Oleic acid-geniposide composite preparation and application thereof

Abstract

According to the oleic acid-geniposide composite preparation and the application thereof, a mixed system of oleic acid and geniposide is constructed and acts on PC12 cells induced by hydrogen peroxide to generate oxidative damage, and the result shows that compared with cells treated by singly using geniposide with the same concentration, the oleic acid-geniposide composite preparation has the advantages that the effect is good, and the cost is low. The mixed system can obviously improve the survival rate of PC12 cells, and the obvious effect is not observed after linoleic acid and linolenic acid are respectively mixed with geniposide. The invention clarifies that oleic acid can enhance the protective effect of geniposide on oxidative damage cells, and provides a brand new thought and technical support for developing products for resisting oxidative damage of cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to an oleic acid-geniopicroside complex preparation and application thereof. BACKGROUND

[0002] In the field of biological medicine, cell oxidative damage is a key link in many physiological and pathological processes. When the generation and clearance of active oxygen in the body or cells are imbalanced, excessive active oxygen will attack biological macromolecules in cells, destroy the structure and function of cells, and further induce or aggravate neurodegenerative diseases, cardiovascular diseases, liver damage and aging-related diseases. Therefore, exploring safe and effective anti-cell oxidative damage strategies is of great significance for the prevention, intervention and treatment of related diseases.

[0003] In vitro cell models are the core tools for analyzing oxidative damage mechanisms and screening antioxidant substances. Among them, PC12 cells have typical biological characteristics of nerve cells and are relatively sensitive to oxidative damage inducers, and are often used to simulate the process of oxidative damage of nerve cells, providing a reliable in vitro experimental basis for in-depth study of antioxidant protection mechanisms and evaluation of candidate antioxidant substances.

[0004] Geniopicroside is a substance with potential biological activity. Existing studies have shown that it has related characteristics such as antioxidant, anti-inflammatory and cell protection, and can theoretically improve cell oxidative damage. However, in in vitro studies on cell oxidative damage, the protective effect of geniopicroside alone on oxidative damage cells cannot be effectively demonstrated, which hinders its application in the field of anti-cell oxidative damage and makes it difficult to fully exert its potential biological value.

[0005] Fatty acids are important material basis for cell life activities. They are not only the core components of cell membranes, but also participate in the regulation of various physiological processes in cells, including the regulation of antioxidant signaling pathways. Existing studies have mostly focused on the independent physiological functions of common fatty acids such as oleic acid, linoleic acid and linolenic acid (such as the direct protective effect of some fatty acids on cell oxidative damage), but have not paid attention to the regulatory effect of fatty acids on the anti-cell oxidative damage effect of geniopicroside, and have not found that specific fatty acids can specifically enhance the protective effect of geniopicroside.

[0006] In summary, there are two major gaps in existing technology: first, the protective effect of geniopicroside alone on oxidative damage cells is not good, and its antioxidant value is difficult to exert; second, there is a lack of substances and application schemes that can specifically enhance the anti-cell oxidative damage effect of geniopicroside. Therefore, developing a technical scheme that can effectively improve the anti-cell oxidative damage ability of geniopicroside has become a problem to be solved in the current field of biological medicine, and has important significance for promoting the research and development of related reagents or compositions. SUMMARY

[0007] Technical problems to be solved: In view of the above technical problems, the purpose of the present application is to build a mixed system of oleic acid and geniposide, act on PC12 cells induced by hydrogen peroxide to produce oxidative damage, and the results show that compared with cells treated with the same concentration of geniposide alone, the mixed system can significantly improve the survival rate of PC12 cells, and no such significant effect is observed when linoleic acid and linolenic acid are mixed with geniposide. The present application clarifies that oleic acid can enhance the protective effect of geniposide on oxidative damage cells, and provides a new idea and technical support for the development of products against cell oxidative damage.

[0008] Technical scheme: An oleic acid-geniposide complex preparation, the composition of the oleic acid-geniposide complex preparation comprises oleic acid, geniposide and pharmaceutically acceptable adjuvants.

[0009] Preferably, the effective concentration of oleic acid in the oleic acid-geniposide complex preparation is 100-600 μg / mL; the effective concentration of geniposide is 5-20 μg / mL.

[0010] Preferably, the effective concentration of oleic acid in the oleic acid-geniposide complex preparation is 300 μg / mL; the effective concentration of geniposide is 20 μg / mL.

[0011] The above-mentioned oleic acid-geniposide complex preparation is used for preparing a product for enhancing the protective effect of oxidative damage PC12 cells.

