Actinidia arguta bee pollen total spermine and preparation method and application thereof

Abstract

The invention discloses actinidia arguta bee pollen total spermine and a preparation method and application thereof, and relates to the technical field of biology. The preparation method of the actinidia arguta bee pollen total spermine comprises the following steps: performing degreasing treatment on actinidia arguta bee pollen to obtain degreased bee pollen; carrying out ultrasonic extraction on the defatted bee pollen by using an ethanol water solution, and centrifuging to obtain a supernatant; extracting the supernate by adopting ethyl acetate, separating to obtain an organic extraction phase, and then drying to obtain the total spermine of the actinidia arguta bee pollen. By adopting the preparation method, the total spermine can be extracted from the actinidia arguta bee pollen, and verification shows that the total spermine can effectively improve liver injury caused by cadmium poisoning.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to total spermine from Actinidia arguta bee pollen and a preparation method and application thereof. Background Art

[0002] Cadmium is a common industrial and environmental pollutant that is widely present in air, soil, sludge and water, and can have serious toxic effects on both animals and humans. Cadmium pollution can lead to the accumulation of cadmium in organisms. Cadmium can enter the human body through a variety of pathways, causing chronic cadmium poisoning. Cadmium pollution is ubiquitous around the world and can be detected in drinking water, food, soil and tobacco. After entering the human body, cadmium is excreted very slowly and is mainly accumulated in organs such as the kidneys, liver and testicles, causing a variety of toxic effects.

[0003] Cadmium can cause liver cell damage through multiple mechanisms, including oxidative stress, mitochondrial dysfunction, and blocked protein synthesis. These injuries can lead to liver cell necrosis and apoptosis, which in turn affects the normal function of the liver. Long-term exposure to cadmium environment can lead to the occurrence of liver fibrosis. Cadmium-induced inflammatory response and excessive deposition of extracellular matrix gradually destroy the liver structure and lead to cirrhosis. The progression of liver fibrosis and cirrhosis can seriously affect the function of the liver and increase the risk of liver failure. Cadmium can interfere with lipid metabolism in the liver, leading to fat accumulation and the occurrence of fatty liver. Fatty liver not only affects the metabolic function of the liver, but may also further develop into hepatitis and cirrhosis. In order to improve liver damage caused by cadmium poisoning, it is necessary to develop new therapeutic drugs. Summary of the invention

[0004] The purpose of the present invention is to provide total spermine from Actinidia arguta bee pollen and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The preparation method can be used to extract total spermine from Actinidia arguta bee pollen, and it has been verified that the total spermine can effectively improve liver damage caused by cadmium poisoning.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a method for preparing total spermine from Actinidia arguta bee pollen, comprising the following steps:

[0007] Degreasing the arguta kiwifruit bee pollen to obtain defatted bee pollen;

[0008] Ultrasonic extraction is performed on the defatted bee pollen using an ethanol aqueous solution, followed by centrifugation to obtain a supernatant;

[0009] The supernatant is extracted with ethyl acetate to separate an organic extract phase, which is then dried to obtain the total spermine in the Actinidia arguta bee pollen.

[0010] Furthermore, the Actinidia arguta bee pollen is defatted by using a Soxhlet extraction method.

[0011] Furthermore, the volume fraction of the ethanol aqueous solution is 80%.

[0012] Furthermore, the mass volume ratio of the defatted bee pollen to the ethanol aqueous solution is 1 g:10 mL.

[0013] Further, the temperature of the ultrasonic extraction is 25°C; and / or

[0014] The ultrasonic extraction time is 30 min; and / or

[0015] The ultrasonic power of the ultrasonic extraction is 600W.

[0016] The present invention also provides total spermine from Actinidia arguta bee pollen prepared according to the above preparation method.

[0017] The invention also provides application of the total spermine of Actinidia arguta bee pollen in preparing a medicine for improving liver damage caused by cadmium poisoning.

[0018] The invention also provides a medicine for liver damage caused by cadmium poisoning, wherein the active ingredient comprises the total spermine of the Actinidia arguta bee pollen.

[0019] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0020] Furthermore, the dosage form of the drug is granules, tablets, capsules, powders, oral liquids or pills.

[0021] The present invention discloses the following technical effects:

[0022] The present invention extracts total spermine from Actinidia arguta bee pollen by degreasing Actinidia arguta bee pollen, and then extracts it by combining ethanol ultrasonic extraction and organic solvent extraction. It has been verified that after low-dose intake of total spermine from Actinidia arguta bee pollen, hepatocytes are still in a state of fatty degeneration, hepatic sinusoids and between hepatocytes are still filled with inflammatory cells, hepatic sinusoids are enlarged, necrotic hepatocytes can also be seen, and no inflammatory cells are seen in the interlobular veins; gradually increase the intake of total spermine from Actinidia arguta bee pollen, at a medium dose, the fat content in the cells has been significantly reduced, and the fatty degenerated hepatocytes are also reduced compared to the lower dose; when a higher dose of total spermine from Actinidia arguta bee pollen is used, the fat content of hepatocytes is extremely low, and the state of hepatocyte nucleoli and hepatic sinusoids is close to normal, without necrotic lobules. It can be seen that total spermine from Actinidia arguta bee pollen can effectively improve liver damage caused by cadmium poisoning.

