A liver protection composition prepared from plant extract and corn-derived polypeptide

Abstract

The present invention provides a liver protection composition prepared from a plant extract and a corn-derived polypeptide, wherein the polypeptide is prepared by enzymolyzing corn residue with alkaline protease and ficin, and centrifuging, decolorizing and drying the enzymolysis product; the enzymatic activity ratio of the alkaline protease and ficin is 2:1; the molecular weight of the enzymolysis product is 500-3000Da; the amino acid sequence of one polypeptide fragment of the polypeptide is KDYASKNCLQSFMLM. The polypeptide provided by the present invention has the effect of protecting the liver of fish, and when used in combination with the plant extract, it can achieve a high concentration effect with a smaller dosage.

Description

Technical Field

[0001] The invention belongs to the technical field of fish breeding, and particularly relates to a liver protection composition prepared from a plant extract and a corn-derived polypeptide. Background Art

[0002] After decades of rapid development, my country ranks first in the world in aquaculture, offshore fishing, aquatic product consumption, and aquatic product exports. However, while intensive aquaculture has effectively promoted the development and progress of the industry, it has also caused serious disease problems. The development of intensive aquaculture and the increase in aquaculture density have led to the deterioration of the fish farming environment. The use of high-protein, high-fat or moldy feed, as well as the abuse of antibiotics and pesticides, have increased the burden on fish livers. The liver is an extremely important organ in the body, and most toxic substances are converted into low-toxic or non-toxic substances through the liver. Therefore, the liver is vulnerable to pathogens and metabolic waste, causing liver damage or severe liver disease. The weakened detoxification function of the liver will cause toxic substances to accumulate in the body, thereby affecting the normal operation of other organs. A series of symptoms caused by liver damage are collectively referred to as "fish hepatobiliary syndrome" in clinical practice, with a mortality rate of 50%-60%, which seriously affects the economic benefits of aquaculture. Moreover, in actual aquaculture production, antibiotics, pesticides, and antiviral drugs have little effect on fish hepatobiliary syndrome.

[0003] The liver of zebrafish with liver damage induced by thioacetamide is significantly smaller than that of normal zebrafish, the liver is degenerated and blackened, and the absorption of yolk sac is delayed (yolk sac is fat, and absorption is related to liver function). Zebrafish larvae show different degrees of deformed phenotypes after administration, abnormal nuclear structure of liver cells, a large number of fat vacuoles in the cytoplasm, and fat accumulation in liver cells. Moreover, the toxic effect of thioacetamide on the liver of zebrafish larvae is similar to the damage to the liver of other fish. Zebrafish can be used as a model animal for rapid evaluation, screening and research of drug liver toxicity.

[0004] Many peptides and plant extracts have the effect of protecting the liver, mainly by protecting the liver cell membrane, resisting lipid peroxidation and free radical damage, improving liver function, promoting liver cell regeneration and improving immunity. However, the production cost of peptide preparations is relatively high, mainly because the synthesis and modification of polypeptide chains are very complicated, requiring the use of expensive raw materials, reagents and equipment, which has become an important factor restricting the development and application of peptide preparations. Therefore, researchers in the field of aquaculture are trying to find new active polypeptide molecules from various resources, in the hope that polypeptides can be widely used and popularized as liver-protecting environmentally friendly feed additives in aquaculture. Summary of the invention

[0005] The present invention provides a liver protection composition prepared from plant extracts and corn-derived polypeptides, that is, on the basis of preparing a polypeptide having a protective effect on fish liver, the polypeptide and the plant extract are used in combination to prepare a product for protecting fish liver.

[0006] The invention firstly provides a polypeptide having a protective effect on fish liver. The polypeptide is prepared by enzymolyzing corn residue with alkaline protease and ficin, and centrifuging, decolorizing and drying the enzymolysis product.

[0007] As a specific record of an embodiment, the enzymatic activity ratio of the alkaline protease to the ficin is 2:1;

[0008] Furthermore, the molecular weight of the enzymatic hydrolysis product is 500-3000Da;

[0009] Furthermore, the amino acid sequence of one polypeptide fragment of the polypeptide is KDYASKNCLQSFMLM (SEQ ID NO: 1);

[0010] The present invention also provides a use of the prepared polypeptide, which is use as a feed additive;

[0011] The present invention also provides a use of the prepared polypeptide, which is use in preparing a product for protecting fish liver.

