A curcumin and blueberry polyphenol composition and its application in improving male reproductive damage induced by microplastics

Abstract

The present application relates to the fields of biological medicine and food technology, and specifically discloses a curcumin and blueberry polyphenol composition and its application in improving male reproductive damage induced by microplastics. The active ingredients of the composition include curcumin and blueberry polyphenol extract, and the mass ratio of the two is 1:1 to 1:10. The blueberry polyphenol extract is obtained by ethanol reflux extraction of blueberries, D101 macroporous adsorption resin adsorption, water and low-concentration ethanol impurity removal, ethanol elution to enrich polyphenols, and vacuum drying. Experimental results show that, in terms of functional indicators such as testosterone level and total sperm motility, both curcumin and blueberry polyphenol extract alone can improve male reproductive function damage induced by microplastics or nanoplastics to a certain extent, and the improvement degree is further improved after combined intervention. Among them, the effect of combined intervention is significantly better than that of curcumin or blueberry polyphenol extract alone in terms of testosterone recovery. The composition can also reduce the level of reactive oxygen species in reproductive-related cells, improve the increase of NAT10 protein expression, and abnormal changes of ac4C / C ratio in RNA. The composition of the present application can be used to prepare drugs or dietary supplements for preventing, reducing or treating male reproductive damage induced by microplastic or nanoplatic exposure.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and food technology, specifically to a composition of curcumin and blueberry polyphenols and its application in improving microplastic-induced male reproductive damage. Background Technology

[0002] With the widespread use of plastic products in production and daily life, and the improper disposal of plastic waste, microplastics and nanoplastics have gradually become important emerging environmental pollutants. Microplastics typically refer to plastic particles with a diameter of less than 5 mm, while nanoplastics typically refer to plastic particles with a diameter at the nanoscale. These particles can exist in water, soil, atmosphere, the food chain, and everyday consumer products, and may enter the body through drinking water, food ingestion, inhalation, or skin contact. Due to their smaller particle size and larger specific surface area, nanoplastics may pose a higher risk in terms of migration within the body, tissue deposition, and crossing biological barriers, thus their potential health hazards have received widespread attention.

[0003] The male reproductive system is highly sensitive to environmental pollutants. Existing research suggests that exposure to microplastics or nanoplastics may affect the testes, epididymis, interstitial cells, and spermatogenesis, leading to decreased sperm quality, weakened sperm motility, and abnormal male hormone secretion. For male reproductive function, testosterone levels and total sperm motility are important indicators for evaluating reproductive damage and its repair effects. Therefore, developing intervention products that can improve the decreased testosterone levels, reduced sperm motility, and increased oxidative stress caused by microplastic or nanoplastic exposure has significant application value.

[0004] Currently, there is a lack of specific, effective, and long-term interventions for reproductive damage caused by exposure to environmental microplastics or nanoplastics. Existing protective strategies mostly focus on reducing oxidative stress, such as using vitamin antioxidants, plant extracts, or other nutritional supplements to remove excess reactive oxygen species within cells. While these methods can alleviate oxidative damage to some extent, most interventions primarily target existing downstream oxidative stress responses and struggle to simultaneously address the comprehensive restoration of reproductive endocrine function, sperm motility, and the homeostasis of reproductive-related cells and molecules.

[0005] Therefore, it is necessary to provide a novel composition and its preparation method to form a composition capable of preventing, mitigating, or improving male reproductive damage induced by microplastic or nanoplastic exposure. This composition not only exerts antioxidant and cytoprotective effects but also helps improve testosterone levels, total sperm motility, and molecular homeostasis of reproductive-related cells, thus providing a new intervention strategy for reproductive health risks associated with environmental microplastic or nanoplastic exposure. Summary of the Invention

[0006] In view of the inadequacy of existing intervention methods for male reproductive damage induced by microplastic or nanoplastic exposure, and the difficulty of simultaneously improving oxidative stress, reproductive endocrine function, sperm motility and abnormal molecular homeostasis of reproductive-related cells with existing common antioxidants or crude plant extracts, the purpose of this invention is to provide a curcumin and blueberry polyphenol composition for preventing, mitigating or improving male reproductive damage induced by microplastic or nanoplastic exposure, as well as its preparation method and application.

[0007] According to a first aspect of the present invention, a curcumin and blueberry polyphenol composition for preventing, mitigating or improving male reproductive damage induced by exposure to microplastics or nanoplastics is provided, wherein the active ingredient of the composition consists of curcumin and blueberry polyphenol extract, wherein the mass ratio of curcumin to blueberry polyphenol extract is 1:1 to 1:10; wherein the blueberry polyphenol extract is a purified extract obtained from blueberries as raw material by ethanol extraction, macroporous adsorption resin adsorption, impurity removal elution and elution with 70% ethanol.

[0008] Preferably, the mass ratio of curcumin to blueberry polyphenol extract is 1:1 to 1:5; preferably, the mass ratio of curcumin to blueberry polyphenol extract is 1:2.

