Method for extracting spermidine from natural source

Abstract

The invention relates to a method for extracting spermidine from a natural source. The method comprises the following steps: (1) extracting a natural source substance to obtain a crude extract; and (2) carrying out hydrophilic interaction liquid chromatography purification on the crude extract to obtain spermidine. According to the method disclosed by the invention, the extract from a natural source is efficiently and accurately separated by utilizing the hydrophilic interaction liquid chromatography, so that the high-purity spermidine with a wide source is obtained to meet the requirement on high-quality spermidine.

Description

Technical Field

[0001] The present application relates to the technical field of natural product chemistry, and in particular to a method for extracting spermidine from natural sources. Background Art

[0002] Spermidine (SPD), N-(3-aminopropyl)-1,4-butanediamine, has a structural formula of H2N(CH2)3NH(CH2)4NH2. It is a typical triamine polyamine substance that is widely present in cells, tissues and organs. It binds to negatively charged molecules such as DNA, RNA and proteins, and participates in many important life processes, such as cell proliferation regulation, cell aging, organ development, immunity, cancer and other physiological and pathological processes. At the cellular level, based on existing research, spermidine can significantly prolong the lifespan and survival rate of yeast, fruit flies, nematodes and human peripheral blood mononuclear cells; in animal models, studies have found that exogenous supplementation of spermidine can improve the compliance and mitochondrial function of the left ventricle of mice, thereby improving the aging state and muscle regeneration ability of mice, which is beneficial to improving memory ability, reducing neurodegenerative lesions, reducing lipid accumulation and necrotic core formation in atherosclerotic plaques of obese mice induced by a high-fat diet, improving blood pressure and reducing the morbidity and mortality of cardiovascular diseases. In addition, studies have shown that spermidine has a wide range of anti-inflammatory and antioxidant properties, which can help reduce age-related oxidative protein damage and the accumulation of excessive reactive oxygen species (ROS). Given these properties, spermidine has great application prospects in the food and pharmaceutical fields, and the development of related products has important commercial value.

[0003] The methods currently used to prepare or extract spermidine on the market mainly include: chemical synthesis, microbial fermentation, and enzymatic methods. Among them, the chemical synthesis method requires multi-step reactions to achieve the synthesis of spermidine, which has the disadvantages of complex steps, high energy consumption, many side reactions, and high pollution. The microbial fermentation method uses specific strains to accumulate and extract spermidine through the fermentation process. This method is more environmentally friendly, but there are problems such as unstable fermentation reaction process, limited cell membrane permeability, easy degradation of products, and accumulation of by-products. The enzymatic method uses specific enzymes to catalyze substrates to generate spermidine. This method requires systematic optimization of the "de novo cascade reaction" to achieve "one-pot" preparation, and also faces problems of enzyme stability and cost.

[0004] The methods for extracting spermidine in the prior art also include methods of extracting from natural products. The natural extraction method has the advantages of wide sources, environmental protection and high bioavailability. In addition, the natural extraction method also meets consumers' preference for healthy food and natural raw materials, which is conducive to market acceptance and market competitiveness of products. However, the existing methods mainly use traditional methods such as heat reflux to extract spermidine from wheat germ, which have problems such as low extraction rate and low purity, and cannot meet the market demand for high-quality spermidine. Summary of the invention

[0005] To solve the above problems, the present invention provides a method for extracting spermidine from natural sources and its application, the method is simple to operate, the extraction time is short, the extraction efficiency is high, the product stability is high, and the obtained spermidine has a high purity, which can meet the demand for high-quality spermidine in the food and pharmaceutical industries.

[0006] One aspect of the present invention provides a method for extracting spermidine from a natural source, the method comprising the following steps:

[0007] (1) extracting a natural source to obtain a crude extract; and

[0008] (2) Purifying the crude extract by hydrophilic interaction liquid chromatography to obtain spermidine.

