Disclosure of Invention
In order to solve at least one technical problem mentioned in the background art, one of the purposes of the present invention is to provide a antarctic krill hypolipidemic peptide which shows significant hypolipidemic activity in both cellular and rat hyperlipidemic models.
The second purpose of the invention is to provide an application of the antarctic krill hypolipidemic peptide, wherein the hypolipidemic peptide can be used as hypolipidemic functional molecules to be applied to special medical foods, health-care products and medicines for treating or assisting in treating hyperlipidemia.
In order to achieve the above object, the present invention provides the following technical solutions.
A antarctic krill hypolipidemic peptide comprising the amino acid sequence shown in SEQ ID No. 1 or a variant thereof.
Preferably, the euphausia superba hypolipemic peptide consists of the amino acid sequence shown in SEQ ID NO. 1.
The application takes euphausia superba protein as a raw material, and separates and prepares euphausia superba hypolipidemic peptide which is a nonapeptide compound, and the amino acid sequence of the euphausia superba hypolipidemic peptide is as follows: ile-Pro-Ala-Phe-Ser-Gly-Arg-Gly-Trp (IPAFSGRGW), ESI-MS has a molecular weight of 990.15Da, and the amino acid sequence is shown as SEQ ID NO. 1. According to the results of an induction model test and an animal modeling test, IPAFSGRGW can obviously reduce the contents of lipid, cholesterol (TC) and Triglyceride (TG) in an Oleic Acid (OA) -induced HepG2 cell accumulation model, has the effect of obviously reducing the contents of cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-c) in serum of a rat with hyperlipidemia, and IPAFSGRGW can obviously improve the content of high-density lipoprotein cholesterol (HDL-c) in serum of the rat with hyperlipidemia, so that the hypolipidemic peptide IPAFSGRGW can be applied to special medical foods, health-care products and medicines for treating or assisting in treating the hyperlipidemia.
The application of the euphausia superba hypolipidemic peptide comprises the preparation of special medical food and/or health-care product and/or medicine for improving hyperlipidemia by taking the hypolipidemic peptide as an effective component.
Preferably, the ameliorating hyperlipidemia includes at least one of preventing, treating, and adjunctively treating hyperlipidemia.
Preferably, the improvement comprises reducing the content of lipids, cholesterol and triglycerides in an oleic acid induced HepG2 cell accumulation model.
Preferably, the improvement comprises reducing the cholesterol, triglyceride, low density lipoprotein cholesterol levels in the serum of hyperlipidemic rats.
Preferably, the improvement comprises increasing the content of high density lipoprotein cholesterol in the serum of the hyperlipidemic rat.
Preferably, the biomedical food and/or health food is at least one of a solid food or a liquid beverage.
Preferably, the medicament is at least one of a liquid dosage form or a lyophilized powder.
A medicament for treating hyperlipidemia, which comprises an effective amount of the euphausia superba hypolipidemic peptide and a pharmaceutically acceptable carrier.
The weight percentage of the antarctic krill hypolipemic peptide in the medicine is 0.01-99.99%.
The weight percentage of the antarctic krill hypolipemic peptide in the medicine is 1.0-55%.
The antarctic krill hypolipemic peptide in the medicament is the only or main effective component in the medicament.
The above-mentioned preferable conditions can be combined with each other to obtain a specific embodiment on the basis of common knowledge in the art.
The raw materials or the reagents involved in the invention are all common commercial products, and the related operations are all routine operations in the field unless specified.
Compared with the prior art, the antarctic krill hypolipemic peptide Ile-Pro-Ala-Phe-Ser-Gly-Arg-Gly-Trp (IPAFSGRGW) provided by the invention can obviously reduce the contents of lipid, total Cholesterol (TC) and Triglyceride (TG) in an Oleic Acid (OA) -induced HepG2 cell lipid accumulation model, obviously reduce TC, TG and LDL-c in experimental hyperlipidemia rat serum, and raise HDL-c. IPAFSGRGW has the advantages of safety, no toxic or side effect, remarkable hypolipidemic activity and the like, and can be applied to special medical foods, health products and medicaments for treating or assisting in treating hyperlipidemia.
The invention adopts the technical proposal to realize the aim, makes up the defects of the prior art, has reasonable design and convenient operation.
Detailed Description
Suitable substitutions and/or modifications of the process parameters will be apparent to those skilled in the art from the disclosure herein, however, it is to be expressly pointed out that all such substitutions and/or modifications are intended to be encompassed by the present invention. While the products and methods of preparation of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the products and methods of preparation described herein without departing from the spirit and scope of the invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present invention uses the methods and materials described herein; other suitable methods and materials known in the art may be used. The materials, methods, and examples described herein are illustrative only and not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein, and the like, are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
All percentages, parts, ratios, etc., are by weight unless otherwise specified; the other specification includes, but is not limited to, "wt%" means weight percent, "mol%" means mole percent, "vol%" means volume percent.
