CN111214474A - Application of Stichopus japonicus sterol sulfate in anti-atherosclerosis product - Google Patents
Application of Stichopus japonicus sterol sulfate in anti-atherosclerosis product Download PDFInfo
- Publication number
- CN111214474A CN111214474A CN202010078292.6A CN202010078292A CN111214474A CN 111214474 A CN111214474 A CN 111214474A CN 202010078292 A CN202010078292 A CN 202010078292A CN 111214474 A CN111214474 A CN 111214474A
- Authority
- CN
- China
- Prior art keywords
- sterol sulfate
- silica gel
- sulfate
- sterol
- stichopus japonicus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- 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
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/616—Echinodermata, e.g. starfish, sea cucumbers or sea urchins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J31/00—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring
- C07J31/006—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring not covered by C07J31/003
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Nutrition Science (AREA)
- Food Science & Technology (AREA)
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention relates to the technical field of food nutrition, in particular to application of sea cucumber sterol sulfate in an anti-atherosclerosis product. Stichopus japonicus sterol sulfate is extracted and separated from Stichopus japonicus and microencapsulated, and experiments prove that the Stichopus sterol sulfate and the microcapsule thereof have the effect of improving atherosclerosis. The invention takes the sea cucumber as the raw material to prepare the sterol sulfate, and experiments prove that the plaque area of atherosclerosis can be effectively reduced, namely the Stichopus japonicus sterol sulfate can obviously inhibit the development of atherosclerosis, which shows that the Stichopus sterol sulfate can effectively prevent, improve or treat atherosclerosis.
Description
The technical field is as follows:
the invention relates to the technical field of food nutrition, in particular to application of sea cucumber sterol sulfate in an anti-atherosclerosis product.
Background art:
atherosclerosis is a disease of the aorta, characterized by the accumulation of lipid and fiber elements in the aorta, and the incidence and mortality of cardiovascular and cerebrovascular diseases show a trend of increasing year by year with the improvement of the living standard of people. Epidemiological studies have revealed that atherosclerosis is considered to be a multifactorial disease. Sea cucumber has been proved to have various nutritional efficacies as a traditional medicinal and edible seafood in China. Stichopus japonicus sterol sulfate is a natural sterol substance existing in Stichopus japonicus. Stichopus japonicus sterol sulfate and phytosterol have similar structures to cholesterol, but their functions are completely different. High cholesterol level is a well-known risk factor for cardiovascular diseases, but phytosterol can be used as an effective component for achieving the purposes of reducing the cholesterol level and resisting atherosclerosis. However, no report is available on the improvement effect of the Stichopus japonicus sterol sulfate on atherosclerotic cardiovascular diseases.
The invention content is as follows:
the invention aims to solve the technical problem of whether natural sterol matters, namely the sea cucumber sterol sulfate, existing in the sea cucumber can improve atherosclerosis cardiovascular diseases.
In order to solve the problems, the invention provides the application of the Stichopus japonicus sterol sulfate in the anti-atherosclerosis product, expands the application range of the Stichopus japonicus sterol sulfate, and also provides a new effective component for the anti-atherosclerosis product.
In order to achieve the purpose, the invention is specifically realized by the following technical scheme:
application of Stichopus japonicus sterol sulfate in preparation of anti-atherosclerosis product is provided. Stichopus japonicus sterol sulfate has higher anti-atherosclerosis activity, probably due to the sulfate group on the branched chain C-3 and the different hydrocarbon side chain on C-17.
Further, the sea cucumber sterol sulfate is sterol sulfate extracted from sea cucumber.
Further, the preparation method of the Stichopus japonicus sterol sulfate comprises the following steps:
(1) vacuum freeze drying and pulverizing the sea cucumber body wall, and grinding into powder (250 meshes);
(2) taking the powder, adding ethanol with a certain volume, keeping the material-liquid ratio at 1:10-1:15(m/v), continuously stirring and leaching for 24h at room temperature to obtain leaching liquor, leaching for three times, mixing the leaching liquor, and concentrating under reduced pressure to obtain crude extract of sea cucumber lipid; when the feed-liquid ratio is lower than 1:10(m/v), the extraction is incomplete, and the holothurin sterol sulfate can not be completely leached and is higher than 1:15(m/v), so that the waste of reagents is caused.
