CN113527423B

CN113527423B - Euphausia superba oligopeptide for adjuvant therapy of NAFLD and application thereof

Info

Publication number: CN113527423B
Application number: CN202110800581.7A
Authority: CN
Inventors: 迟长凤; 王斌; 王玉梅
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2021-07-15
Filing date: 2021-07-15
Publication date: 2023-03-07
Anticipated expiration: 2041-07-15
Also published as: CN113527423A

Abstract

The invention discloses an antarctic krill oligopeptide for adjuvant therapy of NAFLD and application thereof, and particularly relates to an antarctic krill oligopeptide for adjuvant therapy of nonalcoholic fatty liver disease (NAFLD) obtained by using the antarctic krill as a raw material through ultrasonic degreasing, enzymolysis, membrane ultrafiltration, chromatographic separation and purification, wherein Phe-Trp-Lys-VaL-VaL-ILe-ALa-Pro-Trp (FWKVWAPW) is used, and the molecular weight of ESI-MS (ESI-MS) is 1145.4Da. The antarctic krill oligo for adjuvant therapy of NAFLD prepared by the invention can obviously reduce lipid accumulation in a NAFLD cell model, obviously reduce the content of active oxygen free Radicals (ROS), triglyceride (TG) and Total Cholesterol (TC) in the NAFLD cell model, and has the obvious effects of reducing blood fat and eliminating oxidative stress injury. In addition, the oligopeptide can also be applied to the preparation of medicaments and health-care products for adjuvant therapy of NAFLD.

Description

Euphausia superba oligopeptide for adjuvant therapy of NAFLD and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an antarctic krill oligopeptide for adjuvant therapy of non-alcoholic fatty liver disease (NAFLD).

Background

Non-alcoholic fatty Liver disease (NAFLD) is a metabolic stress Liver injury that is associated with excessive accumulation of fat (greater than or equal to 5% of the weight of Liver cells) and is closely related to InsuLin Resistance (IR) and genetic susceptibility. NAFLD not only can cause disability and death of liver diseases, but also is closely associated with high incidence of metabolic syndrome, type 2 diabetes, cardiovascular disease, colorectal tumor, etc., and NAFLD is frequently incorporated in patients chronically infected with Hepatitis B Virus (HBV). It is estimated that about 25% of the population worldwide in 2018 suffer from NAFLD, of which 10% -20% are NASH, the latter having a incidence of cirrhosis of the liver as high as 25% within 10 years. With the rapid increase of people with obesity and metabolic syndrome in China, NAFLD has become the leading cause of the abnormality of biochemical indexes of the first chronic liver disease and health physical examination liver in China, but no medicine for effectively treating NAFLD exists clinically. Therefore, the development of highly effective therapeutic drugs aiming at the molecular mechanisms (lipid deposition, oxidative damage, inflammatory response) developed by NAFLD is the focus of research.

According to statistics of two times of southern American biological resource reserves survey of China participated in 1981-1990, the conservation of the Antarctic krill resources is estimated to be 6-10 million tons, and some people estimate to be as much as 50 million tons. Based on the conserved reserves of 6-10 million tons, 0.6-1.0 million tons of antarctic krill resources are harvested in the south ocean every year by human beings without influencing the balance of the biological chain of the south ocean, and the annual harvesting amount is twice of the annual harvesting amount of ocean aquatic products all over the world and is the resource which is firstly provided for the development and utilization of the human beings in the south Pole. Currently, russia, japan, polish, norway, etc. have led to the first commercial fishing of antarctic krill in the southern ocean. Therefore, the antarctic krill resource is like a future protein resource warehouse of human beings. The domestic antarctic krill industry leading enterprises are mainly concentrated on Shandong, liaoning, shanghai and the like for a while. The Liaoning sailing fishery mainly processes edible products such as shrimp balls, shrimp meat sausages and the like, and the technology is also in rapid improvement. The Shanghai Euphausia superba enterprises are mainly in export trade. The development of the antarctic krill industry in China is good, and the antarctic krill industry becomes one of the main development areas of the antarctic krill industry in the world. However, no medicine or functional product related to NAFLD treatment or adjuvant treatment by using antarctic krill is known at present.