[0023] The invention provides a new therapeutic drug for improving liver damage caused by cadmium poisoning and has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is a liver tissue section of mice in the blank control group; the scale bar is 100 μm;

[0026] Figure 2 This is a liver tissue section of mice in the medium-dose drug group; the scale bar is 100 μm;

[0027] Figure 3 This is a liver tissue section of mice in the VC drug control group; the scale bar is 100 μm;

[0028] Figure 4 This is a liver tissue section of mice in the cadmium poisoning control group; the scale bar is 100μm;

[0029] Figure 5 This is a liver tissue section of mice in the low-dose drug treatment group; the scale bar is 100 μm;

[0030] Figure 6 This is a liver tissue section of mice in the medium-dose drug treatment group; the scale bar is 100 μm;

[0031] Figure 7 Liver tissue sections of the high-dose drug treatment group; scale bar is 100 μm. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1 Extraction of total spermine from Actinidia arguta bee pollen

[0038] (1) Degreasing

[0039] The Soxhlet extraction method is used to defatted the arguta kiwifruit bee pollen to obtain defatted bee pollen. The specific method is as follows:

[0040] The bee pollen is packaged with filter paper, the size is just right to fit into the extraction tube, and the length cannot exceed the siphon tube. Assemble the Soxhlet extractor, connect it to the condensation system with a hose, let water enter the lower end of the condenser and discharge water from the upper end, and put the packaged bee pollen into the extractor. Add petroleum ether to the extraction bottle, turn on the water bath, set the temperature to 40℃, slowly increase the temperature until the petroleum ether in the extraction bottle is slightly boiling, the Soxhlet extractor refluxes normally, and stops heating. At this time, the temperature is 45℃, the petroleum ether vaporizes, enters the condenser, condenses into liquid and drips into the extraction tube, immersing the bee pollen wrapped in filter paper. The degreasing time of each package of bee pollen is about two hours. When the petroleum ether in the extraction tube returns to a transparent state, it means that the degreasing is completed, and the next package of bee pollen can be replaced for degreasing. Finally, dry the degreased bee pollen to evaporate the petroleum ether.

[0041] (2) Extraction of total spermine

[0042] a. Weigh 200g of defatted bee pollen, add 80% ethanol aqueous solution at a solid-liquid ratio of 1g:10mL, mix well and then perform ultrasonic treatment for 30min, set the temperature to 25℃ and the power to 600W.

[0043] b. After the ultrasound is completed, centrifuge at 3500 rpm for 10 min to obtain the supernatant.

[0044] c. Add the remaining precipitate again into 80% ethanol aqueous solution (solid-liquid ratio 1 g:10 mL), repeat steps a and b to perform a second extraction.

[0045] d. After the second extraction is completed, the two supernatants are combined and the combined solution is concentrated under reduced pressure at 40°C. When the solution is concentrated to 700 mL, the concentration is stopped to obtain a concentrated solution for subsequent extraction operations.

[0046] e. The concentrate was mixed with an equal volume of ethyl acetate for extraction, and the upper organic extract phase was taken and placed in a vacuum drying oven for drying at 40° C. Finally, 5.15 g of total spermine was obtained.

[0047] Example 2

[0048] 1. Grouping

[0049] 42 male Kunming mice (average weight of 19.46±1.25g) were divided into 7 groups (6 mice in each group), namely blank control group (Control), cadmium poisoning control group (Cadmium), VC drug control group (VC), medium-dose simple drug group (MJA), low-dose drug treatment group (Low), medium-dose drug treatment group (Mid), and high-dose drug treatment group (High). The blank control group did not receive any intervention. CdCl2 was dissolved in 0.9% normal saline, and the cadmium poisoning control group mice, low-dose drug treatment group, medium-dose drug treatment group, and high-dose drug treatment group were intraperitoneally injected with the same concentration and dose (1.5mg / kg) of CdCl2 on the first day. The VC drug control group was orally administered 2.0mg / kg. The dosage details of each group are shown in Table 1 below.

[0050] Table 1 Toxin and drug dosage of each group of mouse models

[0051]

[0052] 2. Preparation and pathological analysis of mouse liver HE-stained paraffin sections

[0053] On the evening of the 7th day, the mice were fasted but not watered. On the morning of the 8th day, after pentobarbital anesthesia, the mice were sacrificed, the abdominal cavity was opened, and the liver was separated. 5mm×5mm×2mm liver tissue was cut. It was immediately immersed in saline. Then it was fixed in sequence, rinsed with running water overnight, dehydrated with gradient concentrations of ethanol, and embedded in paraffin after being transparent. The embedded wax block was cut on a microtome and stained with hematoxylin and eosin respectively. Finally, it was dehydrated and transparent and gum was dripped on it. It can be observed under a microscope.