[0009] Preferably, the purity of the curcumin is not less than 95%.

[0010] Preferably, the preparation method of the blueberry polyphenol extract includes the following steps:

[0011] (1) Take blueberry fruit, add 80% ethanol solution for reflux extraction, filter and combine the filtrates;

[0012] (2) The ethanol in the filtrate is recovered to obtain concentrated aqueous solution, which is then diluted with water to obtain the column loading solution;

[0013] (3) The liquid in the column is adsorbed through D101 macroporous adsorption resin;

[0014] (4) Use pure water and 10% ethanol solution to remove impurities in sequence;

[0015] (5) Elute with 70% ethanol solution and collect the 70% ethanol eluent;

[0016] (6) The 70% ethanol eluent was dried under reduced pressure to obtain the blueberry polyphenol extract.

[0017] Preferably, in step (1), the ratio of blueberry fruit to 80% ethanol solution is 1:4 g / mL, and the extraction is carried out by reflux twice for 30 minutes each time; in step (4), the amount of pure water used is 3 BV, and the amount of 10% ethanol solution used is 2 BV; in step (5), the amount of 70% ethanol solution used is 3 BV; in step (6), the vacuum drying temperature is 40℃.

[0018] Preferably, the blueberry polyphenol extract contains at least two of the following: chlorogenic acid, cyanidin-3-O-glucoside, rutin, proanthocyanidin B1, D-catechin, L-epicatechin, and quercetin.

[0019] Preferably, the blueberry polyphenol extract contains at least chlorogenic acid and cyanidin-3-O-glucoside.

[0020] Preferably, the composition further includes pharmaceutically acceptable or food-acceptable excipients.

[0021] Preferably, the excipients include one or more of suspending agents, encapsulating materials, fillers, disintegrants, binders, stabilizers, and flavoring agents; preferably, the suspending agent includes sodium carboxymethyl cellulose.

[0022] Preferably, the dosage form of the composition is tablets, capsules, granules, powders, oral liquids, suspensions, functional beverages, or solid beverages.

[0023] According to another aspect of the present invention, a method for preparing the curcumin and blueberry polyphenol composition is provided, comprising the following steps:

[0024] S1, Preparation of blueberry polyphenol extract;

[0025] S2, Curcumin and the blueberry polyphenol extract are mixed at a mass ratio of 1:1 to 1:10 to obtain a mixture of active ingredients;

[0026] S3, the active ingredient mixture is mixed with pharmaceutically acceptable or food-acceptable excipients to prepare the curcumin and blueberry polyphenol composition.

[0027] Preferably, step S1 includes: taking blueberry fruit, adding 80% ethanol solution for reflux extraction, filtering and combining the filtrates, recovering ethanol under reduced pressure and diluting with water to obtain a column loading solution; adsorbing the column loading solution through a D101 macroporous adsorption resin, removing impurities sequentially with pure water and 10% ethanol solution, eluting with 70% ethanol solution, collecting the 70% ethanol eluent and drying under reduced pressure to obtain the blueberry polyphenol extract.

[0028] According to another aspect of the invention, the use of the curcumin and blueberry polyphenol composition described above in the preparation of products for the prevention, mitigation or treatment of male reproductive damage induced by exposure to microplastics or nanoplastics is provided.

[0029] Preferably, the product is a medicine, dietary supplement, or functional food.

[0030] Preferably, the male reproductive damage includes one or more of the following: decreased serum testosterone levels, decreased total sperm motility, abnormal male sex hormone secretion, increased reactive oxygen species levels in germ cells, increased NAT10 protein expression, and abnormal ac4C / C ratio in RNA.

[0031] Preferably, the microplastic or nanoplastic is a polystyrene microplastic or a polystyrene nanoplastic.

[0032] Preferably, the curcumin and blueberry polyphenol composition is used to increase serum testosterone levels and / or increase total sperm motility after exposure to microplastics or nanoplastics.

[0033] Preferably, the curcumin and blueberry polyphenol composition is used to reduce the level of reactive oxygen species in reproductive cells induced by exposure to microplastics or nanoplastics.

[0034] Preferably, the curcumin and blueberry polyphenol composition is used to reduce NAT10 protein expression induced by microplastic or nanoplastic exposure and / or regulate the ac4C / C ratio in RNA.

[0035] Preferably, the ac4C / C ratio is detected by liquid chromatography-tandem mass spectrometry; the detection includes: extracting total RNA from cells treated with the composition, enzymatically digesting the total RNA into mononucleotides, adding MTBSTFA for derivatization, and monitoring the ion signals of ac4C and C using liquid chromatography-tandem mass spectrometry.

[0036] Preferably, the ratio of curcumin to blueberry polyphenol extract is 50 mg / kg curcumin to 100 mg / kg blueberry polyphenol extract.

[0037] According to another aspect of the present invention, a method for non-diagnostic in vitro evaluation of the curcumin and blueberry polyphenol composition on the repair of germ cell damage induced by microplastics or nanoplastics is provided, comprising the following steps:

[0038] A1. Use polystyrene microplastics or polystyrene nanoplastics to treat reproductive-related cells and establish a cell damage model caused by microplastics or nanoplastics.