Disclosure of Invention

The invention aims to provide an antarctic krill oligopeptide which can be used for preparing medicines and used as a food additive for adjuvant therapy of NAFLD. In order to achieve the purpose of the invention, the following technical scheme is adopted:

the invention discloses an amino acid sequence of an antarctic krill oligopeptide for adjuvant therapy of non-alcoholic fatty liver disease (NAFLD), which is Phe-Trp-Lys-VaL-VaL-ILe-ALa-Pro-Trp, and the single letter is as follows: FWKVVIAPW. The antarctic krill oligopeptide can obviously reduce the content of reactive oxygen Radicals (ROS), triglyceride (TG) and Total Cholesterol (TC) in a NAFLD cell model, has obvious effects of reducing blood fat and eliminating oxidative stress damage, has the advantages of safety, no toxic or side effect, strong lipid-reducing activity and the like, and can be used for preparing medicines and health-care foods.

Preferably, the relative molecular mass of the oligopeptide is 1145.4Da.

The invention discloses an application of the antarctic krill oligopeptide, which comprises at least one of the following components:

(1) Reducing the level of reactive oxygen radicals in the cell;

(2) Reducing the level of triglycerides in the cell;

(3) Reducing the level of total cholesterol in the cell;

(4) Reducing blood fat;

(5) Eliminating oxidative stress injury;

(6) Preparing a medicine for adjuvant therapy of NAFLD;

(7) Can be used as food additive.

The invention also discloses a method for preparing antarctic krill oligopeptide for adjuvant therapy of NAFLD, which comprises the following steps:

(1) Pretreating Antarctic krill;

(2) Enzymolysis of the euphausia superba powder;

(3) Ultrafiltering and collecting different components;

(4) Preparing an ultrafiltration zymolyte;

(5) Performing gel column chromatography;

(6) Purifying by reversed phase high performance liquid chromatography.

Preferably, the pretreatment in step (1) comprises degreasing.

More preferably, the antarctic krill is unfrozen, the head and the skin and the tissues are removed and minced, a 92-98% ethanol solution is added according to the material-liquid ratio of 1-3g to 5-40 mL, a tissue triturator is used for triturating, 110-120W ultrasonic waves are carried out for 45-60min for degreasing, the steps are repeated for three times, filtering is carried out, and the solid is dried, so that the degreased shrimp meal is obtained.

Even more preferably, the antarctic krill pretreatment comprises the following steps:

thawing Antarctic krill, removing heads, peeling and mincing tissues, adding 95% ethanol solution according to the material-liquid ratio of 1g to 5-8 mL, mashing by using a tissue mashing machine, defatting by 120W ultrasound for 45-60min, repeating the steps for three times, filtering, and drying solids to obtain defatted shrimp meal.

Preferably, the step (2) is carried out by enzymolysis by using neutral protease, and the enzyme activity of the neutral protease is more than or equal to 5.0 multiplied by 10 ⁴ 。

More preferably, the enzymolysis of the euphausia superba powder comprises the following steps:

adding the euphausia superba powder into a phosphate buffer solution (0.5 moL/L, pH7.0) according to the feed-liquid ratio of 1g, uniformly stirring, adjusting the temperature of the solution to 45-50 ℃, adding protease accounting for 2.0-3.0% of the weight of the euphausia superba powder, carrying out enzymolysis for 5-8 h, carrying out heat preservation on the solution in a water bath at the temperature of 90-100 ℃ for 5-30 min, cooling to normal temperature, centrifuging, and collecting supernatant, namely euphausia superba protein enzymolysis liquid.

Still more preferably, the enzymatic hydrolysis of the euphausia superba meal comprises the following steps:

adding 8-10 mL of the euphausia superba powder into phosphate buffer solution (0.5 moL/L, pH7.0) according to a feed-to-liquid ratio of 1g, uniformly stirring, adjusting the temperature of the solution to 45-50 ℃, adding protease accounting for 2.0-3.0% of the weight of the euphausia superba powder, carrying out enzymolysis for 5-8 h, preserving the solution in a water bath at 90-100 ℃ for 10min, cooling to normal temperature, centrifuging for 15min at 9000rmp, and collecting supernatant, namely euphausia superba protein enzymolysis liquid.