[0009] The existing methods for obtaining spermidine from natural sources mainly have problems such as low extraction efficiency and low product purity. Since spermidine is mainly used in the food and pharmaceutical fields, there are high requirements for the purity of raw materials. Therefore, it is very important to obtain high-purity spermidine based on natural extraction methods. However, due to the high polarity and strong water solubility of spermidine, the traditional methods such as reversed-phase liquid chromatography are not ideal for the separation of spermidine, and it is easy to mix with other highly polar compounds in the extract.

[0010] In view of the demand for high-purity spermidine in practical applications, the present invention creatively uses hydrophilic interaction liquid chromatography (HILIC) for separation in the process of obtaining spermidine. Among them, hydrophilic interaction liquid chromatography is a high performance liquid chromatography (HPLC) technology for separating polar and hydrophilic compounds. HILIC-HPLC uses the interaction between the hydrophilic stationary phase and the polar compounds in the solvent to achieve separation. The stationary phase used in HILIC-HPLC is usually based on silica gel or siloxane, and the surface is covered with polar functional groups such as hydroxyl or amino groups, which can form hydrogen bonds or electrostatic interactions with polar groups in water molecules and solutes, thereby achieving effective separation of polar and hydrophilic compounds. This separation mechanism is completely different from reversed-phase liquid chromatography. Compared with traditional methods such as reversed-phase chromatography, HILIC has significant advantages in separating highly polar, ionic, and water-soluble compounds such as amino acids, sugars, and nucleic acid bases, and is more suitable for separating spermidine with strong polarity and good water solubility. The present invention adopts HILIC technology with good separation effect, high reproducibility and fast analysis speed to separate spermidine, thereby realizing high-purity preparation of spermidine, a polar compound.

[0011] In one embodiment, in step (1), the extraction is performed using ultrasound-assisted extraction.

[0012] The extraction step is a key link in the recovery process of bioactive compounds. The ideal extraction step should ensure the effective separation of the target compound from the raw material while maintaining its purity and biological activity, which is crucial for achieving high-efficiency and high-yield recovery and will directly affect subsequent analysis, purification and application. Traditional methods for extracting bioactive compounds from natural sources include: direct solvent extraction, hot reflux extraction, distillation, mechanical pressing and supercritical fluid extraction. Among them, the direct solvent extraction method uses the solvent's solubility for the target compound to extract it through soaking and leaching. It is simple to operate and has a wide range of applications, but it has the disadvantages of long extraction time and high solvent consumption, and it is very easy to introduce solvent residues. The hot reflux extraction method has a higher extraction efficiency than the direct solvent extraction method through the circulation of the solvent, but the need to ensure the circulation of the solvent makes it complicated to operate and has high energy consumption. The distillation method vaporizes the volatile components in the natural source by heating, and then completes the extraction by condensation collection. It has the advantage of high purity, but it is only applicable to components with good thermal stability. Mechanical pressing uses mechanical pressure to squeeze out the oil or liquid components from natural sources. This method does not introduce solvents and can avoid solvent residues, but it is only applicable to natural sources rich in oil or juice, and only to bioactive compounds present in the oil or juice of natural sources. Supercritical fluid extraction uses fluid in a supercritical state for extraction. The advantages are good selectivity and high extraction efficiency, but the equipment cost is high and the operation is complicated.

[0013] Different from the above methods, the ultrasound-assisted extraction technology (UAE) used in the present invention utilizes the cavitation effect, mechanical action and thermal effect generated by ultrasound to destroy the tissue in the natural source, while enhancing the penetration capacity of the solvent, thereby improving the dissolution efficiency of the target compound (such as spermidine in the present invention), effectively shortening the time required for the extraction process while improving the extraction rate, improving the utilization rate of natural sources and saving processing time and operating costs. In addition, ultrasound-assisted extraction can reduce the use of organic solvents. Compared with traditional methods such as heat reflux, ultrasound-assisted extraction consumes less solvent, is green and environmentally friendly, and improves the purity of bioactive compounds, which is conducive to improving product stability.

[0014] In a specific embodiment, the ultrasonic assisted extraction comprises the following steps:

[0015] (1-1) mixing the natural source, preferably in powder form, with an extraction solution and extracting using an ultrasonic disruptor; and

[0016] (1-2) Separation, collecting the supernatant and drying to obtain the crude extract.