[0039] A2, the damaged cell model was treated with the curcumin and blueberry polyphenol composition;

[0040] A3, detect one or more of the following in the cells after treatment: NAT10 protein expression level, ac4C / C ratio in RNA, testosterone secretion level, and reactive oxygen species level;

[0041] A4. Evaluate the repair effect of the curcumin and blueberry polyphenol composition on microplastic or nanoplastic-induced germ cell damage based on the detection results of step A3.

[0042] Preferably, in step A1, the reproductive-related cells are mouse testicular interstitial cells TM3; the treatment concentration of the polystyrene nanoplastic is 50 μg / mL, and the treatment time is 24 hours.

[0043] Preferably, in step A2, the final concentration of curcumin is 3.7 μg / mL, the final concentration of blueberry polyphenol extract is 7.4 μg / mL, and the treatment time is 24 hours.

[0044] Compared with existing technologies, this invention, by combining curcumin (Cur) with blueberry polyphenol extract (BE) prepared through a specific process in an appropriate ratio, obtains a composition that can be used to improve male reproductive damage induced by microplastic or nanoplastic exposure. This composition helps reduce the impact of some water-soluble impurities and highly polar non-target components in ordinary crude blueberry extract on subsequent applications, making polyphenols or anthocyanins such as chlorogenic acid and cyanidin-3-O-glucoside more suitable for synergistic effects with curcumin. Furthermore, experimental results show that both curcumin and blueberry polyphenol extract, alone, can improve microplastic-induced male reproductive function damage to a certain extent, and the protective effect is further enhanced after combined intervention. In particular, regarding TM3 cell testosterone secretion and the recovery of serum testosterone in microplastic-exposed mice, the combined group showed significantly better improvement than the curcumin-only group and the blueberry polyphenol extract-only group; in terms of total sperm motility, the recovery degree of the combined group was also higher than that of the corresponding single-drug groups. Furthermore, the composition of this invention can reduce the level of reactive oxygen species induced by microplastics or nanoplastics in cells, improve the elevated expression of NAT10 protein, and alleviate abnormal changes in the ac4C / C ratio in RNA. This indicates that the composition of this invention can comprehensively improve male reproductive damage induced by microplastics or nanoplastics from multiple levels, including oxidative stress, reproductive endocrine function, sperm motility, and reproductive-related cellular molecular homeostasis. Moreover, the raw materials are readily available, and the preparation process is relatively simple, making it suitable for further development into a drug or dietary supplement. Attached Figure Description

[0045] Figure 1This is a flow chart of the preparation process of blueberry polyphenol extract in Example 1 of the present invention. The chart shows the process in sequence: pretreatment of fresh blueberries, reflux extraction with 80% ethanol, solvent recovery under reduced pressure, adsorption with D101 macroporous adsorption resin, removal of impurities with water and low concentration ethanol, elution and enrichment of polyphenols with 70% ethanol, and drying under reduced pressure to obtain powdered blueberry polyphenol extract.

[0046] Figure 2 This is a graph showing the regulatory effect of the composition of the present invention on microplastic-induced abnormal NAT10 / ac4C molecular homeostasis in cells. Figure 2 A shows a representative extracted ion chromatogram of intracellular ac4C-NMP detected by LC-MS / MS, indicating the changes in ac4C-NMP signal in the normal control group, PS-NPs model group, and PS-NPs+Cur / BE combination treatment group; Figure 2 B is an immunoblot image of Western blotting detection of NAT10 protein expression level, with GAPDH as an internal control protein; Figure 2 C is a quantitative statistical graph of intracellular ac4C modification levels, used to characterize changes in RNA modification levels after microplastic exposure and composition intervention.

[0047] Figure 3 This is a graph showing the effect of the composition of the present invention on the recovery of testosterone levels induced by microplastics. Among them, Figure 3 A shows the serum testosterone level detection results of C57BL / 6 male mice, indicating the changes in serum testosterone levels in the normal control group, PS-NPs model group, PS-NPs+Cur treatment group, PS-NPs+BE treatment group, and PS-NPs+Cur / BE combination treatment group. Figure 3 B shows the testosterone level in the TM3 cell culture supernatant, illustrating the effects of microplastic exposure and different interventions on testosterone secretion function in testicular interstitial cells. (Figure 1 shows...) P<0.001 (compared with the normal control group); # and ## represent P<0.05 and P<0.01 respectively (compared with the PS-NPs model group); @ represents P<0.05 (compared with the single-drug group shown in parentheses).

[0048] Figure 4 This is a graph showing the inhibitory effect of the composition of the present invention on the increase of reactive oxygen species levels in cells induced by microplastics. Among them, Figure 4 A is a fluorescence micrograph of intracellular reactive oxygen species (ROS) levels detected using the DCFH-DA fluorescent probe, including a fluorescence channel map, a bright field map, and a combined map, used to show the changes in the intensity of intracellular ROS signals in the normal control group, PS-NPs model group, PS-NPs+Cur treatment group, and PS-NPs+Cur / BE combination treatment group. Figure 4 B is Figure 4The quantitative statistical graph of fluorescence signal in A is used to characterize the relative changes in intracellular reactive oxygen species levels in each treatment group.

