Application of protocatechuic acid in promoting mouse embryonic developmental capacity

Abstract

The invention provides an application of protocatechuic acid in promoting the developmental capacity of a mouse embryo, belongs to the technical field of bioengineering, and relates to the application of protocatechuic acid in embryo in-vitro culture, high-purity protocatechuic acid (greater than or equal to 95%) is obtained by optimizing a preparation process, the blastocyst formation rate of the mouse embryo is remarkably increased (83.4% vs 61.5%) under the concentration of 10-100 mu M, and the ROS level is reduced; protocatechuic acid activates an Nrf2 / ARE anti-oxidation pathway, and the protocatechuic acid is non-toxic and suitable for wide popularization and application.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to the application of protocatechuic acid in promoting the developmental ability of mouse embryos. Background Art

[0002] Protocatechuic acid (PCA), with the chemical name of 3,4-dihydroxybenzoic acid, CAS registration number 99-50-3, molecular formula (HO)2C6H3COOH, and molecular weight: 154.12. The product is a white to brown crystalline powder and changes color in air. Density 1.54 g / cm 3 ; melting point about 200 °C (decomposed); solubility: soluble in 50 parts of water, soluble in ethanol and ether. Protocatechuic acid has diverse biological activities against different molecular targets and has antibacterial, antioxidant, anti-inflammatory, anti-hyperglycemic, and neuroprotective effects.

[0003] In in vitro culture of mouse embryos, oxidative stress caused by the accumulation of reactive oxygen species (ROS) is one of the main reasons for embryonic developmental failure. The blastocyst formation rate of traditional culture media (such as KSOM, G1 / G2) is usually lower than 65%. Existing antioxidants (such as vitamin C, glutathione) have problems of poor stability, low toxicity threshold, or limited effects. For example, vitamin C may induce DNA damage at a concentration > 100 μM. Therefore, there is an urgent need for an antioxidant additive that is highly efficient, low-toxic, and can be stably integrated into the existing culture system to improve the embryonic development rate and clinical application potential. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of protocatechuic acid in promoting the developmental ability of mouse embryos and solve the technical problems mentioned in the background art.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present invention is as follows:

[0006] The application of protocatechuic acid in promoting the developmental ability of mouse embryos, wherein the mouse embryos are pre-blastocyst stage mouse embryos and the culture time is 24 - 96 hours.

[0007] Further, the protocatechuic acid is a culture medium containing protocatechuic acid, and the purity of the protocatechuic acid is 95% - 99%.

[0008] Further, the culture medium is used to improve the embryonic survival rate, blastocyst formation rate, or reduce the intracellular reactive oxygen species level.

[0009] Further, the culture medium is KSOM, mHTF, or G1 / G2 series culture medium, the culture time is 24 - 96 hours, and it is applicable to early mouse embryos.

[0010] Furthermore, the concentration of protocatechuic acid in the culture medium is 10 - 100 μM.

[0011] Furthermore, it is preferred that the concentration of protocatechuic acid in the culture medium is 20 - 50 μM

[0012] Furthermore, the protocatechuic acid is prepared by a plant extraction method or by a chemical synthesis method.

[0013] Furthermore, the specific process of the plant extraction method is as follows: using Salvia miltiorrhiza or olive leaves as raw materials, extracting by ultrasonic-assisted ethanol reflux, and purifying by AB-8 macroporous resin to obtain protocatechuic acid with a purity ≥ 95%.

[0014] Furthermore, the specific process of the chemical synthesis preparation method is as follows: using vanillin as a precursor, and obtaining protocatechuic acid through an alkaline hydrolysis reaction.

[0015] Due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0016] The present invention prepares high-purity protocatechuic acid (≥ 95%), significantly improves the blastocyst formation rate of mouse embryos and reduces the intracellular reactive oxygen species level at a concentration of 10 - 100 μM; thereby promoting the embryonic development ability of mice, and having no toxicity, being suitable for wide promotion and use. Specific Embodiments

[0017] To make the purpose, technical solution and advantages of the present invention clearer, the following preferred embodiments are given to further illustrate the present invention in detail. However, it should be noted that many details listed in the specification are only for enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be realized even without these specific details.

