CN105030837A - Method of extracting and isolating three-spot seahorse liposoluble components - Google Patents

Abstract

The invention discloses a method for extracting and separating fat-soluble components of hippocampus three-spotted hippocampus, specifically: first extracting the fat-soluble components, and measuring the wavelength of its maximum ultraviolet absorption peak, and then taking the wavelength of the maximum ultraviolet absorption peak as the detection wavelength, and comparing the Perform medium-pressure preparation and purification chromatography on silica gel column chromatography to separate and elute, and obtain a fat-soluble isolate of three-spotted hippocampus from which cholesterol is removed. The fat-soluble components extracted in the present invention are subjected to medium-pressure preparation and purification chromatography on silica gel column chromatography, step/linear gradient elution with different polar solvents, and separated components with different polarities are obtained. Among them, the content of physiologically active substances in the relatively polar component FrⅣ was relatively high, and cholesterol was not detected in this component; at the same time, this component could inhibit lipopolysaccharide from stimulating the mouse macrophage RAW264.7 inflammatory factor nitric oxide, and It has no toxic and side effects on cells and has no significant impact on cell viability.

Description

A method for extracting and separating fat-soluble components of three-spotted hippocampus

technical field

The invention belongs to the technical field of biological extraction, and relates to a method for extracting and separating fat-soluble components of three-spotted hippocampus.

Background technique

Inflammation is the body's defense response to infection, pathogen invasion, or cell damage. Its basic biological process is the most common signal of disease. Inflammation is alleviated in most cases by the accumulation of endogenous anti-inflammatory mediators and intracellular negative regulators. However, continued accumulation and activation, hallmarks of chronic inflammation, will lead to dysfunction of these negative regulatory mechanisms. Therefore, strengthening and accelerating the limitation and resolution of inflammation is the key to the development of anti-inflammatory drugs.

In recent years, the extraction of anti-inflammatory active substances from natural products has attracted more and more attention because of its safe and extensive sources and low side effects. Marine organisms live in complex habitats, produce secondary metabolites, etc., and contain a unique series of biomolecules, so marine resources are a huge treasure trove for anti-inflammatory drugs.

Three-spotted hippocampus is a precious medicinal material in some Asian countries including China. According to ancient Chinese medical records, it has the functions of nourishing kidney and strengthening yang, warming blood vessels, calming nerves, dispelling stagnation and swelling, and relaxing tendons and activating collaterals. Modern pharmacological research shows that it has anti-oxidation, anti-tumor, anti-bacterial and other physiological activities, which are related to phospholipids, sterols, amino acids, trace elements, etc. Among them, the fat-soluble component is the main functional component, but it contains a lot of cholesterol. At present, the main three-spotted hippocampus products on the market are hippocampus wine, hippocampus chasing wind oil, etc. The fat-soluble components of animal tissues generally contain cholesterol.

Cholesterol exists in all tissues of animals and is necessary for maintaining normal physiological activities of animals. However, excessive intake of cholesterol by the human body will cause hyperlipidemia, and then lead to a series of cardiovascular diseases, so controlling the intake of cholesterol in the diet is the way advocated by the modern diet.

The methods for removing cholesterol from animal tissues currently include physical methods such as organic solvent extraction, supercritical carbon dioxide extraction, β-cyclodextrin embedding, and biological methods such as decomposition of lactobacilli, which are costly or have unclear mechanisms, and other A series of unfavorable factors such as metabolites interfering with the absorption of other nutrients limit its use.

Contents of the invention

The purpose of the present invention is to provide a method for extracting and separating fat-soluble components of hippocampus three-spotted, which is used to screen and extract fat-soluble bioactive substances with strong anti-inflammatory activity from three-spotted hippocampus and remove the cholesterol therein.

