CN112778138A - Method for extracting spermidine from animal muscle tissue - Google Patents
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- CN112778138A CN112778138A CN202110032398.7A CN202110032398A CN112778138A CN 112778138 A CN112778138 A CN 112778138A CN 202110032398 A CN202110032398 A CN 202110032398A CN 112778138 A CN112778138 A CN 112778138A
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Abstract
The invention discloses a method for extracting spermidine from animal muscle tissues, which comprises the following steps: s1, homogenizing animal muscle tissues, adding sodium chloride, and performing ultrasonic extraction to obtain a mixture; s2, centrifugally separating the mixture obtained in the step S1 to obtain a precipitate and a supernatant; s3, adding the supernatant obtained in the step S2 into 60% ethanol, and filtering to obtain a first filtrate; s4, adding 60% ethanol into the precipitate obtained in the step S2, then carrying out ultrasonic extraction, and filtering to obtain a second filtrate; s5, combining the first filtrate obtained in the step S3 and the second filtrate obtained in the step S4, performing 60% ethanol reflux extraction on the combined filtrates, then performing reduced pressure concentration, purifying the obtained concentrated solution, and performing freeze drying to obtain the product. The method can quickly and efficiently extract and separate the spermidine from the animal muscle tissue, and has the advantages of simple and convenient operation, high yield and good purity.
Description
Technical Field
The invention belongs to the technical field of detection and analysis, and particularly relates to a method for extracting spermidine from animal muscle tissues.
Background
Spermidine is a low molecular weight aliphatic compound with 3 amino groups, one of the natural polyamines widely distributed in all organisms. Spermidine is widely distributed in various tissues and organs such as brains, cerebellums, hearts, kidneys, lungs, intestinal tracts and the like of animal bodies, and has important regulation and control effects on cell proliferation, differentiation, aging and death processes. Studies show that spermidine can reduce inflammatory cell infiltration, inhibit inflammatory factor expression, and induce autophagy. In addition, spermidine also has important functions of resisting oxidation, depression and tumor, relieving brain injury and the like. Therefore, the search for a rapid and efficient spermidine extraction method has important theoretical and practical significance for clarifying the biological functions and action mechanisms of natural spermidine for regulating animal growth and development, and preventing and treating cancers.
Spermidine is widely distributed in organisms such as animals, plants, bacteria and fungi, and has important regulation and control effects on maintaining homeostasis. Among them, animal muscle and visceral tissues, soybeans and fermented bean products, and some mushrooms have a high spermidine content. Spermidine in the organism is mostly ingested from food, and a small part is synthesized and biosynthesized by intestinal flora. Research shows that the polyamine content in animals and human bodies is reduced along with the increase of age, but the polyamine content in the elderly with long life is close to that in the young and the middle-aged. Increasing polyamine intake can increase endogenous polyamine content in organism, and relieve cardiovascular diseases caused by aging by inducing autophagy, thereby achieving antiaging and life prolonging effects. Therefore, the supplement of spermidine in the daily diet can prevent diseases and prolong the life. In addition, studies have shown that oral administration of spermidine can reduce the incidence of chemically induced hepatocellular carcinoma and liver fibrosis in mice and can prolong the lifespan by 25%. Studies have shown that adequate supplementation of mice and the elderly with spermidine-rich extracts is safe and well tolerated. Therefore, the natural spermidine extract has wide application prospect in the fields of disease prevention and aging delay. The search for a rapid and efficient spermidine extraction method has important theoretical significance and practical significance. However, the methods for extracting spermidine from animal tissues are currently lacking.
Disclosure of Invention
The invention aims to: in view of the above-mentioned deficiencies of the prior art, a method for extracting spermidine from animal muscle tissue is provided.
