CN110835299B - Seaweed functional oil and application thereof - Google Patents
Seaweed functional oil and application thereof Download PDFInfo
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- CN110835299B CN110835299B CN201911144533.6A CN201911144533A CN110835299B CN 110835299 B CN110835299 B CN 110835299B CN 201911144533 A CN201911144533 A CN 201911144533A CN 110835299 B CN110835299 B CN 110835299B
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- 241001474374 Blennius Species 0.000 title claims abstract description 102
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 57
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 36
- 230000000694 effects Effects 0.000 claims abstract description 35
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 32
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- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
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- OUDSBRTVNLOZBN-UHFFFAOYSA-N tolazamide Chemical compound C1=CC(C)=CC=C1S(=O)(=O)NC(=O)NN1CCCCCC1 OUDSBRTVNLOZBN-UHFFFAOYSA-N 0.000 description 1
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/03—Phaeophycota or phaeophyta (brown algae), e.g. Fucus
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Abstract
The invention discloses seaweed functional oil and application thereof. The invention firstly provides seaweed functional oil, which is obtained by eluting a crude extract of sargassum fusiforme by a mixed solution of petroleum ether and ethyl acetate, eluting by a mixed solution of petroleum ether, chloroform and methanol, and collecting active components which meet the requirements that a thin-layer chromatography specific transfer value is 0.1-0.4 and acetylcholinesterase is subjected to biological self-development to form white spots. The seaweed functional oil has the functions of obviously inhibiting the activity of acetylcholinesterase, eliminating the activity of DPPH free radicals, inhibiting the generation of NO induced by inflammatory factors in cells and eliminating the level of ROS in the cells, and has NO toxic or side effect on normal cells; in addition, the preparation method has high yield and natural material sources, and the obtained seaweed functional oil has no toxicity, does not cause adverse reactions and has good application prospect in the aspect of preparing medicines for treating Alzheimer's disease.
Description
Technical Field
The invention belongs to the technical field of essential oil extraction. More particularly, relates to seaweed functional oil and application thereof.
Background
Alzheimer's Disease (AD), commonly known as senile dementia, is one of the major diseases endangering human health in the 21 st century, is a neurodegenerative disease accompanied by cognitive, behavioral and functional disorders, and is frequently seen in elderly people over 65 years old. The existing research shows that AD is closely related to oxidative stress in the brain, neurodegeneration caused by neuroinflammation, cholinergic neuron injury and the like. According to 2016 world AD report, the number of people suffering from AD in the current year is up to 4700 thousands, and with the increasing aging progress of population, the number of AD patients in China increases at the fastest global speed, and related experts predict that 2200 thousands of people will suffer from AD in the world by 2025 years. Meanwhile, the cost of AD patients for medicine and health care reaches trillion annually, which has great influence on the physical and mental health of the patients and the national economic development.
Currently, drugs for treating AD include acetylcholinesterase inhibitors and vaccines such as tacrine, tolazamide, rivastigmine, galantamine, memantine hydrochloride, and the like. However, the drugs or vaccines are expensive, have great toxic and side effects, have limited effects on treating AD, can only improve AD symptoms but cannot prevent disease progression, and can cause a plurality of adverse reactions.
The marine organisms generate and accumulate a large amount of secondary metabolites with new structures and strong activity in the growth and metabolism process due to the special living environment of the marine organisms, and the marine organisms are important resources for researching and discovering novel marine medicines. Research shows (Zhang Gan et al, 2005, research on inhibition of acetylcholinesterase activity by seaweed components), some kinds of seaweed components have activity of inhibiting acetylcholinesterase; but the content difference of the effective components of different algae is obvious, the related research is less at present, and the extraction results of the related active components of different algae by different extraction processes show great difference. Therefore, the medicine for treating Alzheimer's disease, which has good activity, high yield, no toxicity and natural sources, is screened out, and has important significance for preventing and treating AD.
