Selenium-rich rape extract for improving cadmium-damaged male reproductive capacity as well as extraction method and application of selenium-rich rape extract

Abstract

The invention provides a selenium-rich rape extract for improving cadmium-damaged male reproductive capacity as well as an extraction method and application of the selenium-rich rape extract, and belongs to the technical field of plant extraction. Comprising the following steps: extracting selenium-rich rape stalks by using a methanol aqueous solution as an extracting agent to obtain the selenium-rich rape extract. According to the invention, the action effect of the selenium-rich brassica campestris extract obtained under different extraction conditions on the aspect of reducing TM4 cell CdCl2 damage is studied, and the optimal extraction condition of active ingredients for improving cadmium-damaged male reproductive capacity is determined. The Brassica campestris extract obtained through extraction can effectively reduce TM4 cell CdCl2 damage, the cell survival rate can still reach 140% or above under the influence of CdCl2 treatment, and the Brassica campestris extract has great application potential in preparation of drugs for improving cadmium damage male reproductive capacity.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and particularly relates to a selenium-enriched rapeseed extract for improving male reproductive capacity damaged by cadmium, and an extraction method and application thereof. Background Art

[0002] Infertility is one of the major health issues affecting human health in the 21st century. Male factors contribute to approximately 50% of infertility, and poor semen quality is a key contributing factor. Statistics show that the causes of poor semen quality are primarily internal (genetic) and external (environmental) factors. In particular, increased environmental pollution from the heavy metal cadmium can severely damage male spermatogenic cells. Cadmium easily accumulates in the gonadal tissue of animals, significantly impacting their reproductive activity. Cadmium significantly affects male reproductive function, disrupting the male reproductive system through various pathways and methods, including the testes (testes), steroid hormone and enzyme levels, and endocrine activity. Studies have shown that cadmium's toxic effects on the testes are more severe than those of other heavy metals such as zinc and lead. Cadmium can cause irreversible changes in testicular germ cells, including apoptosis and necrosis, leading to gonadal hypoplasia, reduced spermatogenic, Sertoli, and Leydig cell numbers, reduced gonadal weight, decreased semen quality, and even azoospermia. Therefore, the improvement of reproductive health and reproductive safety should focus on research to improve cadmium-damaged male reproductive capacity.

[0003] The applicant previously provided a method for extracting active ingredients from selenium-enriched rapeseed that enhance male fertility (CN117462582 B). While this method disclosed a selenium-enriched rapeseed extract capable of enhancing male fertility and studied its effect on TM4 cell survival, it did not investigate its effect on damaged TM4 cells. Furthermore, treatments for male fertility impairment vary depending on the cause in the field. The present application aims to provide an effective treatment for male fertility impairment caused by cadmium pollution. Summary of the Invention

[0004] The purpose of the present invention is to obtain a selenium-enriched rapeseed extract from selenium-enriched rapeseed that can effectively improve the reproductive capacity of males damaged by cadmium, aiming to solve the problem of severe damage to male spermatogenic cells caused by the aggravation of heavy metal cadmium environmental pollution.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a method for extracting a selenium-rich rapeseed extract for improving male reproductive capacity damaged by cadmium: the method comprises extracting selenium-rich rapeseed stalks with a methanol aqueous solution as an extractant to obtain the selenium-rich rapeseed extract.

[0007] Preferably, the volume fraction of the methanol aqueous solution is 30-70%.

[0008] Preferably, the solid-to-liquid ratio of the selenium-enriched rapeseed stems to the methanol aqueous solution is: 1 g: (10-25) ml.

[0009] Preferably, the extraction time is 6 to 18 minutes.

[0010] The invention provides a selenium-enriched rapeseed extract capable of improving male reproductive ability damaged by cadmium.

[0011] The present invention also provides the use of the selenium-rich rapeseed extract in preparing a medicine for improving male reproductive capacity damaged by cadmium.

[0012] Preferably, the concentration of the selenium-enriched rapeseed extract in the drug or reagent is 2-6 mg / mL.

[0013] The present invention studies the effects of selenium-enriched rapeseed stalk extracts obtained under different extraction conditions in reducing CdCl2 damage in TM4 cells, and determines the optimal extraction conditions for the active ingredient for improving male reproductive ability damaged by cadmium: a methanol concentration of 50%, a solid-liquid ratio of 1g:15mL, and an extraction time of 9min.