Preferably, the ultrafiltration membrane used in step (3) has a molecular weight cut-off of 3.5kDa.

More preferably, the collection of the different components by ultrafiltration comprises the steps of:

and (3) classifying the euphausia superba protease hydrolysate by an ultrafiltration membrane with the molecular weight cutoff of 3.5kDa, and collecting a classification component.

Preferably, the NAFLD cell model used in step (4) is a HepG2 cell line.

More preferably, the preparation of the ultrafiltrate substrate comprises the steps of:

measuring the ability of each component obtained by ultrafiltration to reduce lipid accumulation and Reactive Oxygen Species (ROS) level in NAFLD cell model, selecting the component with best activity, and lyophilizing to obtain ultrafiltration zymolyte.

More preferably, the gel column chromatography comprises the steps of:

dissolving the ultrafiltration zymolyte in double distilled water to prepare a solution with the concentration of 20 to 25mg/mL, separating by Sephadex G-25 column chromatography through Sephadex, eluting by the double distilled water with the flow rate of 0.5 to 0.8mL/min, preparing a gel chromatography chromatogram according to the absorbance under 220nm, collecting various chromatographic peaks, determining the capacity of various chromatographic peak components for reducing the lipid accumulation and the active oxygen free radical level in a NAFLD cell model, selecting the chromatographic peak component with the best activity, and freeze-drying to obtain the gel chromatography zymolyte.

More preferably, the RP-HPLC purification comprises the following steps:

preparing the gel chromatography zymolyte into a solution of 45 to 50 mu g/mL by using double distilled water, purifying by using RP-HPLC, and obtaining 1 oligopeptide Phe-Trp-Lys-VaL-VaL-ILe-ALa-Pro-Trp with high activity for adjuvant therapy of NAFLD according to the activity of prepared peptide, wherein the molecular weight is 1145.4Da by ESI-MS determination.

Even more preferably, the RP-HPLC conditions are:

the sample size is 10 to 15 mu L; column Kromasil C18 (250 mm. Times.4.6 mm,5 μm); mobile phase: 60% acetonitrile; the elution speed is 0.5 to 0.8mL/min; the ultraviolet detection wavelength is 220nm.

Compared with the prior art, the invention has the beneficial effects that:

the antarctic krill oligopeptide can obviously reduce the content of active oxygen free radicals, triglyceride and total cholesterol in a NAFLD cell model, has the obvious effects of reducing blood fat and eliminating oxidative stress injury, has the advantages of safety, no toxic or side effect, strong blood fat reducing activity and the like, and can be used for preparing medicines and health-care foods.

Drawings

FIG. 1 is a graph of the effect of the ultrafiltration component of antarctic krill hydrolysate on lipid packing in NAFLD cell models at a concentration of 10 mg/mL;

FIG. 2 shows the effect of ultrafiltration components of antarctic krill enzymatic hydrolysate on the content of active oxygen free radicals in NAFLD cell model at a concentration of 10 mg/mL;

FIG. 3 is a Sephadex G-25 chromatogram of Sephadex;

FIG. 4 is a graph of the effect of Sephadex G-25 preparation of the zymolyte fraction on lipid packing in the NAFLD cell model at a concentration of 10 mg/mL;

FIG. 5 is a graph of the effect of Sephadex G-25 prepared zymolyte fraction on the content of active oxygen radicals in the NAFLD cell model at a concentration of 10 mg/mL;

FIG. 6 shows RP-HPLC analysis of the zymolyte prepared from Sephadex G-25;

FIG. 7 is a graph of the effect of RP-HPLC fractions on lipid packing in NAFLD cell models at a concentration of 10 mg/mL;

FIG. 8 is a graph of the effect of RP-HPLC fractions on active oxygen free radical content in NAFLD cell models at a concentration of 10 mg/mL;

FIG. 9 is a structure of FWKVVIAPW;

FIG. 10 is a mass spectrum of FWKVVIAPW;

figure 11 is the effect of FWKVVIAPW on triglyceride content in NAFLD cell models;

figure 12 is the effect of FWKVVIAPW on total cholesterol content in NAFLD cell model.

