CN118324643A - Method for extracting spermidine from lycium ruthenicum and application of method - Google Patents
Method for extracting spermidine from lycium ruthenicum and application of method Download PDFInfo
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Abstract
The invention relates to the technical field of natural product chemistry and pharmacology, in particular to a method for extracting spermidine from lycium ruthenicum and application thereof, at least comprising the following steps: (1) Preprocessing the dried lycium ruthenicum fruit to obtain lycium ruthenicum powder; (2) Adding the lycium ruthenicum powder into distilled water, extracting with water and filtering to obtain lycium ruthenicum water extract; (3) Vacuum concentrating the Lycium ruthenicum Murr water extract to obtain concentrated solution, adding ethanol into the concentrated solution, controlling ethanol content in the concentrated solution, standing, and filtering to obtain precipitate and ethanol extract; (4) Vacuum concentrating the ethanol extract to obtain Lycium ruthenicum Murr extract; (5) Dissolving the lycium ruthenicum extract into a disperse phase to obtain a lycium ruthenicum extract solution, separating the lycium ruthenicum extract solution through macroporous resin column chromatography to obtain a lycium ruthenicum spermidine crude extract, and the method is simple and convenient to operate and high in extraction rate and product stability.
Description
Technical Field
The invention relates to the technical field of natural product chemistry and pharmacology, in particular to a method for extracting spermidine from lycium ruthenicum and application thereof.
Background
Lycium ruthenicum (school name: lycium ruthenicum Murr.) is a shrub plant grown in the alpine region of Asia and its fruit is rich in various nutrients including various vitamins, minerals and bioactive compounds. Traditionally, lycium ruthenicum is widely applied to the fields of foods and traditional Chinese medicines to enhance immunity, improve eyesight and the like.
Spermidine, a polyamine compound that was originally isolated from semen, is also present in many plants and plays an important physiological role in the organism. Past studies have shown that spermidine has a promoting effect on the growth and development of eukaryotic cells, and at the same time plays a positive role in cell metabolism, antioxidation, anti-inflammatory and the like. Recent researches show that spermidine can exhibit various physiological effects through induction of autophagy, which is a process of cell self-degradation, besides the traditional effect of promoting cell growth, and is important in the aspects of eliminating damaged organelles, inhibiting inflammatory reactions, maintaining intracellular homeostasis and the like. Spermidine is thought to promote lipid metabolism and enhance antioxidant activity by activating autophagy, thereby positively affecting the health and life of the body. Researchers have found that spermidine can effectively inhibit oxidative stress and extend the life of mice by administering spermidine in a mouse experiment. In addition, oral spermidine also activates microtubule-associated protein MAP 1S-mediated autophagy, prevents liver fibrosis and further prolongs the lifespan of the mice.
Although the extraction of spermidine and its derivatives from plants has been widely studied in the past, the study on lycium ruthenicum spermidine has been relatively weak, and the method still needs further optimization and improvement, for example, chinese patent (publication No. CN117304054 a) discloses a method for enriching caffeoylspermidine compound, its anti-aging property and a method for inhibiting histone deacetylase, and 75% aqueous ethanol solution is used as an extractant for enriching caffeoylspermidine compound from dry lycium ruthenicum fruits, but the yield of the extract is lower and stability is still to be further improved.
Disclosure of Invention
In order to solve the problems, the invention provides a method for extracting spermidine from lycium ruthenicum and application thereof, the method is simple and convenient to operate, the extraction efficiency is high, the spermidine extract can be effectively obtained from the lycium ruthenicum, and convenience is provided for further development and utilization of pharmacological activity of the lycium ruthenicum.