Disclosure of Invention
The invention aims to provide the seaweed functional oil with the functions of inhibiting acetylcholinesterase, resisting oxidation and inflammation and the preparation method thereof.
The invention aims to provide seaweed functional oil.
The invention also aims to provide the application of the seaweed functional oil in preparing the medicine for treating the Alzheimer's disease.
The invention further aims to provide application of the seaweed functional oil in preparing a product for preventing and/or assisting in improving Alzheimer's disease.
The above purpose of the invention is realized by the following technical scheme:
the invention firstly provides seaweed functional oil, which is obtained by eluting a crude extract of sargassum fusiforme by a mixed solution of petroleum ether and ethyl acetate, eluting by a mixed solution of petroleum ether, chloroform and methanol, and collecting active components which meet the requirements that a thin-layer chromatography specific transfer value is 0.1-0.4 and acetylcholinesterase is subjected to biological self-development to form white spots.
The main components of the seaweed functional oil are myristic acid, (Z) -9-hexadecenoic acid, palmitic acid, phytol, arachidonic acid and 11,14, 17-eicosatrienoic acid, and the structures of the seaweed functional oil are respectively shown as follows:
the inventor obtains the seaweed functional oil when the thin-layer chromatography-biological self-development method and the 96-pore plate method are utilized to carry out activity tracing, organic solvent extraction and column chromatography are utilized to purify the active components of the seaweed sargassum fusiforme; through analysis and experimental verification, the seaweed functional oil has the functions of obviously inhibiting the activity of acetylcholinesterase, eliminating the activity of DPPH free radicals, inhibiting the generation of NO induced by inflammatory factors in brain-derived BV-2 microglia, eliminating the ROS level in the brain-derived BV-2 microglia, and has NO toxic or side effect on normal cells; therefore, the seaweed functional oil has good application prospect in preparing medicines for treating Alzheimer's disease and products for preventing and/or assisting in improving Alzheimer's disease.
Preferably, the preparation method of the seaweed functional oil comprises the following steps:
s1, drying and crushing sargassum fusiforme, adding a mixed solution of methanol and chloroform for extraction, and concentrating under reduced pressure to obtain a sargassum fusiforme crude extract;
s2, subjecting the crude extract of the seaweed sargassum fusiforme obtained in the step S1 to reduced pressure column chromatography, and performing column chromatography by using a solvent with a volume ratio of 8: 2 and 7: 3, eluting the mixed solution of petroleum ether and ethyl acetate to obtain a crude component 1 and a crude component 2;
s3, mixing the crude component 1 and the crude component 2 obtained in the step S2, and treating the mixture by a silica gel column at a volume ratio of 9: 1, eluting with a mixed solution of petroleum ether and ethyl acetate, and passing through a gel column by using a solvent with a volume ratio of 2: 1: eluting with mixed solution of petroleum ether, chloroform and methanol to obtain the seaweed functional oil.
Preferably, the filler of the reduced pressure column in the step S2 is 100-200 mesh silica gel.
Preferably, the filler of the silica gel column in the step S3 is 200-300 mesh silica gel.
Preferably, the filler of the gel column of step S3 is sephadex.
Preferably, the volume ratio of methanol to chloroform in step S1 is 1: 1 to 3.
More preferably, the volume ratio of methanol to chloroform in step S1 is 1: 2.
in addition, the application of the seaweed functional oil in preparing the medicine for treating the Alzheimer's disease and the product for preventing and/or assisting in improving the Alzheimer's disease are both within the protection scope of the invention.
Preferably, the product is a medicament, food or health product.
Preferably, the use is in the manufacture of a medicament for inhibiting acetylcholinesterase activity, in the manufacture of a medicament for scavenging DPPH free radical activity, and in the manufacture of a medicament for inhibiting the production of NO and ROS levels in a cell.