[0012] Preferably, the product enhances the protective effect of geniposide on oxidative damage PC12 cells.

[0013] Beneficial effects

[0014] 1. The present application effectively breaks through the application limitations of geniposide alone, and significantly improves the protective effect on oxidative damage cells. Geniposide has no protective effect on H2O2-induced PC12 cell oxidative damage in the range of 0.0271-0.1625 μg / mL, while the mixed system composed of 300 μg / mL oleic acid and 5 μg / mL geniposide can increase the cell survival rate by 6.20%, and the mixed system composed of 300 μg / mL oleic acid and 20 μg / mL geniposide can increase the cell survival rate by 5.90%, which indicates that oleic acid can significantly enhance the protective effect of geniposide on H2O2-damaged PC12 cells, successfully solves the technical problem of poor anti-cell oxidative damage effect of geniposide, and fully releases its potential antioxidant value.

[0015] 2. The experimental data of this invention clearly verify that only oleic acid can enhance the protective efficacy of genipin against oxidatively damaged cells; while linoleic acid and linolenic acid, when mixed with genipin respectively, did not show similar enhancing effects. This difference fully reflects the specific characteristics of oleic acid's action, providing a reliable basis and data support for the research and development of anti-cellular oxidative damage products. Attached Figure Description

[0016] Figure 1 The effects of oleic acid, linoleic acid, and linolenic acid on the cytotoxicity of PC12 cells are represented by A, B, and C, respectively. Figure 2 The effect of different concentrations of H2O2 on the survival rate of PC12 cells; Figure 3 The diagram shows the cell protection effects of oleic acid, linoleic acid, and linolenic acid on PC12 cells, where A represents oleic acid, B represents linoleic acid, and C represents linolenic acid. Figure 4 This image shows the cell protection effect of genipin on PC12 cells. Figure 5 The cell protection effect of 300 μg / mL oleic acid mixed with different concentrations of genipin is shown in the figure. Figure 6 The cell protection effect of 400 μg / mL linoleic acid mixed with different concentrations of genipin is shown in the figure. Figure 7 The cell protection effect of 5 μg / mL linolenic acid mixed with different concentrations of genipin is shown in the figure. Figures 1-7 The asterisk (*) indicates a significant difference compared to the H2O2 treatment group. P <0.05,** P <0.01), ## indicates a significant difference compared to the control group (# P <0.05; ## P <0.01). Detailed Implementation

[0017] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments: Example 1

[0018] This embodiment investigated the effects of oleic acid, linoleic acid, and linolenic acid on the cytotoxic effects of PC12 cells.

[0019] (1) PC12 cell culture PC12 cells were cultured in high-sugar DMEM medium containing 1% penicillin-streptomycin mixture and 10% fetal bovine serum in a culture box with a condition setting of 5% CO2 and 37°C, and the PC12 cells were observed under a microscope to be in an adherent state. The PC12 cells were placed in a freezing tube for storage. The freezing tube containing the PC12 cells was taken out from liquid nitrogen and immediately placed in a 37°C water bath for thawing. After thawing, the surface of the freezing tube was wiped with alcohol, transferred to a clean bench, and the PC12 cell suspension in the freezing tube was transferred to a sterile centrifuge tube and centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was discarded, 1 mL of complete medium was added to resuspend the cells and transferred to a culture dish, and cultured in a 5% CO2, 37°C incubator. When the cell density in the culture dish reached about 85%, the cells were passaged. The waste liquid in the culture dish was discarded, the cells were washed with sterile PBS for 2-3 times, the PBS was aspirated, 1 mL of 0.25% trypsin was added, and the culture dish was placed in the incubator for 1 min to digest the cells, and then 1.5 mL of high-sugar DMEM medium containing 1% penicillin-streptomycin mixture and 10% fetal bovine serum was immediately added to terminate the digestion. The cells in the culture dish were blown into the culture medium with a pipette gun and transferred to a sterile centrifuge tube, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were passaged at a ratio of 1:3. The PC12 cells were digested in the logarithmic growth phase, and the steps were the same as the above passage. After the supernatant was discarded, 1 mL of cell freezing solution was added to resuspend the cells and transferred to a freezing tube. The cell freezing tube was programmed to cool down and then transferred to liquid nitrogen for storage.

