Oleic acid-crocetin compound preparation and application thereof

Abstract

The invention discloses an oleic acid-crocetin compound preparation and application thereof, oleic acid and crocetin are adopted to form a mixed system to act on PC12 cells damaged by H2O2 induced oxidation, the survival rate of the PC12 cells can be obviously improved compared with the single use of crocetin with the same concentration, and the obvious effect does not exist after linoleic acid and linolenic acid are mixed with crocetin. The invention reveals that oleic acid can enhance the cell oxidative damage resistance of crocetin, provides a brand new scheme for research and development of reagents or compositions for resisting cell oxidative damage, and has important biomedical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a oleic acid-crocetin 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 production and clearance of active oxygen in the body or cells are imbalanced, excessive active oxygen will attack biological macromolecules in cells, destroy cell structure and function, and further induce or aggravate neurodegenerative diseases, cardiovascular diseases, liver damage and aging-related diseases and other 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 antioxidants. Among them, PC12 cells have typical biological characteristics of nerve cells and are 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 further research on anti-oxidative damage mechanisms and candidate substance evaluation.

[0004] Crocetin, as one of the main active ingredients of saffron, has been shown to have potential antioxidant, anti-inflammatory and neuroprotective properties, and theoretically has the potential to improve cell oxidative damage. However, in in vitro studies on cell oxidative damage, the protective effect of crocetin alone on oxidative damage cells has not been effectively demonstrated. This limitation has greatly limited the application of crocetin in the field of anti-cell oxidative damage, making it difficult to fully realize its potential value.

[0005] Fatty acids are an important material basis for cell life activities, not only being a key component of cell membranes, but also participating in the regulation of various physiological processes in cells, including the regulation of antioxidant signaling pathways. Common fatty acids such as oleic acid, linoleic acid, and linolenic acid have been the focus of existing research on their individual physiological functions, such as the direct protective effect of some fatty acids on cell oxidative damage. However, no research has focused on the regulatory effect of fatty acids on the anti-cell oxidative damage effect of crocetin, and no specific fatty acid has been found to specifically enhance the protective effect of crocetin on oxidative damage cells.

[0006] In summary, there are two major gaps in existing technology: first, the protective effect of crocetin alone on oxidative damage cells is not good, and its potential anti-oxidative value is difficult to realize; second, there is a lack of substances and related application programs that can specifically enhance the anti-cell oxidative damage effect of crocetin. Therefore, developing a technical solution that can effectively improve the anti-cell oxidative damage ability of crocetin is a problem that needs to be solved in the current field of biological medicine, and it is of great significance for the development of anti-cell oxidative damage-related reagents or compositions. SUMMARY

[0007] Technical problem to be solved: In view of the above technical problems, the purpose of the present application is to use oleic acid and saffloric acid to form a mixed system to act on H2O2-induced oxidative damage PC12 cells, which can significantly improve the survival rate of PC12 cells compared with the use of the same concentration of saffloric acid alone, and linoleic acid and linolenic acid mixed with saffloric acid have no such significant effect. The present application discloses that oleic acid can enhance the cell antioxidant damage effect of saffloric acid, which provides a new scheme for the development of cell oxidative damage reagents or compositions, and has important biological and medical application value.

[0008] Technical scheme: The application of an oleic acid-saffloric acid complex preparation in the preparation of a product for enhancing the protection of oxidative damage PC12 cells.

[0009] Preferably, the composition of the oleic acid-saffloric acid complex preparation comprises oleic acid, saffloric acid and pharmaceutically acceptable excipients.

[0010] Preferably, the effective concentration of oleic acid in the oleic acid-saffloric acid complex preparation is 100-600 μg / mL; and the effective concentration of saffloric acid is 0.1-1 μg / mL.

[0011] Preferably, the effective concentration of oleic acid in the oleic acid-saffloric acid complex preparation is 300 μg / mL; and the effective concentration of saffloric acid is 1 μg / mL.

[0012] Preferably, the product enhances the protection of oxidative damage PC12 cells by saffloric acid.