The invention has the following beneficial effects:
the invention provides seaweed functional oil and application thereof. Experiments prove that the seaweed functional oil provided by the invention has obvious acetylcholinesterase activity inhibition, antioxidant activity and anti-inflammatory activity, specifically has the functions of inhibiting the activity of acetylcholinesterase, eliminating the activity of DPPH free radicals, inhibiting the generation of NO induced by inflammatory factors in cells and eliminating the ROS level in the cells, has NO toxic or side effect on normal cells, and can be used as a medicine which has high activity, high yield, NO toxicity and natural source and inhibits the activity of acetylcholinesterase;
in addition, the preparation method of the seaweed functional oil has high yield, the seaweed functional oil takes natural seaweed and sargassum fusiforme as raw materials, the material source is natural and wide, the obtained seaweed functional oil has no toxicity, adverse reaction can not be caused, and the method for preparing the seaweed functional oil is simple and convenient and has low cost; therefore, the seaweed functional oil provided by the invention has good application prospects in preparation of medicines for treating Alzheimer's disease and products for preventing and/or assisting in improving Alzheimer's disease.
Drawings
FIG. 1 is a graph showing the results of tests on the main components of the functional oil of seaweeds of the present invention; wherein, A is the gas chromatogram of the non-methyl esterification of the seaweed functional oil; b is a gas chromatogram of the methyl esterification of the seaweed functional oil.
FIG. 2 is a graph showing the results of the acetylcholinesterase inhibition test using the algal oil of the present invention.
FIG. 3 is a graph showing the results of the antioxidant property test of the functional oil of seaweed of the present invention.
FIG. 4 is a graph showing the results of the toxicity test of the algal functional oils of the present invention to cells.
FIG. 5 is a graph showing the results of the activity test of the algal functional oils of the present invention for inhibiting the production of NO in cells.
FIG. 6 is a graph showing the results of the inhibition of LPS-induced ROS in BV-2 cells by algal functional oil of the present invention.
FIG. 7 is a graph showing the results of the activity test of the algal functional oils of the present invention to inhibit intracellular ROS levels.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 preparation of seaweed functional oil
First, experiment method
1. A preparation method of seaweed functional oil comprises the following steps:
s1, removing impurities from purchased or collected seaweed sargassum fusiforme, drying the seaweed sargassum fusiforme in a 40 ℃ oven for 1h or drying the seaweed sargassum fusiforme in the sun, drying the seaweed sargassum fusiforme in the 40 ℃ oven for 1h, crushing the seaweed sargassum fusiforme in a crusher, adding 2 times of methanol: chloroform-1: 2 soaking overnight, filtering, concentrating under reduced pressure, repeating for 3 times to obtain crude extract of Sargassum fusiforme;
s2, subjecting the crude extract of the sargassum fusiforme obtained in the step S1 to a reduced pressure column (the filler of the reduced pressure column is silica gel with 100-200 meshes), and respectively eluting and separating by using the following reagents:
(1) using petroleum ether: ethyl acetate (vol) ═ 8: 2. 7: 3. 1: 1. 3: 7, eluting and separating to obtain 4 crude components Fr 1-Fr 4;
(2) eluting with pure ethyl acetate to obtain 1 crude component Fr 5;
(3) using chloroform: methanol (volume ratio) 5: 1. 2: 1, eluting and separating to obtain 2 crude components Fr 6-Fr 7;
(4) eluting with pure methanol to obtain 1 crude component Fr 8;
co-separating to obtain 8 crude components Fr 1-Fr 8, and developing with acetylcholinesterase inhibiting biological autography to obtain crude components Fr1 and Fr2 with white spots;