The technical scheme adopted in the present invention is a method for extracting and separating the fat-soluble components of hippocampus three-spotted, specifically implemented according to the following steps:

Step 1, extract fat-soluble components:

1.1: Using ethanol solution to reflux extract the three-spotted hippocampus powder to obtain crude ethanol extract;

1.2: Carry out suction filtration on the ethanol crude extract, repeat the suction filtration operation of the filter residue several times, combine the filtrate to remove the solvent by evaporating under reduced pressure, and obtain the extract-like ethanol crude extract;

1.3: Extract the extract-like ethanol crude extract with an organic solvent to obtain the fat-soluble components of hippocampus three-spotted;

Step 2, determining the wavelength of the maximum ultraviolet absorption peak of the fat-soluble component;

Step 3, using the maximum ultraviolet absorption peak wavelength of the three-spotted hippocampus fat-soluble component measured in the step as the detection wavelength, the fat-soluble component is subjected to medium-pressure preparation and purification chromatography silica gel column chromatography to separate and elute, and then collect each elution peak , concentrated to an extract, and freeze-dried at 0°C to obtain a fat-soluble isolate of hippocampus three-spotted without cholesterol.

The present invention is also characterized in that,

Step 1.1 is specifically: washing the dried body of the three-spotted hippocampus with tap water to remove sludge and sand, drying, crushing, and sieving to obtain the three-spotted hippocampus powder, and mixing the three-spotted hippocampus powder with ethanol with a mass fraction of 70%-95% The solutions were mixed and refluxed at 80°C for 1h-2h to obtain crude ethanol extract.

The drying temperature is 50-60°C, and the drying time is 24-36 hours; the sieving specification is 20-40 mesh.

The solid-liquid ratio of three-spot hippocampus powder and ethanol solution is 1:8-12g/ml.

Step 1.3 is specifically: mix the extract-like ethanol crude extract with deionized water, then add petroleum ether, extract, take the upper petroleum ether phase, repeat the extraction several times, combine all the extracted petroleum ether phases, 45-55°C After the extract was concentrated by evaporation under reduced pressure, it was freeze-dried at 0°C to obtain the fat-soluble component of hippocampus three-spotted.

The mass volume ratio of extract-like ethanol crude extract to deionized water is 1:8-12g/ml, and the amount of petroleum ether added is equal to the mixture of extract-like ethanol crude extract and deionized water.

Step 2 is specifically as follows: take the fat-soluble components of hippocampus three-spots obtained in step 1, dissolve them in petroleum ether, vortex and mix them evenly, use the solvent petroleum ether as a blank, scan the ultraviolet spectrum at 400-190nm, obtain the scanning spectrum, and obtain hippocampus three-spots The fat-soluble component has a maximum absorption peak at a wavelength of 205nm.

In step 3, the medium pressure preparation and purification chromatography silica gel column chromatography separation parameters are as follows: injection volume: 0.5-2.0mL; detection wavelength: 205nm, monitoring wavelength: 210nm.

The elution in step 3 is a step/linear gradient elution.

Step/linear gradient elution is specifically as follows: the elution solvent uses petroleum ether as mobile phase A, ethanol as mobile phase B, and the ratio of mobile phase A and B by volume percentage is: 45-95% mobile phase A, 5-55% %Mobile phase B, the sum of the volume percentages of the above components is 100%, and the elution flow rate: 8-10mL/min.

The beneficial effect of the present invention is that the method of the present invention adopts ethanol solution to reflux to extract the three-spotted hippocampus to obtain the ethanol crude extract, and then adopts the system solvent extraction method of liquid-liquid separation to obtain the petroleum ether extract with low polarity from the ethanol crude extract , The petroleum ether extract was subjected to medium-pressure preparation and purification chromatography on silica gel column chromatography, and step/linear gradient elution was performed with different polar solvents to obtain separated components with different polarities. Among them, the content of physiologically active substances in the relatively polar component FrⅣ was relatively high, and cholesterol was not detected in this component; at the same time, this component could inhibit lipopolysaccharide from stimulating the mouse macrophage RAW264.7 inflammatory factor nitric oxide, and It has no toxic and side effects on cells and has no significant impact on cell viability.

