Spermine, chemically known as N-(3-aminopropyl)-1,4-butanediamine, is a naturally occurring polyamine compound found in living organisms, widely distributed in animal and plant cells, tissues, and organs. As an important metabolic regulator in vivo, spermine participates in regulating various key physiological and pathological processes, such as cell proliferation, differentiation, aging, autophagy, and immune responses, by binding to negatively charged biomolecules such as DNA, RNA, and proteins.

[0003] Spermine has demonstrated its value in multiple pharmaceutical fields. Specifically, in the treatment of ulcerative colitis, it can reduce colonic inflammation and damage, and related clinical trials have been initiated; in anti-aging, it can extend the lifespan of model organisms and improve the bodily functions of the elderly; in cardiovascular protection, it can delay cardiac aging and improve arteriosclerosis; and in neuroprotection, it helps alleviate cognitive decline symptoms such as Alzheimer's disease. Given these applications, spermine has broad prospects in the pharmaceutical field, and the development of related products has significant commercial value.

[0004] However, an existing domestic patent discloses a method for extracting spermidine from natural sources (application publication number CN 119431156 A). This extraction method uses a high proportion of acetonitrile and corrosive trifluoroacetic acid, which has high solvent toxicity and high residual risk. In addition, it relies on expensive liquid chromatography systems, has small processing capacity, and is difficult to scale up industrially, which does not meet the requirements of large-scale production for green pharmaceuticals.

[0005] Therefore, there is an urgent need to design a method for preparing high-purity spermidine that uses readily available raw materials, is green and safe, has a mild process, is cost-controllable, and is easy to industrialize. Summary of the Invention

[0006] One object of this invention is to provide spermidine and its extraction method, which uses readily available plant raw materials, is green and safe, has a mild process, controllable cost, is easy to industrialize, and can produce high-quality spermidine. Spermine of pure purity.

[0007] An object of a first aspect of the present invention is to provide a method for extracting spermidine, comprising the following steps: S1. The plant material is pulverized under a low-temperature protective atmosphere and mixed with an acidic stabilizer to obtain a pretreated raw material; wherein the plant material is defatted wheat germ meal, defatted soybean germ meal or corn germ meal; S2. Use a natural eutectic solvent to extract the pretreated raw material obtained in step S1, and obtain the extract after solid-liquid separation; S3. The extract obtained in step S2 is adsorbed through a hydrogen-type cation exchange resin column and treated with detergent and eluent in sequence to collect the eluent containing spermidine. S4. After concentrating the eluent obtained in step S3, add anhydrous ethanol for crystallization, separate the solid and liquid, and dry to obtain spermidine.

[0008] The defatted wheat germ meal, defatted soybean germ meal, or corn germ meal selected in this invention are all by-products of the oil and fat industry. They are widely available and inexpensive, realizing the reuse of waste resources and avoiding the adverse effects of oil in the raw materials on the extraction process and resin adsorption efficiency.

[0009] In some embodiments, the low-temperature protective atmosphere in step S1 is a nitrogen atmosphere or liquid nitrogen protection, and the pulverization temperature is -30°C to -10°C.

[0010] Spermine is highly sensitive to heat and oxygen. Frictional heat generated during material grinding at room temperature or high temperatures can easily lead to its oxidative degradation. This invention uses nitrogen or liquid nitrogen to create an inert protective atmosphere, effectively isolating oxygen. Simultaneously, the grinding temperature is controlled within a low-temperature range of -30℃ to -10℃, suppressing the thermal effects on the spermine structure during grinding and reducing the loss of active ingredients. Furthermore, the low-temperature environment increases the brittleness of the plant raw materials, which is beneficial for obtaining a uniform and fine powder, improving subsequent extraction efficiency. In addition, this temperature range balances protective effect with energy costs, avoiding the excessive liquid nitrogen consumption problem associated with extreme low temperatures (such as below -50℃), ensuring the feasibility and economy of the grinding process.

[0011] In some embodiments, the acidic stabilizer is selected from one or more of citric acid, phytic acid, ascorbic acid, and tartaric acid, and its addition amount is 0.2%-1.0% of the mass of the plant material.

