Method for extracting agmatine from lycium ruthenicum murr and application thereof

Abstract

The present application relates to the technical field of natural product chemistry and pharmacology, in particular to a method for extracting agmatine from Lycium ruthenicum Murr and application thereof, which comprises at least the following steps: (1) obtaining Lycium ruthenicum Murr powder by pretreating Lycium ruthenicum Murr dry fruit; (2) adding the Lycium ruthenicum Murr powder into distilled water, water extracting and filtering to obtain Lycium ruthenicum Murr water extract; (3) vacuum concentrating the Lycium ruthenicum Murr water extract to obtain a concentrated solution, adding ethanol into the concentrated solution, controlling the ethanol content in the concentrated solution, and then 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 Murr extract into a dispersion phase to obtain Lycium ruthenicum Murr extract solution, and separating the Lycium ruthenicum Murr extract solution through macroporous resin column chromatography to obtain Lycium ruthenicum Murr agmatine crude extract, which is simple in operation, high in extraction rate and product stability.

Description

Technical Field

[0001] This invention relates to the fields of natural product chemistry and pharmacology, specifically to a method for extracting spermidine from black goji berries and its application. Background Technology

[0002] Black goji berries (scientific name: Lycium ruthenicum Black goji berries (Murr.) are a shrub that grows in the cold, high-altitude regions of Asia. Their fruit is rich in various nutrients, including vitamins, minerals, and bioactive compounds. Traditionally, black goji berries have been widely used in the food and traditional Chinese medicine fields to enhance immunity and improve eyesight, among other benefits.

[0003] Spermine is a polyamine compound initially isolated from semen. It is also found in many plants and plays important physiological roles in organisms. Past research has shown that spermine promotes cell growth and development in eukaryotes, while also playing a positive role in cell metabolism, antioxidation, and anti-inflammation. Recent studies have revealed that, in addition to its traditional cell growth-promoting effects, spermine can also exert various physiological effects by inducing autophagy. Autophagy is a cellular self-degradation process crucial for clearing damaged organelles, suppressing inflammatory responses, and maintaining intracellular homeostasis. By activating autophagy, spermine is believed to promote lipid metabolism and enhance antioxidant activity, thus positively impacting health and lifespan. Researchers, through administering spermine to mice, found that it effectively inhibits oxidative stress and prolongs lifespan. Furthermore, oral administration of spermine can activate microtubule-associated protein MAP1S-mediated autophagy, preventing liver fibrosis and further extending lifespan in mice.

[0004] Although the extraction of spermidine and its derivatives from plants has received extensive attention in past studies, research on spermidine from black goji berries is relatively weak, and the methods still need further optimization and improvement. For example, Chinese patent (publication number CN117304054A) discloses a method for enriching caffeoyl spermidine compounds, its anti-aging properties, and a method for inhibiting histone deacetylase. It uses a 75% ethanol aqueous solution as the extractant to enrich caffeoyl spermidine compounds from dried black goji berry fruits, but the yield of the extract is low and the stability needs to be further improved. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for extracting spermidine from black goji berries and its application. This method is simple to operate and has high extraction efficiency, effectively obtaining spermidine extract from black goji berries, thus facilitating the further development and utilization of the pharmacological activity of black goji berries.

[0006] The application provides a method for extracting agmatine from Lycium ruthenicum Murr, and at least comprises the following steps:

[0007] (1) obtaining Lycium ruthenicum Murr powder after pretreatment of Lycium ruthenicum Murr dry fruit;

[0008] (2) adding Lycium ruthenicum Murr powder into distilled water, obtaining Lycium ruthenicum Murr water extract after water extraction and filtration;

[0009] (3) obtaining concentrated solution after vacuum concentration of the Lycium ruthenicum Murr water extract, adding ethanol into the concentrated solution, controlling the ethanol content in the concentrated solution, and then obtaining precipitate and ethanol extract after standing and filtration;

[0010] (4) obtaining Lycium ruthenicum Murr extract after vacuum concentration of the ethanol extract;

[0011] (5) dissolving the Lycium ruthenicum Murr extract into a dispersed phase to obtain Lycium ruthenicum Murr extract solution, and obtaining Lycium ruthenicum Murr agmatine crude extract through macroporous resin column chromatography separation of the Lycium ruthenicum Murr extract solution.

[0012] As a preferred technical solution, the pretreatment in step (1) is: after washing, removing impurities and crushing the Lycium ruthenicum Murr dry fruit, sieving through an 80-120 mesh sieve, and drying to obtain the Lycium ruthenicium Murr powder.