Description of drawings

Fig. 1 is a flow chart of the inventive method;

Fig. 2 is the ultraviolet spectrum scanning collection of petroleum ether extracts in the present invention;

Fig. 3 is the chromatogram of the silica gel column chromatogram for rapid preparation and separation of the fat-soluble components of hippocampus three-spotted in the present invention under medium pressure;

Fig. 4 is the cholesterol thin-layer chromatogram spectrum that the present invention separates;

Fig. 5 is a schematic diagram of the effect of the lipid-soluble components of the three-spotted hippocampus in Example 1 of the present invention on the viability of mouse macrophage RAW264.7 cells;

Fig. 6 is a graph showing the inhibition of the production of nitrite in mouse macrophage RAW264.7 by the isolated fraction of the fat-soluble component of the three-spotted hippocampus in Example 1 of the present invention.

In the figure, 1. standard cholesterol, 2. FrI component, 3. FrII component, 4. FrIII component, 5. FrIV component.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

The invention provides a method for extracting and separating the fat-soluble components of hippocampus three-spotted, as shown in Figure 1, specifically implemented according to the following steps:

Step 1, extract fat-soluble components:

1.1 Rinse the dried three-spotted hippocampus with tap water to remove sludge and sand, and then place it in a constant temperature drying oven at 50-60°C for 24-36h. After pulverized by a Chinese herbal medicine grinder, pass through a 20-40 mesh sieve to obtain the three-spotted hippocampus powder, and mix the three-spotted hippocampus powder with an ethanol solution with a mass fraction of 70%-95% at a material-to-liquid ratio of 1:8-12g/ml, Reflux at 80°C for 1h-2h to obtain crude ethanol extract.

1.2 The ethanol crude extract of hippocampus three-spotted was suction-filtered in a Buchner funnel, and the filter residue was suction-filtered three times. The combined filtrates were evaporated under reduced pressure to remove the solvent to obtain extract-like ethanol crude extract.

1.3 Mix the extract-like ethanol crude extract with deionized water at a ratio of 1:8-12g/ml, then add an equal volume of petroleum ether II (boiling range: 60-90°C) to the above mixture, and put it in a pear-shaped funnel Extraction was carried out, and the upper petroleum ether phase was taken, and the extraction was repeated 3 times. All the extracted petroleum ether phases were combined, evaporated and concentrated under reduced pressure at 45-55°C to form an extract, and then freeze-dried at 0°C to obtain the fat-soluble component of hippocampus three-spotted hippocampus, which was stored in a -20°C refrigerator.

Step 2, determine the wavelength of the maximum UV absorption peak of the fat-soluble components:

Take the fat-soluble component of hippocampus three spots obtained in step 1, dissolve it in petroleum ether, vortex and mix well, use the solvent petroleum ether as a blank, and scan the ultraviolet spectrum at 400-190nm. Obtain the scanning spectrum, and detect the maximum absorption wavelength of the fat-soluble components of the three-spotted hippocampus. The 400-190nm ultraviolet spectrum scanning results of the petroleum ether extract showed that the fat-soluble components of the three-spotted hippocampus had a maximum absorption peak at a wavelength of 205nm, so 205nm was used as the detection wavelength, as shown in Figure 2.

Step 3, medium-pressure preparation and purification of the fat-soluble components of hippocampus three-spotted chromatographic separation by silica gel column chromatography:

3.1 The fat-soluble components of the three-spotted hippocampus were separated by medium-pressure preparation and purification chromatography equipped with a silica gel column. Injection volume: 0.5-2.0mL; detection wavelength: 205nm, monitoring wavelength: 210nm;

3.2 Use the elution solvent to carry out step/linear gradient elution of the isolate. The mobile phase A in the elution solvent is petroleum ether, and the mobile phase B is ethanol. The ratio of mobile phase A and B is 45-95% by volume Mobile phase A, 5-55% mobile phase B, the total volume percentage of the above components is 100%; elution flow rate: 8-10mL/min. Each eluted peak was collected, concentrated to an extract with a rotary evaporator, and freeze-dried at 0°C to obtain a fat-soluble isolate of hippocampus three-spotted from cholesterol.