s3, combining the crude components Fr1 and Fr2, passing through a silica gel column (the filler of the silica gel column is 200-300 meshes of silica gel), and passing through petroleum ether: ethyl acetate (vol) ═ 98: 2(7 column volumes), 95: 5(5 column volumes), 90: 10(5 column volumes), 85: 15(5 column volumes), 80: 20(5 column volumes) and pure ethyl acetate (5 column volumes) are eluted and concentrated to obtain a component Fr ' 1-Fr ' 1-32, and the component Fr ' 1-17 with white spots is obtained by utilizing the biological autoradiography color development of acetylcholinesterase inhibition activity;
s4, passing the components Fr' 1-17 through a gel column (the filler of the gel column is sephadex), and passing the gel column through a reactor with the volume ratio of petroleum ether: chloroform: methanol 2: 1: 1 at a flow rate of 0.5mL/min, performing TLC analysis, combining the same components to obtain components Fr ' 1-17-1-Fr ' 1-17-6, performing ultraviolet absorption on the components Fr ' 1-17-1-Fr ' 1-17-6 on a thin-layer chromatography plate (Silica gel 60F254 produced by Merck, a developing agent is petroleum ether and ethyl acetate is 19: 1) under ultraviolet irradiation at 254nm, enabling sulfuric acid-anisaldehyde to be purple in color, and performing thin-layer chromatography with a specific shift value of 0.1-0.4, and spraying a biological self-developing solution containing acetylcholinesterase, a substrate of thioacetyl choline and 5, 5-dithiodinitrobenzoic acid on the thin-layer chromatography plate to obtain a component Fr ' 1-17-3 with white spots, namely the seaweed functional oil.
2. Then, the main components of the obtained seaweed functional oil are measured, the seaweed functional oil sample is dissolved in normal hexane (1mg/mL), the column incubator is 80 ℃, the injection port temperature is 280 ℃, the split ratio is 5, the carrier gas is He, and the temperature rise program is as follows: 0-2 min at 80 ℃; 2-6 min at 80-120 ℃; 6-11 min at 120 ℃; 11-16 min at 120-180 ℃; 16-22 min at 180 ℃; 22-32 min at 180-280 ℃; 32-60 min at 280 ℃; the ion source temperature is 250 ℃, the interface temperature is 280 ℃, and the m/z range is 50-500; measuring the retention time in gas chromatography (GCMS-QP2010 Ultra, column model: SH-Rxi-5Sil MS, size 30m × 0.25mm, film thickness 0.25 μm); the main components of the seaweed functional oil were determined using National Institute of Standards and Technology (NIST) library and GC-MS system library of the south sea institute of science and technology in comparison with mass spectra.
Second, experimental results
The test results of the main components of the seaweed functional oil are shown in fig. 1, and it can be seen that the main components of the seaweed functional oil are tetradecanoic acid, (Z) -9-hexadecenoic acid, hexadecanoic acid, phytol, arachidonic acid and 11,14, 17-eicosatrienoic acid; the retention times in the gas chromatograph are respectively:
18.50min (tetradecanoic acid, corresponding to peak 1 in FIG. 1, panel A, retention time 18.50 min),
23.60min (hexadecanoic acid, corresponding to peak 3 at retention time 23.60min in panel A of FIG. 1),
22.82min ((Z) -9-hexadecenoic acid, corresponding to peak 2 at retention time 22.82min in panel A of FIG. 1),
29.03min (arachidonic acid, corresponding to peak 5 in panel A of FIG. 1, retention time 29.03 min) (non-methyl esterified),
17.83min (tetradecanoic acid, corresponding to peak 2 in the methyl esterification product of Panel B of FIG. 1, retention time 17.83 min),
22.14min (hexadecanoic acid, corresponding to peak 4 in the methyl esterification product of Panel B of FIG. 1, retention time 22.14 min),
21.63min ((Z) -9-hexadecenoic acid, corresponding to peak 3 in the methyl esterification product of Panel B of FIG. 1, retention time 21.63 min),
26.32min (11,14, 17-eicosatrienoic acid, peak 7 corresponding to retention time 26.32min in the sample after the methyl esterification treatment in Panel B of FIG. 1),
26.51min (phytol, corresponding to peak 8 of retention time 26.51min in the sample after the methyl esterification treatment in panel B of FIG. 1),
28.49min (arachidonic acid, peak 10 in panel B methyl esterification product of FIG. 1, retention time 28.49 min) (methyl esterification).