In one aspect, the invention provides a method for extracting spermidine from lycium ruthenicum, which at least comprises the following steps:
(1) Preprocessing the dried lycium ruthenicum fruit to obtain lycium ruthenicum powder;
(2) Adding the lycium ruthenicum powder into distilled water, extracting with water and filtering to obtain lycium ruthenicum water extract;
(3) Vacuum concentrating the Lycium ruthenicum Murr water extract to obtain concentrated solution, adding ethanol into the concentrated solution, controlling ethanol content in the concentrated solution, standing, and filtering to obtain precipitate and ethanol extract;
(4) Vacuum concentrating the ethanol extract to obtain Lycium ruthenicum Murr extract;
(5) Dissolving the lycium ruthenicum extract into a disperse phase to obtain a lycium ruthenicum extract solution, and separating the lycium ruthenicum extract solution through macroporous resin column chromatography to obtain a lycium ruthenicum spermidine crude extract.
As a preferable technical scheme, the pretreatment in the step (1) is as follows: cleaning dried Lycium ruthenicum Murr, removing impurities, pulverizing, sieving with 80-120 mesh sieve, and drying to obtain Lycium ruthenicum Murr powder.
As a preferable technical scheme, in the step (2), the mass ratio of the dried lycium ruthenicum to the distilled water is 1: (10-20), preferably 1:10.
As a preferable technical scheme, the temperature of the water extraction in the step (2) is 70-90 ℃ and the time is 2-3h.
As a preferable technical scheme, the volume ratio of the lycium ruthenicum water extract and the concentrated solution in the step (3) is (8-12): 1.
Preferably, the ethanol content in the concentrated solution in the step (3) is 30-60wt%, and the standing time is more than or equal to 12h.
Preferably, the dispersed phase in the step (5) is at least one of formic acid aqueous solution, acetic acid aqueous solution and citric acid aqueous solution, preferably formic acid aqueous solution, and the formic acid aqueous solution is preferably 0.5-1wt% formic acid aqueous solution.
As a preferable technical scheme, the macroporous resin column chromatography separation in the step (5) is as follows: and (3) after pretreatment of macroporous resin, loading columns by adopting a wet method, loading the lycium ruthenicum extract solution onto columns for adsorption, and eluting by adopting an eluent to obtain the lycium ruthenicum spermidine crude extract.
Preferably, the eluting with the eluent comprises impurity eluting and lycium ruthenicum mill spermidine crude extract eluting, wherein the eluent in the impurity eluting is ethanol water solution containing 1wt% of formic acid, and the concentration of ethanol in the ethanol water solution is controlled to be 10wt%; the eluent in the elution of the lycium ruthenicum mill spermidine crude extract is ethanol water solution containing 1% formic acid, and the concentration of ethanol in the ethanol water solution is controlled to be 20-40wt%.
In order to realize the extraction of spermidine from lycium ruthenicum, the application designs the dry lycium ruthenicum as a raw material, and sequentially carries out dosage form pretreatment, water extraction, alcohol extraction and macroporous resin column chromatography separation to obtain the crude extract of the spermidine from lycium ruthenicum, and the crude extract of the spermidine from lycium ruthenicum can be effectively applied to the preparation of anti-aging products. The parameter conditions of each step are further controlled, so that the method has higher stability on the basis of ensuring the yield of the prepared lycium ruthenicum spermidine crude extract, and can realize large-scale industrial production and better meet the subsequent application requirements. The conventional water extraction method, ethanol thermal reflux method and supercritical fluid extraction method are used for guaranteeing the stability of spermidine while not obtaining higher yield when extracting spermidine from dried lycium ruthenicum, and the ethanol thermal reflux method and the supercritical fluid extraction method have higher cost and are difficult to realize large-scale industrial production application. Finally, dissolving the lycium ruthenicum mill extract into a disperse phase, and then separating by adopting macroporous resin column chromatography to obtain a lycium ruthenicum mill spermidine crude extract, particularly, eluting by adopting a 10wt% ethanol aqueous solution containing 1wt% of formic acid to remove impurities, and eluting by adopting a 20-40wt% ethanol aqueous solution containing 1wt% of formic acid to obtain the lycium ruthenicum mill spermidine crude extract, thereby reducing the influence of impurities on spermidine stability and obtaining higher yield.