Detailed Description

The exemplary embodiments will be described herein in detail, and the implementations described in the exemplary embodiments below do not represent all implementations consistent with the present disclosure. Rather, they are merely examples of methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.

The experimental procedures in the following examples are, unless otherwise specified, either conventional or according to the manufacturer's recommendations. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

Establishment and experimental grouping of human liver cancer cell HepG2 cell model

Human hepatoma cells HepG2 cells were cultured in RPMI1640 medium containing 10% Fetal Bovine Serum (FBS). Preparing HL7702 cell in logarithmic growth phase into suspension, inoculating to 96-well plate at a temperature of 37 deg.C and 5% CO content, and culturing at 200 μ L per well ₂ Adherence in incubator 5h. Cultured cells were divided into 4 groups: (1) blank control group: culturing in RPMI1640 medium containing 10% Fetal Bovine Serum (FBS) for 24 hr; (2) model group: 1.0mM free fatty acid (oleic acid: palmitic acid = 2:1) in RPMI1640 medium for 24h; (3) positive control group: 1.0mM of FFA and GSH (20 mu M) in RPMI1640 medium for 24h; (4) sample group: 1.0mM free fatty acid and the sample was incubated for 24h in RPMI1640 medium.

Example 2

The preparation method of the antarctic krill oligopeptide for adjuvant therapy of NAFLD comprises the following specific steps:

1) Pretreating Antarctic krill: thawing Antarctic krill, removing head and skin, mincing, adding 95% ethanol solution according to the material-liquid ratio of 1g to 6mL, mashing with a tissue triturator, defatting with 120W ultrasound for 60min, repeating for three times, filtering, and drying the solid to obtain defatted shrimp powder.

2) Enzymolysis of Antarctic krill powder: adding the degreased shrimp powder into a phosphate buffer (0.5 moL/L, pH7.0) according to a feed-to-liquid ratio of 1g ⁴ ) And performing enzymolysis for 6h, preserving the solution in a water bath at 95 ℃ for 10min, cooling to the normal temperature, centrifuging for 15min at 9000rmp, and collecting supernatant, namely euphausia superba protease hydrolysate (APH).

3) Preparation of antarctic krill oligopeptides: classifying the euphausia superba protease hydrolysate by an ultrafiltration membrane with the molecular weight cutoff of 3.5kDa, collecting classification components APH1 (MW is less than or equal to 3.5 kDa) and APH2 (MW is more than or equal to 3.5 kDa), and determining the capacity of each component for reducing the lipid accumulation and the content of active oxygen free radicals in a NAFLD cell model; the determination steps are as follows:

the ability of each component to reduce lipid packing in a cell model was determined:

incubating HepG2 cells of a blank control group, a model group, a positive control group and a sample group for 24 hours according to the method described in example 1, discarding the culture medium, washing the cells with PBS (room temperature) buffer solution for 1 time, discarding PBS, adding 70 mu L of 4% paraformaldehyde fixing solution into each hole, fixing the cells in a dark place at room temperature for 30min, discarding paraformaldehyde, washing the cells with PBS for 1 time, discarding PBS,60% isopropanol 60 mu L/Kong Runxi min, discarding isopropanol, adding 60 mu L of 0.3% oil red O staining solution into each hole, dyeing the cells in a dark place at room temperature for 1 hour, discarding oil red O, washing the cells with PBS buffer solution for 3 times, and discarding PBS; dissolved in DMSO, 100. Mu.L/well, OD was measured at 358nm using a microplate reader. The blank group was set to 100%, and the lipid accumulation level (% blank) = (OD sample group/OD blank) × 100% for the remaining groups. The experimental results are calculated and arranged and then drawn into a graph 1, and the graph 1 shows that the APH1 has the best activity and the strongest capacity of reducing the lipid content.