[0014] The rapeseed stalk extract obtained by the present invention can still achieve a cell survival rate of more than 140% under the influence of CdCl2 treatment, promote cell proliferation, effectively reduce CdCl2 damage of TM4 cells, and has great application potential in preparing drugs for improving male reproductive capacity damaged by cadmium. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Effects of different extraction solvents on cadmium damage in TM4 cells: (a) Effect of selenium-enriched rapeseed stalk extract on cell survival; (b) Effect of selenium-enriched rapeseed stalk extract combined with chromium chloride on cell survival; *P<0.05, **P<0.01, and ***P<0.001 compared with the control group or CdCl2 group;

[0016] Figure 2 Figure 4 Effects of methanol concentration on TM4 cells treated with different groups: (a) Effect of selenium-enriched rapeseed stalk extract on cell survival; (b) Effect of selenium-enriched rapeseed stalk extract combined with chromium chloride on cell survival; *P<0.05, **P<0.01, and ***P<0.001 compared with the control group or CdCl2 group;

[0017] Figure 3Figure 4: Effects of solid-liquid ratio on TM4 cells treated with different treatments. (a) Effect of selenium-enriched rapeseed stalk extract on cell viability; (b) Effect of selenium-enriched rapeseed stalk extract combined with chromium chloride on cell viability. *P<0.05, **P<0.01, and ***P<0.001 compared with the control group or CdCl2 group.

[0018] Figure 4 Figure 4 Effects of extraction time on TM4 cells in different treatment groups: (a) Effect of selenium-enriched rapeseed stalk extract on cell survival rate; (b) Effect of selenium-enriched rapeseed stalk extract and chromium chloride co-treatment on cell survival rate; *P<0.05, **P<0.01 and ***P<0.001 compared with the control group or CdCl2 group. DETAILED DESCRIPTION

[0019] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1

[0021] A method for extracting selenium-enriched rapeseed extract for improving cadmium-damaged male reproductive ability:

[0022] Selenium-rich rapeseed stalks (from the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences) were crushed and mixed with 50% (v / v) methanol aqueous solution at a ratio of 1 g:15 ml, and then ultrasonically extracted at 360W for 9 minutes to obtain selenium-rich rapeseed extract.

[0023] Example 2

[0024] Effects of extraction solvent types on the effect of selenium-enriched rapeseed on reducing chromium chloride (CdCl2) damage in TM4 cells

[0025] The solid-liquid ratio of selenium-enriched rapeseed stems to the extraction solvent was fixed at 1g:15ml, the extraction time was 9min, and pure water, 50% (v / v) methanol, and 50% (v / v) ethanol were used as extraction solvents to study the effect of the selenium-enriched rapeseed stem extract on reducing CdCl2 damage in TM4 cells. TM4 cells (commercially available) that had been cultured and digested were prepared into 1×10 5 Cell suspensions of 100 μL / well were added to a 96-well plate. After 24 hours of incubation, the plates were removed from the incubator and observed for cell adhesion. The cell culture medium was aspirated from each well and 100 μL / well of rapeseed stalk extract (diluted to various concentrations in cell culture medium) was added to each well. TM4 cells were treated for 24 hours and the survival rate of TM4 cells was calculated using a CCK-8 assay.

[0026] Experimental group settings:

[0027] (a) Selenium-enriched rapeseed stalk extract-treated (CE) group: the concentrations of rapeseed stalk extract were 0 (control group), 0.5, 2, 4, and 6 mg / mL, respectively;

[0028] (b) Group treated with selenium-enriched rapeseed stalk extract and chromium chloride: The final concentration of CdCl2 in each well was 7 μmol / L, and the rapeseed stalk extract concentration was the same as that in group (a).

[0029] The results are as follows Figure 1 As shown in (a), compared with the control group, when pure water was used as the extraction solvent, the selenium-enriched rapeseed stalk extract at a concentration of 4 mg / mL could significantly promote the proliferation of TM4 cells; while when methanol and ethanol were used as the extraction solvents, the selenium-enriched rapeseed stalk extract had no effect on the survival rate of TM4 cells within the concentration range studied.