(3) Weighing 200-300 mesh silica gel, activating at 110 deg.C for 10-12h, and cooling at room temperature; the method comprises the steps of filling a column by a dry method, dissolving silica gel by chloroform, filling the column after bubbles are removed by ultrasonic treatment, pressing the column by chloroform with the volume of 3 times after the silica gel is completely settled, so that the silica gel in the column is more compact, the bubbles in the silica gel column are discharged, if the silica gel is not compact enough or the bubbles exist, the separation surface of substances is easy to bend, the separation degree is reduced, even a plurality of substances can not be separated, and the yield of the holothurin sterol sulfate is reduced. Dissolving appropriate amount of crude extract of sea cucumber lipid with appropriate amount of chloroform, mixing with silica gel, quickly pouring into silica gel column, and purifying with silica gel column chromatography.
Further, the silica gel column chromatography method comprises the following steps: using chloroform: washing 3 column volumes with a mixture of methanol (7-9: 1, v/v), and then washing with chloroform: washing 1 column volume with methanol (4-5: 1, v/v), collecting eluent, performing thin-layer silica gel plate chromatography (TLC) on the eluent one by one, wherein a developing agent is chloroform: methanol: water (80:15:1, v/v/v), using 90% sulfuric acid ethanol color developing agent to carry out color development, then confirming the components, combining the collected eluents, and carrying out reduced pressure concentration to remove the organic reagent, namely the holothurin sterol sulfate.
Further, the sea cucumber is a cucumis metuliferus.
A microcapsule containing the Stichopus japonicus selenka sulfate is prepared by the following steps:
(1) preparing a microcapsule wall material: weighing a proper amount of maltodextrin and Arabic gum, dispersing in water according to a solid-to-liquid ratio (g: g: l) of (0.4-0.8) to (0.6-0.8) to 8 until the maltodextrin and the Arabic gum are completely dissolved, and hydrating at room temperature for 5-10 hours to obtain a wall material solution; the low solid-liquid ratio can cause excessive water content, high molecules in the dilute solution are dispersed in a coil form, the molecules are far away from each other and cannot form a stable three-dimensional space structure, so that the embedding rate of the core material is reduced, and the high solid-liquid ratio can cause overlarge viscosity of the solution, so that the core material is not easy to uniformly mix and is difficult to process;
(2) preparing a microcapsule core material: weighing a certain amount of Stichopus japonicus sterol sulfate, dispersing in vegetable oil according to a solid-to-liquid ratio (g: g) of 0.4-0.8: 0.8, and fully dissolving; too high a proportion leads to insufficient dissolution of the Stichopus japonicus sterol sulfate, too low a proportion leads to too low loading capacity of Stichopus japonicus sterol sulfate in the core material, and simultaneously causes waste of vegetable oil;
(3) mixing: under the condition of stirring, dropwise adding the prepared core material into the prepared wall material solution according to the mass ratio of (5-10) to (100), and uniformly stirring and mixing to obtain a mixed solution; the low proportion leads to the reduction of the loading capacity of the Stichopus japonicus sterol sulfate in the core material, and the high proportion leads to the reduction of the embedding rate of the Stichopus japonicus sterol sulfate;
(4) encapsulation: homogenizing and high-pressure micro-jet processing the prepared mixed solution to form micro-emulsion, and carrying out spray drying on the obtained micro-emulsion to obtain the sea cucumber sterol sulfate microcapsule. The spray drying method has the advantages of simple operation, low comprehensive cost, large embedding amount, high production capacity and simple process, and is suitable for continuous and automatic industrial mass production.
In the preparation process of the holothurin sterol sulfate microcapsules, the loss rate of the holothurin sterol sulfate is controlled to be 5-10%.
Further, the vegetable oil in the step (2) is soybean oil.
An application of the above Stichopus japonicus sterol sulfate microcapsule in food, health product and medicine additive is provided.
The invention has the beneficial effects that:
(1) the invention extracts and prepares the holothurin sterol sulfate from the sea cucumber, and finally proves the improvement effect of the holothurin sterol sulfate on atherosclerosis.
(2) Animal experiments prove that compared with phytosterol, the Stichopus japonicus sterol sulfate can obviously inhibit the development of atherosclerosis, and the Stichopus japonicus sterol sulfate can effectively prevent, improve or treat atherosclerosis.
(3) The invention microencapsulates the Stichopus japonicus sterol sulfate, and the provided Stichopus sterol sulfate microcapsule has the effect of treating atherosclerosis, and can be used for preparing health-care medicines and medicines for treating or preventing atherosclerosis diseases and the like.
Drawings
FIG. 1 is a chemical structure diagram of Stichopus japonicus sterol sulfate.
FIG. 2 is a graph showing the effect of Stichopus japonicus sterol sulfate and phytosterol on the improvement of atherosclerotic plaque area in mice.
FIG. 3 shows the effect of Stichopus japonicus sterol sulfate and phytosterol on mouse serum lipids.