The invention also provides an application of the spermidine extracted by the method in preparing an anti-aging product, wherein the anti-aging product is one of an oral preparation, an external preparation and an injection.
Advantageous effects
1. The invention provides a method for extracting spermidine from lycium ruthenicum, which is simple and convenient to operate and high in extraction efficiency, can effectively obtain the spermidine extract from the lycium ruthenicum, and provides convenience for further developing and utilizing the pharmacological activity of the lycium ruthenicum.
2. In order to realize the extraction of spermidine from lycium ruthenicum, the invention designs the dry lycium ruthenicum as a raw material, and sequentially carries out dosage form pretreatment, water extraction, alcohol extraction and macroporous resin column chromatography separation to obtain the crude extract of the spermidine from lycium ruthenicum, and the crude extract of the spermidine from lycium ruthenicum can be effectively applied to the preparation of anti-aging products.
3. The method has higher stability on the basis of ensuring the yield of the prepared lycium ruthenicum mill spermidine crude extract by controlling the parameter conditions of each step, and can realize large-scale industrial production and better meet the subsequent application requirements.
4. According to the application, the lycium ruthenicum powder is obtained after pretreatment of the lycium ruthenicum dry fruits, the feed-liquid ratio, the extraction temperature and the extraction time are controlled, and the active ingredients in the lycium ruthenicum are dissolved out as much as possible on the basis of guaranteeing the extraction efficiency and the extract activity, so that the lycium ruthenicum water extract is obtained.
5. The ethanol extraction liquid containing the lycium ruthenicum extract is obtained by further concentrating the lycium ruthenicum aqueous extract and then carrying out ethanol extraction, wherein the ethanol content in the concentrated solution is controlled to be 30-60wt% and the concentrated solution is kept stand for 24 hours at normal temperature (20-30 ℃), so that the high cost caused by the use of a large amount of high-purity high-concentration ethanol is avoided, and the influence of high-concentration ethanol hot reflux extraction and high-temperature water extraction on the stability of the extract is avoided.
6. Dissolving the lycium ruthenicum mill extract into a disperse phase, and then separating by adopting macroporous resin column chromatography to obtain a lycium ruthenicum mill spermidine crude extract, particularly, eluting by adopting a 10wt% ethanol aqueous solution containing 1wt% of formic acid to remove impurities, and eluting by adopting a 20-40wt% ethanol aqueous solution containing 1wt% of formic acid to obtain the lycium ruthenicum mill spermidine crude extract, thereby reducing the influence of impurities on spermidine stability and simultaneously obtaining higher yield.
Detailed Description
Example 1
Embodiment 1 of the present invention provides a method for extracting spermidine from lycium ruthenicum, comprising the following steps:
(1) Preprocessing 500g of dried lycium ruthenicum murr to obtain lycium ruthenicum powder;
(2) Adding the lycium ruthenicum powder into 5L of distilled water, extracting with water and filtering to obtain 4.5L of lycium ruthenicum water extract; (3) Vacuum concentrating the Lycium ruthenicum Murr water extract to obtain 500mL of concentrate, adding 1.3L of ethanol into the concentrate, controlling the ethanol content in the concentrate, standing, and filtering to obtain 100g of precipitate and 1.4L of ethanol extract;
(4) Concentrating the ethanol extract in vacuum to obtain 50g of lycium ruthenicum extract;
(5) Dissolving the lycium ruthenicum extract into a disperse phase to obtain a lycium ruthenicum extract solution, and separating the lycium ruthenicum extract solution through macroporous resin column chromatography to obtain 5g of lycium ruthenicum spermidine crude extract.
The pretreatment in the step (1) is as follows: cleaning dried Lycium ruthenicum Murr, removing impurities, pulverizing, sieving with 100 mesh sieve, and drying to obtain Lycium ruthenicum Murr powder.