[0049] Figure 5 This is a statistical graph showing the effect of the composition of the present invention on the total sperm motility of mice exposed to microplastics. The graph shows the changes in total sperm motility of mice in the normal control group, nanoplastic model group, nanoplastic + Cur treatment group, nanoplastic + BE treatment group, and nanoplastic + Cur / BE combination treatment group, used to compare the effects of curcumin and blueberry polyphenol extract alone and in combination on improving the decreased sperm motility caused by nanoplastic exposure. Detailed Implementation

[0050] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Conventional adjustments made by those skilled in the art to the source of raw materials, extraction scale, formulation, detection equipment model, and experimental parameters without departing from the concept of the present invention should all be included within the scope of protection of the present invention.

[0051] Example 1: Preparation of blueberry polyphenol extract and a combination of curcumin and blueberry polyphenols

[0052] This embodiment illustrates a specific preparation method of the blueberry polyphenol extract and the curcumin and blueberry polyphenol composition described in this invention. The preparation process is as follows: Figure 1 As shown.

[0053] Fresh blueberries were taken, washed, and crushed. An 80% (v / v) ethanol solution was added for reflux extraction. The mass-to-volume ratio of fresh blueberries to 80% ethanol solution was 1:4 g / mL. In one specific embodiment, 375 g of fresh blueberries were added to 1500 mL of 80% ethanol solution, and extracted twice under reflux conditions for 30 min each time. After extraction, the mixture was filtered, and the two filtrates were combined. The ethanol was recovered under reduced pressure to obtain approximately 300 mL of concentrated aqueous solution. The concentrated aqueous solution was diluted with pure water to make the volume ratio of concentrated aqueous solution to the total volume of the diluted solution for column loading approximately 3:10. In this embodiment, approximately 300 mL of concentrated aqueous solution was diluted to 1000 mL to obtain the solution for column loading.

[0054] The resulting column buffer was adsorbed onto a pretreated D101 macroporous adsorption resin column. After adsorption, elution was performed sequentially: first, pure water was used to remove some water-soluble components; then, 10% ethanol solution was used to further remove some highly polar non-target components; finally, 70% ethanol solution was used to collect the 70% ethanol eluent. In one specific embodiment, the elution volume ratio of pure water, 10% ethanol solution, and 70% ethanol solution was 2:1:2; in this example, 1000 mL of pure water, 500 mL of 10% ethanol solution, and 1000 mL of 70% ethanol solution were used sequentially for elution. The 70% ethanol eluent was dried under reduced pressure at 40°C to obtain 2.207 g of a purple-red powdered blueberry polyphenol extract.

[0055] The components of the obtained blueberry polyphenol extract were analyzed by liquid chromatography-tandem mass spectrometry. A Sepax GP-C18 column was used for detection, with a mobile phase of 0.1% formic acid aqueous solution and acetonitrile, and gradient elution was employed for separation. The obtained blueberry polyphenol extract and mixed standards were analyzed under the same chromatographic and mass spectrometric conditions, and comparisons were made based on retention time and secondary mass spectrometry fragmentation information. The results showed that chlorogenic acid, cyanidin-3-O-glucoside, rutin, proanthocyanidin B1, D-catechin, L-epicatechin, and quercetin, among other polyphenols and anthocyanins, were detected in the obtained blueberry polyphenol extract. Chlorogenic acid and cyanidin-3-O-glucoside were the representative components of this blueberry polyphenol extract.

[0056] A standard of curcumin with a purity of not less than 95% was weighed and mixed with the above-mentioned blueberry polyphenol extract at a mass ratio of 1:2. Sodium carboxymethyl cellulose (0.5% of the total mass of the composition) was added as a suspending agent. The mixture was then thoroughly ground and mixed in a mortar to obtain the curcumin and blueberry polyphenol composition. That is, in this embodiment, the mass ratio of curcumin, blueberry polyphenol extract, and sodium carboxymethyl cellulose is 1:2:0.015. The resulting composition can be directly used in subsequent cell and animal experiments, or, as needed, can be further formulated with pharmaceutically acceptable or food-acceptable excipients to prepare capsules, tablets, granules, powders, oral liquids, suspensions, solid beverages, or functional beverages.

[0057] Depend on Figure 1As can be seen, this embodiment employs a process route of 80% ethanol reflux extraction, D101 macroporous adsorption resin adsorption, water and low-concentration ethanol elution, 70% ethanol elution, and low-temperature vacuum drying to obtain blueberry polyphenol extract. This blueberry polyphenol extract is then further combined with curcumin to form a composition. This process can reduce the impact of some water-soluble impurities and highly polar non-target components on subsequent applications, and helps to obtain a refined blueberry extract containing polyphenols or anthocyanins such as chlorogenic acid and cyanidin-3-O-glucoside.