[0017] In a specific embodiment, in step (1-1), the ultrasonic disruptor is selected from a water bath ultrasonic device or a probe ultrasonic device, preferably a probe ultrasonic device, such as Ningbo Xinzhi, JY92-IIN ultrasonic device; and / or

[0018] The extraction solution is 50% ethanol.

[0019] Among them, the ultrasonic equipment commonly used in related experiments is generally a water bath ultrasonic equipment and a probe ultrasonic equipment. When the ultrasonic equipment is applied to the ultrasonic-assisted extraction step of the present invention, the energy output of the water bath ultrasonic equipment is relatively weak and unevenly distributed, which may cause the extraction efficiency and effect to vary at different positions in the container, and the consistency of the extraction results is poor. In comparison, the probe ultrasonic equipment can achieve higher liquid cavitation efficiency and more efficient extraction effect by concentrating ultrasonic energy on the sample area, and the extraction results are more consistent and controllable. Therefore, this scheme adopts a probe ultrasonic equipment to further explore the method of extracting spermidine from wheat germ powder using ultrasonic-assisted extraction technology.

[0020] In general, compared with the common extraction methods in the prior art, the ultrasound-assisted extraction technology used in the present invention is not only conducive to improving the extraction efficiency, reducing the use of organic solvents, and improving product stability, but also has simple equipment and convenient operation, which is conducive to its wide application in the production of spermidine raw materials.

[0021] In a specific embodiment, in step (1-2),

[0022] The separation was performed by centrifugation at 4°C and 18,000 x g for 10 minutes; and / or

[0023] The drying is performed by freeze drying.

[0024] In one embodiment, in step (1), the conditions of the ultrasound-assisted extraction step are as follows:

[0025] The ultrasonic power is 250-400W; the ultrasonic frequency is 1.5 seconds for each ultrasonication and 1.5 seconds for rest; the total ultrasonic time is 10-20 minutes.

[0026] In one embodiment, in step (2), the conditions of the hydrophilic interaction liquid chromatography are as follows:

[0027] A Shimadzu LC-20AR liquid phase system was used; a Hungpu XBT Amide column was selected as the chromatographic column; the chromatographic column size was 250 mm*4.6 mm, 5 μm; the column temperature was 25°C; the mobile phase included 95% acetonitrile, 5% water and 0.1% trifluoroacetic acid; the flow rate was 1 mL / min; isocratic elution was adopted; and the elution time was 20 minutes.

[0028] In a specific embodiment, the step (2) comprises the following steps:

[0029] (2-1) mixing and dissolving the crude extract obtained in step (1) with a mobile phase;

[0030] (2-2) centrifuging and collecting the supernatant, followed by filtration; and

[0031] (2-3) Centrifuging the supernatant, loading the supernatant onto a chromatography column, and performing isocratic elution to obtain spermidine.

[0032] In a specific embodiment, in the step (2-1), the volume / weight ratio of the mobile phase to the natural source is 1:35 to 1:45; and / or

[0033] In the step (2-1), the dissolution is performed by ultrasound, preferably water bath ultrasound for 5 minutes; and / or

[0034] In the step (2-2), the centrifugation condition is 4°C, 18000xg for 5 minutes; and / or

[0035] In the step (2-2), the filtration is performed using a filter membrane, preferably a 0.45 μm filter membrane, and preferably a PTFE filter membrane; and / or

[0036] In the step (2-3), the centrifugation condition is 4°C, 18000xg for 3 minutes.

[0037] In one embodiment, the natural source is selected from wheat germ, natto and animal liver, preferably wheat germ.

