CN116173151A - Application of ceramide compound in antioxidation stress - Google Patents
Application of ceramide compound in antioxidation stress Download PDFInfo
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- CN116173151A CN116173151A CN202310430387.3A CN202310430387A CN116173151A CN 116173151 A CN116173151 A CN 116173151A CN 202310430387 A CN202310430387 A CN 202310430387A CN 116173151 A CN116173151 A CN 116173151A
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Abstract
The invention provides application of ceramide compounds in preparation of anti-oxidative stress drugs, wherein the ceramide compounds are extracted from red rice bran and used for reducing toxic effects and cell damage caused by oxidative stress. Experiments show that the ceramide compound can prolong the average life of caenorhabditis elegans, can improve the activities of SOD, CAT and GSH in caenorhabditis elegans, reduces the content of MDA and can reduce the level of ROS in caenorhabditis elegans. The ceramide compound extracted by the invention can reduce toxic effect and cell injury caused by oxidative stress, and enhance the antioxidant capacity of caenorhabditis elegans.
Description
Technical Field
The invention belongs to the technical field of marine organism application, relates to biological function application of a seawater rice extract, and in particular relates to application of ceramide compounds in red rice bran to antioxidation stress.
Background
The red rice is a new type of salt-tolerant rice grown in coastal areas and contains high levels of minerals, proteins and lipid compounds. Particularly, the red rice bran contains more abundant ceramide compounds (Ceramides, cers) than common rice bran.
Ceramide compounds are considered to be lipid secondary causes and play an important role in various information transmission processes such as cell proliferation, differentiation and apoptosis. At present, the action mechanism of ceramide compounds as second messengers in apoptosis is relatively deeply studied. It was found that ceramide compounds are involved in the apoptosis process of intracellular mitochondria primarily by activating specific downstream targeting of pro-apoptotic protein Bcl-2 and other related proteins to promote changes in mitochondrial cytochrome C and outer membrane permeability proximally. In mammalian cells and animal models, ceramides can also activate several Reactive Oxygen Species (ROS) producing enzymes, such as NADPH oxidase, uncoupled eNOS, xanthine oxidase, etc.
Caenorhabditis elegans (L.) LindlC.elegans) Is a well-known in vivo model organism, has high genetic homology with mammals, and is sensitive to environmental stress. Under normal conditions, various stress stimuli (e.g., radiation, toxins, and ultraviolet light) can be used to induce apoptosis and injury in caenorhabditis elegans cells. The caenorhabditis elegans mutant can be used as an in vivo experimental model for researching apoptosis and apoptosisSignal transduction mechanisms of inflammatory factors.
Caenorhabditis elegans (L.) LindlC.elegans) Is a well-known in vivo model organism, has high genetic homology with mammals, and is sensitive to environmental stress. Under normal conditions, various stress stimuli (e.g., radiation, toxins, and ultraviolet light) can be used to induce apoptosis and injury in caenorhabditis elegans cells. The caenorhabditis elegans mutant can be used as an in vivo experimental model for researching the signal transduction mechanism of apoptosis and inflammatory factors.
Disclosure of Invention
The inventor of the application researches and discovers that the ceramide compound has the effect of relieving the oxidative stress of caenorhabditis elegans under different polar environments.
The application provides ideas for researching application of the ceramide compounds in resisting oxidative stress, and has substantial reference significance for improving organism antioxidation and anti-aging.
The invention aims to research the function of the ceramide compounds in the red rice bran to resist oxidative stress, provide a new thought for the application of the ceramide compounds, and provide a new material and reference for improving the antioxidant and anti-aging capacities of organisms.
In order to achieve the above purpose, the invention adopts the following technical scheme: the application of the ceramide compound in preparing the antioxidant stress medicament is that the ceramide compound is extracted from the red rice bran and is used for reducing the toxic effect and the cell damage caused by the oxidative stress.
Experiments prove that the ceramide compound can prolong the average service life of caenorhabditis elegans, can improve the activities of SOD, CAT and GSH in caenorhabditis elegans, reduces the content of MDA and can reduce the level of ROS in caenorhabditis elegans. Based on the experience of the caenorhabditis elegans with a high degree of genetic homology to mammals and its superior in vivo test model, the claimed application of the invention can be supported by the specific examples and results of the invention.
Compared with the prior art, the invention has the following beneficial effects or advantages:
the ceramide compound in the rhodomyrtus tomentosa bran can effectively prolong the service life of caenorhabditis elegans.