[0058] Example 2: Regulatory effect of curcumin and blueberry polyphenol composition on microplastic-induced abnormal cell molecular homeostasis.

[0059] This embodiment illustrates the regulatory effect of the composition of the present invention on microplastic-induced increases in NAT10 expression and abnormal RNA ac4C modification in cells. The detection results are as follows: Figure 2 As shown.

[0060] TM3 interstitial testicular cells from mice in the logarithmic growth phase were seeded in cell culture plates and incubated at 37°C. Cells were cultured under controlled conditions until they adhered to the culture vessel and reached a suitable degree of confluence. A polystyrene nanoplastic suspension was added to both the model group and the drug-treated group to a final concentration of 50 μg / mL, and the cells were incubated for another 24 h to establish a microplastic-induced reproductive-related cell damage model. This model can simulate the molecular homeostasis imbalance that occurs in germ cells or reproductive-related cells after microplastic exposure.

[0061] After the model was established, the curcumin and blueberry polyphenol composition prepared in Example 1 was added to the cells in the drug-treated group to achieve a final concentration of 3.7 μg / mL for curcumin and 7.4 μg / mL for blueberry polyphenol extract, and the cells were cultured for another 24 h. After culture, the cells were collected and the cell samples were divided into two parts: one part was used for total protein extraction and Western blot detection, and the other part was used for total RNA extraction and LC-MS / MS detection.

[0062] For Western blotting, cells were lysed using RIPA lysis buffer and total protein was extracted. After determining the protein concentration, SDS-PAGE electrophoresis, cell transfer, blocking, and antibody incubation were performed to detect NAT10 protein expression levels, with GAPDH used as an internal control protein. Results are as follows: Figure 2 As shown in Figure B, compared with the normal control group, the expression level of NAT10 protein in cells was significantly increased after treatment with polystyrene nanoplastics; after intervention with the composition of the present invention, the expression level of NAT10 protein decreased, indicating that the composition of the present invention can inhibit or alleviate the abnormal expression of NAT10 induced by microplastics.

[0063] For RNA modification detection, after extracting total RNA from cells, 10 μg of total RNA was taken and hydrolyzed with S1 nuclease and snake venom phosphodiesterase at 37°C for 2 h to obtain a mononucleotide mixture. Subsequently, MTBSTFA was added for derivatization, and the reaction was carried out at 37°C with constant temperature shaking for 30 min. After derivatization, the ion signals of ac4C and C were detected by liquid chromatography-tandem mass spectrometry, and the ac4C / C ratio was calculated.

[0064] The liquid chromatography conditions were as follows: a C18 reversed-phase column was used; mobile phase A was 0.1% formic acid aqueous solution; mobile phase B was acetonitrile; and a gradient elution program was employed for separation. Mass spectrometry was performed using an electrospray ionization source in negative ion mode, monitoring the characteristic ion pairs of ac4C and C separately. Standard curves were established based on ac4C and C standards, and the molar percentage of ac4C / C in the sample was calculated. The calculation formula is:

[0065] ac4C level (%) = [Molar(ac4C) / (Molar(C)+Molar(ac4C))]×100%.

[0066] like Figure 2 A and Figure 2 As shown in Figure C, polystyrene nanoplastic treatment increased the level of ac4C modification in cells, suggesting that microplastic exposure can lead to abnormal RNA acetylation modification. After intervention with the composition of the present invention, the ac4C / C ratio decreased and tended to normal levels. The above results indicate that the composition of the present invention not only has antioxidant protective effects, but can also regulate microplastic-induced cell damage at the level of NAT10 / ac4C-related molecular homeostasis.

[0067] Example 3: The effect of a combination of curcumin and blueberry polyphenols on improving microplastic-induced testosterone decline.

[0068] This embodiment compares the effects of curcumin and blueberry polyphenol extract alone, as well as their combined intervention, on improving microplastic-induced testosterone decline. The test results are as follows: Figure 3 As shown. Figure 3 A shows the serum testosterone level in C57BL / 6 male mice. Figure 3 B shows the testosterone level in the TM3 cell culture supernatant.

[0069] I. Cell-level detection

[0070] A microplastic injury model of TM3 cells was established according to the method in Example 2. After the model was established, normal control group, microplastic model group, curcumin alone group, blueberry polyphenol extract alone group, and curcumin and blueberry polyphenol combination group were set up respectively. Among them, the microplastic model group was treated with polystyrene nanoplastics; the curcumin alone group was treated with curcumin in addition to microplastic treatment, so that the final concentration of curcumin was 3.7 μg / mL; the blueberry polyphenol extract alone group was treated with blueberry polyphenol extract prepared in Example 1 in addition to microplastic treatment, so that the final concentration of blueberry polyphenol extract was 7.4 μg / mL; the combination group was treated with both curcumin and blueberry polyphenol extract in addition to microplastic treatment, with the final concentration of curcumin being 3.7 μg / mL and the final concentration of blueberry polyphenol extract being 7.4 μg / mL. Each treatment group was cultured for another 24 h.