FIG. 4 shows the effect of Stichopus japonicus sterol sulfate and phytosterol on liver lipid metabolism in mice.
FIG. 5 is a graph showing the effect of Stichopus japonicus sterol sulfate microcapsules on the improvement of atherosclerotic plaque area in mice.
FIG. 6 shows the effect of Stichopus japonicus sterol sulfate microcapsules on mouse serum lipids.
FIG. 7 shows the effect of Stichopus japonicus sterol sulfate microcapsules on mouse liver lipids.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: preparation of sea cucumber sterol sulfate
The structural formula of the sea cucumber sterol sulfate is shown in figure 1, and the preparation process is as follows:
(1) vacuum freeze drying and pulverizing body wall of radix Panacis Quinquefolii, and grinding into powder (250 mesh);
(2) taking dry sea cucumber powder, adding ethanol with a certain volume, keeping the material-liquid ratio at 1:10(m/v), continuously stirring and leaching for 24h at room temperature to obtain leaching liquor, leaching for three times, combining the leaching liquor, and concentrating under reduced pressure to obtain crude sea cucumber lipid extract;
(3) weighing silica gel 200-300 meshes, activating at 110 ℃ for 10h, and cooling at room temperature; filling the column by a dry method, dissolving silica gel by chloroform, filling the column after bubbles are removed by ultrasonic treatment, and pressing the column by chloroform with 3 times of volume after the silica gel is completely settled so as to enable the silica gel in the column to be more compact; dissolving a proper amount of crude sea cucumber lipid extract by using a proper amount of chloroform, uniformly mixing the dissolved crude sea cucumber lipid extract with silica gel, quickly pouring the mixture into a silica gel column, and adding chloroform: 3 column volumes were washed with a mixture of methanol (7:1, v/v) and then chloroform: washing 1 column volume with methanol (4:1, v/v), collecting eluate, subjecting to thin layer silica gel plate chromatography (TLC), developing solvent chloroform: methanol: water (80:15:1, v/v/v), using 90% sulfuric acid ethanol color developing agent to carry out color development, then confirming the components, combining the collected eluents, and carrying out reduced pressure concentration to remove the organic reagent, namely the holothurin sterol sulfate.
Example 2: preparation of sea cucumber sterol sulfate
(1) Vacuum freeze drying and pulverizing the sea cucumber body wall, and grinding into powder (250 meshes);
(2) taking the powder, adding ethanol with a certain volume, keeping the material-liquid ratio at 1:12(m/v), continuously stirring and leaching for 24h at room temperature to obtain leaching liquor, leaching for three times, mixing the leaching liquor, and concentrating under reduced pressure to obtain crude extract of sea cucumber lipid;
(3) weighing 200-300 mesh silica gel, activating at 110 deg.C for 12 hr, and cooling at room temperature; loading the column by a dry method, dissolving silica gel with chloroform, loading the column after removing bubbles by ultrasound, pressing the column with 3 times of chloroform after the silica gel is completely settled, so that the silica gel in the column is more compact, discharging the bubbles in the silica gel column, dissolving a proper amount of crude extract of sea cucumber lipid with a proper amount of chloroform, uniformly mixing the dissolved crude extract with the silica gel, quickly pouring the mixture into the silica gel column, and purifying the mixture by a silica gel column chromatography method.
Further, the silica gel column chromatography method comprises the following steps: using chloroform: 3 column volumes were washed with a mixture of methanol (8:1, v/v) and then chloroform: washing 1 column volume with methanol (5:1, v/v), collecting eluate, subjecting to thin layer silica gel plate chromatography (TLC), developing solvent chloroform: methanol: water (80:15:1, v/v/v), using 90% sulfuric acid ethanol color developing agent to carry out color development, then confirming the components, combining the collected eluents, and carrying out reduced pressure concentration to remove the organic reagent, namely the holothurin sterol sulfate.
Example 3: preparation of sea cucumber sterol sulfate
(1) Vacuum freeze drying and pulverizing the sea cucumber body wall, and grinding into powder (250 meshes);
(2) taking the powder, adding ethanol with a certain volume, keeping the material-liquid ratio at 1:15(m/v), continuously stirring and leaching for 24h at room temperature to obtain leaching liquor, leaching for three times, mixing the leaching liquor, and concentrating under reduced pressure to obtain crude extract of sea cucumber lipid;
(3) weighing 200-300 mesh silica gel, activating at 110 deg.C for 12 hr, and cooling at room temperature; loading the column by a dry method, dissolving silica gel with chloroform, loading the column after removing bubbles by ultrasound, pressing the column with 3 times of chloroform after the silica gel is completely settled, so that the silica gel in the column is more compact, discharging the bubbles in the silica gel column, dissolving a proper amount of crude extract of sea cucumber lipid with a proper amount of chloroform, uniformly mixing the dissolved crude extract with the silica gel, quickly pouring the mixture into the silica gel column, and purifying the mixture by a silica gel column chromatography method.