Application example 1 algal functional oil inhibition of acetylcholinesterase Activity test
The seaweed functional oil prepared in the embodiment 1 of the invention is used as a sample, and a commonly used electroseel acetylcholinesterase (AChE) is screened by an acetylcholinesterase resisting drug to carry out an in vitro acetylcholinesterase resisting activity experiment on the seaweed functional oil, wherein the specific experimental method and the experimental result are as follows:
1. experimental methods
Dissolving the seaweed functional oil by using methanol, volatilizing the dry methanol after half dilution in a 96-pore plate, adding DMSO for redissolving, and setting the final concentration of the seaweed functional oil as follows: 1.024mg/mL, 0.512mg/mL, 0.256mg/mL, 0.128mg/mL, 0.064mg/mL, 0.032mg/mL, 0.016mg/mL, 0.008mg/mL, 0.004mg/mL, 0.002mg/mL, 0.001mg/mL, 0.0005mg/mL, two replicates per concentration;
the experiment was performed in 4 groups, each of which was a sample group (sample + 49. mu.L PBS + 1. mu.L DMSO + 10. mu.L AChE + 20. mu.L DTNB, incubation at 37 ℃ for 10min, addition of 20. mu.L ATCH, incubation at 37 ℃ for 20 min);
sample blank (sample + 49. mu.L PBS + 1. mu.L DMSO + 10. mu.L BSA + 20. mu.L DTNB, incubation at 37 ℃ for 10min, addition of 20. mu.L ATCH, incubation at 37 ℃ for 20 min);
control group (49. mu.L PBS + 1. mu.L DMSO + 10. mu.L AChE + 20. mu.L DTNB, incubation 10min at 37 ℃, addition of 20. mu.L ATCH, incubation 20min at 37 ℃);
blank group (49 μ L PBS +1 μ L DMSO +10 μ L BSA +20 μ L DTNB, incubation 10min at 37 ℃, adding 20 μ L ATCH, incubation 20min at 37 ℃), measuring OD value at 405nm of enzyme labeling instrument, calculating the inhibition rate of the seaweed functional oil on acetylcholinesterase according to OD value;
inhibition rate [ (OD)Control group-ODBlank group)-(ODSample set-ODSample blank set)]/(ODControl group-ODBlank group)×100％；
The inhibition rate-concentration is plotted, a logarithmic trend line is taken, and the half-inhibition concentration IC is calculated50。
2. Results of the experiment
The test result of the seaweed functional oil for inhibiting the activity of acetylcholinesterase is shown in figure 2, and it can be seen that when the concentration of the seaweed functional oil is 1.024mg/mL, the inhibition rate of the seaweed functional oil on the activity of acetylcholinesterase reaches more than 50%, which indicates that the seaweed functional oil prepared by the invention has the activity of inhibiting the acetylcholinesterase.
Application example 2 testing of antioxidant Properties of seaweed functional oil
The seaweed functional oil prepared in the embodiment 1 of the invention is used as a sample, an antioxidant is adopted to screen commonly used DPPH free radicals, and an in-vitro antioxidant activity experiment of the seaweed functional oil is carried out, and the specific experimental method and the experimental result are as follows:
1. experimental methods
Dissolving the seaweed functional oil by using methanol, volatilizing the dry methanol after half dilution in a 96-pore plate, adding DMSO for redissolving, and setting the final concentration of the seaweed functional oil as follows: 1.024mg/mL, 0.512mg/mL, 0.256mg/mL, 0.128mg/mL, 0.064mg/mL, 0.032mg/mL, 0.016mg/mL, 0.008mg/mL, 0.004mg/mL, 0.002mg/mL, 0.001mg/mL, 0.0005mg/mL, two replicates per concentration;
the experiment is totally set to 4 groups, which are respectively a sample group (the sample is added with 50 microliter DMSO and 50 microliter DPPH, and is placed in a dark place for 30 min); sample blank (sample + 50. mu.L DMSO + 50. mu.L methanol, left in the dark for 30 min);
control group (50. mu.L DMSO + 50. mu.L DPPH, left in dark for 30 min);
blank (50. mu.L DMSO + 50. mu.L methanol, left in the dark for 30 min); measuring OD value at 517nm of an enzyme labeling instrument, and calculating the removal rate of the seaweed functional oil on DPPH free radicals according to the OD value;
clearance rate [ [ (OD)Control group-ODBlank group)-(ODSample set-ODSample blank set)]/(ODControl group-ODBlank group)×100％。
2. Results of the experiment
The result of the antioxidant performance test of the seaweed functional oil is shown in figure 3, and it can be seen that when the concentration of the seaweed functional oil is 1.024mg/mL, the clearance rate of DPPH free radicals is above 37%, which indicates that the seaweed functional oil prepared by the invention has antioxidant activity.