[0071] After processing, the cell culture supernatant was collected, and testosterone levels were detected using an enzyme-linked immunosorbent assay (ELISA). A standard curve was established according to the kit instructions, and the testosterone concentration in each group of samples was calculated.

[0072] like Figure 3 As shown in Figure B, compared with the normal control group, the testosterone level in the supernatant of TM3 cells in the microplastic model group was significantly reduced, indicating that microplastic exposure can inhibit the testosterone secretion function of testicular interstitial cells. Compared with the microplastic model group, both curcumin intervention alone and blueberry polyphenol extract intervention alone could increase the testosterone secretion level of TM3 cells, indicating that both active components have a certain protective effect. Further comparison revealed that after combined intervention with curcumin and blueberry polyphenol extract, the testosterone level of TM3 cells further increased, and its recovery degree was significantly higher than that of the curcumin single-use group and the blueberry polyphenol extract single-use group. The above results indicate that under the dosage conditions used in this embodiment, the combined use of curcumin and blueberry polyphenol extract can produce a more significant testosterone secretion recovery effect than either component alone, suggesting that the combined use of the two active components has a significant synergistic effect.

[0073]

[0074] The King's Law formula is used to determine the final effect after combined drug administration. King's Law formula: q = E(A + B) / (EA + EB - EA·EB). In this formula, the numerator represents the measured combined effect, and the denominator is the expected combined effect.

[0075] Substituting the testosterone secretion recovery rate into the formula, the recovery rate of curcumin alone was 0.183, the recovery rate of blueberry polyphenol extract alone was 0.136, and the recovery rate of combined curcumin and blueberry polyphenol extract intervention was 0.419. According to the King's Law, the synergy index was calculated to be 1.426. When the synergy index is >1.15, it indicates that the two drugs exert a synergistic effect when used together. Therefore, the combined intervention of curcumin and blueberry polyphenol extract exerts a synergistic effect, thus producing a better therapeutic effect.

[0076] II. Animal-level testing

[0077] SPF-grade male C57BL / 6 mice, 6-8 weeks old and weighing 20±2 g, were selected. After one week of acclimatization, the mice were randomly divided into 5 groups: normal control group, microplastic model group, curcumin alone group, blueberry polyphenol extract alone group, and curcumin and blueberry polyphenol combination group, with 10 mice in each group. The normal control group was given an equal amount of solvent daily; the microplastic model group was given polystyrene nanoplastic suspension at a concentration of 1 mg / L in drinking water for 70 consecutive days; the curcumin monotherapy group was given polystyrene nanoplastic suspension in drinking water and was given curcumin 50 mg / kg by gavage daily; the blueberry polyphenol extract monotherapy group was given polystyrene nanoplastic suspension in drinking water and was given blueberry polyphenol extract 100 mg / kg prepared in Example 1 by gavage daily; the combination group was given polystyrene nanoplastic suspension in drinking water and was given the combination prepared in Example 1 by gavage daily, wherein the curcumin dose was 50 mg / kg and the blueberry polyphenol extract dose was 100 mg / kg, for 70 consecutive days.

[0078] Blood samples were collected from mice after the last administration, serum was separated, and serum testosterone levels were detected by ELISA.

[0079] like Figure 3 As shown in Figure A, compared with the normal control group, the serum testosterone level in the microplastic model group was significantly lower, indicating that long-term microplastic exposure can cause endocrine dysfunction in male mice. Compared with the microplastic model group, both curcumin alone and blueberry polyphenol extract alone could restore serum testosterone levels in mice to some extent; after combined intervention with curcumin and blueberry polyphenol extract, the serum testosterone level in mice further increased, and its restorative effect was significantly better than that of the curcumin alone group or the blueberry polyphenol extract alone group. This result is consistent with the results of the TM3 cell experiment, indicating that the combined use of curcumin and blueberry polyphenol extract can produce a more significant testosterone restoration effect than the use of either component alone.

[0080] Example 4: Inhibitory effect of curcumin and blueberry polyphenol composition on microplastic-induced cellular oxidative stress.

[0081] This embodiment illustrates the inhibitory effect of the composition of the present invention on the increase of intracellular reactive oxygen species induced by microplastics. The detection results are as follows: Figure 4 As shown.

[0082] TM3 cells were cultured according to the method in Example 2 to establish a polystyrene nanoplastic damage model. The experiment included a normal control group, a microplastic model group, a curcumin-only group, and a curcumin-blueberry polyphenol combination group. The microplastic model group was treated with polystyrene nanoplastics; the curcumin-only group received curcumin in addition to the microplastic treatment; and the combination group received the curcumin-blueberry polyphenol combination prepared in Example 1 in addition to the microplastic treatment.