[0013] As a preferred technical solution, the mass ratio of the Lycium ruthenicum Murr dry fruit to the distilled water in step (2) is 1: (10-20), preferably 1:10.

[0014] As a preferred technical solution, the water extraction temperature in step (2) is 70-90 ℃, and the time is 2-3 h.

[0015] As a preferred technical solution, the volume ratio of the Lycium ruthenicum Murr water extract to the concentrated solution in step (3) is (8-12):1.

[0016] Preferably, the ethanol content in the concentrated solution in step (3) is 30-60 wt%, and the standing time is ≥12 h.

[0017] Preferably, the dispersed phase in step (5) is at least one of formic acid aqueous solution, acetic acid aqueous solution and citric acid aqueous solution, and the dispersed phase is preferably formic acid aqueous solution, and the formic acid aqueous solution is preferably 0.5-1 wt% formic acid aqueous solution.

[0018] As a preferred technical solution, the macroporous resin column chromatography separation in step (5) is: after pretreatment of the macroporous resin, wet column packing is adopted, the Lycium ruthenicum Murr extract solution is column adsorbed, and the Lycium ruthenicium Murr agmatine crude extract is obtained after elution with an eluent.

[0019] Preferably, the eluent elution includes impurity elution and Lycium ruthenicum Mill agmatine crude extract elution, the eluent in the impurity elution is 1 wt% formic acid-containing ethanol aqueous solution, the concentration of ethanol in the ethanol aqueous solution is controlled to be 10 wt%; the eluent in the Lycium ruthenicum Mill agmatine crude extract elution is 1 wt% formic acid-containing ethanol aqueous solution, the concentration of ethanol in the ethanol aqueous solution is controlled to be 20-40 wt%.

[0020] In order to realize the extraction of agmatine from Lycium ruthenicum Mill, the present application designs Lycium ruthenicum Mill dried fruit as raw material, and sequentially performs dosage form pretreatment, water extraction, alcohol extraction, and macroporous resin column chromatography separation to obtain Lycium ruthenicum Mill agmatine crude extract, which can be effectively applied to the preparation of anti-aging products. Further control of the parameter conditions of each step has higher stability on the basis of ensuring the yield of the prepared Lycium ruthenicum Mill agmatine crude extract, can realize large-scale industrial production and better meet the subsequent application requirements. The conventional water extraction method, ethanol hot reflux method and supercritical fluid extraction method cannot obtain high yield while ensuring the stability of agmatine when used for extracting agmatine from Lycium ruthenicum Mill dried fruit, and the ethanol hot reflux method and supercritical fluid extraction method have high cost and are difficult to realize large-scale industrial production and application. The present application obtains Lycium ruthenicum Mill powder after pretreatment of Lycium ruthenicum Mill dried fruit, controls the solid-liquid ratio, extraction temperature and extraction time, and dissolves the effective components in Lycium ruthenicum Mill as much as possible to obtain Lycium ruthenicum Mill water extract on the basis of ensuring the extraction efficiency and activity of the extract. Further concentration of the Lycium ruthenicum Mill water extract is performed for alcohol extraction, only the ethanol content in the concentrated solution is controlled to be 30-60 wt% and the ethanol extraction liquid containing Lycium ruthenicum Mill extract is obtained by standing at room temperature (20-30℃) for 24 h, which avoids the high cost caused by the use of a large amount of high-purity and high-concentration ethanol and avoids the influence of high-concentration ethanol hot reflux extraction and high-temperature water extraction on the stability of the extract. Finally, the Lycium ruthenicum Mill extract is dissolved into a dispersion phase, and macroporous resin column chromatography separation is performed to obtain Lycium ruthenicum Mill agmatine crude extract, especially 10 wt% ethanol aqueous solution containing 1 wt% formic acid is used for elution to remove impurities, and then 20-40 wt% ethanol aqueous solution containing 1 wt% formic acid is used for elution to obtain Lycium ruthenicum Mill agmatine crude extract, which reduces the influence of impurities on the stability of agmatine and obtains high yield.

[0021] Advantages

[0022] 1. The present application provides a method for extracting agmatine from Lycium ruthenicum Mill, which is simple in operation, high in extraction efficiency, and can effectively obtain agmatine extract from Lycium ruthenicum Mill, thereby providing convenience for further development and utilization of the pharmacological activity of Lycium ruthenicum Mill.