The ceramide compound in the rhodometer chaff effectively relieves the oxidative stress of caenorhabditis elegans, improves the injury of the oxidative stress of caenorhabditis elegans, obviously improves the antioxidant enzyme activity (SOD, CAT, GSH) of caenorhabditis elegans, obviously reduces the Metabolite (MDA), and obviously reduces the ROS level in the body.
The invention provides a new thought for application of ceramide compounds and provides a new material and reference for improving the antioxidant and anti-aging capacities of organisms.
Drawings
FIG. 1 is a graph showing the longevity of caenorhabditis elegans intake of ceramide compounds at various concentrations under a stress condition of 30% hydrogen peroxide solution. The blank group represents the ceramide compound with the concentration of 0, namely, the control group, and 0.10 mg/mL, 0.25 mg/mL and 0.50 mg/mL respectively represent the ceramide compound with the corresponding concentration.
FIG. 2 is a graph showing the longevity of caenorhabditis elegans intake of varying concentrations of ceramide compounds under stress conditions of 5mM paraquat. The blank group represents the ceramide compound with the concentration of 0, namely, the control group, and 0.10 mg/mL, 0.25 mg/mL and 0.50 mg/mL respectively represent the ceramide compound with the corresponding concentration.
FIG. 3 shows the effect of ceramide compounds on the SOD activity of caenorhabditis elegans. The blank represents a concentration of 0, i.e., the control, and 0.10, 0.25, and 0.50 represent the ceramide compound concentration (mg/mL), respectively.
FIG. 4 shows the effect of ceramide compounds on CAT activity in caenorhabditis elegans. The blank represents a concentration of 0, i.e., the control, and 0.10, 0.25, and 0.50 represent the ceramide compound concentration (mg/mL), respectively.
FIG. 5 is a graph showing the effect of ceramide compounds on GSH activity in caenorhabditis elegans. The blank represents a concentration of 0, i.e., the control, and 0.10, 0.25, and 0.50 represent the ceramide compound concentration (mg/mL), respectively.
Figure 6 is the effect of ceramide compounds on the MDA activity of caenorhabditis elegans. The blank represents a concentration of 0, i.e., the control, and 0.10, 0.25, and 0.50 represent the ceramide compound concentration (mg/mL), respectively.
FIG. 7 is a graph showing the effect of varying concentrations of ceramide compounds on ROS formation in caenorhabditis elegans. The blank represents the concentration of 0, i.e., the control, and 0.10, 0.25, and 0.50 represent the corresponding concentrations of ceramide compound (mg/mL), respectively, and the ordinate represents the relative content (%) of ROS.
Detailed Description
The following describes the technical aspects of the present invention with reference to examples, but the present invention is not limited to the following examples.
The experimental methods and the detection methods in the following embodiments are all conventional methods unless otherwise specified; the medicament and the material can be purchased in the market if no special description exists, and all reagents are analytically pure; the index data are all conventional measurement methods unless specified.
Example 1
The method for extracting ceramide compounds from the red rice bran in this embodiment refers to the application CN114807258A filed by the inventor of this patent.
The raw material of the red rice bran described in this example was produced in Zhanzhan, guangdong, and purchased from Hubei Jusheng technology Co.
Removing impurities from the dried red rice bran raw material, crushing and sieving to obtain 20-40 mesh bran raw material powder. 100g of red rice bran powder is taken, added into 300mL of enzymolysis liquid (calculated by enzyme activity unit, the proportion of sphingomyelinase and cellulase is 4:6, the concentration is 0.2 w/v%) of the preparation, and the mixture is subjected to enzymolysis for 60min at 35 ℃ with pH adjusted to 5, and filtered to obtain the enzymolysis Pi Kang (containing water). Taking 1g of enzymolysis Pi Kang (containing water), adding 500mL of 75% ethanol solution, stirring for 20 minutes, standing and soaking for 1 hour, sealing, placing into an ultrasonic cleaner, starting a procedure, selecting ultrasonic power to be 360w, heating at 45 ℃, and extracting for 1 hour. After the extraction is finished, filtering, transferring ethanol extract of ceramide into a rotary evaporator for concentration until the ethanol concentration is 10%, transferring the concentrated solution into a clean three-mouth flat-bottomed flask, adding 2 times of petroleum ether according to the volume of the concentrated solution, stirring for 0.5h, transferring into a separating funnel for standing for 0.5h, layering, and collecting an ether phase at the upper layer for extraction for 2 times. The ether phases were combined and petroleum ether recovered and concentrated to an extract to give a brown ceramide sample I, weighing 16.4g.