[0013] Beneficial effects

[0014] 1. The present application effectively breaks through the application limitations of saffloric acid alone, and significantly improves the protection effect on oxidative damage cells. Saffloric acid has no protective effect on H2O2-induced PC12 cell oxidative damage in the range of 0.0158-0.0947 μg / mL, while the mixed system composed of 300 μg / mL oleic acid and 0.1 μg / mL saffloric acid can increase the cell survival rate by 7.62% when acting on H2O2-induced PC12 cells, and the mixed system composed of 300 μg / mL oleic acid and 1 μg / mL saffloric acid can increase the cell survival rate by 6.58% when acting on H2O2-induced PC12 cells, which indicates that oleic acid can significantly enhance the protection of saffloric acid on H2O2-damaged PC12 cells, successfully solves the technical problem of poor anti-cell oxidative damage effect of saffloric acid, and fully releases its potential antioxidant value.

[0015] 2. The experimental results of this invention confirm that only oleic acid can enhance the protective effect of crocin against oxidative damage to cells, while the combination of linoleic acid, linolenic acid and crocin does not have the corresponding effect, demonstrating the specificity of oleic acid's effect; it can provide a solid reference and data support for the development of anti-oxidative damage products for cells, and promote the development of antioxidant protection technology in the biomedical field. Attached Figure Description

[0016] Figure 1 The effect of different concentrations of H2O2 on the survival rate of PC12 cells is shown, where ** indicates a significant difference compared to the control group. P <0.01); Figure 2 This is a diagram showing the cytotoxicity of oleic acid on PC12 cells. Figure 3 This is a diagram showing the cytotoxicity of linoleic acid on PC12 cells. Figure 4 This is a diagram showing the cytotoxicity of linolenic acid on PC12 cells. Figure 5 This image shows the cell-protective effect of oleic acid on PC12 cells. Figure 6 This image shows the cell protection effect of linoleic acid on PC12 cells. Figure 7 This is a diagram showing the cytotoxicity of linolenic acid on PC12 cells. Figure 8 This image shows the cell-protective effect of crocin on PC12 cells. Figure 9 The cell protection effect of 300 μg / mL oleic acid mixed with different concentrations of crocin is shown in the figure. Figure 10 The cell protection effect of 400 μg / mL linoleic acid mixed with different concentrations of crocin is shown in the figure. Figure 11 The cell protection effect of 5 μg / mL linolenic acid mixed with different concentrations of crocin is shown in the figure. Figures 2-11 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] Establishment of PC12 cell injury model induced by H2O2 1.1 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 incubator with a condition setting of 5% CO2, 37°C. PC12 cells were observed under a microscope to be in an adherent state. (1) PC12 cell recovery: the cryopreservation tube containing 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 cryopreservation tube was wiped with alcohol, and the tube was transferred to a clean bench. The PC12 cell suspension in the cryopreservation tube was transferred to a sterile centrifuge tube and centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was discarded, and 1 mL of complete culture medium was added to resuspend the cells and transferred to a culture dish for culture in a 5% CO2, 37°C incubator. (2) PC12 cell passage: when the cell density in the culture dish reached about 85%, the cells were passaged. The waste liquid in the culture dish was discarded, and 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 for digestion. The cells gradually detached from the wall of the culture dish, and 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. Centrifugation was performed at 1000 rpm for 5 min, and the supernatant was discarded. The cells were subcultured at a ratio of 1:3. (3) PC12 cell cryopreservation: PC12 cells were digested in the logarithmic growth phase, and the steps were consistent with the above passage. After the supernatant was discarded, 1 mL of cell cryopreservation solution was added to resuspend the cells and transferred to a cryopreservation tube. After the cell cryopreservation tube was programmed to cool, it was transferred to liquid nitrogen for storage.

[0019] 1.2 Establishment of PC12 cell injury model induced by H2O2 PC12 cells were seeded in a 96-well plate at a density of 1×10 5 After 48 h of adherent culture (the original culture medium was replaced with a new culture medium after 24 h of culture), 100 μL of complete culture medium was added to each well of the control group, and 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 of the H2O2 injury group. The cells were cultured in the incubator for 2 h. After the culture was completed, the cell survival rate was detected by MTT method to determine the concentration of H2O2 for modeling.

[0020] 1.3 MTT detection of cell viability PC12 cells were seeded in a 96-well plate at a density of 1×10 5Cells were seeded in 96-well plates and cultured adherently for 24 hours. After treatment with the drug for a certain period of time, the waste liquid was discarded. 100 μL of MTT prepared with high-glucose DMEM containing 1% penicillin-streptomycin at a concentration of 1 mg / mL was added to each well. After incubation in an incubator for 4 hours, the waste liquid was aspirated, and 150 μL of LDMSO was added to each well. The mixture was slowly shaken for 10 minutes to dissolve the blue-purple formazan crystals. The OD value of each well was measured at 490 nm using a microplate reader, and the cell viability was calculated.