[0054] like Figure 1 As shown, in the normal mouse hepatocytes in the blank control group, the liver tissue structure was complete, the cell cords were neatly arranged (as shown by the black arrows), the liver lobules had clear outlines, clear cell boundaries, round and clear cell nuclei, and abundant cytoplasm.

[0055] like Figure 2 As shown in the figure, after treatment with medium-dose total spermine from Actinidia arguta bee pollen, lipid droplets in and around hepatocytes almost disappeared (as shown by arrows). Hepatic sinusoids were obvious, nucleolus staining deepened, interlobular veins were filled with red blood cells, and no inflammatory cells were seen.

[0056] like Figure 3 As shown, after the VC drug control group mice ingested a dose of VC equivalent to a high dose of total spermine from Actinidia arguta bee pollen, the nucleoli were deeply stained, the hepatic sinusoids were obvious, and there were no obvious lipid droplets in the hepatocytes compared with the normal mice.

[0057] like Figure 4 As shown in the figure, the hepatocytes of the mice in the cadmium poisoning control group had fatty degeneration, necrotic spots between the lobules, infiltration of inflammatory cells between the hepatic sinusoids and hepatocytes, collapse of the nucleus, and dissolution of the nuclear membrane. There were a large number of inflammatory cells and red blood cells in the interlobular veins, and large lipid droplets in the hepatocytes.

[0058] like Figure 5 As shown, after the mice in the low-dose drug treatment group ingested a low dose of total spermine from Actinidia arguta bee pollen, the liver cells were still in a state of fatty degeneration, the hepatic sinusoids and the spaces between the liver cells were still filled with inflammatory cells, the hepatic sinusoids were enlarged, necrotic hepatocytes were also seen, and no inflammatory cells were seen in the interlobular veins.

[0059] Gradually increase the intake of total spermine from Actinidia arguta bee pollen. At medium doses, Figure 6 As shown in the figure, the fat content in hepatocytes has been significantly reduced, and the number of fatty degenerated hepatocytes has also decreased compared with that at lower doses. There is no obvious inflammatory cell infiltration in the hepatic sinusoids, and the nuclei are obvious.

[0060] When using higher doses of total spermine from Actinidia arguta bee pollen, Figure 7 As shown, the fat content of hepatocytes in the slice field of view is extremely low, the state of hepatocyte nucleoli and hepatic sinusoids is close to normal, and there are no necrotic lobules. Figure 7 The middle arrows show obvious hepatic cords, no obvious necrotic spots in the lobules, no inflammatory cells, obvious hepatocyte morphology, and intact membrane structure.

[0061] The levels of liver enzymes in the serum of mice in each group, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were detected, and the results are shown in Table 2. The results showed that compared with the blank group, the activities of AST and ALT in the serum of mice in the cadmium poisoning control group were significantly increased, and the reaction mice showed liver damage. The levels of AST and ALT in the serum of mice in the VC drug control group and the low, medium, and high drug treatment groups and the medium-dose simple drug group were significantly reduced, indicating that total spermine from Actinidia arguta bee pollen has a strong protective effect on liver cell damage in mice poisoned by cadmium.

[0062] Table 2 Effects of total spermine from Actinidia arguta bee pollen on serum enzymes in mice poisoned by cadmium

[0063]

[0064] The above experimental results show that total spermine from Actinidia arguta bee pollen can effectively improve liver damage caused by cadmium poisoning.

[0065] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing total spermine from Actinidia arguta bee pollen, characterized in that: The following steps are involved: Degreasing the arguta kiwifruit bee pollen to obtain defatted bee pollen; Ultrasonic extraction is performed on the defatted bee pollen using an ethanol aqueous solution, followed by centrifugation to obtain a supernatant; The supernatant is extracted with ethyl acetate to separate an organic extract phase, which is then dried to obtain the total spermine in the Actinidia arguta bee pollen.

2. The preparation method according to claim 1, characterized in that: The Actinidia arguta bee pollen is defatted by using a Soxhlet extraction method.

3. The preparation method according to claim 1, characterized in that: The volume fraction of the ethanol aqueous solution is 80%.

4. The preparation method according to claim 1, characterized in that: The mass volume ratio of the defatted bee pollen to the ethanol aqueous solution is 1 g:10 mL.

5. The preparation method according to claim 1, characterized in that: The temperature of the ultrasonic extraction is 25°C; and / or The ultrasonic extraction time is 30 min; and / or The ultrasonic power of the ultrasonic extraction is 600W.

6. Total spermine from Actinidia arguta bee pollen prepared according to the preparation method according to any one of claims 1 to 5.

7. Use of the total spermine from Actinidia arguta bee pollen as claimed in claim 6 in the preparation of a medicament for improving liver damage caused by cadmium poisoning.

8. A drug for liver damage caused by cadmium poisoning, characterized in that: The active ingredients include the total spermine from the Actinidia arguta bee pollen according to claim 6.

9. The drug according to claim 8, characterized in that The drug also includes pharmaceutically acceptable excipients.

10. The drug according to claim 9, characterized in that The dosage form of the medicine is granules, tablets, capsules, powders, oral liquids or pills.

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