The mass ratio of the dried lycium ruthenicum fruit to the distilled water in the step (2) is 1:10.
The temperature of the water extraction in the step (2) is 70 ℃ and the time is 3 hours.
In the step (3), the volume ratio of the lycium ruthenicum water extract to the concentrated solution is 9:1.
The ethanol content in the concentrated solution in the step (3) is 60 weight percent, and the standing time is 24 hours.
The disperse phase in the step (5) is formic acid aqueous solution, and the formic acid aqueous solution is 1wt% formic acid aqueous solution.
The macroporous resin column chromatography separation in the step (5) is as follows: and (3) after pretreatment of macroporous resin, loading columns by adopting a wet method, loading the lycium ruthenicum extract solution onto columns for adsorption, and eluting by adopting an eluent to obtain the lycium ruthenicum spermidine crude extract.
The eluting with eluent comprises impurity eluting and lycium ruthenicum mill spermidine crude extract eluting, wherein the eluent in the impurity eluting is ethanol water solution containing 1wt% of formic acid, and the concentration of ethanol in the ethanol water solution is controlled to be 10wt%; the eluent in the elution of the lycium ruthenicum mill spermidine crude extract is ethanol water solution containing 1wt% of formic acid, and the concentration of ethanol in the ethanol water solution is controlled to be 20-40wt%.
Comparative example 1
Comparative example 1 of the present invention provides a method for extracting spermidine from lycium ruthenicum, which comprises adding 500g of dried lycium ruthenicum into 10 times of water, heating to boil in water bath, keeping boiling for 30 minutes, filtering to obtain an aqueous extract, concentrating the aqueous extract in vacuum, and drying in vacuum to obtain 3g of spermidine crude extract.
Comparative example 2
Comparative example 2 of the present invention provides a method for extracting spermidine from lycium ruthenicum, which comprises adding 10 times mass of carbon dioxide into 500g of dried lycium ruthenicum, adjusting the temperature to 40 ℃ and the pressure to 30MPa, performing supercritical fluid extraction for 60 minutes, collecting the extract, and performing vacuum concentration to obtain 4.0g of spermidine crude extract.
Performance test method
1. The yields of crude spermidine extracts (yield refers to the ratio of the weight of crude spermidine extract to the weight of dried lycium ruthenicum fruit) obtained by the processes of examples and comparative examples were calculated and the results are shown in table 1.
2. After the crude spermidine extracts prepared by the processes of examples and comparative examples were stored at 25 ℃ for 30 days, stability (stability means the ratio of the spermidine content to the initial content of the crude spermidine extract after the crude spermidine extract was stored at normal temperature for 30 days) was calculated, and the results are shown in table 1.
TABLE 1
| Yield (%) | Stability (%) | |
| Example 1 | 1.0 | 95.6 |
| Comparative example 1 | 0.6 | 82.3 |
| Comparative example 2 | 0.8 | 88.7 |
As can be seen from Table 1, the yield and stability of the extracted spermidine are higher than those of comparative examples 1 and 2, which shows that the method has higher efficiency and better protection effect, and can effectively extract spermidine components with high purity and high activity from Lycium ruthenicum.
3. Evaluation of the effects of the lycium ruthenicum spermidine provided in example 1 on the intervention of the D-galactose-induced aging model in mice.
The purpose is as follows: the intervention effect of lycium ruthenicum mill spermidine on the D-galactose-induced mouse aging model is observed and compared with a positive control drug vitamin E.