[0021] Cell viability (%) = (A2 - A0) / (A1 - A0) × 100% In the formula: A0 represents the OD value of the blank group, A1 represents the OD value of the control group, and A2 represents the OD value of the drug group.

[0022] Depend on Figure 1 It was found that, compared with the control group, the cell survival rate decreased significantly with the increase of H2O2 concentration. When the H2O2 concentration was 100 μmol / L, the cell survival rate was 57.53 ± 1.02%, which was close to the half-lethal rate. Therefore, 100 μmol / L was selected for the construction of the H2O2 oxidative damage PC12 cell model in subsequent experimental studies.

[0023] Example 2

[0024] Verification experiment on the toxicity of different fatty acids to PC12 cells 2.1 Verification experiment on the toxicity of oleic acid to PC12 cells PC12 cells were spaced at 1 × 10⁶ cells per well. 5 PC12 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 oleic acid solution of different concentrations (100, 200, 300, 400, 500, 600, 700 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.

[0025] 2.2 Verification experiment on the toxicity of linoleic acid to PC12 cells PC12 cells were spaced at 1 × 10⁶ cells per well. 5 PC12 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 linoleic acid solution of different concentrations (100, 200, 300, 400, 500, 600, 700 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.

[0026] 2.3 Verification experiment on the toxicity of linolenic acid to PC12 cells PC12 cells were seeded at 1 x 10 5 After 24 h of adherent culture, the waste liquid was removed, 100 μL of complete medium was added to each well of the control group, and 100 μL of different concentrations of linolenic acid solution (5, 10, 50, 100, 150, 200, 300 μg / mL) diluted with complete medium was added to each well of the sample group. After 24 h of treatment, the cytotoxicity range of oleic acid was determined by the MTT method.

[0027] From the above, Figure 2 , Figure 3 and Figure 4 it can be seen that oleic acid did not show toxic effects on PC12 cells in the range of 0-600 μg / mL, and this concentration range was used in the subsequent experiment of determining the protective effect of oleic acid on H2O2-damaged PC12 cells; linoleic acid did not show toxic effects on PC12 cells in the range of 0-400 μg / mL, and linolenic acid did not show toxic effects on PC12 cells in the range of 0-200 μg / mL.

[0028] Example 3

[0029] Determination of the protective effect of different fatty acids on H2O2-induced PC12 cells 3.1 Determination of the protective effect of oleic acid on H2O2-induced PC12 cells PC12 cells were seeded at 1 x 10 5 After 24 h of adherent culture, the waste liquid was removed, 100 μL of complete medium was added to each well of the control group, and 100 μL of different concentrations of linolenic acid solution (5, 10, 50, 100, 150, 200, 300 μg / mL) diluted with complete medium was added to each well of the sample group. After 24 h of treatment, the cytotoxicity range of oleic acid was determined by the MTT method.

[0030] 3.2 Determination of the protective effect of linoleic acid on H2O2-induced PC12 cells PC12 cells were seeded at 1 x 10 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 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.

[0031] 3.3 Determination of the protective effect of linolenic acid on H2O2-induced PC12 cells PC12 cells were spaced at 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.

[0032] Depend on Figure 5 It was found that oleic acid at 500 μg / mL had 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 6 It can be seen that linoleic acid at 200 μg / mL has a certain protective effect against H2O2-induced PC12 cells; Figure 7 It is known that linolenic acid has a cell-protective effect on H2O2-induced PC12 cells in the range of 5-200 μg / mL, especially in the concentration range of 50-200 μg / mL, where it has a very significant cell-protective effect.

[0033] Example 4

[0034] Determination of the protective effect of crocin on H2O2-induced PC12 cells PC12 cells were spaced at 1 × 10⁶ 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 crocin (concentrations of 0.0158, 0.0316, 0.0473, 0.0631, 0.0789, and 0.0947 μg / mL) was added to each well and cultured for 24 hours, followed by another 2 hours of 100 μmol / L H2O2. Cell viability was assessed using the MTT assay after culture.