The technical scheme adopted by the invention is as follows:
a method for extracting spermidine from animal muscle tissue, comprising the steps of:
s1, mixing animal muscle tissue with water, homogenizing at-4 ℃ for 1-2h, adding sodium chloride, mixing uniformly, performing ultrasonic extraction at 40-70 ℃ for 10-30min, and standing at-4 ℃ for 10-20 min; ultrasonic extracting at 40-70 deg.C for 10-30min, and standing at-4 deg.C for 10-20 min; then carrying out ultrasonic extraction at 40-70 ℃ for 10-30min to obtain a mixture;
s2, centrifugally separating the mixture obtained in the step S1 to obtain a precipitate and a supernatant;
s3, adding the supernatant obtained in the step S2 into 60% ethanol, stirring for 10-15min, and filtering to obtain a first filtrate;
s4, adding 60% ethanol into the precipitate obtained in the step S2, then carrying out ultrasonic extraction for 30-60min at the temperature of 40-70 ℃, repeating the ultrasonic extraction process for 2-3 times, and filtering to obtain a second filtrate;
s5, combining the first filtrate obtained in the step S3 and the second filtrate obtained in the step S4, performing reflux extraction on the combined filtrates by 60% ethanol for 2-3 times, then performing reduced pressure concentration, purifying the obtained concentrated solution, and performing freeze drying.
The invention fully grinds and crushes animal muscle tissues, puts the animal muscle tissues in a salt solution, and then uses the salt solution to facilitate the extraction of spermidine through the alternate low-temperature standing and ultrasonic extraction technology, on the one hand, the low temperature can maintain the activity of active ingredients in the muscle tissues to a certain degree, and the reduction of yield caused by the combination or reaction of the inactivation or deterioration of the active ingredients and the spermidine is avoided, meanwhile, the ultrasonic extraction further enhances the extraction effect, and more spermidine can be released in the muscle tissues. And after centrifugal separation, removing the salt in the precipitate and the supernatant respectively, further extracting spermidine, and then refluxing, concentrating and drying to obtain high-purity spermidine.
Further, the mass ratio of the animal muscle tissue to the water in S1 is 1: 1-2; preferably 1:1.
Further, the concentration of sodium chloride in the mixture is 50-70 wt%; preferably 60 wt%.
Further, the centrifugal separation conditions in S2 were: 3000-.
Further, the centrifugal separation conditions in S2 were: 3000rpm, 15 min.
Further, the volume ratio of the supernatant to the 60% ethanol in S3 is 1: 3-5; preferably 1: 4.
Further, the mass ratio of the precipitate in the S4 to the 60% ethanol is 1: 2-3; preferably 1: 3.
Further, the volume ratio of the filtrate to the ethanol in the S5 is 1: 3-6; preferably 1: 5.
Further, the temperature of reflux extraction is 75-85 ℃, and the time of each reflux extraction is 3-4 h.
Further, the temperature of the reflux extraction was 80 ℃ and the time of each reflux extraction was 3 hours.
Further, the temperature of freeze-drying in S5 was-80 ℃.
Further, the concentrated solution was subjected to medium pressure rapid purification through a medium pressure preparative column of C18.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
the invention is beneficial to enhancing the extraction rate of the salt solution, simultaneously adopts low temperature to avoid the inactivation or deterioration of active ingredients and the combination or reaction of spermidine to cause the reduction of the yield, and ultrasonic extraction further enhances the extraction effect and more spermidine can be released in muscle tissues. The product obtained by the method has the advantages of high yield of about 95.33 percent through detection, few detected hybrid peaks and high purity of spermidine. In addition, the method has the advantages of simple operation, high purity, high repeatability, safe and controllable extraction reagent, low cost, short extraction time, high efficiency, large amount of extracted samples in unit time and high purity of spermidine.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a liquid chromatogram of a sample prepared in example 1;
fig. 2 is a graph of the operating curve.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The preferred embodiment of the present invention provides a method for extracting spermidine from animal muscle tissue, comprising the following steps:
s1, mixing animal muscle tissue and water according to a mass ratio of 1:1, homogenizing at-4 ℃ for 1h, adding sodium chloride, uniformly mixing to enable the concentration of the sodium chloride in the mixture to be 60 wt%, then carrying out ultrasonic extraction at 50 ℃ for 15min, and then placing at-4 ℃ for 15 min; ultrasonic extracting at 50 deg.C for 15min, and standing at-4 deg.C for 15 min; then carrying out ultrasonic extraction at 50 ℃ for 15min to obtain a mixture;
s2, centrifuging the mixture obtained in the step S1 at 3000rpm for 15min to obtain a precipitate and a supernatant;
s3, adding the supernatant obtained in the step S2 into 60% ethanol, stirring for 12min, and filtering to obtain a first filtrate; wherein the volume ratio of the supernatant to 60% ethanol is 1: 4;
s4, adding 60% ethanol into the precipitate obtained in the step S2, then carrying out ultrasonic extraction for 45min at the temperature of 60 ℃, repeating the ultrasonic extraction process for 2 times, and filtering to obtain a second filtrate; wherein the mass ratio of the precipitate to the 60% ethanol is 1: 3;
s5, combining the first filtrate obtained in the step S3 and the second filtrate obtained in the step S4, performing reflux extraction on the combined filtrates for 3h and 2 times at 80 ℃ by using 60% ethanol, then performing reduced pressure concentration, performing medium pressure rapid purification on the obtained concentrated solution by using a C18 medium pressure preparation column, and performing freeze drying at-80 ℃ to obtain the compound; wherein the volume ratio of the filtrate to the ethanol is 1: 5.
