Application of cyanidin in improving reproductive and growth development abilities

Abstract

The present invention discloses the use of cyanidin in the preparation of a drug for improving reproductive and / or growth and development ability. The compound has a significant effect on promoting the reproductive ability, motility and growth and development of Caenorhabditis elegans. By measuring the egg production, body bending frequency, head swing frequency, pharyngeal pumping frequency and body length of Caenorhabditis elegans after administration and in the control group, in the reproductive ability test, cyanidin increased the egg production of Caenorhabditis elegans and improved the reproductive ability. In the motility test, cyanidin increased the body bending, head swing frequency and pharyngeal pumping frequency of Caenorhabditis elegans, and enhanced the motility. In the body length test, cyanidin increased the body length of Caenorhabditis elegans. The above results show that cyanidin has a significant promoting effect on the growth and development and reproductive ability of Caenorhabditis elegans.

Description

Technical Field

[0001] The present invention relates to the field of pharmacy, and in particular to application of cyanidin in improving reproductive and growth and development abilities. Background Art

[0002] Growth and development are crucial to the body's life cycle, and delayed growth and development can easily lead to intellectual and behavioral problems. Reproductive health is a key topic in global reproductive research. Impaired reproductive function can lead to emotional distress and even depression in patients, making it a key focus and hotspot in reproductive medicine. The model organism Caenorhabditis elegans, with its well-defined genome, low cost, ease of reproduction, and reliable results, is widely used in growth and development and reproductive research.

[0003] Natural products have long been an important source for discovering new drugs or lead compounds due to their novel chemical structures, drug-like properties, and unique pharmacological effects. texasin, also known as texasin, is an isoflavone isolated from the xylem heartwood of the medicinal plant Caragana fasciata. Its structural formula is:

[0004]

[0005] Research on the biological activity of cyanidin is very limited. Currently, it is only reported to inhibit tyrosinase and leukocyte 5-esteroxygenase activity. Whether cyanidin can promote growth and reproductive capacity has not been reported.

[0006] Generally speaking, in vitro cell model drugs require less, but are easily interfered with, and the results are often not accurate enough. Conventional animal models such as mice and rats require a large amount of drugs, and the operation is cumbersome, the workload is large, and they also have limitations. Caenorhabditis elegans is a multicellular model organism with a known entire genome. It has many advantages such as strong reproductive capacity, easy to culture, easy to observe, sensitive to multi-system toxicity, stable behavioral response patterns and short testing cycles. It is an in vivo experimental model animal with low drug demand, sensitivity and easy operation. Caenorhabditis elegans can be used to quickly and effectively determine the pharmacological effects of cyanobacterium indigoferum. However, the function of cyanobacterium indigoferum on Caenorhabditis elegans is still unclear. Summary of the Invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of cyanidin in the preparation of drugs for promoting growth and development and improving reproductive ability.

[0008] Technical solution: In order to solve the above technical problems, the present invention provides the use of cyanidin in the preparation of drugs for improving reproductive and / or growth and development capabilities.

[0009] Wherein, the medicine is a single component or a compound preparation.

[0010] The dosage forms of the drug include tablets, capsules, granules, suspensions, pills, solutions, syrups or injections.

[0011] The present invention uses the Caenorhabditis elegans model organism to preliminarily verify the application of cyanobacterium pseudoindigo in promoting growth and reproductive ability in a rapid and low-consumption manner.

[0012] The application includes an experiment to measure the egg-laying capacity of the Caenorhabditis elegans model organism.

[0013] The application includes conducting an experiment to measure the movement ability of the Caenorhabditis elegans model organism.

[0014] The application includes an experiment to measure the body length of the Caenorhabditis elegans model organism.

[0015] Wherein, the concentration of the cyanus pseudoindigo is 12.5-50 μM.

[0016] Specifically, the present invention is further verified through the following aspects:

[0017] 1) The egg-laying capacity of Caenorhabditis elegans was compared with that of the blank control group by using an egg-laying capacity measurement experiment, preliminarily verifying the promoting effect of cyanobacterium on reproductive capacity.