The ability of each component to reduce reactive oxygen radicals in a cell model was determined:

after incubation of HepG2 cells of the blank control, model, positive control and sample groups for 24h as described in example 1, 10. Mu.L of fluorescent probe DCFH-DA was added at 37 ℃ and 5% CO ₂ And incubating for 2h in a constant-temperature cell incubator in a dark place. Cells were washed gently with cold PBS buffer and repeated twice. The cell morphology was observed and photographed by a fluorescence inverted microscope (excitation wavelength 485 nm/emission wavelength 535 nm) in a dark room. The light intensity was measured using a fluorescent microplate reader and the data statistically analyzed. The test results are plotted in FIG. 2 after treatment, and it can be seen from FIG. 2 that APH1 has the best activity and the strongest ability to reduce reactive oxygen species.

According to the determination result, the component APH1 is taken to carry out the subsequent preparation steps. After APH1 is freeze-dried, sequentially performing gel column chromatography and reversed-phase high performance liquid chromatography purification to obtain the antarctic krill oligopeptide for adjuvant therapy of the non-alcoholic fatty liver disease. The method comprises the following specific steps:

gel chromatography: preparing the APH1 into a solution with the concentration of 20mg/mL by using double distilled water, performing Sephadex LH-20 column chromatography separation, eluting by using double distilled water, collecting elution components APH1-A, APH-B and APH1-C according to an absorbance curve at 220nm, wherein the chromatography result is shown in figure 3; the influence of the 3 components on lipid accumulation and active oxygen free radical level in the NAFLD cell model is measured, the measurement results are shown in figure 4 and figure 5, and the component APH1-B with the best activity of chromatographic peak is selected and freeze-dried to obtain the gel chromatography zymolyte.

RP-HPLC refining: preparing the APH1-B into a solution with the concentration of 50 mug/mL by using double distilled water, purifying the solution by using RP-HPLC (the sample injection amount is 20 mug, a chromatographic column Kromasil C18 (250 mm multiplied by 4.6mm,5μm), a mobile phase of 60% acetonitrile, an ultraviolet detection wavelength of 220 nm), collecting oligopeptides APH-P1, APH-P2, APH-P3, APH-P4 and APH-P5 according to an absorbance curve under 220nm, and obtaining the RP-HPLC determination result shown in figure 6; determining the effect of 5 oligopeptides on lipid accumulation and active oxygen radical levels in a NAFLD cell model; the results are shown in FIG. 7 and FIG. 8, and it is known that APH-P3 has the best effect of reducing lipid accumulation and scavenging active oxygen free radicals in cell models, so that APH-P3 is taken as a high-activity oligopeptide for adjuvant therapy of NAFLD.

According to the preparation process, the prepared antarctic krill oligopeptide has good capability of reducing intracellular lipid accumulation level and reducing active oxygen free radicals, and further shows that the antarctic krill oligopeptide has the effect of eliminating oxidative stress damage.

Test example 1

Determination of oligopeptide amino acid sequence and molecular weight of antarctic krill

The amino acid sequence of the resulting polypeptide was determined by Edman degradation using the ABI 494 protein/polypeptide sequencer using APH-P3 from example 2, and the results are shown in FIG. 9. The molecular weight was measured by ESI-MS, and the results are shown in FIG. 10. The amino acid sequence determined was Phe-Trp-Lys-VaL-VaL-ILe-ALa-Pro-Trp, expressed in single letter: FWKVVIAPW with molecular weight of 1145.4Da.

Test example 2

Effect of oligopeptide properties of Antarctic krill on triglyceride and total cholesterol content in cells

The amounts of intracellular triglycerides and total cholesterol were performed using the cellular total cholesterol and triglyceride quantification kit according to the instructions provided by the manufacturer and the protein content in the sample was determined using the BCA protein quantification kit according to the instructions provided by the manufacturer. And then calculating the content of triglyceride and total cholesterol in the cells. The specific operation steps are as follows:

1) Measurement of Triglyceride (TC) content

(1) Sample preparation: preparing cell lysate containing 5% of NP-40 by double-distilling with water, and collecting 10 ⁶ And taking one cell as a sample, adding 100 mu L of lysis solution, putting the cell into a metal bath, starting to heat, and maintaining for 5min after the temperature reaches 90 ℃ until the lysis solution is turbid (at the moment, the standard substance can be heated at the same time). Then cooled to room temperature and heated repeatedly once at 90 ℃ for 5min. Centrifuging for 2min at low speed on a miniature centrifuge. Meanwhile, parallel samples were taken for BCA protein assay. And taking a proper amount of the centrifuged sample, diluting the sample by 10 times by using double distilled water, taking 20 mu L of the diluted sample, adding the sample into a 96-well plate, and complementing the volume to 50 mu L by using a buffer solution.