Further, the silica gel column chromatography method comprises the following steps: using chloroform: 3 column volumes were washed with a mixture of methanol (8:1, v/v) and then chloroform: washing 1 column volume with methanol (5:1, v/v), collecting eluate, subjecting to thin layer silica gel plate chromatography (TLC), developing solvent chloroform: methanol: water (80:15:1, v/v/v), using 90% sulfuric acid ethanol color developing agent to carry out color development, then confirming the components, combining the collected eluents, and carrying out reduced pressure concentration to remove the organic reagent, namely the holothurin sterol sulfate.
Example 4: anti-atherosclerosis effect of Stichopus japonicus sterol sulfate and phytosterol
1. Experimental Material
phytosterols are available from Shanxi Pannier Biotech, Inc. as a mixture of β -sitosterol, stigmasterol, campesterol, and campesterol.
ApoE-/-mice (C57 BL/6J mice), male, SPF grade, 9-10 weeks old, body weight 20-22g, purchased from Nanjing cyan violet technologies, Inc., license number: SCXK (Su) 2010-0006.
2. Experimental methods
(1) Animal feeding
After apoE-/-mice are adaptively fed for one week, dividing the mice into 3 groups according to weight, a model control group, a Stichopus japonicus sterol sulfate group and a phytosterol group, wherein each group comprises 8 mice, feeding corresponding feeds respectively, improving the feeds according to an AIN-93M animal feed formula, and adding 1% Stichopus japonicus sterol sulfate and phytosterol into experimental groups respectively. The mice freely eat and drink water, are fed under the conditions of room temperature of 23 +/-2 ℃, 12:12 hours of light and shade alternation and humidity of 65 +/-15 percent, change animal feed every day, record food intake, record body weight every other day and continuously feed for 8 weeks under the conditions.
(2) Atherosclerotic plaque area analysis
The liver of the mouse was peeled off, the right auricle of the heart was cut with an ophthalmic scissors to allow a small amount of blood to flow out, then a 10mL syringe was used to aspirate the physiological saline, and the remaining blood in the heart and the aorta was washed, and after 3 times of washing with the physiological saline, the heart and the aorta were fixed and washed with 4% neutral formaldehyde for 3 times. Finally, the heart and the whole aorta are stripped, the fat and the connective tissue adhered to the aorta are cut off by using an ophthalmic scissors, the aorta is placed into Sudan red dye solution and dyed for 10min at the temperature of 37 ℃, and the redundant dye solution on the tube wall is washed away by using ultrapure water until the tube wall of the aorta is transparent. Finally, the whole aorta is cut off longitudinally by an ophthalmologic scissors, the aorta is fixed on a foam plate by an acupuncture needle, the accumulation of lipid is observed, and the aorta image is collected by a digital camera.
(3) Measurement of blood lipid
Serum TC, TG and HDL-c were assayed according to the kit instructions
(4) Determination of liver fat
0.25g of liver was weighed, 8mL of methanol and 16mL of chloroform were added for homogenization, the homogenate was transferred to a 25mL volumetric flask, and the mixture was purified by chloroform: methanol (2:1, v/v) is metered to 25mL, the plug is plugged, the homogenate is mixed evenly, and the mouth is sealed by a preservative film. Placing the volumetric flask with the constant volume in a water bath shaker at 37 deg.C for 45 min. The volumetric flask was removed and the homogenate filtered through a funnel into a 25mL stoppered graduated cylinder. According to chloroform: methanol: adding ultrapure water according to the proportion of 10:5:3, covering a stopper tightly, sealing the mixture by using a preservative film after uniformly mixing, and standing the mixture at room temperature overnight. Discarding the uppermost layer of the solution after overnight, reserving the middle layer and the lower layer, pouring the middle layer and the lower layer into a rotary bottle for vacuum concentration, drying, metering the volume to 25mL by using petroleum ether, packaging the liquid after metering the volume to obtain the liver lipid solution, and storing in a refrigerator at-20 ℃.
1mL of liver lipid solution is taken, dried by nitrogen, added with 50 mu L of Triton-isopropanol solution, and liver lipids TC and TG are measured according to the kit instructions.