[0038] In summary, the present invention provides a method for extracting spermidine from natural sources, creatively utilizing hydrophilic interaction liquid chromatography to purify the extract from the natural sources, significantly improving the purity of the spermidine product to meet the market demand for high-quality spermidine. By further combining ultrasound-assisted extraction technology, the present invention can also improve the extraction efficiency of spermidine from natural sources while ensuring product stability and ease of operation, thereby improving raw material utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 shows a chromatogram of spermidine extracted by HILIC-HPLC purification;

[0040] Figure 2 HILIC-HPLC chromatogram of spermidine standard is shown;

[0041] Figure 3The mass spectrum of the fraction obtained in Example 1 is shown. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0043] Example 1

[0044] Embodiment 1 of the present invention provides a method for extracting spermidine from wheat germ, comprising the following steps:

[0045] 1. Extraction of spermidine

[0046] 0.5 g of wheat germ powder was weighed and fully mixed with 20 mL of 50% ethanol solution. The mixed solution was subjected to ultrasonic-assisted extraction using a probe ultrasonic disruptor (Ningbo Xinzhi, JY92-IIN) under ice bath conditions.

[0047] Among them, the ultrasonic-assisted extraction technology using a probe-type ultrasonic disruptor can utilize the cavitation effect and mechanical action generated by ultrasound to effectively destroy the cell structure of wheat germ, thereby greatly improving the solvent's leaching and dissolution of spermidine contained in wheat germ while controlling the amount of organic solvent used, thereby significantly improving the extraction efficiency.

[0048] Specifically, in this embodiment, the ultrasonic parameters are set as: 6 mm horn probe, power 250W, ultrasonic 1.5s, stop 1.5s, and the total ultrasonic time is 10min.

[0049] After the ultrasonic extraction, the obtained solution was centrifuged at 4°C and 18000 xg for 10 min, and the supernatant was collected and freeze-dried to obtain a crude extract in a dry powder state.

[0050] 2. Purification of spermidine

[0051] The crude extract was mixed with 5 mL of mobile phase and dissolved by water bath sonication for 5 min. The mobile phase refers to the mobile phase used for subsequent HILIC-HPLC purification, specifically, the mobile phase is 95% ACN, 5% H2O, 0.1% TFA.

[0052] After dissolution, the resulting solution was centrifuged at 4°C and 18000 x g for 5 min, the supernatant was collected, and then filtered. The filtration was performed using a 0.45 μm filter membrane. The solution was centrifuged at 4°C and 18000 x g for 3 min, and the supernatant was subjected to hydrophilic interaction liquid chromatography for purification to obtain spermidine.

[0053] Specifically, the liquid phase system used for purification by hydrophilic interaction liquid chromatography is Shimadzu LC-20AR, and the hydrophilic interaction chromatography column is Hungpu XBT Amide (Guangzhou Hengpu); the column size is 250mm*4.6mm, 5μm; the column temperature is 25°C; the mobile phase is 95% ACN, 5% H2O, 0.1% TFA, and the flow rate is 1mL / min; the elution condition adopts isocratic elution.

[0054] Polar solutions are used as mobile phases in HILIC, and the mobile phase used in most HILIC separations is a mixture containing a small amount of water / buffer and an organic phase. Generally, according to the retention mechanism of HILIC chromatography, the proportion of H2O is usually not less than 3%. In the present application, since the target spermidine to be extracted has a strong hydrophilicity, the proportion of aqueous phase in the mobile phase used for elution should not be too high, and specifically 5% H2O is used in this embodiment. For the organic phase, compared with methanol, acetonitrile has a weaker elution ability in HILIC, so 95% ACN is used as the organic phase in this embodiment. In addition, TFA is used as a buffer in this embodiment to maintain the degree of ionization of spermidine. The inventors found that compared with common buffer systems such as ammonium acetate, mobile phases containing TFA have better purification performance.

[0055] The detector is an ultraviolet detector, and the data is obtained by detection at a wavelength of 200 nm.

[0056] 3. Results Analysis

[0057] According to the liquid phase external standard method, the purity of spermidine obtained after extraction and purification in this embodiment is 90.2%, and the recovery rate is 49.6%. Among them, the spermidine standard used in the liquid phase external standard method is from Macklin, S817735-1g, with a purity of 99%.

[0058] The HILIC-HPLC chromatogram of spermidine obtained by the above method is shown in Figure 1 Correspondingly, the HILIC-HPLC chromatogram obtained using spermidine standard (Macklin, S817735-1g, 99% purity) as reference is shown in Figure 2 .