Application example 3 toxicity test of algal functional oils on cells
Cytotoxicity is a simple cell killing event caused by cells or chemical substances, does not depend on apoptosis or necrotic cell death mechanisms, and sometimes needs to be detected by cytotoxicity of specific substances, such as drug screening; therefore, the cytotoxicity test can screen the toxicity of the compound, and the specific experimental method and experimental results of the cytotoxicity test by using the seaweed functional oil prepared in the embodiment 1 of the invention as a sample are as follows:
1. experimental methods
(1) Preparing BV-2 cells into cell suspension, adding 100 μ L of cell suspension into 96-well plate with density of 5 × 103Tapping the pore plate to uniformly distribute the cells at the bottom of the pore plate, and putting the cell into a carbon dioxide incubator to continue culturing;
(2) after 24h, taking out the pore plate, discarding the old culture medium, uniformly mixing the seaweed functional oil sample mother solution with a serum-free culture medium to ensure that the final concentration of the seaweed functional oil is 5 mug/mL, 10 mug/mL and 20 mug/mL, adding the mixture into corresponding pores, enabling each group to be parallel for 3 times, and putting the mixture into an incubator for culture;
(3) after 24h, taking out the 96-well plate, sucking out the culture medium, adding 100 mu L of MTT (1mg/mL) solution into each well, and putting the well into an incubator for continuous culture; after 4h, the well plate was removed, the MTT solution was carefully aspirated, and then 100. mu.L of DMSO solution was added to each well; placing on shaking table, shaking for 30min to dissolve crystal violet, and determining OD540Absorbance of (a);
relative cell viability (%) ═ (OD)Experimental group－ODBlank hole)/(ODBlank group－ODBlank hole)×100％。
2. Results of the experiment
The result of the toxicity test of the seaweed functional oil on cells is shown in figure 4, and the seaweed functional oil has no toxicity on BV-2 cells.
Application example 4 anti-inflammatory Activity test of seaweed functional oil
Activity test experiment for seaweed functional oil to inhibit generation of NO in cells
LPS is added to induce BV-2 cells to trigger inflammation so that NO can be generated and released, and when anti-inflammatory drugs are added, the amount of generated and released NO is reduced; therefore, by using the amount of intracellular NO as a screening model for screening anti-inflammatory drugs and using the seaweed functional oil prepared in the embodiment 1 of the present invention as a sample, specific experimental methods and experimental results are as follows:
1. experimental methods
(1) Preparing BV-2 cells into cell suspension, adding 100 μ L of cell suspension into 96-well plate with density of 5 × 104Tapping the pore plate to uniformly distribute the cells at the bottom of the pore plate, and putting the cell into a carbon dioxide incubator to continue culturing;
(2) after 24h, taking out the pore plate, and discarding the old culture medium; uniformly mixing the seaweed functional oil sample mother solution with a serum-free culture medium to ensure that the final concentration of the seaweed functional oil is 5 mug/mL, 10 mug/mL and 20 mug/mL, then adding the mixture into corresponding holes, wherein each group is parallel for 3 times, and putting the mixture into an incubator for culture;
(3) after 24h, taking out the 96-well plate, and sucking 50 mu L of cell supernatant in each well;
(4) measuring the NO content according to Progema Griess Reagent System instruction;
(5) OD of wells in accordance with standard curve regression equation and experimental set524The corresponding NO content was calculated.