When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a series of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5 (1 to 5)" is described, the described range should be understood to include ranges of "1 to 4 (1 to 4)", "1 to 3 (1 to 3)", "1 to 2 (1 to 2) and 4 to 5 (4 to 5)", "1 to 3 (1 to 3) and 5", and the like. Where a range of values is described herein, unless otherwise stated, the range includes the range endpoints and all integers and fractions within the range.
Unless specifically stated otherwise, the materials, methods, and examples described herein are illustrative only and not intended to be limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
It should be known in the art that the amino acid sequence of the antarctic krill hypolipidemic peptide provided by the invention is Ile-Pro-Ala-Phe-Ser-Gly-Arg-Gly-Trp, abbreviated as IPAFSGRGW, wherein Ile corresponds to I, i.e. isoleucine; pro corresponds to P, i.e., proline; ala corresponds to A, and alanine; phe corresponds to F, phenylalanine; ser corresponds to S, serine; gly corresponds to G, namely ammonium glycoside; arg corresponds to R, arginine; trp corresponds to W, tryptophan.
It should be understood in the art that OA is oleic acid, TC is total cholesterol, TG is triglyceride, LDL-c is low density lipoprotein cholesterol, HDL-c is high density lipoprotein cholesterol.
The invention provides antarctic krill hypolipidemic peptide which is a nonapeptide compound, and the amino acid sequence of the antarctic krill hypolipidemic peptide is as follows: ile-Pro-Ala-Phe-Ser-Gly-Arg-Gly-Trp (IPAFSGRGW), the amino acid sequence table is shown as SEQ ID NO. 1, and the molecular weight measured by ESI-MS is 990.15Da.
The invention also provides a preparation method of the antarctic krill hypolipidemic peptide, which comprises the following steps: pretreatment, enzymolysis, separation and extraction, specifically comprising:
1) Pretreatment: removing heads and shells of euphausia superba, cleaning, homogenizing to paste, adding absolute ethyl alcohol according to a feed-liquid ratio of 1g to 5-8 mL, performing 500W ultrasonic degreasing for 30-60 min, filtering, repeating the above operation for 2 times, and thoroughly removing lipid in euphausia superba; drying and filtering the obtained solid to obtain defatted euphausia superba powder;
2) Enzymolysis: adding buffer solution into defatted antarctic krill powder according to the feed-liquid ratio of 1 g:5-8 mL, regulating pH value to 1.5-2.5, adding pepsin with 2-3% of the weight of the shrimp powder (enzyme activity is more than or equal to 3.0X10) 4 U/g), enzymatic hydrolysis is carried out for 3 to 4 hours at the temperature of 35 to 40 ℃, enzyme is inactivated for 10 to 15 minutes at the temperature of 90 to 100 ℃, the temperature is cooled to 45 to 50 ℃, the pH value is regulated to 6.5 to 8.0, and neutral protease with the weight of 1.5 to 2.5 percent of the shrimp meal (the enzyme activity is more than or equal to 1.0 multiplied by 10) is added 5 U/g), enzymatic hydrolysis for 3-4 h, enzyme deactivation at 90-100 ℃ for 10-15 min, centrifugation at 6000-9000 rmp for 25-30 min, collection and collectionClear liquid is obtained to obtain euphausia superba enzymolysis liquid; the euphausia superba polypeptide is obtained by using a double-enzyme composite enzymolysis technology, and no toxic or side organic solvent is added, so that the safety is high, the preparation cost is low, and the reaction condition is mild; pepsin and neutral protease can quickly degrade defatted euphausia superba powder into small-molecule high-activity oligopeptides, and the yield is high;
3) Separating and extracting: grading the euphausia superba proteolysis solution by an ultrafiltration membrane with a molecular weight cutoff of 3kDa, collecting grading components, determining lipid reduction capacity (expressed by a% blank group) of each component in an Oleic Acid (OA) -induced HepG2 cell lipid accumulation model, and sequentially enriching the ultrafiltration component with the strongest lipid reduction capacity by macroporous resin, performing gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC purification) to obtain euphausia superba lipid-lowering peptide.