The silica gel column chromatography chromatogram of the fat-soluble components of the three-spotted hippocampus is shown in Figure 3. FrI, FrII, FrIII, and FrIV are the different polar isolates obtained after silica gel column chromatography of the petroleum ether extract, and the isolates are Viscous oil with certain fluidity. Among them, the more polar component FrⅣ had a higher content of physiologically active substances, and no cholesterol was detected in this component.

The method of the present invention adopts the reflux of ethanol solution to extract the three-spotted hippocampus to obtain the ethanol crude extract, and then adopts the system solvent extraction method of liquid-liquid separation to obtain the petroleum ether extract with low polarity from the ethanol crude extract, and the petroleum ether extract is processed through the Pressure preparation and purification chromatography on silica gel column chromatography, step/linear gradient elution with different polar solvents, to obtain separated components with different polarities. Among them, the content of physiologically active substances in the relatively polar component FrⅣ was relatively high, and cholesterol was not detected in this component; at the same time, this component could inhibit lipopolysaccharide from stimulating the mouse macrophage RAW264.7 inflammatory factor nitric oxide, and It has no toxic and side effects on cells and has no significant impact on cell viability.

Example 1

Step 1, extract fat-soluble components:

1.1 Rinse the dried body of three-spotted hippocampus with tap water to remove sludge and sand, and then place it in a constant temperature drying oven at 60°C for 24 hours. After being pulverized by a Chinese herbal medicine pulverizer, pass through a 20-40 mesh sieve to obtain a three-spot hippocampus powder. The three-spot hippocampus powder is mixed with an ethanol solution with a mass fraction of 70%-95% at a material-to-liquid ratio of 1:8g/ml. Reflux at ℃ for 1 h to obtain crude ethanol extract.

1.2 The ethanol crude extract of hippocampus three-spotted was suction-filtered in a Buchner funnel, and the filter residue was suction-filtered three times. The combined filtrates were evaporated under reduced pressure to remove the solvent to obtain extract-like ethanol crude extract.

1.3 Mix the extract-like ethanol crude extract with deionized water at a ratio of 1:8g/ml, then add petroleum ether equal to the volume of the above mixture, extract in a pear-shaped funnel, take the upper petroleum ether phase, and repeat the extraction for 3 Second-rate. All the extracted petroleum ether phases were combined, evaporated and concentrated under reduced pressure at 45°C to an extract, and then freeze-dried to obtain the fat-soluble component of hippocampus three-spotted.

Step 2, determine the wavelength of the maximum UV absorption peak of the fat-soluble components:

Take the fat-soluble component of hippocampus three spots obtained in step 1, dissolve it in petroleum ether, vortex and mix well, use the solvent petroleum ether as a blank, and scan the ultraviolet spectrum at 400-190nm. The scanning spectrum was obtained, and the maximum absorption wavelength of the fat-soluble components of the three-spotted hippocampus was detected at 205nm.

Step 3, medium-pressure preparation and purification of the fat-soluble components of hippocampus three-spotted chromatographic separation by silica gel column chromatography:

3.1 The fat-soluble components of the three-spotted hippocampus were separated by medium-pressure preparation and purification chromatography equipped with a silica gel column. Injection volume: 0.5mL; detection wavelength: 205nm, monitoring wavelength: 210nm;

3.2 Use the elution solvent to carry out step/linear gradient elution of the isolate. The mobile phase A in the elution solvent is petroleum ether, and the mobile phase B is ethanol solution. The volume percentage of mobile phase A and B is: 45% mobile phase A , 55% mobile phase B, the sum of the volume percentages of the above components is 100%; elution flow rate: 10mL/min. Each eluted peak was collected, concentrated to an extract with a rotary evaporator, and freeze-dried at 0°C to obtain a fat-soluble isolate of hippocampus three-spotted from cholesterol.