[0012] Acid stabilizers can provide an acidic microenvironment for spermidine, inhibiting its extraction during step S2. Oxidation and degradation occur, and the amount of acidic stabilizer added is limited to 0.2%-1.0% of the plant raw material mass. This ensures sufficient protection while avoiding the introduction of impurities or increased costs due to excessive addition. Within this mass range, the best balance between stability protection and extraction economy can be achieved.

[0013] In some embodiments, the natural eutectic solvent is composed of a hydrogen bond acceptor, a hydrogen bond donor, and water in a molar ratio of 1:(1.5-2.5):(0.5-1.5); the hydrogen bond acceptor is one or more of choline chloride, betaine, acetylcholine, proline, and alanine; and the hydrogen bond donor is a polyol or urea.

[0014] Choline chloride, acting as a hydrogen bond acceptor, forms a natural eutectic solvent network with polyols or urea (hydrogen bond donors) through hydrogen bonding. This lowers the melting point of the natural eutectic solvent, enhances its penetration into plant cell walls, efficiently releases spermidine, and reduces the release of co-soluble impurities such as proteins and polysaccharides. Furthermore, this solvent system exhibits good biocompatibility and is completely degradable, avoiding the toxicity and residue problems associated with organic solvents (such as acetonitrile and trifluoroacetic acid).

[0015] In some embodiments, the ratio of the plant material to the natural eutectic solvent is 1:(10-20).

[0016] The material-to-liquid ratio is limited to 1:10-1:20 to ensure that the natural eutectic solvent and the plant raw materials in the pretreated raw materials are in full contact, so that spermidine can be efficiently dissolved, while avoiding excessive solvent which would lead to increased energy consumption and cost in subsequent concentration, thus achieving a balance between extraction efficiency and economy.

[0017] In some embodiments, the extraction in step S2 is performed using ultrasound-assisted extraction, with an ultrasound power of 200-400W, a frequency of 30-50kHz, a temperature of 25-40℃, and a time of 20-60min.

[0018] In some embodiments, the hydrogen-form cation exchange resin in the hydrogen-form cation exchange resin column is any one of D152, D113, D151, D155, AmberLite IRC-86, Dowex MAC-3, Lewatit CNP 80, or Purolite C104.

[0019] Hydrogen-form cation exchange resins such as D152 and D113 are selected, which exhibit high selective adsorption capacity for positively charged spermidine, effectively capturing the target component and eliminating most neutral or anion-positive impurities. Hydrogen-form cation exchange resins have large adsorption capacity, are easy to regenerate, and are reusable, reducing purification costs and improving product purity. Among these, the H... + With H in the extract + Together, they maintain a stable acidic microenvironment, ensuring that spermidine always exists in a positively charged form.

[0020] In some embodiments, the detergent is an aqueous solution of ethanol with a volume fraction of 30-70%; the eluent is an aqueous solution of ammonium chloride or sodium chloride of 0.5-2M, and the pH is adjusted to 3.5-5.0 with acid.

[0021] In step S3, after adsorption through the hydrogen-form cation exchange resin column, a small amount of polar impurities (such as flavonoid glycosides, phenolic acids, etc.) remain and are non-specifically adsorbed onto the resin through hydrogen bonding or hydrophobic interactions, coexisting with spermidine. Therefore, a 30%-70% (v / v) ethanol aqueous solution is used as a washing agent to effectively remove the polar impurities adsorbed on the hydrogen-form cation exchange resin, while spermidine, due to its positive charge, remains on the hydrogen-form cation exchange resin. Subsequently, a eluting agent is used to gently elute spermidine through a cation competitive exchange mechanism. This pH range ensures the stability of spermidine without degradation while achieving efficient desorption. The eluent has a simple composition, facilitating subsequent crystallization and purification.

[0022] In some embodiments, the crystallization step in step S4 is as follows: the eluent is concentrated under reduced pressure to 1 / 5-1 / 3 of its original volume, then anhydrous ethanol at a temperature of -20℃-0℃ is added, and the mixture is allowed to stand for crystallization at a temperature of 0℃-10℃ for a time of 4h-24h.