Example 2
The preferred embodiment of the present invention provides a method for extracting spermidine from animal muscle tissue, comprising the following steps:
s1, mixing animal muscle tissue and water according to a mass ratio of 1:1.5, homogenizing at-4 ℃ for 2h, adding sodium chloride, uniformly mixing to make the concentration of the sodium chloride in the mixture to be 55 wt%, then carrying out ultrasonic extraction at 60 ℃ for 10min, and then placing at-4 ℃ for 15 min; ultrasonic extracting at 60 deg.C for 10min, and standing at-4 deg.C for 15 min; then carrying out ultrasonic extraction at 60 ℃ for 10min to obtain a mixture;
s2, centrifuging the mixture obtained in the step S1 at 4000rpm for 12min to obtain a precipitate and a supernatant;
s3, adding the supernatant obtained in the step S2 into 60% ethanol, stirring for 10min, and filtering to obtain a first filtrate; wherein the volume ratio of the supernatant to 60% ethanol is 1: 3;
s4, adding 60% ethanol into the precipitate obtained in the step S2, then carrying out ultrasonic extraction for 35min at the temperature of 60 ℃, repeating the ultrasonic extraction process for 3 times, and filtering to obtain a second filtrate; wherein the mass ratio of the precipitate to the 60% ethanol is 1: 2;
s5, combining the first filtrate obtained in the step S3 and the second filtrate obtained in the step S4, performing reflux extraction on the combined filtrates for 3 hours and 3 times at 80 ℃ by using 60% ethanol, then performing reduced pressure concentration, performing medium pressure rapid purification on the obtained concentrated solution by using a C18 medium pressure preparation column, and performing freeze drying at-80 ℃ to obtain the compound; wherein the volume ratio of the filtrate to the ethanol is 1: 4.
Example 3
The preferred embodiment of the present invention provides a method for extracting spermidine from animal muscle tissue, comprising the following steps:
s1, mixing animal muscle tissue and water according to a mass ratio of 1:2, homogenizing at-4 ℃ for 1h, adding sodium chloride, uniformly mixing to enable the concentration of the sodium chloride in the mixture to be 65 wt%, then carrying out ultrasonic extraction at 65 ℃ for 20min, and then placing at-4 ℃ for 15 min; ultrasonic extracting at 65 deg.C for 20min, and standing at-4 deg.C for 15 min; then carrying out ultrasonic extraction at 65 ℃ for 20min to obtain a mixture;
s2, centrifuging the mixture obtained in the step S1 at 5000rpm for 10min to obtain a precipitate and a supernatant;
s3, adding the supernatant obtained in the step S2 into 60% ethanol, stirring for 15min, and filtering to obtain a first filtrate; wherein the volume ratio of the supernatant to 60% ethanol is 1: 3;
s4, adding 60% ethanol into the precipitate obtained in the step S2, then carrying out ultrasonic extraction at 65 ℃ for 55min, repeating the ultrasonic extraction process for 2 times, and filtering to obtain a second filtrate; wherein the mass ratio of the precipitate to the 60% ethanol is 1: 3;
s5, combining the first filtrate obtained in the step S3 and the second filtrate obtained in the step S4, performing reflux extraction on the combined filtrates for 3h and 2 times at 80 ℃ by using 60% ethanol, then performing reduced pressure concentration, performing medium pressure rapid purification on the obtained concentrated solution by using a C18 medium pressure preparation column, and performing freeze drying at-80 ℃ to obtain the compound; wherein the volume ratio of the filtrate to the ethanol is 1: 6.