[0012] In another aspect, the present invention also provides a composition for protecting fish liver, wherein the composition comprises the polypeptide;

[0013] Furthermore, the composition also contains plant extracts; the plant extracts are water extracts of Apocynum venetum, Licorice and Ebony plum.

[0014] The polypeptide provided by the present invention has the effect of protecting fish liver, and when used in combination with plant extracts, a high-concentration effect can be achieved with a smaller dosage. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Changes in liver morphology and fluorescence in the liver area of ​​zebrafish larvae after treatment with different fractions of corn peptides, where Ctl is the control group and MC is the modeling group;

[0016] Figure 2 : HPLC spectrum of corn polypeptide;

[0017] Figure 3 : HPLC spectrum of corn liver protection peptide;

[0018] Figure 4: Changes in liver morphology and liver fluorescence of zebrafish larvae after treatment with corn peptide T1, where Ctl is the control group and MC is the model group;

[0019] Figure 5 : Changes in liver morphology and fluorescence in the liver area of ​​zebrafish larvae after treatment with different concentrations of T1, where Ctl is the control group and MC is the model group;

[0020] Figure 6 : Changes in liver morphology and fluorescence in the liver area of ​​zebrafish larvae after treatment with different concentrations of Y1, where Ctl is the control group and MC is the model group;

[0021] Figure 7 : Changes in liver morphology and fluorescence in the liver area of ​​zebrafish larvae after treatment with different concentrations of Y2, where Ctl is the control group and MC is the model group;

[0022] Figure 8 : Relative fluorescence area of ​​zebrafish liver in different treatment groups, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Ctl is the control group, and MC is the model group. DETAILED DESCRIPTION

[0023] The invention provides a corn liver-protecting peptide prepared by using corn residues, wherein the corn residues are byproducts obtained from corn starch production; the corn liver-protecting peptide is a polypeptide with liver-protecting function obtained by enzymatic hydrolysis of protein in the corn residues.

[0024] The alkaline protease and ficin used in the embodiments of the present invention are enzymes specified in the National Food Safety Standard for the Use of Food Additives (GB2760-2014).

[0025] The transgenic zebrafish Tg (L-FABP: EGFP) strain was provided by the zebrafish drug screening platform of the Institute of Biology, Shandong Academy of Sciences; thioacetamide (TAA) was purchased from Sigma-Alrdich, phenylthiourea (PTU) was purchased from ALDRICH, methylcellulose (MC) and other reagents and culture plates were domestically produced. SZX16 fluorescence microscope and DP2-BSW image acquisition system (Olympus, Japan); X51 inverted microscope (Olympus, Japan); Forma 3111 water-jacketed CO2 incubator (Forma, USA); zebrafish breeding equipment (Beijing Aisheng Technology Co., Ltd.)

[0026] In the embodiment, male and female zebrafish were raised in zebrafish breeding solution with a conductivity of 500-550 μS / cm (oxygenated, water temperature 28.5° C., pH 7.5±0.5), with a photoperiod of 14 h light / 10 h dark, and were fed with artificial granular bait and newly hatched Artemia nauplii at regular intervals every day.

[0027] Before ovulation, select normal adult zebrafish that have not ovulated within 2 weeks, place them in a mating tank at a female-to-male ratio of 1:1, separate the female and male fish with a partition, and raise them overnight. Remove the partition in the morning of the next day and let the zebrafish mate and lay eggs. Collect healthy fertilized eggs after 3 hours (blow away the clumped egg masses), add E3 culture medium (5mM NaCl, 0.17mM KCl, 0.33mM CaCl2 and 0.33mM MgSO4), place them in a 28.5℃ incubator for culture, and select dead eggs after 10 hours. Change the culture medium once a day.

[0028] Thioacetamide (TAA) stock solution was prepared by using zebrafish feeding water to prepare a 120 mmol / L stock solution according to the experimental dosage. The stock solution was then divided into portions and stored in the freezer. The stock solution was diluted to a specified final concentration of 6 mmol / L before use.