[0083] After treatment, the cell culture medium was aspirated, and the cells were washed once with PBS. A final concentration of 10 μM DCFH-DA fluorescent probe was added, and the cells were incubated at 37°C in the dark for 20 min. After incubation, the cells were washed three times with serum-free medium to thoroughly remove any uninfiltrated DCFH-DA. Subsequently, fluorescence images of the cells were captured using a fluorescence microscope, and the green fluorescence intensity at a 488 nm excitation wavelength could be detected using flow cytometry, or the average fluorescence intensity could be analyzed based on the fluorescence images.

[0084] like Figure 4 As shown in Figure A, the normal control group cells showed weak fluorescence signals, while the microplastic model group cells exhibited significantly enhanced green fluorescence, indicating that microplastic treatment induced a large accumulation of intracellular ROS. The fluorescence intensity of the curcumin-only group was lower than that of the model group, while the fluorescence intensity of the composition group of this invention was further reduced. Figure 4 Quantitative results for B also showed that, compared with the microplastic model group, the ROS level in the cells of the composition group of the present invention was significantly reduced, and showed a further decreasing trend compared with curcumin intervention alone. These results indicate that, under the experimental conditions of this embodiment, the combination of curcumin and blueberry polyphenol extract can further enhance the inhibitory effect on microplastic-induced oxidative stress and improve the germ cell microenvironment.

[0085] Example 5: Effect of curcumin and blueberry polyphenol composition on improving total sperm motility in mice exposed to microplastics.

[0086] This example compares the effects of curcumin and blueberry polyphenol extract alone, as well as their combined intervention, on reducing the decline in total sperm motility in mice exposed to nanoplastics. The results are as follows: Figure 5 As shown.

[0087] SPF-grade male C57BL / 6 mice, 6-8 weeks old and weighing 20±2 g, were selected. After one week of acclimatization, the mice were randomly divided into 5 groups of 10 mice each: normal control group, microplastic model group, curcumin alone group, blueberry polyphenol extract alone group, and curcumin and blueberry polyphenol combination group.

[0088] The normal control group was given an equal amount of solvent daily; the microplastic model group was given polystyrene nanoplastic suspension at a concentration of 1 mg / L in drinking water for 70 consecutive days; the curcumin monotherapy group was given polystyrene nanoplastic suspension in drinking water and was given curcumin 50 mg / kg by gavage daily; the blueberry polyphenol extract monotherapy group was given polystyrene nanoplastic suspension in drinking water and was given blueberry polyphenol extract 100 mg / kg prepared in Example 1 by gavage daily; the combination group was given polystyrene nanoplastic suspension in drinking water and was given curcumin and blueberry polyphenol combination prepared in Example 1 by gavage daily, wherein the curcumin dose was 50 mg / kg and the blueberry polyphenol extract dose was 100 mg / kg, for 70 consecutive days.

[0089] Twenty-four hours after the last administration, mice were sacrificed. Under aseptic conditions, the bilateral epididymal tail tissue was isolated, minced, and incubated in preheated PBS buffer (37°C) to allow sperm to fully disperse, preparing a sperm suspension. 10 μL of the sperm suspension was added to a preheated glass slide, covered with a coverslip, and observed under an optical microscope at 400× magnification. At least five fields of view were randomly selected on a temperature-controlled stage, and the total number of sperm and the number of motile sperm in each field were counted. Total sperm motility was calculated using the following formula:

[0090] Total sperm motility (%) = (Total number of motile sperm / Total number of counted sperm) × 100%.

[0091] like Figure 5 As shown, compared with the normal control group, the total sperm motility of mice in the microplastic model group was significantly reduced, indicating that long-term microplastic exposure can lead to significant male reproductive toxicity. Compared with the microplastic model group, the total sperm motility of mice treated with curcumin alone and blueberry polyphenol extract alone was improved; after combined intervention with curcumin and blueberry polyphenol extract, the total sperm motility of mice was further improved, and the degree of recovery was higher than that of the corresponding single-drug groups. These results indicate that the curcumin and blueberry polyphenol composition of the present invention can effectively alleviate the decline in sperm motility caused by nanoplastic exposure and shows a synergistic effect superior to single-component intervention.

[0092] Example 6: Conversion of the effective dose of curcumin and blueberry polyphenol composition to the human equivalent dose

[0093] This embodiment illustrates the method for converting the effective dose of the composition of the present invention to the human equivalent dose based on the effective dose in animal experiments.

[0094] According to the animal experiment dosing regimen of Example 5, the dose of curcumin in the composition group was 50 mg / kg, and the dose of blueberry polyphenol extract was 100 mg / kg. Using the body surface area conversion method, the conversion factor between humans and mice (Km) was 12.3. The human equivalent dose (HED) was calculated using the following formula:

[0095] Human equivalent dose HED (mg / kg) = mouse dose (mg / kg) ÷ 12.3.

[0096] For a 60 kg adult, the equivalent daily intake of curcumin is:

[0097] (50÷12.3)×60≈244 mg / day.

[0098] The human equivalent daily intake of blueberry polyphenol extract is:

[0099] (100÷12.3)×60≈488 mg / day.