Comparative example 1
A method for extracting spermidine from animal muscle tissue comprises the following steps:
mixing animal muscle tissue and water at a mass ratio of 1:1, homogenizing at-4 deg.C for 1h, adding sodium chloride, mixing to make the concentration of sodium chloride in the mixture 60 wt%, ultrasonic extracting at 50 deg.C for 15min, and standing at-4 deg.C for 15 min; ultrasonic extracting at 50 deg.C for 15min, and standing at-4 deg.C for 15 min; then carrying out ultrasonic extraction at 50 ℃ for 15min to obtain a mixture; adding the mixture into 60% ethanol, stirring for 15min, and filtering to obtain filtrate; reflux-extracting the filtrate with 60% ethanol at 80 deg.C for 3 hr and 2 times, concentrating under reduced pressure, subjecting the concentrated solution to medium-pressure rapid purification with C18 medium-pressure preparative column, and freeze-drying at-80 deg.C; wherein the volume ratio of the filtrate to the ethanol is 1: 5.
Comparative example 2
A method for extracting spermidine from animal muscle tissue comprises the following steps:
s1, mixing animal muscle tissue and water according to a mass ratio of 1:1, homogenizing at-4 ℃ for 1h, adding sodium chloride, uniformly mixing to enable the concentration of the sodium chloride in the mixture to be 60 wt%, and performing ultrasonic extraction at 50 ℃ for 15min to obtain a mixture;
s2, centrifuging the mixture obtained in the step S1 at 3000rpm for 15min to obtain a precipitate and a supernatant;
s3, adding the supernatant obtained in the step S2 into 60% ethanol, stirring for 12min, and filtering to obtain a first filtrate; wherein the volume ratio of the supernatant to 60% ethanol is 1: 4;
s4, adding 60% ethanol into the precipitate obtained in the step S2, then carrying out ultrasonic extraction for 45min at the temperature of 60 ℃, repeating the ultrasonic extraction process for 2 times, and filtering to obtain a second filtrate; wherein the mass ratio of the precipitate to the 60% ethanol is 1: 3;
s5, combining the first filtrate obtained in the step S3 and the second filtrate obtained in the step S4, performing reflux extraction on the combined filtrates for 3h and 2 times at 80 ℃ by using 60% ethanol, then performing reduced pressure concentration, performing medium pressure rapid purification on the obtained concentrated solution by using a C18 medium pressure preparation column, and performing freeze drying at-80 ℃ to obtain the compound; wherein the volume ratio of the filtrate to the ethanol is 1: 5.
Comparative example 3
A method for extracting spermidine from animal muscle tissue comprises the following steps:
mixing animal muscle tissue and water according to a mass ratio of 1:1, homogenizing at-4 deg.C for 1h, adding sodium chloride, mixing to make the concentration of sodium chloride in the mixture be 60 wt%, and performing ultrasonic extraction at 50 deg.C for 15min to obtain a mixture; adding the mixture into 60% ethanol, stirring for 15min, and filtering to obtain filtrate; reflux-extracting the filtrate with 60% ethanol at 80 deg.C for 3 hr and 2 times, concentrating under reduced pressure, subjecting the concentrated solution to medium-pressure rapid purification with C18 medium-pressure preparative column, and freeze-drying at-80 deg.C; wherein the volume ratio of the filtrate to the ethanol is 1: 5.
Examples of the experiments
Accurately weighing 0.0158g of spermidine standard from a spermidine standard stock solution, dissolving the spermidine standard in water, respectively metering the volume to 10mL volumetric flasks, accurately transferring 1mL of the two solutions into 2 10mL volumetric flasks, metering the volume to the scale by using water, and taking the solution as a standard use solution, wherein the content of spermidine in the stock solution is 1mmoL/mL.