[0035] Depend on Figure 8 It can be seen that crocin did not show a cell-protective effect on H2O2-induced PC12 cells in the range of 0.0158-0.0947 μg / mL.

[0036] Example 5

[0037] Application of oleic acid in enhancing the protective effect of crocin against oxidative damage to cells An oleic acid-crocin mixture was prepared by mixing oleic acid at a concentration of 300 μg / mL and crocin at a concentration of 0.1 μg / mL; its protective effect on H2O2-induced PC12 cells was determined as follows: PC12 cells were spaced at 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 an oleic acid-crocin mixture 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.

[0038] Example 6

[0039] Application of oleic acid in enhancing the protective effect of crocin against oxidative damage to cells An oleic acid-crocin mixture was prepared by mixing oleic acid at a concentration of 300 μg / mL and crocin at a concentration of 1 μg / mL; the protective effect of H2O2 on PC12 cells was determined, as follows: PC12 cells were spaced at 1 × 10⁶ 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 an oleic acid-crocin mixture 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.

[0040] Comparative Example 1

[0041] The difference between this comparative example and Example 5 is that oleic acid was not added. The protective effect against H2O2-induced PC12 cells was measured, as follows: PC12 cells were spaced at 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 0.1 μg / mL crocin 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.

[0042] Comparative Example 2

[0043] The difference between this comparative example and Example 6 is that oleic acid was not added. The protective effect against H2O2-induced PC12 cells was measured, as follows: PC12 cells were spaced at 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 1 μg / mL crocin 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.

[0044] Comparative Example 3

[0045] Use of linoleic acid in enhancing the protective effect of crocetin on oxidative damage cells The difference between this comparative example and Example 5 is that the concentration of 300 μg / mL oleic acid is replaced by the concentration of 400 μg / mL linoleic acid.

[0046] The concentration of 400 μg / mL linoleic acid is mixed with the concentration of 0.1 μg / mL crocetin to prepare a linoleic acid-crocetin mixed system; the protective effect on H2O2-induced PC12 cells is determined, and the details are as follows: PC12 cells are inoculated in a 96-well plate at 1×10 5 After adherent culture for 24 h, the waste liquid is removed, and the following groups are added: the control group is added with 100 μL of complete culture medium for 24 h, and then 100 μL of complete culture medium is added for 2 h of continuous culture; the H2O2 damage group is 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 is added for 2 h of continuous culture; the sample group is added with 100 μL of complete culture medium containing the linoleic acid-crocetin mixed system for 24 h, and then 100 μmol / L of H2O2 is added for 2 h of continuous culture. After the culture is completed, the cell survival rate of each group is detected by the MTT method.

[0047] Comparative Example 4

[0048] Use of linoleic acid in enhancing the protective effect of crocetin on oxidative damage cells The difference between this comparative example and Example 6 is that the concentration of 300 μg / mL oleic acid is replaced by the concentration of 400 μg / mL linoleic acid.

[0049] The concentration of 400 μg / mL linoleic acid is mixed with the concentration of 1 μg / mL crocetin to prepare a linoleic acid-crocetin mixed system; the protective effect on H2O2-induced PC12 cells is determined, and the details are as follows: PC12 cells are inoculated in a 96-well plate at 1×10 5 After adherent culture for 24 h, the waste liquid is removed, and the following groups are added: the control group is added with 100 μL of complete culture medium for 24 h, and then 100 μL of complete culture medium is added for 2 h of continuous culture; the H2O2 damage group is 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 is added for 2 h of continuous culture; the sample group is added with 100 μL of complete culture medium containing the linoleic acid-crocetin mixed system for 24 h, and then 100 μmol / L of H2O2 is added for 2 h of continuous culture. After the culture is completed, the cell survival rate of each group is detected by the MTT method.

[0050] Comparative Example 5

[0051] Use of linoleic acid in enhancing the protective effect of crocetin on oxidative damage cells The difference between this comparative example and Example 5 is that the concentration of 300 μg / mL oleic acid is replaced by the concentration of 5 μg / mL linolenic acid.