[0018] Example 1

[0019] A preparation method of protocatechuic acid, comprising the following steps:

[0020] (1) Raw material treatment: crushing and sieving the dried roots of Salvia miltiorrhiza through a 60-mesh sieve to obtain Salvia miltiorrhiza powder;

[0021] (2) Ethanol extraction: adding 200 g of Salvia miltiorrhiza powder to ethanol with a concentration of 80% (material-liquid ratio 1:12), refluxing and extracting 3 times at a temperature of 80 °C, each time for 1.5 hours;

[0022] (3) Purification: combining the three extraction solutions to obtain a mixed extraction solution, concentrating the mixed extraction solution under reduced pressure until there is no alcohol smell, and then performing column chromatography on an AB-8 macroporous resin column, eluting successively with 20%, 30%, and 50% ethanol gradients, and collecting the 30% ethanol elution fraction;

[0023] (4) Drying: The eluate was freeze-dried to obtain a white powder (protocatechuic acid), and the purity was detected by HPLC to be ≥95% (chromatographic conditions: C18 column, gradient elution with acetonitrile - 0.1% phosphoric acid, flow rate 1.0 mL / min, detection wavelength 260 nm).

[0024] Example 2

[0025] Another preparation method of protocatechuic acid, comprising the following steps:

[0026] (1) Reaction: 10 g of vanillin was dissolved in 10% NaOH solution, and stirred at 100 °C for 4 hours to obtain a reaction solution;

[0027] (2) Acidification: The pH value of the reaction solution was adjusted to 2.0 with concentrated hydrochloric acid, precipitates were formed, filtered and washed with water until neutral to obtain a crude product;

[0028] (3) Purification: The crude product was recrystallized from ethanol three times to obtain needle-shaped crystals (protocatechuic acid), which were verified as PCA by 1H nuclear magnetic resonance spectrum (1HNMR, DMSO-d6) (δ 7.35 (d, 1H), 6.77 (d, 1H), 6.65 (s, 1H)), and the purity was 99.1%.

[0029] Example 3

[0030] A comparative experiment was conducted to detect the effect of protocatechuic acid on mouse embryonic development.

[0031] Embryo source: After superovulation of ICR mice, fertilized eggs were collected (n = 200) and randomly divided into 6 groups:

[0032] Control group: Mouse embryos were placed in a conventional KSOM medium and cultured at 37 °C and 5% CO2 for 72 hours, and the blastocyst formation rate and cell number were counted.

[0033] Experimental group 1: The protocatechuic acid with a concentration of 20 μM obtained in Example 1 was added to the KSOM medium, and then the mouse embryos were placed in the KSOM medium containing protocatechuic acid and cultured at 37 °C and 5% CO2 for 72 hours, and the blastocyst formation rate and cell number were counted.

[0034] Experimental group 2: The protocatechuic acid with a concentration of 50 μM obtained in Example 1 was added to the KSOM medium, and then the mouse embryos were placed in the KSOM medium containing protocatechuic acid and cultured at 37 °C and 5% CO2 for 72 hours, and the blastocyst formation rate and cell number were counted.

[0035] Experimental group 3: Add protocatechuic acid with a concentration of 50 μM obtained in Example 2 to the KSOM medium, then place the mouse embryos in the KSOM medium containing protocatechuic acid and culture them for 72 hours under the conditions of 37 °C and 5% CO2, and count the blastocyst formation rate and the number of cells.

[0036] Experimental group 4: Add protocatechuic acid with a concentration of 210 μM obtained in Example 1 to the KSOM medium, then place the mouse embryos in the KSOM medium containing protocatechuic acid and culture them for 72 hours under the conditions of 37 °C and 5% CO2, and count the blastocyst formation rate and the number of cells.

[0037] Experimental group 5: Add protocatechuic acid with a concentration of 210 μM obtained in Example 2 to the KSOM medium, then place the mouse embryos in the KSOM medium containing protocatechuic acid and culture them for 72 hours under the conditions of 37 °C and 5% CO2, and count the blastocyst formation rate and the number of cells.

[0038] Control group 1: Add 100 μM vitamin C (crude extract of Salvia miltiorrhiza PCA, purity < 30%) to the KSOM medium, then place the mouse embryos in the KSOM medium containing vitamin C and culture them for 72 hours under the conditions of 37 °C and 5% CO2, and count the blastocyst formation rate and the number of cells.

[0039] Control group 2: Add 50 μM gallic acid to the KSOM medium, then place the mouse embryos in the KSOM medium containing gallic acid and culture them for 72 hours under the conditions of 37 °C and 5% CO2, and count the blastocyst formation rate and the number of cells.