[0018] 2) Using a motor ability measurement experiment, the body bending frequency, head swing frequency, and pharyngeal pumping frequency of C. elegans after drug administration were compared with those of the blank control group, preliminarily verifying the growth-promoting effect of cyanobacterium pseudoindigofera on growth and development.

[0019] 3) Using a body length measurement experiment, the body length of C. elegans after drug administration was compared with that of the blank control group, preliminarily verifying the growth-promoting effect of cyanidin on growth and development.

[0020] Beneficial effects: The present invention only requires a small amount of cyanidin and a short time to verify the effect of cyanidin in promoting growth and reproduction. Experimental verification shows that cyanidin has a significant effect in promoting growth and reproduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 , UPLC analysis chart of blue flower pseudoindigo standard.

[0022] Figure 2 , the results of the optimal concentration evaluation experiment of blue flower pseudoindigo

[0023] Figure 3 , the effect of blue flower pseudoindigo on increasing the egg production of Caenorhabditis elegans.

[0024] Figure 4 , the promoting effect of blue flower pseudoindigo on the body bending frequency of Caenorhabditis elegans.

[0025] Figure 5 , the promoting effect of blue flower pseudoindigo on the head shaking frequency of Caenorhabditis elegans.

[0026] Figure 6 , the promoting effect of blue flower pseudoindigo on the pharyngeal pumping frequency of Caenorhabditis elegans.

[0027] Figure 7 , the effect of blue flower pseudoindigo on the body length increase of Caenorhabditis elegans. DETAILED DESCRIPTION

[0028] The cyanidin used in the present invention is homemade in the laboratory, and its purity is 98.01% as analyzed by HPLC. It is dissolved in DMSO and diluted with M9 buffer solution.

[0029] Preparation of M9 buffer: 0.9 g potassium dihydrogen phosphate, 4.5 g disodium hydrogen phosphate, 1.5 g sodium chloride, and 300 mL deionized water.

[0030] NGM medium preparation: 1.5 g sodium chloride, 1.25 g peptone, 8 g agar powder, 500 mL deionized water, 500 μL each of 1 M magnesium sulfate, 1 M calcium chloride, and 5 mg / mL cholesterol solution, and 12.5 mL potassium phosphate buffer.

[0031] The Escherichia coli strain used for the culture of Caenorhabditis elegans was E. coli OP50, which was a gift from Professor Wang Dayong's laboratory at Southeast University.

[0032] Example 1 Evaluation of the reproductive effects of cyanobacterium pseudoindigoferum on Caenorhabditis elegans

[0033] Evaluation of the optimal concentration: Caenorhabditis elegans were cultured for 24 hours after synchronization (L1 stage) to the L2 stage. L2 stage Caenorhabditis elegans were divided into the following groups for administration: blank control group, high (50μM), medium (25μM), and low (12.5μM) dose groups of cyanidin. The blank control group was added with 200μL M9 buffer and 100μL Escherichia coli OP50. Each experimental group was added with 200μL cyanidin and 100μL Escherichia coli OP50. The eggs were cultured for 24 hours and the egg production was determined. The results are shown in the figure. Figure 2 The results showed that the optimal concentration group was the medium (25 μM) dose group of cyanidin.

[0034] Effect on Egg Production in Caenorhabditis elegans: C. elegans were treated with the same treatment (L1 stage) and cultured at 20°C for 24 hours. After culture, C. elegans were divided into the following groups: a blank control group and a group treated with 25 μM cyanidin. Each experimental group received 200 μL of cyanidin and 100 μL of E. coli OP50, while the blank control group received 200 μL of M9 buffer and 100 μL of E. coli OP50. After treatment, the cells were cultured at 20°C for 48 hours until the C. elegans entered the L4 egg-laying stage. Thirty C. elegans were randomly selected from each group during the egg-laying stage and picked with a needle. The culture medium used was a 2 cm diameter blank culture medium. 10 μL of E. coli solution was dropped in the center and dried. The C. elegans were then placed in the center of the E. coli culture medium, using one culture medium per elegans. The day of picking was designated as day 1. Observe the culture medium every 12 hours thereafter and transfer the C. elegans to fresh culture medium every 24 hours using a pick. For the three days before peak egg-laying, record the total number of eggs laid by each C. elegans daily. Each group consisted of 10 animals, with three parallel groups.