Materials and methods: 60 healthy male Kunming mice of 8 weeks of age were selected and randomly divided into 6 groups of 10 animals each, which were control group, model group, positive control group, low dose group, medium dose group and high dose group, respectively. Except for the control group, mice of each group were daily intraperitoneally injected with D-galactose (100 mg/kg) to prepare an aging model, and the injections were continued for 4 weeks. Meanwhile, the positive control mice were given vitamin E (100 mg/kg) daily by intragastric administration, the low dose group, the medium dose group and the high dose group mice were given spermidine of the present invention (50 mg/kg, 100mg/kg and 200 mg/kg) daily by intragastric administration, and the control group and model group mice were given an equivalent amount of physiological saline daily by intragastric administration for 4 weeks. The weight change of the mice is recorded during the experiment period, blood, liver and brain tissue samples of the mice are collected after the experiment is finished, and indexes such as blood biochemical indexes (total cholesterol, triglyceride, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase and the like), liver antioxidant enzyme activities (superoxide dismutase, catalase, glutathione peroxidase and the like), brain tissue acetylcholinesterase activities and the like are measured.
Results and analysis: table 2 shows the weight change of mice in different treatment groups.
TABLE 2
| Group of | Before the experiment (g) | After the experiment (g) | Weight gain Rate (%) |
| Control group | 22.5 | 28.3 | 25.8 |
| Model group | 22.4 | 24.1 | 7.6 |
| Positive control group | 22.6 | 26.8 | 18.6 |
| Low dose group | 22.5 | 25.9 | 15.1 |
| Medium dose group | 22.7 | 27.2 | 19.8 |
| High dose group | 22.6 | 28.1 | 24.3 |
As can be seen from Table 2, the weight gain of the mice in the model group was significantly lower than that of the control group, indicating that D-galactose induced aging in the mice. The weight gain of mice in the positive control group and the spermidine treatment group with each dose is higher than that of mice in the model group, which indicates that the vitamin E and the spermidine can improve the growth state of the mice and delay aging. The weight gain of the mice in the high-dose spermidine treatment group is equivalent to that of the mice in the control group, and even slightly higher than that of the mice in the positive control group, so that the spermidine compound has a strong anti-aging effect.
Table 3 shows the blood biochemical index (mmol/L or U/L) of mice in different treatment groups.
TABLE 3 Table 3
As can be seen from Table 3, the total cholesterol, triglyceride, glutamic pyruvic transaminase and glutamic oxaloacetic transaminase levels of mice in the model group were higher than those of the control group, indicating that D-galactose damages liver function of mice, resulting in lipid metabolism disorder. The indexes of the positive control group and the mice in the spermidine treatment group with each dose are lower than those of the model group, which indicates that the vitamin E and spermidine compound can protect the liver function of the mice and improve the blood lipid metabolism. The indexes of the mice in the high-dose spermidine treatment group are similar to those of the mice in the control group, and even slightly lower than those of the mice in the positive control group, so that the spermidine has stronger anti-aging effect.
Table 4 shows the liver antioxidant enzyme activity (U/mg) of mice from different treatment groups.
TABLE 4 Table 4
As can be seen from Table 4, the liver antioxidant enzyme activities of the mice in the model group were lower than those of the control group, indicating that D-galactose increased the oxidative stress of the mice, resulting in peroxidative damage of the cell membrane. The liver antioxidant enzyme activity of mice in the positive control group and the spermidine treatment group with each dose is higher than that of mice in the model group, which indicates that the vitamin E and the spermidine can enhance the antioxidant capacity of the mice and reduce the peroxidation damage of cell membranes. The liver antioxidant enzyme activity of the mice in the high-dose spermidine treatment group is similar to that of the mice in the control group, and even slightly higher than that of the mice in the positive control group, so that the spermidine compound has a strong anti-aging effect.
Table 5 shows the activity (U/mg) of acetylcholinesterase in brain tissue of mice from different treatment groups.