Putting 40uL of spermidine standard use solution into a 10mL plastic centrifuge tube with a cover, adding 7uL of benzoyl chloride, adding 1mL of 2mol/LNaOH, swirling for 20s, reacting in a water bath at 37 ℃ for 20min, adding 2mL of saturated NaCI solution, uniformly mixing, extracting with 2mL of diethyl ether, centrifuging on a centrifuge (1500/min) for 5min, taking 1mL of ether phase into a 1mL centrifuge tube, and drying in a water bath at 37 ℃. Dissolving with 100uL methanol by vortex, taking filtrate to prepare a mixed solution standard series with spermidine concentrations of 0.020, 0.010, 0.007, 0.005 and 0.004nmo/uL, taking 10 uL of sample, analyzing and measuring by liquid chromatography, and drawing a working curve by peak area to concentration.
According to the detection, 0.2207mg of spermidine can be extracted from 1.0g of pig muscle tissue by the method of example 1, and the actual content of spermidine detected by high performance liquid chromatography in the same pig muscle tissue sample is 0.2315mg, 95.33% of spermidine can be extracted from pig muscle tissue by the method, while the yield of spermidine extracted in general is about 85%, the yield of comparative example 1 is 90.25%, the yield of comparative example 2 is 88.65%, and the yield of comparative example 3 is 86.04%, and the yield obtained by the method is high, and in addition, the detected impurity peaks are few, and the purity is high.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A method for extracting spermidine from animal muscle tissue, comprising the steps of:
s1, mixing animal muscle tissue with water, homogenizing at-4 ℃ for 1-2h, adding sodium chloride, mixing uniformly, performing ultrasonic extraction at 40-70 ℃ for 10-30min, and standing at-4 ℃ for 10-20 min; ultrasonic extracting at 40-70 deg.C for 10-30min, and standing at-4 deg.C for 10-20 min; then carrying out ultrasonic extraction at 40-70 ℃ for 10-30min to obtain a mixture;
s2, centrifugally separating the mixture obtained in the step S1 to obtain a precipitate and a supernatant;
s3, adding the supernatant obtained in the step S2 into 60% ethanol, stirring for 10-15min, and filtering to obtain a first filtrate;
s4, adding 60% ethanol into the precipitate obtained in the step S2, then carrying out ultrasonic extraction for 30-60min at the temperature of 40-70 ℃, repeating the ultrasonic extraction process for 2-3 times, and filtering to obtain a second filtrate;
s5, combining the first filtrate obtained in the step S3 and the second filtrate obtained in the step S4, performing reflux extraction on the combined filtrates by 60% ethanol for 2-3 times, then performing reduced pressure concentration, purifying the obtained concentrated solution, and performing freeze drying.
2. The method for extracting spermidine from animal muscle tissue according to claim 1, wherein the mass ratio of animal muscle tissue to water in S1 is 1: 1-2.
3. A process for the extraction of spermidine from animal muscle tissue as claimed in claim 2, characterized in that the concentration of sodium chloride in the mixture is comprised between 50 and 70% by weight.
4. The method for extracting spermidine from animal muscle tissue as claimed in claim 1, wherein the centrifugation conditions in S2 are: 3000-.
5. The method for extracting spermidine from animal muscle tissue as claimed in claim 1, wherein the volume ratio of supernatant to 60% ethanol in S3 is 1: 3-5.
6. The method for extracting spermidine from animal muscle tissue as claimed in claim 1, wherein the mass ratio of the precipitate in S4 to 60% ethanol is 1: 2-3.
7. The method for extracting spermidine from animal muscle tissue as claimed in claim 1, wherein the volume ratio of the filtrate to ethanol in S5 is 1: 3-6.
8. A process for the extraction of spermidine from animal muscle tissue as claimed in claim 7 wherein the temperature of the reflux extraction is 75-85 ℃ and the time of each reflux extraction is 3-4 hours.
9. The method for extracting spermidine from animal muscle tissue as claimed in claim 1, wherein the temperature of freeze drying in S5 is-80 ℃.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114751830A (en) * | 2022-05-19 | 2022-07-15 | 珀莱雅化妆品股份有限公司 | Method for extracting high-purity spermidine from cowpea seedlings |
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