The sample was purified by silica gel chromatography with a loading of 1g of crude product (ceramide sample I) in combination with 100g of silica gel (purchased from Shanghai Yao trade Co., ltd.). 100g of 300-mesh silica gel is weighed and activated for 1h at 105-110 ℃, then the activated silica gel is put into a container, a proper amount of petroleum ether is added, stirring is carried out along a fixed direction, when no bubbles exist in the silica gel, the silica gel is packed into a silica gel chromatographic column by a wet method, the silica gel column is phi 40 mm, and the column volume is 250mL. The column was then equilibrated with 500mL of initial eluent at a flow rate of 4 mL/min.
Dissolving the crude ceramide product I by an initial mobile phase, loading the ceramide product I by a wet method, respectively carrying out gradient elution by using petroleum ether/ethyl acetate (v/v) as eluent in the ranges of 95:5, 85:15, 60:40 and 40:60, wherein the use amount of the two kinds of eluent is 1BV, the use amount of the two kinds of eluent is 2BV, collecting effluent liquid step by step, detecting the effluent liquid by TLC, combining the effluent liquid with the same component, recovering the solvent, and drying to obtain a ceramide sample II, and the weight of the ceramide sample II is 0.45g.
0.1g of ceramide sample II is wet loaded into a medium pressure chromatographic column, then compressed by a pressurizing device, the working pressure of the compressed column body under normal flow rate is 10bar, and the chromatographic column is balanced by an initial eluent. And (3) performing gradient elution with ethyl acetate and acetone according to the volume ratio of 6:4 and 1:1, wherein the dosage of the ethyl acetate and the acetone is 2BV, the eluting speed is 1BV/h, collecting effluent in steps, detecting the effluent by TLC, combining the effluent with the same components, recovering the solvent, drying to obtain the ceramide compound, and preserving for later use.
Example 2
The example provides a survival test of ceramide compounds applied to caenorhabditis elegans antioxidant stress.
The C.elegans strain N2 (wild type) and E.coli OP50 described in this example were purchased from C.elegans Genetics Center (CGC) at university of Minnesota; hydrogen peroxide, paraquat and Nematode Growth Medium (NGM) were all purchased from Sigma-Aldrich (St.Louis, MO, USA); the ceramide compound was derived from example 1.
Caenorhabditis elegans were inoculated onto NGM plates, escherichia coli OP50 was uniformly coated on NGM plates for nematode growth and grown as sole food for nematodes, grown in culture at 20 ℃, after L4 stage caenorhabditis elegans were rinsed with M9 buffer and lysed with bleaching solution (1 mol/L sodium hypochlorite+2 mol/L sodium hydroxide solution) to obtain the contemporaneous nematodes.
Setting 8 groups of experiments, wherein each group is repeated for 3 times, and 80 caenorhabditis elegans are used repeatedly; the caenorhabditis elegans obtained by the previous culture are respectively ingested with 0, 0.10, 0.25 and 0.50 mg/mL ceramide compound, and after 5 days of culture, the nematodes at each concentration are divided into two groups. Nematodes were exposed to NGM containing 30% hydrogen peroxide or 5mM paraquat, respectively, every other day and counted until all deaths.
The nematodes were gently detected with platinum wires, and the failure to give a response to external physical stimuli within 5s of the nematodes was noted as death, the dead nematodes were counted and cleared every day, and the average of 3 replicates was taken for each set of data.
The statistical results of the life distribution of caenorhabditis elegans ingesting ceramide compounds at different concentrations under different stress conditions are shown in figure 1, figure 2 and table 1.
TABLE 1 statistics of the effect of different concentrations of ceramide compounds on the longevity of C.elegans under different stressors
As can be seen from fig. 1, 2 and table 1, the survival rate of caenorhabditis elegans that ingest ceramide compounds is higher, the survival time is longer and the total death time is longer under the same stress condition than that of the control treatment that does not ingest ceramide compounds. In the concentration range of 0.10-0.50 mg/mL, the higher the concentration of the ceramide compound ingested by caenorhabditis elegans, the more remarkable the survival rate is improved, and the longer the total death time is. Ingestion of the ceramide compound increases the mean life of caenorhabditis elegans in a dose-dependent manner, with a concentration of 0.50 mg/mL extending the mean life of the nematode by 66.6% when stressed at 30% hydrogen peroxide. The above results demonstrate that ingestion of ceramide compounds significantly reduces the toxic effects and cell damage caused by paraquat and hydrogen peroxide to varying degrees.
Example 3
The example provides an antioxidant enzyme activity assay of ceramide compounds applied to antioxidant stress of caenorhabditis elegans.