[0020] (2) Effect of oleic acid, linoleic acid and linolenic acid on the toxicity of PC12 cells ①PC12 cells were seeded in 96-well plates at 1×10 5 After 24 h of adherent culture, the waste liquid was aspirated, 100 μL of complete medium was added to each well of the control group, and 100 μL of oleic acid solution of different concentrations diluted with complete medium was added to each well of the sample group. After 24 h of treatment, the cell toxicity range of oleic acid was determined by MTT method; ②PC12 cells were seeded in 96-well plates at 1×10 5 After 24 h of adherent culture, the waste liquid was aspirated, 100 μL of complete medium was added to each well of the control group, and 100 μL of oleic acid solution of different concentrations diluted with complete medium was added to each well of the sample group. After 24 h of treatment, the cell toxicity range of oleic acid was determined by MTT method; ③PC12 cells were seeded in 96-well plates at 1×10 5PC12 cells were seeded in 96-well plates and cultured adherently for 24 h. After the waste liquid was removed, 100 μL of complete culture medium was added to each well of the control group, and 100 μL of linolenic acid solution of different concentrations (5, 10, 50, 100, 150, 200, 300 μg / mL) diluted with complete culture medium was added to each well of the sample group. After 24 h of treatment, the cytotoxic range of oleic acid was determined by the MTT assay.

[0021] Depend on Figure 1 It was found that oleic acid did not show toxicity to PC12 cells in the range of 0-600 μg / mL, and this concentration range was used for subsequent experiments to determine the protective effect of H2O2-damaged PC12 cells; linoleic acid did not show toxicity to PC12 cells in the range of 0-400 μg / mL, and linolenic acid did not show toxicity to PC12 cells in the range of 0-200 μg / mL.

[0022] Example 2

[0023] This embodiment investigated the effects of oleic acid, linoleic acid, and linolenic acid on the protective effects of H2O2-induced PC12 cells.

[0024] (1) Establishment of H2O2-induced PC12 cell damage model The cultured PC12 cells were divided into 1×10⁶ cells per well. 5 Cells were seeded in 96-well plates and cultured adherently for 48 hours (the original culture medium was removed and replaced with fresh culture medium after the first 24 hours of culture). In the control group, 100 μL of complete culture medium was added to each well. In the H2O2 damage group, 100 μL of H2O2 diluted with complete culture medium at different concentrations (40, 60, 80, 100, 120, 140, 160 μmol / L) was added to each well. The cells were then cultured in an incubator for another 2 hours. After culture, cell viability was detected using the MTT assay to determine the appropriate H2O2 concentration for modeling. Depend on Figure 2 It can be seen that when the H2O2 concentration is 100 μmol / L, the cell survival rate is 57.53 ± 1.02%, which is close to the half-lethal rate. Therefore, 100 μmol / L was selected as the concentration of H2O2 oxidative damage PC12 cell model. (2) Protective effects of oleic acid, linoleic acid and linolenic acid on H2O2-induced PC12 cells ①Place 1×10⁶ PC12 cells per well 5Cells were seeded in 96-well plates and cultured adherently for 24 hours. After removing the waste liquid, samples were added according to the following groups: Control group: 100 μL of complete culture medium was added to each well and cultured for 24 hours, followed by another 100 μL of complete culture medium and culturing for 2 hours. H2O2 damage group: 100 μL of complete culture medium was added to each well and incubated for 24 hours, followed by another 100 μL of 100 μmol / L H2O2 and culturing for 2 hours. Sample group: 100 μL of complete culture medium containing oleic acid (concentrations of 50, 100, 300, 500, and 600 μg / mL) was added to each well and cultured for 24 hours, followed by another 100 μmol / L H2O2 and culturing for 2 hours. Cell viability was assessed using the MTT assay after culture.

[0025] ②Place 1×10⁶ PC12 cells per well 5 Cells were seeded in 96-well plates and cultured adherently for 24 hours. After removing the waste liquid, samples were added according to the following groups: Control group: 100 μL of complete culture medium was added to each well and cultured for 24 hours, followed by another 100 μL of complete culture medium and culturing for 2 hours. H2O2 damage group: 100 μL of complete culture medium was added to each well and incubated for 24 hours, followed by another 100 μL of 100 μmol / L H2O2 and culturing for 2 hours. Sample group: 100 μL of complete culture medium containing linoleic acid (concentrations of 50, 100, 200, 300, and 400 μg / mL) was added to each well and cultured for 24 hours, followed by another 100 μmol / L H2O2 and culturing for 2 hours. Cell viability was assessed using the MTT assay after culture.