Example 2

Step 1, extract fat-soluble components:

1.1 Rinse the dried body of three-spotted hippocampus with tap water to remove sludge and sand, and then place it in a constant temperature drying oven at 50°C for 36 hours. After being pulverized by a Chinese herbal medicine grinder, pass through a 20-40 mesh sieve to obtain the three-spot hippocampus powder. The three-spot hippocampus powder is mixed with an ethanol solution with a mass fraction of 70%-95% at a material-to-liquid ratio of 1:12g/ml. Reflux at ℃ for 2h to obtain crude ethanol extract.

1.2 The ethanol crude extract of hippocampus three-spotted was suction-filtered in a Buchner funnel, and the filter residue was suction-filtered three times. The combined filtrates were evaporated under reduced pressure to remove the solvent to obtain extract-like ethanol crude extract.

1.3 Mix the extract-like ethanol crude extract with deionized water at a ratio of 1:12g/ml, then add petroleum ether equal to the volume of the above mixture, extract in a pear-shaped funnel, take the upper petroleum ether phase, and repeat the extraction for 3 Second-rate. All the extracted petroleum ether phases were combined, evaporated and concentrated under reduced pressure at 55°C to an extract, and then freeze-dried to obtain the fat-soluble component of hippocampus three-spotted.

Step 2, determine the wavelength of the maximum UV absorption peak of the fat-soluble components:

Take the fat-soluble component of hippocampus three spots obtained in step 1, dissolve it in petroleum ether, vortex and mix well, use the solvent petroleum ether as a blank, and scan the ultraviolet spectrum at 400-190nm. The scanning spectrum was obtained, and the maximum absorption wavelength of the fat-soluble components of the three-spotted hippocampus was detected at 205nm.

Step 3, medium-pressure preparation and purification of the fat-soluble components of hippocampus three-spotted chromatographic separation by silica gel column chromatography:

3.1 The fat-soluble components of the three-spotted hippocampus were separated by medium-pressure preparation and purification chromatography equipped with a silica gel column. Injection volume: 0.5mL; detection wavelength: 205nm, monitoring wavelength: 210nm;

3.2 Use the elution solvent to carry out step/linear gradient elution of the isolate. The mobile phase A in the elution solvent is petroleum ether, and the mobile phase B is ethanol solution. The volume percentage of mobile phase A and B is: 95% mobile phase A , 5% mobile phase B, the sum of the volume percentages of the above components is 100%; elution flow rate: 8mL/min. Each eluted peak was collected, concentrated to an extract with a rotary evaporator, and freeze-dried at 0°C to obtain a fat-soluble isolate of hippocampus three-spotted from cholesterol.

Example 3

Step 1, extract fat-soluble components:

1.1 Rinse the dried three-spotted hippocampus with tap water to remove sludge and sand, and then place it in a constant temperature drying oven at 55°C for 30 hours. After being pulverized by a Chinese herbal medicine pulverizer, pass through a 20-40 mesh sieve to obtain the three-spot hippocampus powder. The three-spot hippocampus powder is mixed with an ethanol solution with a mass fraction of 70%-95% at a material-to-liquid ratio of 1:10g/ml. Reflux at ℃ for 1 h to obtain crude ethanol extract.

1.2 The ethanol crude extract of hippocampus three-spotted was suction-filtered in a Buchner funnel, and the filter residue was suction-filtered three times. The combined filtrates were evaporated under reduced pressure to remove the solvent to obtain extract-like ethanol crude extract.

1.3 Mix the extract-like ethanol crude extract with deionized water at a ratio of 1:10g/ml, then add petroleum ether equal to the volume of the above mixture, extract in a pear-shaped funnel, take the upper petroleum ether phase, and repeat the extraction for 3 Second-rate. All the extracted petroleum ether phases were combined, evaporated and concentrated under reduced pressure at 50°C to an extract, and then freeze-dried to obtain the fat-soluble component of hippocampus three-spotted.