[0040] The results obtained are shown in the following table:

[0041] Group Blastocyst formation rate (%) Number of cells (mean ± SD) ROS level (%) Apoptosis rate (%) Control group 61.5 43.8±5.2 100 12.3 Experimental group 1 76.8 57.3±6.5 68 9.1 Experimental group 2 83.4 63.1±7.2 55 7.5 Experimental group 3 82.1 62.7±6.8 57 7.8 Experimental group 4 56.5 42.8±6.5 80 19.3 Experimental group 5 55.6 41.8±6.7 83 19.5 Comparative example 1 70.2 50.1±5.9 82 18.6 Comparative example 2 72.5 53.4±6.3 70 14.2

[0042] As can be seen from the above table, experimental groups 1 - 3 (protocatechuic acid 20 - 50 μM) are significantly superior to the existing antioxidants (control group 2) in improving the blastocyst formation rate and the number of cells, and reducing the intracellular reactive oxygen species (ROS) level and apoptosis rate; in control group 1, the crude extract of Salvia miltiorrhiza (PCA purity < 30%) is used, and the blastocyst formation rate and the number of cells are lower than those in experimental groups 1 - 3 (protocatechuic acid 20 - 50 μM); thus, it can be obtained that the KSOM medium containing protocatechuic acid with a purity ≥ 95% can improve the blastocyst formation rate of mouse embryos and reduce the intracellular reactive oxygen species level.

[0043] Example 4

[0044] Conduct mechanism of action and safety verification

[0045] (1) Western blot: Detect the protein expression of Nrf2, HO-1, and Caspase-3 in embryos;

[0046] (2) TUNEL staining: Analyze the apoptosis rate of embryos;

[0047] (3) Long-term toxicity: Continuously culture with PCA (50 μM) for 120 hours and observe the developmental potential after embryo implantation.

[0048] The results showed that in experimental groups 1-3 (protocatechuic acid 20-50 μM), the nuclear translocation of Nrf2 increased by 2.5 times, the expression of HO-1 was upregulated by 3.1 times, and Caspase-3 was downregulated by 60%; the apoptosis rate in experimental groups 1-3 (protocatechuic acid 20-50 μM) was 7.5%, the apoptosis rate in control group 1 was 14.2%, and the apoptosis rate in control group 2 was 18.6%; the apoptosis rate in experimental groups 1-3 (protocatechuic acid 20-50 μM) was significantly lower than that in control groups 1-2; the blastocyst formation rates in experimental groups 4-5 (protocatechuic acid > 200 μM) decreased to 55.6% and 56.5%, and there was no difference in the birth rate after embryo transfer between experimental groups 1-3 (protocatechuic acid 20-50 μM) and the control group. The concentration window of protocatechuic acid at 10-100 μM is the most suitable for promoting the developmental ability of mouse embryos.

[0049] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. The application of protocatechuic acid in promoting the embryonic development ability of mice is characterized by: The mouse embryos are mouse pre-blastocyst stage embryos, and the culture time is 24-96 hours.

2. The use of protocatechuic acid in promoting the embryonic development ability of mice according to claim 1, characterized in that: The protocatechuic acid is a culture medium containing protocatechuic acid, and the purity of the protocatechuic acid is 95%-99%.

3. The use of protocatechuic acid in promoting the embryonic development ability of mice according to claim 2, characterized in that: The culture medium is used for improving embryo survival rate, blastocyst formation rate or reducing intracellular active oxygen level.

4. The use of protocatechuic acid in promoting the embryonic development ability of mice according to claim 2, characterized in that: The culture medium is KSOM, mHTF or G1 / G2 series culture medium, the culture time is 24-96 hours, and is suitable for early mouse embryos.

5. The use of protocatechuic acid in promoting the embryonic development ability of mice according to claim 2, characterized in that: The concentration of protocatechuic acid in the culture medium is 10-100 μM.

6. The use of protocatechuic acid in promoting the embryonic development ability of mice according to claim 5, characterized in that: The protocatechuic acid is prepared by a plant extraction method or a chemical synthesis method.

7. The use of protocatechuic acid in promoting the embryonic development ability of mice according to claim 6, characterized in that: The specific process of the plant extraction method is: using salvia miltiorrhiza or olive leaves as raw materials, extracting with ethanol reflux assisted by ultrasound, and purifying with AB-8 macroporous resin to obtain protocatechuic acid with a purity of ≥95%.

8. The use of protocatechuic acid in promoting the embryonic development ability of mice according to claim 6, characterized in that: The specific process of the chemical synthesis preparation method is: using vanillin as a precursor, and preparing protocatechuic acid through alkaline hydrolysis reaction.