(5) Real-Time PCR detection of liver tissue lipid metabolism related gene expression
Weighing 0.1g of liver tissue, adding 1mL of Trizol, quickly homogenizing under ice bath condition, transferring to a 1.5mL enzyme-inactivating centrifuge tube, standing at room temperature for 5min, adding 0.2mL of chloroform, fully mixing, and standing for 10 min. Centrifuging at 4 deg.C and 12000r/min for 15min, transferring 500 μ L of supernatant into a new 1.5mL enzyme-inactivating centrifuge tube, adding 500 μ L of isopropanol, and mixing. Standing for 10min, centrifuging at 12000r/min for 15min at 4 deg.C, discarding the upper layer liquid, and keeping the bottom precipitate. Adding 75% glacial ethanol solution, slowly blowing, centrifuging at 4 deg.C and 10000r/min for 15min, removing supernatant, adding appropriate amount of DEPC water to dissolve precipitate, and obtaining liver RNA sample. The RNA was analyzed for degradation by agarose gel electrophoresis and RNA concentration was determined by reading the A260/280 ratio using a Nanodrop 2000.
Liver sample RNA was diluted to the appropriate concentration, 2. mu.g of RNA was added to 2. mu.L of random primer, and DEPC water was added to make up to 12.5. mu.L. After metal bath at 70 ℃ for 5min, the mixture is cooled in an ice bath, 5 mu L of 5 xbuffer, 1.25 mu L of dNTP, 0.625 mu L of RNaseInhibitor, 1 mu L of LM-MLV reverse transcriptase and 4.625 mu L of LDEPC water are added, and then the mixture is put into a PCR instrument and is subjected to temperature regulation at 30 ℃ for 10 min; 37 ℃ for 60 min; the 25uL cDNA was obtained by inversion at 90 deg.C, 5min and 4 deg.C.
The cDNA was diluted to the appropriate concentration and 2.5. mu.L of sample cDNA, 0.75. mu.L of the forward primer, 0.75. mu.L of the reverse primer, 12.5. mu.L of SYBR and 8.5. mu.L of DEPC water were added as required by Fast Start Universal SYBR Green Master (Rox) protocol to form a 25. mu.L reaction system under the following conditions: pre-denaturation at 95 ℃ for 10min, then continuous denaturation at 95 ℃ for 10s, low-temperature annealing at 60 ℃ for 20s, and extension at 72 ℃ for 30s are taken as a cycle period, and 45 cycles are operated. After the PCR amplification reaction was completed, the dissolution curve was analyzed to determine the identity of the product. The expression level of the target gene was measured by using the expression level of 18s mRNA as an internal control. The primer sequences used were all BLAST-verified and synthesized by Shanghai Bioengineering, Inc., and the primer sequences used were as follows:
18S(forward,5’-GTAACCCGTTGAACCCCATT-3’；reverse,5’-CCATCCAATCGGTAGTAGCG-3’)；NR1H3，(forward,5'-CCTTCAGAACCCACAGAGATCC-3'；reverse,5'-ACGCTGCATAGCTCGTTCC-3')；ABCA1(forward,5’-TAGCAGCACCGTGTCTTGTC-3’；reverse,5’-TACGGCAGCACATAGGTCAG-3’)；ABCG8(forward,5’-GACATCTGGCACCCCTATCTAC-3’；reverse,5’-GTTCCTTTGCCTCAGCTTTC-3’)；ABCG5(forward,5’-TCTCCGCGTCCAGAACAAC-3’；reverse,5’-CATTGAGCATGCCGGTGTAT-3’)；SREBF2，(forward,5′-GCAGTGGTGGTAGTGGTAGCA-3′；reverse,5′-GTGGGAACTGAGGTGGGAGAAA-3′)；HMGCR(forward,5’-GGCTGGTGAGTTGTCCTTGAT-3’；reverse,5’-TCTAAAGAGCCAGAAACCAAGC-3’)；
ACAT2，LDLR(forward,5’-GAGGAACTGGCGGCTGAA-3’；reverse,5’-GTGCTGGATGGGGAGGTCT-3’)；VLDLR(forward,5′-CTGCAGGGACTGGAGTGATGAG-3′,reverse5′-GCAGATTCCTGGATTTTGGCA-3’)
(6) statistical treatment
Experimental data are expressed as x ± SEM, Tukey's test analysis with SPSS 18.0 software with significant differences of P < 0.05.
