Silymarin peptide, and preparation method and application thereof

Abstract

The embodiment of the present application discloses a kind of silybum marianum peptide and its preparation method and application, belong to the technical field of bioactive peptide.The silybum marianum peptide includes peptide chain 1, peptide chain 2 and peptide chain 3, the peptide chain 1 includes the amino acid sequence shown in SEQ ID NO:1, peptide chain 2 includes the amino acid sequence shown in SEQ ID NO:2, and peptide chain 3 includes the amino acid sequence shown in SEQ ID NO:3.The silybum marianum peptide of the present application has the effect of significantly improving or relieving non-alcoholic liver injury, can effectively regulate lipid metabolism, reduce oxidative stress and inflammatory response, and provides a new effective means for the treatment of non-alcoholic liver injury.The silybum marianum peptide of the present application is derived from natural silybum marianum, has high safety, small side effect, can be used in medicine, health food and other fields, has good social and economic benefits.The preparation method of the present application is simple, low in cost, suitable for large-scale industrial production, and has wide market application prospect.

Description

TECHNICAL FIELD

[0001] The embodiment of the present application relates to the technical field of bioactive peptides, in particular to a silybum marianum peptide and a preparation method and application thereof. BACKGROUND

[0002] Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide, and its incidence is increasing year by year. NAFLD is mainly caused by insulin resistance and genetic susceptibility, leading to excessive deposition of fat in the liver, and is not related to the amount of alcohol consumed by patients. At present, the treatment of non-alcoholic liver injury in clinic is limited, mainly relying on lifestyle intervention and drug treatment. However, traditional drug treatment has the problems of large side effects and long-term use of other adverse effects on the body. Therefore, it is urgent to develop safe and effective methods or substances for treating non-alcoholic liver injury.

[0003] Silybum marianum is a traditional medicinal plant, and its main active ingredient silymarin has the effects of antioxidant, anti-inflammatory, and protection of hepatocytes, and shows good application prospect in the treatment of liver diseases. However, direct use of silymarin has the problems of low bioavailability and limited efficacy. It is expected to improve the bioactivity and utilization rate of silymarin by converting the effective components in silybum marianum into bioactive peptides, and to provide a new solution for the treatment of non-alcoholic liver injury. SUMMARY

[0004] To this end, the embodiment of the present application provides a silybum marianum peptide and a preparation method and application thereof.

[0005] In order to achieve the above-mentioned purpose, the embodiment of the present application provides the following technical scheme:

[0006] According to the first aspect of the embodiment of the present application, the silybum marianum peptide comprises a peptide chain 1, a peptide chain 2 and a peptide chain 3, the peptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 1, the peptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the peptide chain 3 comprises the amino acid sequence shown in SEQ ID NO: 3.

[0007] According to the second aspect of the embodiment of the present application, the present application provides a preparation method of the silybum marianum peptide as described above, which comprises:

[0008] The silybum marianum seed is used as raw material, and is sequentially pretreated, subjected to first enzymolysis using papain, first enzyme inactivation, second enzymolysis using acid protease, second enzyme inactivation, and separation and purification.

[0009] Further, the pretreatment comprises: after the silybum marianum seed is soaked, pure water is added, homogenized, and extracted by a flash extractor for 5-10 min.

[0010] Further, the one enzymolysis condition is that the dosage of papain is 3 ‰ of the net weight of the S. mooreana seed, and the reaction is carried out at 50 DEG C for 2 hours.

[0011] The one enzyme inactivation condition is that the reaction system of the one enzymolysis is heated to 80 DEG C and kept for 10 minutes.

[0012] Further, the two enzymolysis condition is that the reaction system of the one enzyme inactivation is adjusted to pH = 3.0 by HCl, the dosage of acid protease is 3 ‰ of the net weight of the S. mooreana seed, and the reaction is carried out at 38 DEG C for 12 hours.

[0013] The two enzyme inactivation condition is that the reaction system of the two enzymolysis is heated to 80 DEG C and kept for 10 minutes.

[0014] Further, the separation and purification includes that the reaction system of the two enzyme inactivation is filtered through a stainless steel plate frame filter while hot, the obtained filtrate is adjusted to pH = 8.0 by NaOH, kept at 90 DEG C for 10 minutes, filtered through a stainless steel plate frame filter again while hot, cooled to 40 DEG C, and ultrafiltrated by using a membrane package with a molecular weight cut-off of 6 KDa, and the obtained ultrafiltrate is filtered through a 0.22 mu m and 0.1 mu m sterilization filter in series to obtain a filtrate.