Step 2, determine the wavelength of the maximum UV absorption peak of the fat-soluble components:

Take the fat-soluble component of hippocampus three spots obtained in step 1, dissolve it in petroleum ether, vortex and mix well, use the solvent petroleum ether as a blank, and scan the ultraviolet spectrum at 400-190nm. The scanning spectrum was obtained, and the maximum absorption wavelength of the fat-soluble components of the three-spotted hippocampus was detected at 205nm.

Step 3, medium-pressure preparation and purification of the fat-soluble components of hippocampus three-spotted chromatographic separation by silica gel column chromatography:

3.1 The fat-soluble components of the three-spotted hippocampus were separated by medium-pressure preparation and purification chromatography equipped with a silica gel column. Injection volume: 0.5mL; detection wavelength: 205nm, monitoring wavelength: 210nm;

3.2 Use the elution solvent to carry out step/linear gradient elution of the isolate. The mobile phase A in the elution solvent is petroleum ether, and the mobile phase B is ethanol solution. The volume percentage of mobile phase A and B is: 70% mobile phase A , 30% mobile phase B, the sum of the volume percentages of the above components is 100%; elution flow rate: 9mL/min. Each eluted peak was collected, concentrated to an extract with a rotary evaporator, and freeze-dried at 0°C to obtain a fat-soluble isolate of hippocampus three-spotted from cholesterol.

1. Qualitative and quantitative analysis of the fat-soluble components of the hippocampus three-spotted hippocampus after separation in Example 1

1. TLC detection of separated component cholesterol

TLC plates coated with silica gel (Siliica pH=5, MF254) were used for TLC analysis. Mobile phase: petroleum ether/dichloromethane/acetic acid (7:3:0.2, v/v/v), TLC spots observed under UV light (λ=254nm), color developer: 1% vanillin concentrated sulfuric acid solution. Apply about 10 μL of 10 mg/mL sample solution with a capillary tube, and the silica gel thin-layer plate migrates 8 cm. Color development conditions: After spraying the color developer, heat in an oven at 105°C for 5 minutes. 1 mg/mL cholesterol standard substance (Rf=0.16) was used as the control substance. The test results are shown in Figure 4, it can be seen that FrIV does not contain cholesterol, indicating that the present invention achieves the purpose of removing cholesterol from components.

2. Determination of Cholesterol Content by High Performance Liquid Chromatography

Cholesterol content was determined by HPLC. Before connection, the components were filtered through a polyvinylidene fluoride membrane with a pore size of 0.22 μm, and a high-performance liquid chromatography system equipped with a binary pump, a heater, an autosampler, and a variable wavelength detector was used. AAC18 reversed-phase column (particle size 5 μm, diameter 2.1 mm, length 200 mm) was used as the stationary phase. The analysis conditions are flow rate: 1mL/min; analysis time: 25min; injection volume: 10μL; elution peak detection wavelength: 205nm; column temperature: 35°C; mobile phase: methanol; elution program: 0%A/100%B , 0-25min. The detection compound content is calculated according to the linear curve obtained from the standard cholesterol concentration and the peak area. Compound identification and quantification of eluting peaks were performed by data acquisition, spectral evaluation, manual integration using ChemStation chromatography software.

The yield and component composition of the components separated by silica gel column chromatography obtained by the inventive method are:

Table 1 The yield and chemical composition of the components separated by silica gel column chromatography of the petroleum ether extract of Hippocampus trispotae

Table 2 Fatty acid composition of fractions separated by silica gel column chromatography of petroleum ether extract of hippocampus three-spotted

It can be seen from Table 1 and Table 2 that no cholesterol was detected in the component IV obtained by the above method, and its total phospholipid content and total phenol content were relatively high, and the ability of this component to inhibit the release of inflammatory factors was relatively strong It may be related to its high content of physiologically active substances.

2. Determining the effect of the fat-soluble components of the three-spotted hippocampus isolated in Example 1 on the viability and nitric oxide production of macrophage RAW264.7 cells stimulated by lipopolysaccharide.