[0100] Therefore, the composition of this invention exhibits the effects of improving serum testosterone levels and increasing total sperm motility in animal experiments. Combined with the aforementioned cell experiments, the composition of this invention also has the effects of reducing ROS levels and regulating NAT10 / ac4C molecular homeostasis. The effective animal doses, when converted to human dosage, represent a low intake level, providing a dosage reference for further development into a drug or dietary supplement.

[0101] As demonstrated in Examples 1 to 6, the present invention prepares a refined blueberry extract rich in polyphenols and anthocyanins using a specific process, and then combines it with curcumin in a specific ratio. This combination can improve reproductive damage caused by microplastic or nanoplastic exposure at both the cellular and animal levels. Its effects are at least manifested in: reducing abnormal expression of NAT10 protein, regulating the ac4C / C ratio in RNA, reducing intracellular ROS levels, restoring testosterone secretion, and improving total sperm motility in mice exposed to microplastics. Therefore, the composition of the present invention can be used to prepare drugs or dietary supplements for the prevention, mitigation, or treatment of male reproductive damage and decreased sperm quality induced by microplastic or nanoplastic exposure.

Claims

1. A composition of curcumin and blueberry polyphenols, characterized in that, The active ingredients of the composition consist of curcumin and blueberry polyphenol extract, wherein the mass ratio of curcumin to blueberry polyphenol extract is 1:1 to 1:

10. The blueberry polyphenol extract is a refined extract obtained from blueberries through ethanol extraction, macroporous adsorption resin adsorption, impurity removal and elution with 70% ethanol.

2. The curcumin and blueberry polyphenol composition according to claim 1, characterized in that, The mass ratio of curcumin to blueberry polyphenol extract is 1:1 to 1:5; preferably, the mass ratio of curcumin to blueberry polyphenol extract is 1:

2.

3. The curcumin and blueberry polyphenol composition according to claim 1 or 2, characterized in that, The purity of the curcumin is not less than 95%.

4. The curcumin and blueberry polyphenol composition according to any one of claims 1 to 3, characterized in that, The preparation method of the blueberry polyphenol extract includes the following steps: (1) Take blueberry fruit, add 80% ethanol solution for reflux extraction, filter and combine the filtrates; (2) The ethanol in the filtrate is recovered to obtain concentrated aqueous solution, which is then diluted with water to obtain the column loading solution; (3) The liquid in the column is adsorbed through D101 macroporous adsorption resin; (4) Use pure water and 10% ethanol solution to remove impurities in sequence; (5) Elute with 70% ethanol solution and collect the 70% ethanol eluent; (6) The 70% ethanol eluent was dried under reduced pressure to obtain the blueberry polyphenol extract.

5. A method for preparing the curcumin and blueberry polyphenol composition according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1, Preparation of blueberry polyphenol extract; S2, Curcumin and the blueberry polyphenol extract are mixed at a mass ratio of 1:1 to 1:10 to obtain a mixture of active ingredients; S3, the active ingredient mixture is mixed with pharmaceutically acceptable or food-acceptable excipients to prepare the curcumin and blueberry polyphenol composition.

6. The preparation method according to claim 5, characterized in that, Step S1 includes: taking blueberry fruits, adding 80% ethanol solution for reflux extraction, filtering and combining the filtrates, recovering ethanol under reduced pressure and diluting with water to obtain the column loading solution; adsorbing the column loading solution through D101 macroporous adsorption resin, removing impurities with pure water and 10% ethanol solution in sequence, eluting with 70% ethanol solution, collecting the 70% ethanol eluent and drying under reduced pressure to obtain the blueberry polyphenol extract.

7. The use of the curcumin and blueberry polyphenol composition according to any one of claims 1 to 3 in the preparation of products for the prevention, mitigation or treatment of male reproductive damage induced by exposure to microplastics or nanoplastics.

8. The application according to claim 7, characterized in that, The male reproductive damage includes one or more of the following: decreased serum testosterone levels, decreased total sperm motility, abnormal male sex hormone secretion, increased reactive oxygen species levels in germ cells, increased NAT10 protein expression, and abnormal ac4C / C ratio in RNA.

9. The application according to any one of claims 7, characterized in that, The microplastics or nanoplastics are polystyrene microplastics or polystyrene nanoplastics.

10. A method for non-diagnostic in vitro evaluation of the curcumin and blueberry polyphenol composition according to any one of claims 1 to 3 on the repair of germ cell damage induced by microplastics or nanoplastics, characterized in that, Includes the following steps: A1. Use polystyrene microplastics or polystyrene nanoplastics to treat reproductive-related cells and establish a cell damage model caused by microplastics or nanoplastics. A2, the damaged cell model was treated with the curcumin and blueberry polyphenol composition; A3, detect one or more of the following in the cells after treatment: NAT10 protein expression level, ac4C / C ratio in RNA, testosterone secretion level, and reactive oxygen species level; A4. Evaluate the repair effect of the curcumin and blueberry polyphenol composition on microplastic or nanoplastic-induced germ cell damage based on the detection results of step A3.

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