[0035] The experimental results can be found in Figure 3 , blue flower pseudoindigo (25μM) can significantly increase the total egg production and enhance the reproductive capacity of Caenorhabditis elegans.

[0036] Example 2 Evaluation of the Effect of Pseudoindigofera on the Movement Ability of Caenorhabditis elegans

[0037] Effects on Caenorhabditis elegans motility: C. elegans were homogenized and cultured at 20°C for 24 hours. The cultured C. elegans were then divided into the following groups: a blank control group and a 25 μM cyanidin group. Each cyanidin group received 200 μL of cyanidin and 100 μL of E. coli OP50, while the blank control group received 200 μL of M9 buffer and 100 μL of E. coli OP50. The cells were exposed to 20°C for 24 hours after dosing. Prepare NGM medium without E. coli, randomly select a C. elegans to be tested and place it on the blank medium. Wait for a period of time until its overreaction due to external stimuli is relieved, and then record the number of head swings and body bends of the C. elegans within 60 seconds (a head swing is defined as a change in the direction of the C. elegans head, and a body bend is defined as a change in the direction of movement of its front part (i.e., pharynx) along the Y axis) and the number of pharyngeal aspirations within 30 seconds. Randomly select 20 C. elegans for each group, and set up 3 parallel groups.

[0038] The experimental results are as follows Figure 4 、 Figure 5 、 Figure 6 As shown in the results, cyanobacterium pseudoindigofera (25 μM) can significantly increase the body bending frequency, head swing frequency and pharyngeal pumping frequency of Caenorhabditis elegans, and enhance the movement ability of Caenorhabditis elegans.

[0039] Example 3 Effects of Pseudoindigofera on the Growth and Development of Caenorhabditis elegans

[0040] Effects on Caenorhabditis elegans Body Length: C. elegans (L1 stage) were homogenized and cultured at 20°C for 24 hours. After culture, C. elegans were divided into the following groups: a blank control group and a 25μM cyanidin group. The cyanidin group received 200μL of cyanidin and 100μL of E. coli OP50, while the blank control group received 200μL of M9 buffer and 100μL of E. coli OP50. After dosing, the C. elegans were exposed at 20°C for 48 hours. After exposure, the C. elegans in each group were washed with M9 buffer at least three times to remove any remaining E. coli. 0.5mL of M9 buffer was added to each group, followed by one drop (approximately 10μL) of levamisole hydrochloride solution. This anesthetized the C. elegans to restrain their movements and facilitate observation and imaging under an upright microscope. Let the C. elegans sit for 1 minute until they are thoroughly anesthetized. Pipette the solution to mix thoroughly. Pipette a droplet of C. elegans at an appropriate density onto the prepared agarose pad to create a temporary slide. Observe the C. elegans using an upright microscope and photograph them. (Each photograph must include at least one intact C. elegans; multiple C. elegans are permitted.) Measure body length using Image J software. Draw a line from the center of the head to the tail of the C. elegans. Measure and calculate the length of the line.

[0041] The experimental results are as follows Figure 7 As shown in the results, cyanobacterium pseudoindigofera (25 μM) can significantly increase the body length of Caenorhabditis elegans and promote the growth and development of Caenorhabditis elegans.

Claims

1. Use of cyanidin in the preparation of drugs for improving reproductive and / or growth and development abilities.

2. The use according to claim 1, characterized in that The medicine is a single component or a compound preparation.

3. The use according to claim 1, characterized in that The dosage forms of the drug include tablets, capsules, granules, suspensions, pills, solutions, syrups or injections.

4. The use according to claim 1, characterized in that The concentration of the cyanidin is 12.5-50 mM.

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