[0026] ③ Place PC12 cells at a density of 1×10⁶ cells per well 5 Cells were seeded in 96-well plates and cultured adherently for 24 hours. After removing the waste liquid, samples were added according to the following groups: Control group: 100 μL of complete culture medium was added to each well and cultured for 24 hours, followed by another 100 μL of complete culture medium and culturing for 2 hours. H2O2 damage group: 100 μL of complete culture medium was added to each well and incubated for 24 hours, followed by another 100 μL of 100 μmol / L H2O2 and culturing for 2 hours. Sample group: 100 μL of complete culture medium containing linolenic acid (concentrations of 5, 10, 50, 100, and 200 μg / mL) was added to each well and cultured for 24 hours, followed by another 100 μmol / L H2O2 and culturing for 2 hours. After culture, cell viability was assessed using the MTT assay.

[0027] Depend on Figure 3 As shown in A, oleic acid at 500 μg / mL has a significant cell protective effect against H2O2-induced PC12 cells, increasing cell survival to 61.85% (compared to 53.17% in the H2O2-damaged group). Figure 3As can be known from B, linoleic acid has a certain cell protection effect on H2O2-induced PC12 cells at 200 μg / mL; as can be known from Figure 3 As can be known from C, linolenic acid has a cell protection effect on H2O2-induced PC12 cells in the range of 5-200 μg / mL, especially in the concentration range of 50-200 μg / mL.

[0028] Example 3

[0029] This example investigates the influence of geniposide on the protection effect of H2O2-induced PC12 cells.

[0030] PC12 cells were inoculated in 96-well plates at 1×10 5 After adherent culture for 24 h, the waste liquid was removed, and the following groups were added: 100 μL of complete culture medium was added to each well of the control group for 24 h, and then 100 μL of complete culture medium was added for 2 h; 100 μL of complete culture medium was added to each well of the H2O2 damage group for 24 h, and then 100 μL of H2O2 with a concentration of 100 μmol / L was added for 2 h; 100 μL of complete culture medium containing geniposide (with concentrations of 0.0271, 0.0542, 0.0812, 0.1083, 0.1353, and 0.1625 μg / mL) was added to each well of the sample group for 24 h, and then 100 μL of H2O2 with a concentration of 100 μmol / L was added for 2 h. After the culture, the cell survival rate of each group was detected by the MTT method.

[0031] As can be known from B, linoleic acid has a certain cell protection effect on H2O2-induced PC12 cells at 200 μg / mL; as can be known from Figure 4 As can be known from B, linoleic acid has a certain cell protection effect on H2O2-induced PC12 cells at 200 μg / mL; as can be known from

[0032] Example 4

[0033] This example investigates the influence of geniposide with a concentration of 300 μg / mL on the protection effect of H2O2-induced PC12 cells.

[0034] Oil acid with a concentration of 300 μg / mL and geniposide with a concentration of 5 μg / mL (or 20 μg / mL) were mixed to prepare an oil acid-geniposide mixed system; the protection effect on H2O2-induced PC12 cells was determined, and the details are as follows: PC12 cells were inoculated in 96-well plates at 1×10 5PC12 cells were inoculated in 96-well plates and adherently cultured for 24 h, after which the waste liquid was removed and the following sample addition was performed: 100 μL of complete medium was added to each well of the control group, which was cultured for 24 h, and then 100 μL of complete medium was added for continued culture for 2 h; 100 μL of complete medium was added to each well of the H2O2 damage group, which was incubated for 24 h, and then 100 μL of H2O2 at a concentration of 100 μmol / L was added for continued culture for 2 h; 100 μL of complete medium containing the oleic acid-geniopicroside mixed system was added to each well of the sample group, which was cultured for 24 h, and then 100 μmol / L of H2O2 was added for continued culture for 2 h; after the culture was completed, the cell survival rate of each group was detected by the MTT method.

[0035] Comparative Example 1

[0036] This comparative example investigated the influence of different concentrations of geniopicroside on the protective effect of H2O2-induced PC12 cells.

[0037] Geniopicroside at a concentration of 5 μg / mL or 20 μg / mL was mixed to form an oleic acid-geniopicroside mixed system; the protective effect on H2O2-induced PC12 cells was determined as follows: PC12 cells were inoculated in 96-well plates at 1×10 5 PC12 cells were inoculated in 96-well plates and adherently cultured for 24 h, after which the waste liquid was removed and the following sample addition was performed: 100 μL of complete medium was added to each well of the control group, which was cultured for 24 h, and then 100 μL of complete medium was added for continued culture for 2 h; 100 μL of complete medium was added to each well of the H2O2 damage group, which was incubated for 24 h, and then 100 μL of H2O2 at a concentration of 100 μmol / L was added for continued culture for 2 h; 100 μL of complete medium containing the oleic acid-geniopicroside mixed system was added to each well of the sample group, which was cultured for 24 h, and then 100 μmol / L of H2O2 was added for continued culture for 2 h; after the culture was completed, the cell survival rate of each group was detected by the MTT method.