The specific processes of macroporous resin enrichment, gel chromatography and RP-HPLC purification in the separation and extraction step are as follows:
enriching with macroporous resin: dissolving the ultrafiltration component with the strongest lipid-lowering capacity in double distilled water to prepare a solution with the concentration of 90-100 mg/mL, slowly adding the sample solution into a resin column according to the ratio of 1g to 10-20 g of sample to D101 macroporous resin, and washing the resin column by double distillation with the volume of 2-3 times of the column volume to remove unadsorbed samples; washing the resin column with 2-3 times of 60% ethanol, collecting the eluting solution, and freeze-drying to obtain an enriched sample;
gel column chromatography: dissolving a D101 macroporous resin enriched sample in double distilled water to prepare a solution with the concentration of 40-50 mg/mL, separating by Sephadex LH-20 column chromatography (2.5 cm multiplied by 160 cm), eluting by double distilled water at the flow rate of 0.5-0.8 mL/min, collecting chromatographic peaks according to a 214nm chromatogram, and determining the lipid-lowering capacity of each chromatographic peak, wherein the highest active chromatographic peak is gel chromatographic zymolyte;
RP-HPLC purification: preparing the gel chromatography zymolyte into a solution with the concentration of 25-30 mug/mL by double distilled water, purifying by RP-HPLC, obtaining 1 high-activity hypolipidemic peptide Ile-Pro-Ala-Phe-Ser-Gly-Arg-Gly-Trp (IPAFSGRGW) according to hypolipidemic activity of the prepared oligopeptide, and measuring the molecular weight of 990.15Da by ESI-MS.
The conditions for the RP-HPLC purification are as follows: the sample injection amount is 15-20 mu L; chromatographic column Kromasil C-18 (250 mm. Times.4.6 mm,5 μm); mobile phase: the acetonitrile concentration is increased from 0 to 100 percent at a constant speed within 0 to 30 minutes; the elution speed is 0.6-0.8 mL/min; the ultraviolet detection wavelength is 214nm.
The present invention is described in detail below.
Example 1:
the embodiment provides a antarctic krill hypolipidemic peptide, which is a nonapeptide compound, and the amino acid sequence of the hypolipidemic peptide is as follows: ile-Pro-Ala-Phe-Ser-Gly-Arg-Gly-Trp (IPAFSGRGW), ESI-MS has a molecular weight of 990.15Da, and the amino acid sequence is shown as SEQ ID NO. 1.
The preparation method of the antarctic krill hypolipidemic peptide provided by the embodiment comprises the following steps:
1) Pretreatment: removing heads and shells of antarctic krill, cleaning, homogenizing to paste, adding absolute ethyl alcohol according to a feed-liquid ratio of 1g to 6mL, performing 500W ultrasonic degreasing for 45min, filtering, repeating the above operation for 2 times, and removing lipid in antarctic krill; drying and filtering the obtained solid to obtain defatted euphausia superba powder;
2) Enzymolysis: adding buffer solution into defatted Euphausia superba powder at a ratio of 1 g/8 mL, adjusting pH to 2.5, and adding pepsin (enzyme activity 3.0X10:3% of shrimp powder weight) 4 U/g), performing enzymolysis at 37deg.C for 4 hr, inactivating enzyme at 95deg.C for 10min, cooling to 50deg.C, adjusting pH to 7.0, adding neutral protease (enzyme activity 1.0X10) accounting for 2.0% of shrimp meal weight 5 U/g), performing enzymolysis for 4 hours, inactivating enzyme at 95 ℃ for 15 minutes, centrifuging at 9000rmp for 25 minutes, and collecting supernatant to obtain euphausia superba enzymolysis liquid (NPH);
3) Separating and extracting: grading the euphausia superba proteolysis solution (NPH) by an ultrafiltration membrane with a cut-off molecular weight of 3kDa, collecting grading components NPH-I (MW less than or equal to 3 kDa) and NPH-II (MW more than 3 kDa), determining a HepG2 cell lipid accumulation model induced by the NPH-I and the NPH-II on Oleic Acid (OA) (model building method is referred to [ Pan Xin ], the preparation of the squid meat and the swim bladder functional polypeptide and the oxidation resistance and lipid reduction activity research [ D ] the ocean university of Zhejiang, 2017 ]) and sequentially carrying out macroporous resin enrichment, gel chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) purification on the NPH-I with the strongest lipid reduction capacity to obtain euphausia superba hypolipemic peptide, and determining the structure by using an amino acid sequence analyzer and mass spectrum, wherein the specific process is as follows:
(1) enriching with macroporous resin: dissolving NPH-I in double distilled water to prepare a solution with the concentration of 100mg/mL, slowly adding the NPH-I solution into a resin column according to the proportion of NPH-I to D101 macroporous resin 1g:15g, and washing the resin column by double distillation with the volume of 3 times of the column volume to remove the NPH-I solution to obtain an adsorption sample; washing the resin column with 3 times of 60% ethanol, collecting the eluting solution, lyophilizing to obtain enriched sample (NPH-I-1), and determining NPH-I-1 lipid-lowering ability (see Table 1);
(2) gel chromatography: dissolving NPH-I-1 in double distilled water to prepare a solution with the concentration of 50mg/mL, separating by Sephadex LH-20 column chromatography (2.5 cm multiplied by 160 cm), eluting by double distilled water at the flow rate of 0.5mL/min, collecting each chromatographic peak NPH-I-1 a-NPH-I-1 c according to a 214nm chromatogram, determining the hypolipidemic capacity of each chromatographic peak (see Table 1), wherein the highest active chromatographic peak is gel chromatography zymolyte NPH-I-1c;
(3) refining by RP-HPLC and detecting the structure: preparing the gel chromatography zymolyte NPH-I-1C into a solution with the concentration of 30 mug/mL by double distilled water, purifying by RP-HPLC, wherein the sample injection amount is 20 mug, and the chromatographic column Kromasil C-18 (250 mm multiplied by 4.6mm,5 mu m; the acetonitrile concentration of the mobile phase is uniformly increased from 0 to 100% in 0-30 min) and the elution speed is 0.8mL/min; collecting active peptides NJP-1-NJP-7 according to 214nm chromatographic peaks, and obtaining 1 high-activity lipid-lowering peptide NJP-3 according to the lipid-lowering activity (see Table 1) of the oligopeptides NJP-1-NJP-7 shown in FIG. 2; the amino acid sequence is Ile-Pro-Ala-Phe-Ser-Gly-Arg-Gly-Trp (IPAFSGRGW), the structural formula is shown in figure 3, and the molecular weight is 990.15Da by ESI-MS, which is shown in figure 4. Table 1 effect of antarctic krill protein hydrolysate, separation fractions and preparation of active peptides on lipid content in Oleic Acid (OA) -induced HepG2 cell lipid accumulation model (n=3)
Example 2:
based on the foregoing examples, this example demonstrates the effect of antarctic krill hypolipidemic peptide on Oleic Acid (OA) -induced content of Triglyceride (TG) and cholesterol (TC) in HepG2 cell lipid accumulation model, specifically comprising:
and (3) building a model: referring to the prior art [ Pan Xin ] preparation of functional polypeptides of the fish meat and swim bladder of the Miichthys miiuy, antioxidant and lipid lowering activity research [ D ] university of ocean in Zhejiang, 2017] to establish an Oleic Acid (OA) -induced HepG2 cell lipid accumulation model;
analysis of results: the effects of IPAFSGRGW on triglyceride and cholesterol in an Oleic Acid (OA) -induced HepG2 cell lipid accumulation model are shown in fig. 5 and 6, respectively, and as can be seen from fig. 5 and 6, the antarctic krill hypolipemic peptides IPAFSGRGW in the application have remarkable inhibitory effects on TG and TC in an Oleic Acid (OA) -induced HepG2 cell lipid accumulation model, and the inhibitory effects are close to that of simvastatin, a positive drug.
Example 3:
based on the foregoing examples, in vivo experiments in rats with hyperlipidemia were performed in this example, and the effects of antarctic krill hypolipidemic peptide on the same were verified, specifically including:
and (3) building a model: referring to the prior art [ Chen Bei ] the lipid regulating effect of the basil extract on the hyperlipidemia rats and the mechanism research [ D ] the university of Xinjiang medical science, 2009] to establish an experimental hyperlipidemia rat model;
analysis of results: effects of IPAFSGRGW on TG, TC, LDL-c and HDL-c in serum of hyperlipidemic rats As shown in FIGS. 7 to 10, respectively, LPGTRIPA was found to significantly reduce the levels of TG, TC and LDL-c and increase the levels of HDL-c in serum of experimental hyperlipidemic rats.
In conclusion, ile-Pro-Ala-Phe-Ser-Gly-Arg-Gly-Trp (IPAFSGRGW) obviously reduces the blood lipid level in cells and animal models, is safe and has no toxic or side effect, and can be applied to special medical foods, health care products and medicaments for treating or assisting in treating hyperlipidemia.
The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.
The specific embodiments described herein are offered by way of example only to illustrate the spirit of the invention. Various modifications or additions to the described embodiments may be made by those skilled in the art to which the invention pertains or may be substituted in a similar manner without departing from the spirit of the invention or beyond the scope of the appended claims.
Finally, it should be noted that the above list is only one embodiment of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
The invention is a well-known technique.