[0029] The present invention is described in detail below in conjunction with embodiments and drawings.

[0030] Example 1: Preparation of corn liver-protecting peptide

[0031] 1.1 Preparation of corn peptides using alkaline protease and ficin

[0032] The corn residue was crushed with a grinder, 100 g of corn residue was weighed, 1000 ml of distilled water was added, and the mixture was homogenized with a high-speed shearing machine for 30 min. 3.0 g of alkaline protease and ficin (enzyme activity ratio of 2:1) were added to the homogenate, and enzymolysis was performed at 55°C for 4 h. After the enzymolysis was completed, the mixture was boiled for 15 min. The enzymolysis solution was centrifuged, decolorized, concentrated and dried to obtain 36.27 g of corn polypeptide with a yield of 36.27% and a polypeptide purity of 49.28%.

[0033] 1.2 Isolation and purification of corn peptides

[0034] The corn polypeptide prepared in 1.1 was formulated into a 100 mg / ml solution and separated using a membrane filtration device, using 500Da, 3000Da and 10000Da membranes for separation, collecting different components and detecting their protective effects against liver damage.

[0035] 1.3 Hepatoprotective activity detection

[0036] Using zebrafish experiments, the activity of corn liver protective peptide was tested and it was found that the 500-3000Da part had obvious liver protective activity. The obtained sample was named corn liver protective peptide. Figure 1 shown.

[0037] 1.4 HPLC analysis

[0038] The corn polypeptide was analyzed by HPLC, and the analysis conditions were as follows: instrument: Agilent 1260 high performance liquid chromatograph, chromatographic column: Elite SimoChrom ODS-BP 5μm (4.6mm×250mm), mobile phase: methanol and 0.1% TFA water, flow rate: 1ml / min, detection wavelength: 220nm, method: 0min (5% methanol), 5min (5% methanol), 40min (95% methanol), 50min (95% methanol). The HPLC spectra of corn polypeptide and corn liver protective peptide are as follows: Figure 2 and Figure 3 shown.

[0039] Example 2: Sequence structure identification of corn liver protective peptide

[0040] The structure of the corn liver-protecting peptide prepared in Example 1 was identified, and the chromatographic conditions were as follows: instrument type: LCMS-9030Q-TOF quadrupole-time-of-flight mass spectrometer, chromatographic column: Inertsil HILIC C18 reverse phase chromatographic column 150 mm×3.0 mm, 3 μm, Shimadzu, Japan), column temperature: 30°C, injection volume: 1.0 μL; mobile phase: A is 0.1% (v / v) formic acid aqueous solution, mobile phase B is 0.1% (v / v) formic acid acetonitrile solution, gradient elution program: 0-5.0 min, 5%, 5.0-10.0 min, 10%, 10.0-15.0 min, 20%, 15.0 -20.0min, 40%; flow rate is 0.3mL / min, mass spectrometry ion mode: ESI source, positive ion mode, ion source interface voltage: 4.0kV, gas flow rate: 10.0L / min, drying gas flow rate: 10.0L / min, heating gas flow rate: 10.0L / min; heating module temperature: 400℃, interface temperature: 300℃, scanning mode: full scan mode (m / z 50-2000).

[0041] The mass spectrometry information obtained was identified using proteomics technology, and the effective polypeptide sequences obtained were (Table 1).

[0042] Table 1: Identification data of corn liver protective peptides

[0043]

[0044] The peptide sequences in Table 1 were synthesized using solid phase peptide synthesis and prepared into a peptide solution with a concentration of 200 μg / mL. Figure 4 As shown, when the modeling drug was added at the same time, 200 μg / mL of corn polypeptide T1 (KDYASKNCLQSFMLM, SEQ ID NO: 1) could significantly reduce the damage to the liver caused by the modeling drug, and there was no significant difference between the other sequence corn polypeptides and the modeling group.