3, analyzing results:
the influence of the Stichopus japonicus sterol sulfate on reduction of the area of atherosclerotic plaques in mice is shown in FIG. 2, and the result shows that the Stichopus sterol sulfate can significantly reduce the area of atherosclerotic plaques in mice compared with a model group, and the effect is better compared with phytosterol. Figure 3 serum lipid results show that holothurin sterol sulfate can significantly reduce serum cholesterol levels compared to phytosterols. Meanwhile, the phytosterol and the Stichopus japonicus sterol sulfate can reduce the content of serum triglyceride, but have no significant change, and the serum high-density lipoprotein has no significant change. Liver lipid results show that compared with a model group, the holothurin sterol sulfate and the phytosterin can reduce the levels of liver cholesterol and triglyceride, and the effect of the holothurin sterol sulfate is more obvious. The results in fig. 4 show that the Stichopus japonicus sterol sulfate can significantly up-regulate ABCG8 to promote the efflux of cholesterol, so that the cholesterol content in the liver is reduced, and the cholesterol level is kept stable due to the significant up-regulation of Nr1h 3. The holothurin and the phytosterol sulfate can both remarkably up-regulate the level of LDLR, thereby reducing the endogenous synthesis of cholesterol, and simultaneously, the level of VLDLR is remarkably increased, thereby increasing the absorption and utilization of liver and peripheral tissues to the cholesterol, reducing the blood fat level, and the effect of the holothurin sulfate is better than that of the phytosterol. The above results suggest that Stichopus japonicus sterol sulfate can prevent, treat or improve atherosclerosis. The Stichopus japonicus sterol sulfate has a similar structure to that of phytosterol, except that C-3 of phytosterol is hydroxyl, and C-3 of Stichopus japonicus sterol sulfate is sulfate. In addition, the carbon-hydrogen side chain of the Stichopus japonicus sterol sulfate and the plant sterol is different at C-17. Therefore, the superior anti-atherosclerosis effect of Stichopus japonicus sterol sulfate compared with phytosterol may be caused by C-3 sulfate group and different carbon-hydrogen side chain on C-17. The phytosterol can reduce cholesterol content because it has a structure similar to that of cholesterol, can compete with cholesterol to bind to a carrier, thereby reducing the content of total cholesterol and triglyceride in blood, and has an anti-atherosclerosis effect; on one hand, the holothurin sterol sulfate is easier to combine with a carrier to reduce the content of cholesterol because of the C-3 sulfate group and different carbon-hydrogen side chains on C-17, on the other hand, the holothurin sterol sulfate can remarkably increase ABCG8 to promote the discharge of cholesterol because of the structural particularity, and can remarkably increase VLDLR to increase the absorption and utilization of the liver and peripheral tissues to the cholesterol, further reduce the cholesterol, and further realize the effect of resisting atherosclerosis.
Example 5: preparation of sea cucumber sterol sulfate microcapsule
A preparation method of the holothurian sterol sulfate microcapsule comprises the following steps:
(1) the preparation of the sea cucumber sterol sulfate is the same as that of the example 1;
(2) preparing a microcapsule wall material: weighing a proper amount of maltodextrin and Arabic gum, dispersing in water according to the solid-to-liquid ratio of 0.4:0.6:8 until the maltodextrin and the Arabic gum are completely dissolved, and hydrating for 5 hours at room temperature to obtain a wall material solution;
(3) preparing a microcapsule core material: weighing a certain amount of Stichopus japonicus sterol sulfate, dispersing in vegetable oil according to a solid-to-liquid ratio (g: g) of 0.4:0.8, and dissolving completely;
(4) mixing: under the condition of stirring, dropwise adding the prepared core material into the prepared wall material solution according to the mass ratio of 5:100, and uniformly stirring and mixing to obtain a mixed solution;
(5) encapsulation: homogenizing and high-pressure micro-jet processing the prepared mixed solution to form micro-emulsion, and carrying out spray drying on the obtained micro-emulsion to obtain the sea cucumber sterol sulfate microcapsule.
Example 6: preparation of sea cucumber sterol sulfate microcapsule
(1) The preparation of the sea cucumber sterol sulfate is the same as that of the example 1;
(2) preparing a microcapsule wall material: weighing a proper amount of maltodextrin and Arabic gum, dispersing in water according to the solid-to-liquid ratio of 0.6:0.8:8 until the maltodextrin and the Arabic gum are completely dissolved, and hydrating at room temperature for 8 hours to obtain a wall material solution;
(3) preparing a microcapsule core material: weighing a certain amount of Stichopus japonicus sterol sulfate, dispersing in vegetable oil according to a solid-to-liquid ratio (g: g) of 0.6:0.8, and dissolving completely;
(4) mixing: under the condition of stirring, dropwise adding the prepared core material into the prepared wall material solution according to the mass ratio of 8:100, and uniformly stirring and mixing to obtain a mixed solution;
(5) encapsulation: homogenizing and high-pressure micro-jet processing the prepared mixed solution to form micro-emulsion, and carrying out spray drying on the obtained micro-emulsion to obtain the sea cucumber sterol sulfate microcapsule.