[0059] according to Figure 1 and Figure 2It can be seen from the retention time shown in that hydrophilic interaction liquid chromatography can accurately separate and purify spermidine from the crude extract of wheat germ, and the acquisition rate of spermidine can be guaranteed.

[0060] Figure 3 To verify the mass spectrum of the components contained in the finally collected fractions, it can be seen from the figure that the fraction obtained after hydrophilic interaction liquid chromatography purification is high-purity spermidine.

[0061] The above data show that Example 1 achieves efficient separation of various components in the crude extract and obtains a high-purity spermidine product.

[0062] In summary, Example 1 first uses ultrasound-assisted extraction technology, and utilizes the mechanical effect and cavitation effect of ultrasound to improve the permeability of the cell wall of wheat germ cells, accelerate the release of spermidine, and greatly improve the leaching and dissolution of spermidine by limited solvents, thereby significantly improving the extraction efficiency. Secondly, the crude extract obtained is purified and separated in combination with HILIC-HPLC technology, which is an efficient technology specifically used for separating and purifying polar compounds. The polar groups such as hydroxyl and amino groups on the surface of the HILIC stationary phase can form hydrogen bonds, ion exchange and other effects with the polar compound spermidine, thereby achieving efficient separation of spermidine with good separation effect. Example 1 organically combines probe-type ultrasound-assisted extraction and HILIC-HPLC purification technology to achieve efficient extraction and separation of high-purity spermidine products from wheat germ, a natural source.

[0063] Example 2

[0064] Embodiment 2 of the present invention provides a method for extracting spermidine from wheat germ, comprising the following steps:

[0065] 1. Extraction of spermidine

[0066] The purification method of Example 2 is consistent with that of Example 1, but the ultrasonic extraction conditions are slightly changed.

[0067] Weigh 2.5 g of wheat germ powder, add 100 mL of 50% ethanol solution and mix thoroughly. Use a probe ultrasonic disruptor (Ningbo Xinzhi, JY92-IIN) to perform ultrasonic-assisted extraction on the mixture under ice bath conditions. The ultrasonic parameters are: 10 mm horn probe, power 400 w, ultrasonic 1.5 s, stop 1.5 s, and the total ultrasonic time is 20 min. Subsequently, centrifuge at 4 ° C, 18000 x g for 10 min, collect the supernatant, and freeze-dry to obtain a crude extract in a dry powder state.

[0068] 2. Purification of spermidine

[0069] The crude extract was mixed with 25 mL of 95% ACN, 5% H2O, 0.1% TFA solution and dissolved by water bath ultrasound for 5 min. Subsequently, the supernatant was collected at 4°C, 18000xg for 5 min. The supernatant was filtered through a 0.45 μm PTFE filter and centrifuged at 4°C, 18000xg for 3 min. The supernatant was loaded onto a chromatographic column (Hungpu XBT Amide, 250 mm*4.6 mm, 5 μm) for purification. 95% ACN, 5% H2O, 0.1% TFA was used as the mobile phase for isocratic elution for 20 min.

[0070] 3. Results Analysis

[0071] According to the liquid phase external standard method, the purity of spermidine obtained after extraction and purification in this embodiment is 89.1%, and the recovery rate is 50.2%. Among them, the spermidine standard used in the liquid phase external standard method is from Macklin, S817735-1g, with a purity of 99%.

[0072] Comparative Example 1

[0073] The comparative example of the present application provides a method for extracting spermidine from wheat germ, comprising the following steps:

[0074] 1. Extraction of spermidine

[0075] The extraction method of Comparative Example 1 is consistent with that of Example 1.

[0076] Weigh 0.5 g of wheat germ powder, add 20 mL of 50% ethanol solution and mix thoroughly. Use a probe ultrasonic disruptor (Ningbo Xinzhi, JY92-IIN) to perform ultrasonic-assisted extraction on the mixture under ice bath conditions. The ultrasonic parameters are: power 250w, ultrasonic 1.5s, stop 1.5s, and total ultrasonic time is 10min. Subsequently, centrifuge at 4°C, 18000xg for 10min, collect the supernatant, and freeze-dry to obtain a crude extract in a dry powder state.