[0030] like Figure 1 As shown in (b), compared with the CdCl2 group, when pure water was used as the extraction solvent, 2 and 4 mg / mL of selenium-enriched rapeseed extract reduced CdCl2 damage in TM4 cells, with cell viability reaching 92.13% and 100.63%, respectively. When methanol was used as the extraction solvent, 2, 4, and 6 mg / mL of selenium-enriched rapeseed extract significantly reduced CdCl2 damage in TM4 cells, with cell viability reaching 132.35%, 140.66%, and 140.40%, respectively. When ethanol was used as the extraction solvent, 2 mg / mL of selenium-enriched rapeseed extract significantly reduced CdCl2 damage in TM4 cells, with a cell viability of 122.41%. Based on these experimental results, methanol extraction solvent was more effective than pure water and ethanol in reducing CdCl2 damage in TM4 cells. Therefore, methanol was selected as the extraction solvent for subsequent experiments.

[0031] Example 3

[0032] Effect of extraction solvent concentration on the effect of selenium-enriched rapeseed on reducing CdCl2 damage in TM4 cells

[0033] A selenium-enriched rapeseed stalk extract treatment group (a) and a selenium-enriched rapeseed stalk extract and chromium chloride co-treatment group (b) were set up respectively. At the same time, the solid-liquid ratio of selenium-enriched rapeseed stalk and methanol aqueous solution was fixed at 1 g:15 ml, the extraction time was 9 min, and the volume fraction of methanol aqueous solution was set at 30%, 50%, 70%, and 90%, respectively. The effect of the selenium-enriched rapeseed stalk extract on reducing CdCl2 damage in TM4 cells was studied. The detection method was the same as in Example 2.

[0034] like Figure 2As shown in (a), compared with the control group, when the methanol concentration was 30%, the selenium-enriched rapeseed stalk extract at concentrations of 2 and 4 mg / mL could promote the survival rate of TM4 cells, which were 147.12% and 134.64% respectively; when the methanol concentration was 50%, the selenium-enriched rapeseed stalk extract had no significant effect on the survival rate of TM4 cells; when the methanol concentration was 70%, the selenium-enriched rapeseed stalk extract at concentrations of 2, 4 and 6 mg / mL could promote the survival rate of TM4 cells, which were 134.73%, 142.47% and 139.42% respectively; when the methanol concentration was 90%, the selenium-enriched rapeseed stalk extract had no significant effect on the survival rate of TM4 cells.

[0035] like Figure 2 As shown in (b), compared with the CdCl2 group, when the methanol concentration was 30%, 2 and 4 mg / mL selenium-enriched rapeseed extract had a significant effect on reducing CdCl2 damage in TM4 cells, with cell survival rates of 129.30% and 106.09%, respectively; when the methanol concentration was 50%, selenium-enriched rapeseed extract had a significant effect on reducing CdCl2 damage in TM4 cells at concentrations of 2, 4, and 6 mg / mL, with cell survival rates of 132.05%, respectively. , 140.30%, and 132.27%. When the methanol concentration was 70%, selenium-enriched rapeseed stalk extract at concentrations of 2, 4, and 6 mg / mL had a significant effect on reducing CdCl2 damage in TM4 cells, with cell viability rates of 77.82%, 112.66%, and 121.83%, respectively. When the methanol concentration was 90%, selenium-enriched rapeseed stalk extract at 4 mg / mL had a significant effect on reducing CdCl2 damage in TM4 cells, with a cell viability rate of 70.00%. In summary, 50% was selected as the optimal methanol concentration for subsequent experiments.

[0036] Example 4

[0037] Effect of solid-liquid ratio on the effect of selenium-enriched rapeseed on reducing CdCl2 damage in TM4 cells

[0038] Selenium-enriched rapeseed stalk extract treatment groups (a) and selenium-enriched rapeseed stalk extract co-treated with chromium chloride (b) were set respectively. At the same time, the extractant was fixed as methanol aqueous solution (50% v / v), the extraction time was 9 min, and different solid-liquid ratios (1:10, 1:15, 1:20, 1:25) were set respectively. The effect of the selenium-enriched rapeseed stalk extract on reducing CdCl2 damage in TM4 cells was studied. The detection method was the same as Example 2.