2. Results of the experiment
The result of the activity test of the seaweed functional oil for inhibiting the generation of NO in cells is shown in figure 5, and it can be seen that when the concentration of the seaweed functional oil is 5 mug/mL, 10 mug/mL and 20 mug/mL, the amount of NO generated by BV-2 cells is obviously reduced along with the increase of the concentration of the seaweed functional oil, which indicates that the seaweed functional oil can obviously inhibit the generation of NO in cells and has anti-inflammatory activity.
Second, activity test experiment of algal functional oil inhibiting intracellular ROS level
Adding LPS to induce BV-2 cells to trigger inflammation can increase the ROS level, and when adding anti-inflammatory drugs, the ROS level is reduced; DCFH-DA has no fluorescence and can freely pass through cell membranes, after entering cells, DCFH without fluorescence can be generated by hydrolysis of intracellular esterase, ROS in the cells can oxidize the DCFH into DCF with fluorescence, and the level of ROS in the cells can be measured by detecting the fluorescence of DCF; therefore, by using the seaweed functional oil prepared in the embodiment 1 of the invention as a sample and measuring the intracellular ROS level as a screening model for screening anti-inflammatory drugs, the specific experimental method and the experimental results are as follows:
1. experimental methods
(1) Preparing BV-2 cells into cell suspension, adding 300 μ L of cell suspension into each well of 24-well plate, wherein the density of each well is 5 × 104Tapping the pore plate to uniformly distribute the cells at the bottom of the pore plate, and putting the cell into a carbon dioxide incubator to continue culturing;
(2) after 24h, taking out the pore plate, and discarding the old culture medium; uniformly mixing the seaweed functional oil sample mother solution with a serum-free culture medium to ensure that the final concentration of the seaweed functional oil is 5 mug/mL, 10 mug/mL and 20 mug/mL, then adding the mixture into corresponding holes, wherein each group is parallel for 3 times, and putting the mixture into an incubator for culture;
(3) after 24h, the well plate was removed, the medium was aspirated away, and the bottom of the well was washed 3 times with serum-free culture;
(4) adding 500 mu L of DCFH-DA solution with the concentration of 10 mu m into each hole, putting the 24-hole plate into an incubator for incubation in a dark place, and shaking and mixing uniformly every 3-5 minutes to ensure that the probe is fully contacted with the cells; after 30min, taking out the 24-hole plate, washing the cells for three times by using a serum-free cell culture solution to fully remove DCFH-DA which does not enter the cells;
(6) observing the fluorescence condition in the experimental hole under an inverted fluorescence microscope, taking a picture, and analyzing the average fluorescence intensity by using Image J software, wherein the average optical density is IntDen/Area;
the experiments were grouped together into 5 groups:
group A: BV-2+ DCFH-DA (10. mu.M), i.e., control group, only cells and active oxygen fluorescent probe DCFH-DA, no addition of seaweed functional oil HFFO and LPS;
group B: BV-2+ LPS (1 μ g/mL) + DCFH-DA (10 μ M), i.e., the model group, with cells, and LPS induced active oxygen, no HFFO, probe DCFH-DA;
group C: BV-2+ LPS (1. mu.g/mL) + HFFO (5. mu.g/mL) + DCFH-DA (10. mu.M), i.e., low dose group, 5. mu.g/mL HFFO was added on the basis of the model formation;
group D: BV-2+ LPS (1. mu.g/mL) + HFFO (10. mu.g/mL) + DCFH-DA (10. mu.M), i.e., the medium dose group, 10. mu.g/mL HFFO is added on the basis of the model forming group;
group E: BV-2+ LPS (1. mu.g/mL) + HFFO (20. mu.g/mL) + DCFH-DA (10. mu.M), i.e., high dose group, 20. mu.g/mL HFFO was added on a model-making basis.