[0077] 2. Purification of spermidine

[0078] In this comparative example, RP-HPLC (Reversed-Phase High-Performance Liquid Chromatography) was used for purification.

[0079] The crude extract was mixed with 5 mL of 5% ACN, 95% H2O, 0.1% TFA solution and dissolved by water bath ultrasound for 5 min. Subsequently, the supernatant was collected at 4°C, 18000xg for 5 min, filtered through a 0.45μm PTFE filter, and then centrifuged at 4°C, 18000xg for 3 min. The supernatant was loaded onto a Shim-pack GIST C18 (250mm*4.6mm, 5μm) reverse phase column for purification. A gradient elution of 5%-50% ACN was used.

[0080] In this comparative example, due to the strong hydrophilicity of spermidine, conventional reverse phase chromatography has poor retention effect on it, and most of the spermidine cannot effectively bind to the chromatographic column. After loading, it elutes together with other impurities, and effective purification is not achieved.

Claims

1. A method for extracting spermidine from a natural source, characterized in that The method comprises the following steps: (1) extracting a natural source to obtain a crude extract; and (2) Purifying the crude extract by hydrophilic interaction liquid chromatography to obtain spermidine.

2. The method according to claim 1, characterized in that In step (1), the extraction is performed using ultrasonic-assisted extraction.

3. The method according to claim 2, characterized in that The ultrasonic assisted extraction comprises the following steps: (1-1) mixing the natural source, preferably in powder form, with an extraction solution and extracting using an ultrasonic disruptor; and (1-2) Separation, collecting the supernatant and drying to obtain the crude extract.

4. The method according to claim 3, characterized in that In step (1-1), the ultrasonic disruptor is selected from a water bath ultrasonic device or a probe ultrasonic device, preferably a probe ultrasonic device, such as Ningbo Xinzhi, JY92-IIN ultrasonic device; and / or The extraction solution is 50% ethanol.

5. The method according to claim 3, characterized in that: In step (1-2), The separation was performed by centrifugation at 4°C and 18,000 x g for 10 minutes; and / or The drying is performed by freeze drying.

6. The method according to claim 2, characterized in that In step (1), the conditions of the ultrasound-assisted extraction step are as follows: The ultrasonic power is 250-400W; the ultrasonic frequency is 1.5 seconds for each ultrasonication and 1.5 seconds for rest; the total ultrasonic time is 10-20 minutes.

7. The method according to any one of claims 1 to 6, characterized in that In step (2), the conditions of the hydrophilic interaction liquid chromatography are as follows: A Shimadzu LC-20AR liquid phase system was used; a Hungpu XBT Amide column was selected as the chromatographic column; the chromatographic column size was 250 mm*4.6 mm, 5 μm; the column temperature was 25°C; the mobile phase included 95% acetonitrile, 5% water and 0.1% trifluoroacetic acid; the flow rate was 1 mL / min; isocratic elution was adopted; and the elution time was 20 minutes.

8. The method according to claim 7, characterized in that The step (2) comprises the following steps: (2-1) mixing and dissolving the crude extract obtained in step (1) with a mobile phase; (2-2) centrifuging and collecting the supernatant, followed by filtration; and (2-3) Centrifuging the supernatant, loading the supernatant onto a chromatography column, and performing isocratic elution to obtain spermidine.

9. The method according to claim 8, characterized in that In the step (2-1), the volume / weight ratio of the mobile phase to the natural source is 1:35 to 1:45 and / or In the step (2-1), the dissolution is performed by ultrasound, preferably water bath ultrasound for 5 minutes; and / or In the step (2-2), the centrifugation condition is 4°C, 18000xg for 5 minutes; and / or In the step (2-2), the filtration is performed using a filter membrane, preferably a 0.45 μm filter membrane, and preferably a PTFE filter membrane; and / or In the step (2-3), the centrifugation condition is 4°C, 18000xg for 3 minutes.

10. The method according to any one of claims 1 to 6, characterized in that: The natural source is selected from wheat germ, natto and animal liver, preferably wheat germ.

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