[0023] Concentrating the eluent under reduced pressure to 1 / 5-1 / 3 of its original volume effectively increases the spermidine concentration and promotes crystallization. Adding pre-cooled anhydrous ethanol at -20℃ to 0℃ as an antisolvent significantly reduces the solubility of spermidine, while the low temperature condition suppresses side reactions. Controlling the crystallization temperature to 0-10℃ and the time to 4-24h is beneficial for slow crystal growth and further eliminates salts, residual solvents, and structural analogs, resulting in high-purity, large-particle, and easily separable spermidine crystals, thus improving product yield and quality.

[0024] A second aspect of the present invention aims to provide a spermidine, which is prepared by the spermidine extraction method described above.

[0025] Compared with the prior art, the present invention has the following effects: First, defatted wheat germ meal, defatted soybean germ meal, or corn germ meal are used as plant raw materials, realizing the reuse of waste products from the oilseed industry, reducing raw material costs, and ensuring wide availability. The degradation of spermidine during the crushing and extraction process is effectively inhibited through low-temperature inert atmosphere pulverization and the synergistic protection of acidic stabilizers, ensuring the bioactivity of the product. Then, a natural eutectic solvent is creatively used for extraction, replacing traditional toxic organic solvents and improving the safety and environmental friendliness of the extraction process. Next, selective enrichment is achieved using hydrogen-form cation exchange resin, utilizing charge selectivity to efficiently capture spermidine. Polar impurities are removed by a detergent, and the eluent containing spermidine is collected using an eluent, eliminating the need for expensive preparative liquid chromatography systems. Finally, anhydrous ethanol is added to precipitate spermidine in high-purity crystal form. The entire process uses conventional industrial equipment, is easy to scale up, and successfully achieves green, efficient, stable, and low-cost industrial production of spermidine. The obtained spermidine has a purity greater than 94% and a relative yield greater than 83%, meeting the requirements for large-scale preparation of pharmaceutical-grade raw materials. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0027] Preparation Example 1 The preparation method of the hydrogen-form cation exchange resin column includes the following steps: A1. Take 15 mL of D152 type hydrogen cation exchange resin and place it in a beaker. Add 50 mL of 1 M hydrochloric acid solution and stir to soak for 2 hours. After soaking for 1 hour, pour off the old acid solution and add 50 mL of 1 M hydrochloric acid solution again to obtain the pretreated hydrogen cation exchange resin. A2. Let the pretreated hydrogen-form cation exchange resin obtained in step A1 stand, pour off the supernatant acid, and repeatedly wash the bottom resin with deionized water until the pH of the deionized water washing solution is 6.5 to obtain hydrogen-form cation exchange resin. A3. The hydrogen-form cation exchange resin obtained in step A2 is packed into a glass column (inner diameter 1.5cm, column height 30cm) using a wet method, with a packing height of 9cm. The resin column is then equilibrated with deionized water to obtain a hydrogen-form cation exchange resin column.

[0028] The following detailed description uses specific embodiments and comparative examples.