[0039] like Figure 3As shown in (a), compared with the control group, when the solid-liquid ratio was 1g:10ml, the selenium-enriched rapeseed stalk extract at concentrations of 4 and 6 mg / mL could promote the survival rate of TM4 cells, with the survival rates being 117.96% and 120.77%, respectively; when the solid-liquid ratio was 1g:15ml and 1g:20ml, the selenium-enriched rapeseed stalk extract at a concentration of 6 mg / mL could promote the survival rate of TM4 cells (119.51% and 127.61%, respectively); when the solid-liquid ratio was 1g:25ml, the selenium-enriched rapeseed stalk extract at concentrations of 2, 4 and 6 mg / mL could promote the proliferation of TM4 cells, with the survival rates being 122.04%, 121.95% and 129.76%, respectively.

[0040] like Figure 3 As shown in (b), compared with the CdCl2 group, the selenium-enriched rapeseed extract at 2, 4, and 6 mg / mL concentrations significantly reduced CdCl2 damage in TM4 cells when treated with 1g:10ml, 1g:15ml, 1g:20ml, and 1g:25ml solid-to-liquid ratios. Considering that treating cells with 1g:15ml of the extract alone had minimal effect, a solid-to-liquid ratio of 1g:15ml was ultimately selected for subsequent experiments.

[0041] Example 5

[0042] Effect of extraction time on the effect of selenium-enriched rapeseed on reducing CdCl2 damage in TM4 cells

[0043] Selenium-enriched rapeseed stalk extract treatment group (a) and selenium-enriched rapeseed stalk extract and chromium chloride co-treatment group (b) were set respectively. At the same time, the solid-liquid ratio of rapeseed stalk to methanol aqueous solution (50% v / v) was fixed at 1 g:15 ml. Different extraction times (6, 9, 12, and 18 min) were set respectively. The effect of the selenium-enriched rapeseed stalk extract on reducing CdCl2 damage in TM4 cells was studied. The detection method was the same as in Example 2.

[0044] like Figure 4 As shown in (a), compared with the control group, when the extraction time was 6 min and 12 min, the selenium-enriched rapeseed stalk extract at a concentration of 6 mg / mL could promote the survival rate of TM4 cells, which were 128.72% and 126.40% respectively; when the extraction time was 9 min, the selenium-enriched rapeseed stalk extract had no significant effect on the survival rate of TM4 cells; when the extraction time was 18 min, the selenium-enriched rapeseed stalk extract at concentrations of 4 and 6 mg / mL promoted the survival rate of TM4 cells, which were 127.95% and 127.77% respectively.

[0045] like Figure 4As shown in (b), compared with the CdCl2 group, the selenium-enriched rapeseed extract at concentrations of 2, 4, and 6 mg / mL significantly reduced CdCl2-induced damage in TM4 cells when the extraction time was 6, 9, 12, and 18 minutes. The highest cell survival rates when the extract and CdCl2 were co-treated were 142.35% (4 mg / mL), 141.47% (6 mg / mL), 146.33% (6 mg / mL), and 147.70% (6 mg / mL), respectively. Taking into account the extraction throughput and the effect of reducing CdCl2-induced damage in TM4 cells, 9 minutes was ultimately selected as the optimal extraction time.

[0046] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for extracting selenium-enriched rapeseed extract for improving cadmium-damaged male reproductive capacity, characterized in that: The following steps are involved: Selenium-enriched rapeseed stems were extracted with methanol-water solution to obtain selenium-enriched rapeseed extract.

2. The extraction method according to claim 1, wherein The volume fraction of the methanol aqueous solution is 30-70%.

3. The extraction method according to claim 2, wherein The solid-liquid ratio of the selenium-enriched rapeseed stems and the methanol aqueous solution is: 1g: (10-25)ml.

4. The extraction method according to claim 3, wherein Extraction time is: 6 to 18 minutes.

5. The selenium-enriched rapeseed extract prepared by the method according to any one of claims 1 to 4.

6. Use of the selenium-enriched rapeseed extract according to claim 5 in the preparation of a drug for improving male reproductive capacity damaged by cadmium.

7. The use according to claim 6, characterized in that The concentration of the selenium-enriched rapeseed extract in the medicine is 2-6 mg / ml.

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