1. The effect of isolated components on the viability of mouse macrophage RAW264.7 cells

Mouse macrophages RAW264.7 were cultured in an incubator at 37° C. in 5% CO ₂ -95% air with Duchenne’s modified medium containing 10% fetal bovine serum, 100 U/mL streptomycin-penicillin. Cell viability was determined by the MTT method. After the cells were subcultured, the density was adjusted to 1×10 ⁵ cells/mL, and 100 μL of the cell suspension was inoculated into a 96-well plate. After culturing overnight, the samples of different components and concentrations prepared in the medium were replaced. After culturing for 24 hours, each Add 20 μL of MTT solution with a final concentration of 5 mg/mL (dissolved in pH7.4 phosphate buffer) to the wells, incubate for 4 hours, carefully remove the liquid, add 100 μL of dimethyl sulfoxide to each well to dissolve the purple crystals, and shake slightly Shake for 15 minutes, and measure the absorbance at _490nm with a microplate reader. The relative viability of the cells in the blank group was counted as 100%, and the relative viability of the cells in the treatment group was expressed as the percentage of the cells in the blank group. The results are shown in Figure 5. In the concentration range of 10-100 μg/mL, none of the samples showed obvious toxic side effects, indicating that the isolate has cell compatibility and can be used for further cell evaluation experiments.

2. The effect of isolated components on the production of nitric oxide stimulated by lipopolysaccharide in mouse macrophage RAW264.7

The production of nitric oxide in cell culture supernatant was measured by Griess reagent. After the RAW264.7 cells were pipetted evenly in the phenol red-free Duchenne's modified medium, the cell suspension was seeded into a 96-well plate at a density of 1×10 ⁴ cells/well. Next, it is divided into normal group, control group and sample group. Cellular nitric oxide production was stimulated by lipopolysaccharide for 48h. 50 μL of supernatant and the same volume of Griess reagent were mixed at room temperature for 15 minutes, and the absorbance at 540 nm was measured on a microplate reader. The content of nitrite (stable oxide of nitric oxide) was obtained according to different concentrations of the standard sodium nitrite. Curve calculation. The results are shown in Figure 6, indicating that FrIV can effectively inhibit lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells in a dose-dependent manner.

Nitric oxide inhibition rate was calculated with the following formula:

Comparing the IC value of the lipopolysaccharide-stimulated mouse macrophage RAW264.7 nitric oxide production of the three-spotted hippocampus fat-soluble component separation fraction obtained by the present invention and dexamethasone, the results are shown in Table ₃ :

Table 3 Separation of lipid-soluble components and dexamethasone in three-spotted hippocampus

IC ₅₀ value of nitric oxide production in mouse macrophage RAW264.7 stimulated by lipopolysaccharide

It can be seen from Table 3 that FrⅣ can effectively inhibit the production of pro-inflammatory cytokines. Although the IC ₅₀ value of FrⅣ is different from the IC ₅₀ value of dexamethasone in the positive control group, it also shows good anti-inflammatory activity.

In summary, the method of the present invention can enrich the active substances of the fat-soluble component of the rare teleost fish three-spotted hippocampus, and FrⅣ removes cholesterol, which meets the requirements of the modern diet for low cholesterol intake; at the same time, the component is within a certain concentration range , significantly inhibited the production of inflammatory factor nitric oxide in mouse macrophage RAW264.7 stimulated by lipopolysaccharide, and showed a dose-dependent relationship, although compared with the positive control product, the traditional anti-inflammatory drug dexamethasone, its activity of inhibiting inflammatory factors was slightly Weak, but the inhibitory effect is also significant, indicating that the consumption of fat-soluble components of hippocampus three-spotted hippocampus can prevent some chronic inflammatory diseases related to inflammation, and improve the application value of hippocampus three-spotted as a precious Chinese medicinal material.