[0038] Comparative Example 2

[0039] This comparative example investigated the influence of linoleic acid at a concentration of 400 μg / mL and different concentrations of geniopicroside on the protective effect of H2O2-induced PC12 cells.

[0040] Linoleic acid at a concentration of 400 μg / mL was mixed with geniopicroside at a concentration of 5 μg / mL (or 20 μg / mL) to form a linoleic acid-geniopicroside mixed system; the protective effect on H2O2-induced PC12 cells was determined as follows: PC12 cells were inoculated in 96-well plates at 1×10 5PC12 cells were inoculated in 96-well plates and adherently cultured for 24 h, after which the waste liquid was removed and the following sample addition was performed according to the following grouping: the control group was added with 100 μL of complete culture medium per well and cultured for 24 h, and then 100 μL of complete culture medium was added for continued culture for 2 h; the H2O2 damage group was first incubated with 100 μL of complete culture medium for 24 h, and then 100 μL of H2O2 with a concentration of 100 μmol / L was added for continued culture for 2 h; the sample group was added with 100 μL of complete culture medium containing the linoleic acid-geniopicroside mixed system per well and cultured for 24 h, and then 100 μmol / L of H2O2 was added for continued culture for 2 h, after which the cell survival rate of each group was detected by the MTT method.

[0041] Comparative Example 3

[0042] This comparative example investigated the influence of linolenic acid with a concentration of 5 μg / mL and different concentrations of geniopicroside on the protective effect of H2O2-induced PC12 cells.

[0043] Linolenic acid with a concentration of 5 μg / mL was mixed with geniopicroside with a concentration of 5 μg / mL (or 20 μg / mL) to prepare a linoleic acid-geniopicroside mixed system; the protective effect on H2O2-induced PC12 cells was determined, and the details are as follows: PC12 cells were inoculated in 96-well plates at 1×10 5 PC12 cells were inoculated in 96-well plates at 1×10 5 PC12 cells were inoculated in 96-well plates at 1×10

[0044] As Figure 5 , Figure 6 and Figure 7As shown, 5 μg / mL or 20 μg / mL geniposide in Comparative Example 1 did not show cell protection effect when applied to H2O2-damaged PC12 cells. However, 300 μg / mL oleic acid + 5 μg / mL geniposide or 300 μg / mL oleic acid + 20 μg / mL geniposide in Example 4 showed significant cell protection effect when applied to H2O2-damaged PC12 cells, compared with the H2O2 treatment group, and the cell survival rate was increased by about 6.20% for the 5 μg / mL geniposide group and 5.90% for the 20 μg / mL geniposide group, compared with Comparative Example 1. Therefore, the results show that oleic acid can significantly enhance the protective effect of geniposide on H2O2-damaged PC12 cells. Comparative Example 3 (400 μg / mL linoleic acid + 5 μg / mL geniposide or 400 μg / mL linoleic acid + 20 μg / mL geniposide) and Comparative Example 4 (5 μg / mL linolenic acid + 5 μg / mL geniposide or 5 μg / mL linolenic acid + 20 μg / mL geniposide) did not show protective effect on H2O2-damaged PC12 cells, indicating that linoleic acid and linolenic acid do not have the effect of enhancing the protective effect of geniposide on H2O2-damaged PC12 cells.

[0045] The above merely describes preferred embodiments of the present application, but does not limit the present application in any form. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application, by using the disclosed methods and technical contents. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present application, without departing from the technical solutions of the present application, still falls within the scope of protection of the technical solutions of the present application.

Claims

1. An oleic acid-jioneprosyl complex preparation, characterized by: The composition of the oleic acid-geniopicroside complex preparation comprises oleic acid, geniopicroside and pharmaceutically acceptable adjuvants; the effective concentration of oleic acid in the oleic acid-geniopicroside complex preparation is 100-600 μg / mL; and the effective concentration of geniopicroside is 5-20 μg / mL.

2. The oleic acid-jioneprosyl complex preparation according to claim 1, characterized by: The effective concentration of oleic acid in the oleic acid-geniopicroside complex preparation is 300 μg / mL; and the effective concentration of geniopicroside is 20 μg / mL.

3. Use of the oleic acid-geniopicroside complex preparation according to claim 1 in the preparation of a product for enhancing the protective effect on oxidative damage PC12 cells.

4. Use according to claim 3, characterized in that: The product enhances the protective effect of geniopicroside on oxidative damage PC12 cells.

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