[0029] Example 1 Example 1 provides a method for extracting spermidine, comprising the following steps: S1. Take 13g of defatted wheat germ meal (fat content is 0.8%), place it in a low-temperature pulverizer, purge it with liquid nitrogen, control the pulverizing temperature at -20℃, and pass it through a 60-mesh sieve after pulverizing; add 0.078g of citric acid to the pulverized material, mix it evenly, and obtain the pretreated raw material; S21. Weigh 104g of choline chloride, 109mL of 1,2-propanediol, and 13.4mL of deionized water, stir in a 60℃ water bath until a clear and transparent solution is formed, and cool to 25±2℃ to obtain 200mL of natural eutectic solvent. S22. Mix the pretreated raw material obtained in step S1 with 200 mL of natural eutectic solvent obtained in step S21, and perform ultrasonic-assisted extraction at an ultrasonic power of 300 W, a frequency of 40 kHz, a temperature of 32 °C, and an extraction time of 40 min. After extraction, centrifuge (8000 rpm, 10 min) and collect the supernatant, which is the extract. S3. After adjusting the pH of the extract obtained in step S22 to 4.5 with 2.5M dilute hydrochloric acid, the extract was loaded into the hydrogen-form cation exchange resin column obtained in Preparation Example 1 at a flow rate of 1 mL / min. After loading, the resin column was washed with 30 mL of 50% ethanol aqueous solution as washing agent at a flow rate of 1.5 mL / min. Then, the column was eluted with 1.2M ammonium chloride aqueous solution (adjusted to pH 4.2 with hydrochloric acid) at a flow rate of 1 mL / min, and the eluent containing spermidine was collected. S4. The eluent collected in step S3 is concentrated under reduced pressure at 50°C to 1 / 4 of its original volume. The concentrate is then slowly added dropwise to anhydrous ethanol pre-cooled to -10°C. After the addition is complete, the eluent is placed in a 4°C refrigerator and allowed to crystallize for 12 hours. After crystallization, the crystals are collected by centrifugation (8000 rpm, 5 min) and then dried in a 50°C vacuum drying oven for 4 hours to obtain spermidine white crystals.

[0030] Example 2 Example 2 provides a method for extracting spermidine, comprising the following steps: S1. Take 13g of defatted soybean germ meal (fat content is 0.9%), place it in a low-temperature pulverizer, purge with nitrogen for protection, control the pulverizing temperature at -30℃, and pass it through an 80-mesh sieve after pulverizing; add 0.104g of phytic acid to the pulverized material, mix evenly, and obtain the pretreated raw material; S21. Weigh 112g betaine, 109mL 1,2-propanediol and 13.4mL deionized water, stir in a 60℃ water bath until a clear and transparent solution is formed, and cool to 25±2℃ to obtain about 200mL of natural eutectic solvent. S22. Mix the pretreated raw material obtained in step S1 with 200 mL of natural eutectic solvent obtained in step S21, and perform ultrasonic-assisted extraction at an ultrasonic power of 250 W, a frequency of 35 kHz, a temperature of 40 °C, and an extraction time of 30 min. After extraction, centrifuge (8000 rpm, 10 min) and collect the supernatant, which is the extract. S3. After adjusting the pH of the extract obtained in step S22 to 4.5 with 2.5M dilute hydrochloric acid, the extract was loaded into the hydrogen-form cation exchange resin column obtained in Preparation Example 1 at a flow rate of 1.5 mL / min. After loading, the resin column was washed with 30 mL of 40% (v / v) ethanol aqueous solution as washing agent at a flow rate of 1.5 mL / min. Then, the column was eluted with 1.0M sodium chloride aqueous solution (pH adjusted to 4.5 with hydrochloric acid) at a flow rate of 1 mL / min, and the eluent containing spermidine was collected. S4. The eluent collected in step S3 is concentrated under reduced pressure at 50°C to 1 / 3 of its original volume. The concentrate is then slowly added dropwise to anhydrous ethanol pre-cooled to -5°C. After the addition is complete, the eluent is placed in an 8°C refrigerator and allowed to crystallize for 18 hours. After crystallization, the crystals are collected by centrifugation (8000 rpm, 5 min) and then dried in a 50°C vacuum drying oven for 4 hours to obtain spermidine white crystals.

[0031] According to the "Residual Solvent Determination Method" in the General Chapter of the 2020 Edition of the Chinese Pharmacopoeia, the residual organic solvents in the spermidine crystals obtained in Examples 1 and 2 were detected by gas chromatography-mass spectrometry (GC-MS). The results showed that the residual ethanol content in the spermidine crystals obtained in Example 1 was 152 ppm, and the residual 1,2-propanediol content was 87 ppm; the residual ethanol content in the spermidine crystals obtained in Example 2 was 168 ppm, and the residual 1,2-propanediol content was 93 ppm. All of these residual amounts are far below the 5000 ppm limit (Class 3 solvents) specified by ICH Q3C, indicating that the spermidine product prepared by this invention has extremely low residual organic solvents, good safety, and is suitable for the pharmaceutical field.