Enzymes (substances) associated with oxidative stress in vivo include superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and Malondialdehyde (MDA) metabolites. This example reflects the antioxidant activity of ceramide compounds against caenorhabditis elegans by measuring the activity level of these enzymes (substances).
The antioxidant enzyme test kit used in this example was purchased from Shanghai mu Biotechnology Co., ltd. And the ceramide compound was obtained from example 1.
Caenorhabditis elegans were inoculated onto NGM plates containing ceramide compounds at concentrations of 0, 0.10, 0.25 and 0.50 mg/mL respectively, and escherichia coli OP50 was evenly coated on the NGM plates for nematode growth and grown as sole food for nematodes, cultured at 20 ℃ for 5d, after which caenorhabditis elegans was rinsed with M9 buffer during the L4 stage.
Crushing the washed caenorhabditis elegans worm body, centrifuging, collecting samples, and respectively using an antioxidant enzyme detection kit to determine the activity of enzymes (substances) related to in-vivo oxidative stress. The measurement results are shown in FIG. 3 to FIG. 6.
Figures 3-6 show the activity of antioxidant stress related enzymes (substances) in caenorhabditis elegans after treatment with different concentrations of ceramide compounds. Compared with the control group, the SOD and CAT activities are obviously improved by 1.43-2.12 times and 1.26-2.31 times respectively, and the GSH activity is improved by 1.15-1.39 times; whereas the metabolite MDA activity was reduced by 67.40%. The results show that the ceramide compound can obviously improve the activities of the antioxidant enzymes (substances) SOD, CAT and GSH of caenorhabditis elegans and reduce the activities of metabolites MDA.
Example 4
This example provides a ceramide compound for use in ROS level assay of caenorhabditis elegans.
ROS play an important role in apoptosis through DNA damage, amino acid oxidation and lipid peroxidation, leading to senescence in organisms. Thus, ROS levels are commonly used to assess the degree of aging and the level of oxidative damage in organisms.
ROS-specific fluorescent probe DCFH-DA (100. Mu.M) used in this example was purchased from Molecular Probes; a tablet reader (Filter Max F5) was purchased from Molecular Devices. The ceramide compound was derived from example 1.
Caenorhabditis elegans were inoculated onto NGM plates containing ceramide compounds at concentrations of 0, 0.10, 0.25 and 0.50 mg/mL respectively, and escherichia coli OP50 was evenly coated on the NGM plates for nematode growth and grown as sole food for nematodes, cultured at 20 ℃ for 5d, after which caenorhabditis elegans was rinsed with M9 buffer during the L4 stage.
The ROS-specific fluorescent probe DCFH-DA was placed in a medium containing 0.5% NaCl to give a final concentration of ROS-specific fluorescent probe DCFH-DA of 500. Mu.M, and stirred for 1 hour in the dark.
After the washed caenorhabditis elegans obtained in the above step were broken up and centrifuged, 50. Mu.L of caenorhabditis elegans tissue centrifugation supernatant was mixed with 50. Mu.L of DCFH-DA-containing medium in 96-well plates, and the fluorescence intensity of each sample was measured with a plate reader (excitation: 485nm; emission: 535 nm). The measurement results are shown in FIG. 7.
FIG. 7 shows the ROS level assay of ceramide compounds applied to caenorhabditis elegans. As can be seen from FIG. 7, the intake of ceramide compound at 0.10-0.50. 0.50 mg/mL decreased ROS level in C.elegans cells by 59.8-34.2% as compared to control groupp<0.05). Notably, within the effective range of ceramide compounds, ROS levels gradually decrease with increasing ceramide compound concentration. This suggests that ingestion of ceramide compounds may slow down the expression of NADPH oxidase on the plasma membrane of C.elegans mitochondrial pre-cell.
The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.
Claims (6)
1. The application of the ceramide compound in preparing the antioxidant stress medicament is that the ceramide compound is extracted from the red rice bran and is used for reducing the toxic effect and the cell damage caused by the oxidative stress.
2. The use according to claim 1, wherein the ceramide compound is capable of increasing SOD activity in an animal.
3. The use according to claim 1, wherein the ceramide compound is capable of increasing CAT activity in an animal.
4. The use according to claim 1, wherein the ceramide compound is capable of increasing GSH activity in an animal.
5. The use according to claim 1, wherein the ceramide compound is capable of reducing the MDA content in an animal.
6. The use according to claim 1, wherein said ceramide compound is capable of reducing ROS content in animals.
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