2. Results of the experiment
The result of the inhibition effect of the seaweed functional oil on the ROS in BV-2 cells induced by LPS is shown in figure 6, and it can be seen that the ROS content (intuitively reflected by fluorescence intensity) in the cells of a control group (group A) without LPS addition is very low; the fluorescence intensity caused by ROS in the cell of the building block (B group) added with LPS is obviously enhanced; when the functional algal oil HFFO is added (groups C, D and E), the fluorescence intensity caused by intracellular ROS shows a dose-dependent decrease with the increase of the concentration of the functional algal oil HFFO.
The activity test result of the seaweed functional oil for inhibiting intracellular ROS level is shown in figure 7, and it can be seen that when the concentration of the seaweed functional oil is 5 mug/mL, 10 mug/mL and 20 mug/mL, the ROS level in BV-2 cells (reflected by the average fluorescence density) is remarkably reduced along with the increase of the concentration of the seaweed functional oil, which indicates that the seaweed functional oil can remarkably inhibit the intracellular ROS level and has antioxidant activity.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The seaweed functional oil is characterized in that a seaweed sargassum fusiforme crude extract is eluted by a mixed solution of petroleum ether and ethyl acetate, then eluted by a mixed solution of petroleum ether, chloroform and methanol, and then an active component which meets the requirements that a thin-layer chromatography specific transfer value is 0.1-0.4 and acetylcholinesterase is subjected to biological self-development to form white spots is collected, so that the seaweed functional oil is obtained;
the main components of the seaweed functional oil are myristic acid, (Z) -9-hexadecenoic acid, palmitic acid, phytol, arachidonic acid and 11,14, 17-eicosatrienoic acid.
2. The seaweed functional oil according to claim 1, characterized in that the preparation method comprises the following steps:
s1, drying and crushing sargassum fusiforme, adding a mixed solution of methanol and chloroform for extraction, and concentrating under reduced pressure to obtain a sargassum fusiforme crude extract;
s2, subjecting the crude extract of the seaweed sargassum fusiforme obtained in the step S1 to reduced pressure column chromatography, and performing column chromatography by using a solvent with a volume ratio of 8: 2 and 7: 3, eluting the mixed solution of petroleum ether and ethyl acetate to obtain a crude component 1 and a crude component 2;
s3, mixing the crude component 1 and the crude component 2 obtained in the step S2, and treating the mixture by a silica gel column at a volume ratio of 9: 1, eluting with a mixed solution of petroleum ether and ethyl acetate, and passing through a gel column by using a solvent with a volume ratio of 2: 1: eluting with mixed solution of petroleum ether, chloroform and methanol to obtain the seaweed functional oil.
3. The seaweed functional oil as claimed in claim 2, wherein the filler of the reduced pressure column in step S2 is silica gel of 100-200 mesh.
4. The seaweed functional oil as claimed in claim 2, wherein the silica gel column of step S3 is filled with 200-300 mesh silica gel.
5. The seaweed functional oil of claim 2, wherein the filler of the gel column of step S3 is sephadex.
6. Use of the seaweed functional oil of any one of claims 1 to 5 in the preparation of a medicament for treating alzheimer's disease.
7. Use of the seaweed functional oil of any one of claims 1 to 5 in the preparation of a product for preventing and/or assisting in the improvement of Alzheimer's disease.
8. The use according to claim 6, in the manufacture of a medicament for inhibiting acetylcholinesterase activity.
9. The use according to claim 6, wherein the use is for the preparation of a medicament having DPPH radical scavenging activity.
10. The use according to claim 6, for the manufacture of a medicament for inhibiting the production of NO and ROS levels in a cell.
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