[0032] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that citric acid is not added in step S1, while the rest of the operation is the same as in Example 1.

[0033] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that in step S1, the pulverization is carried out directly in a normal temperature air atmosphere instead of under a low temperature protective atmosphere. The rest of the operation is the same as in Example 1.

[0034] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the natural eutectic solvent in step S2 is replaced with an aqueous ethanol solution of the same dosage and volume fraction of 75%, while the rest of the operation is the same as in Example 1.

[0035] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the hydrogen-type cation exchange resin is replaced with D101 type macroporous adsorption resin, while the rest of the operation is the same as in Example 1.

[0036] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that the anhydrous ethanol in step S4 is replaced with acetone, while the rest of the operation is the same as in Example 1.

[0037] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that the hydrogen-form cation exchange resin in the hydrogen-form cation exchange resin column in step S3 is Na. + The rest of the operation is the same as in Example 1.

[0038] Performance testing: The purity and relative yield of spermidine obtained in each example and comparative example were determined by high performance liquid chromatography (HPLC). The specific test methods are as follows: Chromatographic conditions: The column was a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm), the column temperature was 30 ℃, the mobile phase was acetonitrile-water (60:40 v / v, containing 0.1% trifluoroacetic acid), the flow rate was 1.0 mL / min, the detection wavelength was 254 nm, and the injection volume was 20 μL.

[0039] Standard curve preparation: Accurately weigh spermidine standard, dissolve and dilute it with deionized water to prepare a series of standard solutions of different concentrations, and inject and determine them under the above chromatographic conditions. Plot the standard curve with peak area as the ordinate and concentration as the abscissa.

[0040] Test sample determination: Take appropriate amounts of spermidine products obtained in Examples 1-2 and Comparative Examples 1-6 (hereinafter referred to as test samples), accurately weigh them, dissolve them in deionized water and dilute to a suitable concentration, filter them through a 0.45 μm filter membrane, and inject them for determination under the above chromatographic conditions, and record the peak area.

[0041] Determination of raw material extract: Take the extracts obtained in Examples 1-2 and Comparative Examples 1-6 respectively, filter them, inject them under the same chromatographic conditions, and record the peak area.

[0042] Calculation: Substitute the peak areas of the sample to be tested and the raw material extract into the standard curve to calculate the mass of spermidine in the sample to be tested (m1) and the total mass of spermidine in the plant raw material (m0). At the same time, accurately weigh the total mass of the sample to be tested (m).

[0043] Spermine purity (%) = (m1 / m) × 100%.

[0044] Relative yield (%) = (m1 / m0) × 100%.

[0045] Table 1 below shows the comprehensive performance comparison results of the test samples obtained from the examples and comparative examples.

[0046] Table 1

[0047] As shown in Table 1, the spermidine purity of Examples 1 and 2 of the present invention reached 94.8% and 94.1%, respectively, and the relative yields were 84.2% and 83.5%, respectively. However, the performance of each comparative example was lower than that of the examples.

[0048] Specifically, in Comparative Example 1, the spermidine purity was 88.7% and the relative yield was 72.3%, indicating that the acidic stabilizer played a key role in inhibiting spermidine degradation and improving the yield. In Comparative Example 2, the spermidine purity was 87.9% and the relative yield was 68.7%, indicating that low-temperature inert atmosphere pulverization effectively prevented spermidine oxidation loss. In Comparative Example 3, the spermidine purity was 84.2% and the relative yield was 62.4%, indicating that the natural eutectic solvent had high extraction selectivity and efficiency. In Comparative Example 4, after replacing the hydrogen-type cation exchange resin with D101 type macroporous adsorption resin, the spermidine purity decreased to 72.3%, indicating that ordinary adsorption resins, due to their lack of charge selectivity, adsorb a large amount of impurities and cannot achieve efficient purification of spermidine. In Comparative Example 5, after replacing anhydrous ethanol with acetone for crystallization, the purity and yield of spermidine decreased to 81.6% and 65.7%, respectively. This indicates that acetone, as an antisolvent, cannot effectively remove impurities and has a low crystallization recovery rate, demonstrating the crucial role of selecting anhydrous ethanol as the crystallization solvent in ensuring both high purity and high yield. In Comparative Example 6, the hydrogen-form cation exchange resin was replaced with Na... + After the process, spermidine purity was 80.6% and yield was 71.2%. The hydrogen-form resin maintained an acidic environment to ensure the protonation of spermidine for selective adsorption. Na + The adsorption efficiency of the hydrogen-form resin decreased due to its lack of pH adjustment capability, indicating that the effect of the hydrogen-form resin could not be achieved by Na+. + Type substitution. The above data fully demonstrates that the synergistic effect of the various technical features of this invention can improve the purity and yield of spermidine.