[0023] 2、The application aims to extract agmatine from Lycium ruthenicum Murr, and designs Lycium ruthenicum Murr dry fruit as raw material, sequentially performs pre-treatment, water extraction, alcohol extraction, and macroporous resin column chromatography separation to obtain Lycium ruthenicium Murr agmatine crude extract, which can be effectively applied to preparation of anti-aging products.

[0024] 3、The application controls parameters of each step, has high stability on the basis of ensuring yield of prepared Lycium ruthenicium Murr agmatine crude extract, can realize large-scale industrial production, and better meets subsequent application requirements.

[0025] 4、The application obtains Lycium ruthenicium Murr powder after pre-treatment of Lycium ruthenicium Murr dry fruit, controls material liquid ratio, extraction temperature and extraction time, and dissolves effective components in Lycium ruthenicium Murr as much as possible to obtain Lycium ruthenicium Murr water extract on the basis of ensuring extraction efficiency and activity of the extract.

[0026] 5、The application further concentrates Lycium ruthenicium Murr water extract to perform alcohol extraction, only needs to control ethanol content in the concentrated solution to be 30-60 wt%, and stands still for 24 h at normal temperature (20-30 ℃) to obtain ethanol extract containing Lycium ruthenicium Murr extract, avoids high cost caused by use of a large amount of high-purity and high-concentration ethanol, and avoids influence of high-concentration ethanol hot reflux extraction and high-temperature water extraction on stability of the extract.

[0027] 6、The application dissolves Lycium ruthenicium Murr extract to a dispersed phase, and adopts macroporous resin column chromatography separation to obtain Lycium ruthenicium Murr agmatine crude extract, especially first adopts 10 wt% ethanol aqueous solution containing 1 wt% formic acid to elute and remove impurities, and then adopts 20-40 wt% ethanol aqueous solution containing 1 wt% formic acid to elute to obtain Lycium ruthenicium Murr agmatine crude extract, which reduces influence of impurities on stability of agmatine and obtains high yield. DETAILED DESCRIPTION

[0028] Example 1

[0029] The application provides a method for extracting agmatine from Lycium ruthenicum Murr, which comprises the following steps:

[0030] (1) 500 g of Lycium ruthenicum Murr dry fruit is pre-processed to obtain Lycium ruthenicium Murr powder;

[0031] (2) The Lycium ruthenicium Murr powder is added into 5 L of distilled water, water extraction is performed, and filtration is performed to obtain 4.5 L of Lycium ruthenicium Murr water extract;

[0032] (3) The Lycium ruthenicium Murr water extract is vacuum concentrated to obtain 500 mL of concentrated solution, 1.3 L of ethanol is added into the concentrated solution, ethanol content in the concentrated solution is controlled, and then standing and filtration are performed to obtain 100 g of precipitate and 1.4 L of ethanol extract;

[0033] (4) vacuum concentration of the ethanol extract to obtain 50 g of Lycium ruthenicum extract;

[0034] (5) dissolving the Lycium ruthenicum extract into the dispersion phase to obtain a Lycium ruthenicum extract solution, and separating the Lycium ruthenicum extract solution by macroporous resin column chromatography to obtain 5 g of Lycium ruthenicum spermidine crude extract.

[0035] In the step (1), the pretreatment is as follows: washing, removing impurities, and crushing the Lycium ruthenicum dried fruit, then passing it through a 100-mesh sieve, and drying to obtain Lycium ruthenicum powder.

[0036] In the step (2), the mass ratio of the Lycium ruthenicum dried fruit to distilled water is 1:10.

[0037] In the step (2), the water extraction temperature is 70°C, and the time is 3 h.

[0038] In the step (3), the volume ratio of the Lycium ruthenicum water extract to the concentrated solution is 9:1.

[0039] In the step (3), the ethanol content in the concentrated solution is 60 wt%, and the standing time is 24 h.

[0040] In the step (5), the dispersion phase is a formic acid aqueous solution, and the formic acid aqueous solution is a 1 wt% formic acid aqueous solution.

[0041] In the step (5), the macroporous resin column chromatography separation is as follows: after pretreatment of the macroporous resin, wet column packing is adopted, the Lycium ruthenicum extract solution is column adsorbed, and after elution with an eluent, the Lycium ruthenicum spermidine crude extract is obtained.

[0042] The elution includes impurity elution and Lycium ruthenicum spermidine crude extract elution, in the impurity elution, the eluent is a 1 wt% formic acid-containing ethanol aqueous solution, and the ethanol concentration in the ethanol aqueous solution is controlled to be 10 wt%; in the Lycium ruthenicum spermidine crude extract elution, the eluent is a 1 wt% formic acid-containing ethanol aqueous solution, and the ethanol concentration in the ethanol aqueous solution is controlled to be 20-40 wt%.