TABLE 5
| Group of | Acetylcholinesterase (U/mg) |
| Control group | 0.8 |
| Model group | 0.4 |
| Positive control group | 0.7 |
| Low dose group | 0.6 |
| Medium dose group | 0.7 |
| High dose group | 0.8 |
As can be seen from Table 5, the brain tissue acetylcholinesterase activity of mice in the model group was lower than that of the control group, indicating that D-galactose impaired the nerve function of mice, resulting in a decrease in memory and learning ability. The brain tissue acetylcholinesterase activity of mice in the positive control group and the spermidine treatment group with each dose is higher than that of mice in the model group, which indicates that vitamin E and spermidine can protect the nerve function of the mice and improve the memory and learning ability. The brain tissue acetylcholinesterase activity of the mice in the high-dose spermidine treatment group is the same as that of the mice in the control group, and even slightly higher than that of the mice in the positive control group, so that the spermidine compound has a strong anti-aging effect.
In conclusion, the lycium ruthenicum mill spermidine disclosed by the invention can effectively interfere with a D-galactose-induced mouse aging model, improve indexes of physiological, biochemical and molecular levels of the D-galactose-induced mouse aging model, delay the aging process and has a quite even better effect compared with a positive control drug vitamin E. These experimental data or examples fully demonstrate the utility value of the spermidine of the invention in terms of anti-aging.
Claims (10)
1. A method for extracting spermidine from lycium ruthenicum, which is characterized by at least comprising the following steps:
(1) Preprocessing the dried lycium ruthenicum fruit to obtain lycium ruthenicum powder;
(2) Adding the lycium ruthenicum powder into distilled water, extracting with water and filtering to obtain lycium ruthenicum water extract;
(3) Vacuum concentrating the Lycium ruthenicum Murr water extract to obtain concentrated solution, adding ethanol into the concentrated solution, controlling ethanol content in the concentrated solution, standing, and filtering to obtain precipitate and ethanol extract;
(4) Vacuum concentrating the ethanol extract to obtain Lycium ruthenicum Murr extract;
(5) Dissolving the lycium ruthenicum extract into a disperse phase to obtain a lycium ruthenicum extract solution, and separating the lycium ruthenicum extract solution through macroporous resin column chromatography to obtain a lycium ruthenicum spermidine crude extract.
2. The method of claim 1, wherein the pretreatment in step (1) is: cleaning dried Lycium ruthenicum Murr, removing impurities, pulverizing, sieving with 80-120 mesh sieve, and drying to obtain Lycium ruthenicum Murr powder.
3. The method for extracting spermidine from lycium ruthenicum according to claim 1 or 2, wherein the mass ratio of the dry lycium ruthenicum to the distilled water in the step (2) is 1: (10-20).
4. A method for extracting spermidine from lycium ruthenicum according to claim 3, wherein the water extraction in step (2) is carried out at a temperature of 70-90 ℃ for a period of 2-3 hours.
5. The method for extracting spermidine from lycium ruthenicum, according to claim 4, wherein the volume ratio of the water extract and the concentrated solution of lycium ruthenicum in the step (3) is (8-12): 1.
6. The method for extracting spermidine from lycium ruthenicum according to claim 5, wherein the ethanol content in the concentrated solution in the step (3) is 30-60wt%, and the standing time is more than or equal to 12 hours.
7. The method according to claim 6, wherein the dispersed phase in the step (5) is at least one of aqueous formic acid, aqueous acetic acid and aqueous citric acid.
8. The method for extracting spermidine from lycium ruthenicum, according to claim 7, wherein the macroporous resin column chromatography separation in the step (5) is as follows: and (3) after pretreatment of macroporous resin, loading columns by adopting a wet method, loading the lycium ruthenicum extract solution onto columns for adsorption, and eluting by adopting an eluent to obtain the lycium ruthenicum spermidine crude extract.
9. The method of extracting spermidine from lycium ruthenicum of claim 8, wherein the eluent elution includes impurity elution and crude extract elution of spermidine from lycium ruthenicum.
10. Use of a method according to any one of claims 1-9 for the extraction of spermidine from lycium ruthenicum, for the preparation of an anti-aging product.
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| Publication Number | Publication Date |
|---|---|
| CN118324643A true CN118324643A (en) | 2024-07-12 |
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