[0049] Therefore, those skilled in the art should recognize that although numerous exemplary embodiments of the present invention have been shown and described in detail herein, many other variations or modifications conforming to the principles of the present invention can be directly determined or derived from the disclosure of the present invention without departing from the spirit and scope of the invention. Thus, the scope of the present invention should be understood and construed as covering all such other variations or modifications.

Claims

1. A method for extracting spermidine, characterized in that, Includes the following steps: S1. The plant material is pulverized under a low-temperature protective atmosphere and mixed with an acidic stabilizer to obtain a pretreated raw material; wherein the plant material is defatted wheat germ meal, defatted soybean germ meal or corn germ meal; S2. Use a natural eutectic solvent to extract the pretreated raw material obtained in step S1, and obtain the extract after solid-liquid separation; S3. The extract obtained in step S2 is adsorbed through a hydrogen-type cation exchange resin column and treated with detergent and eluent in sequence to collect the eluent containing spermidine. S4. After concentrating the eluent obtained in step S3, add anhydrous ethanol to crystallize, separate the solid and liquid, and dry to obtain spermidine.

2. The spermidine extraction method according to claim 1, characterized in that, The low-temperature protective atmosphere in step S1 is a nitrogen atmosphere or liquid nitrogen protection, and the pulverization temperature is -30℃ to -10℃.

3. The spermidine extraction method according to claim 1, characterized in that, The acidic stabilizer is selected from one or more of citric acid, phytic acid, ascorbic acid, and tartaric acid, and its addition amount is 0.2%-1.0% of the mass of the plant raw material.

4. The spermidine extraction method according to claim 1, characterized in that, The natural eutectic solvent is composed of a hydrogen bond acceptor, a hydrogen bond donor, and water in a molar ratio of 1:(1.5-2.5):(0.5-1.5); the hydrogen bond acceptor is one or more of choline chloride, betaine, acetylcholine, proline, and alanine; and the hydrogen bond donor is a polyol or urea.

5. The spermidine extraction method according to claim 1, characterized in that, The ratio of the plant material to the natural eutectic solvent is 1:(10-20).

6. The spermidine extraction method according to claim 1, characterized in that, The extraction in step S2 is performed using ultrasound-assisted extraction, with an ultrasound power of 200-400W, a frequency of 30-50kHz, a temperature of 25-40℃, and a time of 20-60min.

7. The spermidine extraction method according to claim 1, characterized in that, The hydrogen-form cation exchange resin in the hydrogen-form cation exchange resin column is any one of D152, D113, D151, D155, AmberLite IRC-86, Dowex MAC-3, Lewatit CNP 80, or Purolite C104.

8. The spermidine extraction method according to claim 1, characterized in that, The detergent is an aqueous solution of ethanol with a volume fraction of 30-70%; the eluent is an aqueous solution of ammonium chloride or sodium chloride with a volume fraction of 0.5-2M, and the pH is adjusted to 3.5-5.0 with acid.

9. The spermidine extraction method according to claim 1, characterized in that, The crystallization step in step S4 is as follows: the eluent is concentrated under reduced pressure to 1 / 5-1 / 3 of its original volume, then anhydrous ethanol at a temperature of -20℃ to 0℃ is added, and the mixture is allowed to stand for crystallization at a temperature of 0-10℃ for 4-24 hours.

10. A spermidine, characterized in that, The spermidine is prepared by any one of the spermidine extraction methods described in claims 1-9.

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