[0043] Comparative Example 1

[0044] Comparative Example 1 of the present application provides a method for extracting spermidine from Lycium ruthenicum, 500 g of Lycium ruthenicum dried fruit is added to 10 times the mass of water, heated to boiling in a water bath, and kept boiling for 30 minutes, and then filtered to obtain a water extract, which is vacuum concentrated and vacuum dried to obtain 3 g of spermidine crude extract.

[0045] Comparative Example 2

[0046] The comparative example 2 of the present application provides a method for extracting agmatine from Lycium ruthenicum, 500 g of Lycium ruthenicum dried fruits is added with 10 times of mass of carbon dioxide, the temperature is adjusted to 40℃, the pressure is 30 MPa, and supercritical fluid extraction is carried out for 60 minutes, the extraction liquid is collected, and vacuum concentration is carried out to obtain 4.0 g of agmatine crude extract.

[0047] Performance test method

[0048] 1. The yield of the agmatine crude extract obtained by the process of the calculation example and the comparative example (the yield refers to the ratio of the weight of the agmatine crude extract to the weight of the Lycium ruthenicum dried fruits), and the results are shown in Table 1.

[0049] 2. The agmatine crude extract prepared by the process of the examples and the comparative examples is stored at 25℃ for 30 days, and the stability (the stability refers to the ratio of the agmatine content of the agmatine crude extract after being stored at room temperature for 30 days to the initial content) is calculated, and the results are shown in Table 1.

[0050] Table 1

[0051]

[0052] As can be seen from Table 1, the yield and stability of the agmatine extracted by the method of the present application are higher than those of comparative example 1 and comparative example 2, which shows that the method of the present application has higher efficiency and better protection effect, and can effectively extract high-purity and high-activity agmatine components from Lycium ruthenicum.

[0053] 3. The intervention effect evaluation of the Lycium ruthenicum agmatine provided by example 1 on the D-galactose-induced mouse aging model.

[0054] Objective: To observe the intervention effect of the Lycium ruthenicum agmatine of the present application on the D-galactose-induced mouse aging model, and to compare with the positive control drug vitamin E.

[0055] Materials and methods: 60 healthy male Kunming mice aged 8 weeks were selected and randomly divided into 6 groups, 10 mice in each group, namely control group, model group, positive control group, low-dose group, medium-dose group and high-dose group. Except for the control group, the rest of the mice in each group were injected intraperitoneally with D-galactose (100 mg / kg) every day to prepare the aging model, and the injection was continued for 4 weeks. At the same time, the mice in the positive control group were given vitamin E (100 mg / kg) by gavage every day, the mice in the low-dose group, the medium-dose group and the high-dose group were given the spermidine of the application (50 mg / kg, 100 mg / kg and 200 mg / kg) by gavage every day, and the mice in the control group and the model group were given the same amount of normal saline by gavage every day, and the drug administration was continued for 4 weeks. During the experiment, the weight changes of the mice were recorded, and after the experiment, the blood, liver and brain tissue samples of the mice were collected, and the blood biochemical indexes (total cholesterol, triglyceride, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, etc.), liver antioxidant enzyme activity (superoxide dismutase, catalase, glutathione peroxidase, etc.), brain tissue acetylcholinesterase activity and other indexes were determined.

[0056] Results and analysis: Table 2 shows the weight changes of the mice in different treatment groups.

[0057] Table 2

[0058]

[0059] As can be seen from Table 2, the weight gain of the mice in the model group was significantly lower than that in the control group, indicating that D-galactose induced aging in mice. The weight gain of the mice in the positive control group and each dose of spermidine treatment group was higher than that in the model group, indicating that vitamin E and spermidine can improve the growth status of mice and delay aging. Among them, the weight gain of the mice in the high-dose spermidine treatment group was comparable to that in the control group, and even slightly higher than that in the positive control group, indicating that the spermidine complex has a strong anti-aging effect.

[0060] Table 3 shows the blood biochemical indexes (mmol / L or U / L) of the mice in different treatment groups.