(2) Making a standard curve: the triglyceride standard was heated at 90 ℃ for 1min until turbid, and centrifuged in a microcentrifuge for 30 seconds until clear. Heating and centrifugation were then repeated once. And adding 40 mu L of triglyceride standard substance into 160 mu L of buffer solution, uniformly mixing, and diluting to 0.2mM. 0, 10, 20, 30, 40 and 50 μ L were added to a 96 well plate and adjusted to a final volume of 50 μ L/well with buffer at final concentrations of 0, 2, 4, 6, 8 and 10 nmoL/well.

(3) Adding lipase: 2 muL of lipase was added to each well, mixed well on a plate shaker and incubated for 20min at room temperature. The enzyme mixture was added to each well, incubated at room temperature for 60min, protected from light, and OD570 was measured.

The results of the experiment are calculated and collated and summarized in FIG. 11.

2) Determination of Total Cholesterol (TG) content

(1) Sample preparation:

chloroform: isopropyl alcohol: NP-40 cell lysates were prepared at a volume ratio of 7. Get 10 ⁶ One sample of each cell was added with 200. Mu.L of lysate and scraped (one well by one well lysis) with the bottom of the tip. Transferring to EP tube, centrifuging at 15000g for 5-10 min, collecting supernatant, transferring to new EP tube, and oven drying at 50 deg.C to remove chloroform. (at the same time, parallel samples were taken for BCA protein assay). After drying, the mixture was left in vacuum for 30min to completely remove the organic solvent (now, a standard curve was drawn). Add 200. Mu.L buffer and mix by vortexing. A42 μ L sample was taken for the experiment and Buffer complemented to 50 uL/well.

A:42 muL sample +8 muL Buffer

B:42 muL sample +8 muL standard (0.25 mug/muL)

(2) Making a standard curve:

and (4) taking 20 muL of the cholesterol standard substance (on ice), adding the cholesterol standard substance into 140 muL of buffer solution, uniformly mixing, and diluting to 0.25 mug/muL. 0, 4, 8, 12, 16 and 20 μ L were added to a 96-well plate and adjusted with buffer solution to 50 μ L/well in a final volume of 50 μ L/well with final concentrations of 0, 1, 2, 3, 4 and 5 μ g/well for 50, 46, 42, 38, 34 and 30 μ L. 50 muL of enzyme mixed liquor needs to be additionally added into each sample hole for incubation for 60min at 37 ℃, and OD570 is measured in a dark place.

The results of the experiment are calculated and collated and summarized in FIG. 12.

As can be seen from fig. 11 and 12, the triglyceride content and total cholesterol content of the FWKVVIAPW group were greatly reduced compared to the model group, and were close to those of normal cells. The antarctic krill oligopeptides can obviously reduce the content of triglyceride and total cholesterol in a NAFLD cell model, thereby demonstrating that the antarctic krill oligopeptides can reduce the accumulation of lipid in cells and play a role in reducing blood fat.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Zhejiang ocean university

<120> antarctic krill oligopeptide for adjuvant therapy of NAFLD and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Phe Trp Lys Val Val Ile Ala Pro Trp

1 5

Claims

1. An antarctic krill oligopeptide for adjuvant therapy of NAFLD, wherein the amino acid sequence of the oligopeptide is Phe-Trp-Lys-VaL-VaL-ILe-ALa-Pro-Trp.

2. Use of the antarctic krill oligopeptide of claim 1, having at least one of:

(1) Preparing a medicine for adjuvant therapy of NAFLD;

(2) Can be used as food additive.