Example 7: preparation of sea cucumber sterol sulfate microcapsule
(1) The preparation of the sea cucumber sterol sulfate is the same as that of the example 1;
(2) preparing a microcapsule wall material: weighing a proper amount of maltodextrin and Arabic gum, dispersing in water according to the solid-to-liquid ratio of 0.8:0.7:8 until the maltodextrin and the Arabic gum are completely dissolved, and hydrating at room temperature for 10 hours to obtain a wall material solution;
(3) preparing a microcapsule core material: weighing a certain amount of Stichopus japonicus sterol sulfate, dispersing in vegetable oil according to the solid-to-liquid ratio (g: g) of 0.8:0.8, and dissolving completely;
(4) mixing: under the condition of stirring, dropwise adding the prepared core material into the prepared wall material solution according to the mass ratio of 10:100, and uniformly stirring and mixing to obtain a mixed solution;
(5) encapsulation: homogenizing and high-pressure micro-jet processing the prepared mixed solution to form micro-emulsion, and carrying out spray drying on the obtained micro-emulsion to obtain the sea cucumber sterol sulfate microcapsule.
Example 8: anti-atherosclerosis effect of Stichopus japonicus sterol sulfate microcapsule
1. Experimental Material
ApoE-/-mice (C57 BL/6J mice), male, SPF grade, 9-10 weeks old, body weight 20-22g, purchased from Nanjing cyan violet technologies, Inc., license number: SCXK (Su) 2010-0006.
2. Experimental methods
(1) Animal grouping and rearing
One week after adaptive feeding of ApoE-/-mice, the mice were divided into 2 groups, a model control group and a Stichopus japonicus sterol sulfate microcapsule group, each group consisting of 8 mice, according to body weight. Two groups were fed with 4 weeks of high-fat diet, and mice were induced to develop an obesity model. The feed is prepared according to the AIN-93M formula. Then, the mice are subjected to test substance intervention, the model control group is perfused with normal saline every day on the basis of feeding high-fat feed, and the Stichopus japonicus sterol sulfate microcapsule group is perfused with 2 Stichopus japonicus sterol sulfate microcapsules every day on the basis of feeding high-fat feed. Dissolving Stichopus japonicus sterol sulfate microcapsule with distilled water, and performing oral gavage for 4 weeks. The mice freely eat and drink water, the mice are fed under the conditions of room temperature of 23 +/-2 ℃, 12:12 hours of light and shade alternation and humidity of 65 +/-15 percent, the animal feed is changed every day, the food intake is recorded, and the body weight is recorded every other day.
(2) Atherosclerotic plaque area analysis
After the liver of the mouse is picked up, the right auricle is cut by an ophthalmological scissors to remove at least a certain amount of blood and flow out, a syringe is inserted from the left ventricle, the blood remained in the heart and the aorta is washed by PBS, after the blood is completely washed, 4% neutral formaldehyde is used for washing, and the heart and the aorta are fixed. Finally, the whole aorta and heart were stripped, the fat and connective tissue adhering to the periphery of the artery were removed with ophthalmic scissors, stained in oil red O stain for 10min, and the excess stain on the vessel wall was washed off with 60% isopropyl alcohol solution until the arterial wall was transparent. Finally, the whole aorta is longitudinally split by ophthalmic scissors, and is fixed on a foam board by an acupuncture needle, the lipid accumulation condition is observed, and an image is acquired by a digital camera.
(3) Measurement of blood lipid
Serum TC, TG and HDL-c were assayed according to the kit instructions
(4) Determination of liver fat
0.25g of liver was weighed, 8mL of methanol and 16mL of chloroform were added for homogenization, the homogenate was transferred to a 25mL volumetric flask, and the mixture was purified by chloroform: methanol (2:1, v/v) is metered to 25mL, the plug is plugged, the homogenate is mixed evenly, and the mouth is sealed by a preservative film. Placing the volumetric flask with the constant volume in a water bath shaker at 37 deg.C for 45 min. The volumetric flask was removed and the homogenate filtered through a funnel into a 25mL stoppered graduated cylinder. According to chloroform: methanol: adding ultrapure water according to the proportion of 10:5:3, covering a stopper tightly, sealing the mixture by using a preservative film after uniformly mixing, and standing the mixture at room temperature overnight. Discarding the uppermost layer of the solution after overnight, reserving the middle layer and the lower layer, pouring the middle layer and the lower layer into a rotary bottle for vacuum concentration, drying, metering the volume to 25mL by using petroleum ether, packaging the liquid after metering the volume to obtain the liver lipid solution, and storing in a refrigerator at-20 ℃.