Claims (10)

1. a method for extracting and separating the three-spotted hippocampus fat-soluble components, is characterized in that, specifically implement according to the following steps: Step 1, extract fat-soluble components: 1.1: Using ethanol solution to reflux extract the three-spotted hippocampus powder to obtain crude ethanol extract; 1.2: Carry out suction filtration on the ethanol crude extract, repeat the suction filtration operation of the filter residue several times, combine the filtrate to remove the solvent by evaporating under reduced pressure, and obtain the extract-like ethanol crude extract; 1.3: Extract the extract-like ethanol crude extract with an organic solvent to obtain the fat-soluble components of hippocampus three-spotted; Step 2, determining the wavelength of the maximum ultraviolet absorption peak of the fat-soluble component; Step 3, using the wavelength of the maximum ultraviolet absorption peak of the fat-soluble components of hippocampus three-spotted hippocampus measured in step 2 as the detection wavelength, the fat-soluble components were subjected to medium-pressure preparation and purification chromatography on silica gel column chromatography, separated and eluted, and then collected each eluted peak, concentrated to an extract, and freeze-dried at 0°C to obtain a fat-soluble isolate of hippocampus three-spotted without cholesterol. 2. A method for extracting and separating fat-soluble components of Hippocampus trispotae according to claim 1, wherein said step 1.1 is specifically: washing the dried body of Hippocampus trispotae with tap water to remove sludge and sand, and drying After crushing and sieving to obtain hippocampus three-spot powder, mix the three-spot hippocampus powder with an ethanol solution with a mass fraction of 70%-95%, and reflux at 80°C for 1h-2h to obtain a crude ethanol extract. 3. A method for extracting and separating fat-soluble components of Hippocampus trispotae according to claim 2, characterized in that, the drying temperature is 50-60°C, and the drying time is 24-36h; the sieving specification is 20-40 head. 4. A method for extracting and separating fat-soluble components of hippocampus trispotae according to claim 2, characterized in that the solid-liquid ratio of hippocampus trispotae powder to ethanol solution is 1:8-12g/ml. 5. A method of extracting and separating the fat-soluble components of Hippocampus trispotae according to claim 1, wherein said step 1.3 is specifically: mixing the extract-like ethanol crude extract with deionized water, and then adding Petroleum ether, extraction, take the upper petroleum ether phase, repeat the extraction several times, combine all the extracted petroleum ether phases, evaporate and concentrate under reduced pressure at 45-55°C to the extract, freeze-dry at 0°C to obtain the fat-soluble component of three-spotted hippocampus . 6. A method of extracting and separating the fat-soluble components of hippocampus trispotae according to claim 5, wherein the mass volume ratio of the extract-like ethanol crude extract to deionized water is 1:8-12g /ml, the amount of petroleum ether added is equal to the volume of the mixture of extract-like ethanol crude extract and deionized water. 7. A method for extracting and separating the fat-soluble components of Hippocampus trispotae according to claim 1, wherein said step 2 is specifically: taking the fat-soluble components of Hippocampus trispotae obtained in step 1, and dissolving them in petroleum ether Dissolve, vortex and mix well, use solvent petroleum ether as a blank, scan the ultraviolet spectrum at 400-190nm, obtain the scanning spectrum, and obtain the maximum absorption peak at the wavelength of 205nm for the fat-soluble components of the three-spotted hippocampus. 8. A method for extracting and separating the fat-soluble components of Hippocampus trispotae according to claim 1, characterized in that, the medium-pressure preparation and purification chromatographic silica gel column chromatography separation parameters described in step 3 are specifically: sample size : 0.5-2.0mL; detection wavelength: 205nm, monitoring wavelength: 210nm. 9. A method for extracting and separating fat-soluble components of hippocampus three-spotted hippocampus according to claim 1, characterized in that the elution in step 3 is step/linear gradient elution. 10. A method of extracting and separating the fat-soluble components of hippocampus three-spotted according to claim 9, wherein the step/linear gradient elution is specifically: the eluting solvent is mobile phase A with petroleum ether, and Ethanol is the mobile phase B, the ratio of mobile phase A and B by volume percentage is: 45-95% mobile phase A, 5-55% mobile phase B, the total volume percentage of the above components is 100%, elution flow rate: 8- 10mL/min.