[0045] Example 3: Liver protection effect of the combination of corn polypeptide T1 and plant extracts

[0046] Three sample experimental groups were set up for the experiment. The specific groupings are as follows:

[0047] Sample 1: polypeptide T1 (KDYASKNCLQSFMLM, SEQ ID NO: 1)

[0048] Sample 2: Plant extract Y1 (30 grams of Apocynum venetum, 10 grams of Licorice, and 10 grams of Plum, added to 1:10 distilled water and soaked for 30 minutes, boiled for 30 minutes, filtered through four layers of gauze, and the filtered components were continued to be boiled with water, repeated twice, and the filtrate was finally combined, concentrated, and dried.)

[0049] Sample 3: Y2 (T1:Y1=1:1)

[0050] Normally developed zebrafish fry at 3 dpf after fertilization were selected and randomly divided into a blank control group (only E3 fish water), a modeling group (TAA with a final concentration of 6 mmol / L prepared with E3 fish water), and a sample treatment group (containing 6 mmol / L TAA and T1, Y1 and Y2 samples with sample concentrations of 50, 100, and 200 μg / mL, respectively). They were added to a 12-well plate for culture, with 10 fish per well, a liquid volume of 3 mL, and 2 replicate wells. All groups were cultured in a 28.5°C incubator for 3 days, and fresh fish water and drugs were replaced every day. After the culture was completed, the residual drug solution was washed off with fish water, and then the zebrafish was sucked out and anesthetized with 0.3% (w / v) tricaine for 1 min. The zebrafish was adjusted to the left side on a slide containing methylcellulose and photographed under a fluorescence microscope. ImageJ-win32 was used to measure the liver area of ​​zebrafish. The liver area of ​​the blank control was taken as 100, and the relative fluorescence area of ​​each treatment group was calculated and statistically compared.

[0051] GraphPad 7.0 software was used to perform statistical analysis on the experimental data, and the sample treatment results were compared with those of the simple modeling group.

[0052] The morphology and liver fluorescence of transgenic zebrafish Tg(L-FABP:EGFP) in each treatment group are shown in Figure 2 Figure 5As shown, the blank control group (represented by Ctl) had normal zebrafish swim bladders, plump liver areas, and the fluorescent images showed that the liver morphology was intact; while the model group (represented by MC) had deformed swim bladders and shrunken liver areas, and the fluorescent images showed that the liver area was reduced, especially the liver area close to the swim bladder was more severely damaged; 30% to 38% of the fish in the 100 and 200μg / mL T1 groups had normal swim bladders and intact liver morphology. Figure 8 It can be seen that the difference between the two concentrations of T1 group and the simple modeling group reached an extremely significant level (p < 0.001), and the 50μg / mL T1 group also had a significant difference compared with the simple modeling group (p < 0.5). Similarly, 100 and 200μg / mL Y1 can also significantly reduce the damage to the liver caused by the modeling drug ( Figure 6 ), compared with the model group, the difference in the 200μg / mL group reached an extremely significant level (p < 0.01), the difference in the 100μg / mL group reached a significant level (p < 0.05), and there was no significant difference between the 50μg / mL Y1 group and the simple modeling group. Figure 7 and Figure 8 It can be seen that the differences in the Y2 groups at 50, 100 and 200 μg / mL were more significant compared with the T1, Y1 groups and the simple modeling group (p < 0.0001).

[0053] Using transgenic zebrafish Tg (L-FABP: EGFP) with green fluorescence in the liver as a model, zebrafish fry born 3 dpf were modeled with 6 mmol / L TAA and samples T1, Y1 and Y2 of different concentrations were added simultaneously. After 3 days of treatment, the three groups of samples had a good protective effect against liver damage caused by 6 mmol / L TAA. The low concentration T1 group could achieve a strong liver protection effect, and the combined use of peptides and plant extracts had a more significant liver protection effect.

Claims

1. A polypeptide having a protective effect on fish liver, characterized in that: The amino acid sequence of the polypeptide is KDYASKNCLQSFMLM.

2. A fish feed, characterized in that: The fish feed contains the polypeptide according to claim 1.

3. Use of the polypeptide according to claim 1 in the preparation of a product for protecting fish liver.

4. A composition for protecting fish liver, characterized in that: The composition comprises the polypeptide according to claim 1.

5. The composition according to claim 4, characterized in that The composition also contains plant extracts with liver-protecting effects.

6. The composition according to claim 5, characterized in that The plant extract is water extract of rhubarb, liquorice and ebony.

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