[0052] The concentration of 5 μg / mL linolenic acid is mixed with the concentration of 0.1 μg / mL crocetin to prepare a linolenic acid-crocetin mixed system; the protective effect on H2O2-induced PC12 cells is determined, and the details are as follows: PC12 cells are inoculated in a 96-well plate at 1×10 5 After adherent culture for 24 h, the waste liquid is removed, and the following groups are added: the control group is added with 100 μL of complete culture medium for 24 h, and then 100 μL of complete culture medium is added for 2 h of continuous culture; the H2O2 damage group is 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 is added for 2 h of continuous culture; the sample group is added with 100 μL of complete culture medium containing the linolenic acid-crocetin mixed system for 24 h, and then 100 μmol / L of H2O2 is added for 2 h of continuous culture. After the culture is completed, the cell survival rate of each group is detected by the MTT method.

[0053] Comparative Example 6

[0054] Use of linoleic acid in enhancing the protective effect of crocetin on oxidative damage cells The difference between this comparative example and Example 6 is that the concentration of 300 μg / mL oleic acid is replaced by the concentration of 5 μg / mL linolenic acid.

[0055] The concentration of 5 μg / mL linolenic acid is mixed with the concentration of 1 μg / mL crocetin to prepare a linolenic acid-crocetin mixed system; the protective effect on H2O2-induced PC12 cells is determined, and the details are as follows: PC12 cells are inoculated in a 96-well plate at 1×10 5 After adherent culture for 24 h, the waste liquid is removed, and the following groups are added: the control group is added with 100 μL of complete culture medium for 24 h, and then 100 μL of complete culture medium is added for 2 h of continuous culture; the H2O2 damage group is 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 is added for 2 h of continuous culture; the sample group is added with 100 μL of complete culture medium containing the linolenic acid-crocetin mixed system for 24 h, and then 100 μmol / L of H2O2 is added for 2 h of continuous culture. After the culture is completed, the cell survival rate of each group is detected by the MTT method.

[0056] ByFigure 5 It can be seen that oleic acid alone at 300 μg / mL does not show a cell protection effect on the damaged cells, and Figure 9 Comparative Example 1 (0.1 μg / mL crocetin) and Comparative Example 2 (1 μg / mL crocetin) also do not show a cell protection effect on the H2O2-damaged PC12 cells. However, Example 5 (300 μg / mL oleic acid + 0.1 μg / mL crocetin) and Example 6 (300 μg / mL oleic acid + 1 μg / mL crocetin) show a significant cell protection effect on the H2O2-damaged PC12 cells compared with the H2O2-treated group, and the cell survival rate of Example 5 is increased by 7.62% compared with Comparative Example 1, and the cell survival rate of Example 6 is increased by 6.58% compared with Comparative Example 2; thus the results show that oleic acid can significantly enhance the protection effect of crocetin on the H2O2-damaged PC12 cells. From the above, Figure 10 and Figure 11 It can be seen that Comparative Example 3 (400 μg / mL linoleic acid + 0.1 μg / mL crocetin), Comparative Example 4 (400 μg / mL linoleic acid + 1 μg / mL crocetin), Comparative Example 5 (5 μg / mL linolenic acid + 0.1 μg / mL crocetin) and Comparative Example 6 (5 μg / mL linolenic acid + 1 μg / mL crocetin) do not show a protection effect on the H2O2-damaged PC12 cells, indicating that linoleic acid and linolenic acid do not have the effect of enhancing the protection of crocetin on the H2O2-damaged PC12 cells.

[0057] The above merely describes the 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 make equivalent embodiments with equivalent changes, without departing from the spirit and technical solutions of the present application. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present application, still falls within the protection scope of the technical solutions of the present application.

Claims

1. Use of oleic acid-crocetin complex preparation in the preparation of a product for enhancing the protective effect of crocetin on oxidative damage PC12 cells.

2. Use according to claim 1, characterized in that: The composition of the oleic acid-crocetin complex preparation comprises oleic acid, crocetin and pharmaceutically acceptable adjuvants.

3. Use according to claim 1, characterized in that: The effective concentration of oleic acid in the oleic acid-crocetin complex preparation is 100-600 μg / mL; the effective concentration of crocetin is 0.1-1 μg / mL.

4. Use according to claim 1, characterized in that: The effective concentration of oleic acid in the oleic acid-crocetin complex preparation is 300 μg / mL; the effective concentration of crocetin is 1 μg / mL.

5. The use according to claim 1, characterized in that: The product enhances the protective effect of crocetin on oxidative damage PC12 cells.

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