[0061] Table 3

[0062]

[0063] As can be seen from Table 3, the total cholesterol, triglyceride, glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase levels of the mice in the model group were higher than those in the control group, indicating that D-galactose damaged the liver function of the mice, leading to blood lipid metabolism disorder. The indexes of the mice in the positive control group and each dose of spermidine treatment group were lower than those in the model group, indicating that vitamin E and spermidine complex can protect the liver function of mice and improve blood lipid metabolism. Among them, the indexes of the mice in the high-dose spermidine treatment group were similar to those in the control group, and even slightly lower than those in the positive control group, indicating that spermidine has a strong anti-aging effect.

[0064] Table 4 shows the liver antioxidant enzyme activity (U / mg) of mice in different treatment groups.

[0065] Table 4

[0066]

[0067] Table 4 shows that the liver antioxidant enzyme activities of mice in the model group were lower than those in the control group, indicating that D-galactose increased oxidative stress in mice, leading to oxidative damage to cell membranes. Conversely, the liver antioxidant enzyme activities of mice in the positive control group and the groups treated with various doses of spermidine were higher than those in the model group, indicating that vitamin E and spermidine can enhance the antioxidant capacity of mice and reduce oxidative damage to cell membranes. In particular, the liver antioxidant enzyme activities of mice treated with high doses of spermidine were similar to, or even slightly higher than, those in the positive control group, suggesting that the spermidine complex has a strong anti-aging effect.

[0068] Table 5 shows the acetylcholinesterase activity (U / mg) in the brain tissue of mice in different treatment groups.

[0069] Table 5

[0070]

[0071] Table 5 shows that the acetylcholinesterase activity in the brain tissue of the model group mice was lower than that of the control group, indicating that D-galactose damaged the neurological function of the mice, leading to a decline in memory and learning ability. Conversely, the acetylcholinesterase activity in the brain tissue of the positive control group and the mice treated with various doses of spermidine was higher than that of the model group, indicating that vitamin E and spermidine can protect the neurological function of mice and improve memory and learning ability. In particular, the acetylcholinesterase activity in the brain tissue of the high-dose spermidine-treated group was the same as that of the control group, or even slightly higher than that of the positive control group, indicating that the spermidine complex has a strong anti-aging effect.

[0072] In summary, the spermidine from black wolfberry of the present invention can effectively intervene in a D-galactose-induced mouse aging model, improving its physiological, biochemical, and molecular indicators, and delaying the aging process. Compared with the positive control drug vitamin E, it has comparable or even better effects. These experimental data and examples fully demonstrate the application value of spermidine in anti-aging.

Claims

1. A method for extracting spermidine from black goji berries, characterized in that, At least the following steps are included: (1) Black goji berry powder was obtained by pre-processing dried black goji berries; (2) Add black goji berry powder to distilled water, extract with water and filter to obtain black goji berry water extract; (3) The water extract of black wolfberry was concentrated under vacuum to obtain a concentrated solution. Ethanol was added to the concentrated solution to control the ethanol content. Then, the solution was allowed to stand and filtered to obtain the precipitate and ethanol extract. (4) The ethanol extract was concentrated under vacuum to obtain black goji berry extract; (5) Dissolve the black wolfberry extract into the dispersed phase to obtain a black wolfberry extract solution, and separate the black wolfberry extract solution by macroporous resin column chromatography to obtain a crude extract of black wolfberry spermidine. The pretreatment in step (1) is as follows: the dried black goji berries are washed, impurities removed, and pulverized, then passed through an 80-120 mesh sieve and dried to obtain black goji berry powder; the mass ratio of dried black goji berries to distilled water in step (2) is 1:(10-20); the water extraction temperature in step (2) is 70-90℃, and the time is 2-3 hours; the volume ratio of black goji berry water extract to concentrated extract in step (3) is (8-12):1; the ethanol content in the concentrated extract in step (3) is... 30-60 wt%, standing time ≥ 12 h; the dispersed phase in step (5) is at least one of formic acid aqueous solution, acetic acid aqueous solution, and citric acid aqueous solution; the macroporous resin column chromatography separation in step (5) is as follows: after macroporous resin pretreatment, wet packing is used, black goji berry extract solution is loaded onto the column for adsorption, and after elution with eluent, crude extract of black goji berry spermidine is obtained; the eluent elution includes impurity elution and black goji berry spermidine crude extract elution; the eluent in the impurity elution is an ethanol aqueous solution containing 1 wt% formic acid, and the concentration of ethanol in the ethanol aqueous solution is controlled at 10 wt%; the eluent in the black goji berry spermidine crude extract elution is an ethanol aqueous solution containing 1% formic acid, and the concentration of ethanol in the ethanol aqueous solution is controlled at 20-40 wt%.

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