1mL of liver lipid solution is taken, dried by nitrogen, added with 50 mu L of Triton-isopropanol solution, and liver lipids TC and TG are measured according to the kit instructions.
(5) Statistical treatment
Experimental data are expressed as x ± SEM, Tukey's test analysis using SPSS 18.0 software was significantly different with P < 0.05.
3. Results of the experiment
The influence of the Stichopus japonicus sterol sulfate microcapsule on reduction of the area of atherosclerotic plaques in mice is shown in FIG. 5, and the result shows that the Stichopus sterol sulfate microcapsule can significantly reduce the area of atherosclerotic plaques in mice compared with a model group, and the serum lipid result in FIG. 6 shows that the Stichopus sterol sulfate microcapsule can significantly reduce the content of serum cholesterol and triglyceride, and the liver lipid result in FIG. 7 shows that the Stichopus sterol sulfate microcapsule can significantly reduce the levels of liver cholesterol and triglyceride compared with the model group. The results suggest that the Stichopus japonicus sterol sulfate microcapsule can prevent, treat or improve atherosclerosis.
The above embodiments are preferred embodiments of the present invention, but the embodiments are not limited to the above embodiments, and any other changes, substitutions, modifications, simplifications, combinations, and equivalents which do not depart from the spirit and principle of the present invention should be construed as being included in the scope of the present invention.
Claims (9)
1. Application of Stichopus japonicus sterol sulfate in preparation of anti-atherosclerosis product is provided.
2. The use of claim 1, wherein: the product is food, feed or medicine.
3. The use of claim 1, wherein: the preparation method of the sea cucumber sterol sulfate comprises the following steps:
(1) vacuum freeze drying and pulverizing sea cucumber body wall, and grinding into powder;
(2) adding ethanol with a certain volume into the powder, continuously stirring and leaching at room temperature to obtain leaching liquor, mixing the leaching liquor, and concentrating under reduced pressure to obtain crude extract of sea cucumber lipid;
(3) weighing silica gel, activating, and cooling at room temperature; filling the column by a dry method, dissolving silica gel by chloroform, removing bubbles by ultrasonic waves, and filling the column; dissolving appropriate amount of crude extract of sea cucumber lipid with appropriate amount of chloroform, mixing with silica gel, quickly pouring into silica gel column, and purifying with silica gel column chromatography.
4. Use according to claim 3, characterized in that: the silica gel column chromatography method comprises the following steps: using chloroform: the column was washed 3 volumes with a mixture of methanol and then with chloroform: washing with methanol for 1 column volume, collecting eluate, subjecting to thin layer silica gel plate chromatography one by one, developing with 90% sulphuric acid ethanol developer, determining components, mixing collected eluates, and concentrating under reduced pressure to remove organic reagent to obtain Stichopus japonicus sterol sulfate.
5. Use according to claim 3, characterized in that: the sea cucumber is a North American watermelon.
6. A microcapsule containing the holothurian sterol sulfate according to claim 1.
7. A process for the preparation of microcapsules according to claim 6, characterized in that it comprises the following steps:
(1) preparing a microcapsule wall material: weighing a proper amount of maltodextrin and Arabic gum, dispersing in water according to a solid-to-liquid ratio (g: g: l) of (0.4-0.8) to (0.6-0.8) to 8 until the maltodextrin and the Arabic gum are completely dissolved, and hydrating at room temperature for 5-10 hours to obtain a wall material solution;
(2) preparing a microcapsule core material: weighing a certain amount of Stichopus japonicus sterol sulfate, dispersing the Stichopus japonicus sterol sulfate in vegetable oil according to the solid-to-liquid ratio (g: g) of 0-0.8: 0.8, and fully dissolving;
(3) mixing: under the condition of stirring, dropwise adding the prepared core material into the prepared wall material solution according to the mass ratio of (5-10) to (100), and uniformly stirring and mixing to obtain a mixed solution;
(4) encapsulation: homogenizing and high-pressure micro-jet processing the prepared mixed solution to form micro-emulsion, and carrying out spray drying on the obtained micro-emulsion to obtain the sea cucumber sterol sulfate microcapsule.
8. The method of claim 7, wherein: the vegetable oil in the step (2) is soybean oil.
9. Use of the microcapsules of claim 6 in food, health care products and pharmaceutical additives.
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