[0015] According to a third aspect of the embodiments of the present application, the present application provides the S. mooreana peptide as described above, or the application of the S. mooreana peptide prepared by any one of the methods described above in the preparation of a drug for improving or relieving non-alcoholic liver injury.

[0016] According to a fourth aspect of the embodiments of the present application, the present application provides the S. mooreana peptide as described above, or the application of the S. mooreana peptide prepared by any one of the methods described above in the preparation of a health food for improving or relieving non-alcoholic liver injury.

[0017] The embodiments of the present application have the following advantages:

[0018] 1. The S. mooreana peptide has a significant effect of improving or relieving non-alcoholic liver injury, can effectively regulate lipid metabolism, reduce oxidative stress and inflammatory response, and provides a new effective means for the treatment of non-alcoholic liver injury.

[0019] 2. The S. mooreana peptide is derived from natural S. mooreana, has high safety and small side effects, can be used in multiple fields such as medicine and health food, and has good social and economic benefits.

[0020] 3. The preparation method is simple, low in cost, suitable for large-scale industrial production, and has a wide market application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only exemplary, and for those skilled in the art, other drawings can also be obtained from the provided drawings without creative labor.

[0022] Figure 1 Mass spectrometry analysis results of the peptide chain 1 of the S. mooreana peptide provided by the present application;

[0023] Figure 2 Safety and biological toxicity detection results of the S. mooreana peptide provided by the present application;

[0024] Figure 3 Protective effects of the S. mooreana peptide on liver and spleen provided by the present application;

[0025] Figure 4 The S. mooreana peptide provided by the present application reduces cell damage by inhibiting the TRAF3 / NIK / NF-κB signaling pathway;

[0026] Figure 5 Research on the S. mooreana peptide regulating intestinal flora provided by the present application. DETAILED DESCRIPTION

[0027] The embodiments of the present application are described below by specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in the description. Obviously, the described examples are part of the examples of the present application, not all. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0028] Example 1

[0029] Preparation of S. mooreana peptide

[0030] (1) Raw material treatment

[0031] The S. mooreana seeds were washed with water to remove foreign matter, and then soaked in pure water for 12 hours for swelling.

[0032] (2) Water preparation

[0033] After swelling, the S. mooreana seeds were poured into a reaction tank, and 3 times of pure water based on the net weight of the seeds was added. The mixture was uniformly grinded by a colloid mill and extracted by a flash extractor for 5 minutes, and then stirred uniformly.

[0034] (3) Temperature rise: the mixture after water preparation was heated to 50℃ under stirring.

[0035] (4) Enzymatic reaction:

[0036] ① First enzymatic hydrolysis. After warming, the milk thistle seed homogenate was weighed, 3‰ papain was added, dissolved in a small amount of water, slowly poured into the reaction tank under stirring, stirred evenly, and incubated at 50°C for 2h. During the incubation, intermittent stirring was performed every 3-5min.

[0037] Enzyme inactivation was then performed. The first enzymatic hydrolysis product was warmed to 80°C under constant stirring, stirred and incubated for 10min, and then cooled to 50°C.

[0038] ② Second enzymatic hydrolysis. After enzyme inactivation and cooling, the first enzymatic hydrolysis product of the milk thistle seed was obtained, the pH was adjusted to 3.0 with HCl, 3‰ acid protease was added, dissolved in a small amount of pure water, slowly poured into the reaction tank under stirring, stirred evenly, and incubated at 38°C for 12h. During the incubation, intermittent stirring was performed every 3-5min.

[0039] Enzyme inactivation was then performed. The second enzymatic hydrolysis product was warmed to 80°C under constant stirring, stirred and incubated for 10min.

[0040] (5) Filtration: after inactivation, the second enzymatic hydrolysis product was obtained, and a stainless steel plate filter was used for hot filtration to obtain the filtrate.

[0041] (6) Removal of basic proteins: the filtrate was adjusted to pH 8.0 with NaOH, stirred and incubated at 90°C for 10min.

[0042] (7) Filtration: after removing the basic proteins from the polypeptide solution, a stainless steel plate filter was used for hot filtration again, and the temperature was reduced to 40°C.

[0043] (8) Ultrafiltration: a membrane with a molecular weight cut-off of 6KDa was selected for ultrafiltration, and the ultrafiltrate was collected.

[0044] (9) Precision filtration: the ultrafiltrate was precision filtered through a 0.22μm, 0.1μm sterilization filter in series to obtain the filtrate.

[0045] Example 2

[0046] Analysis of milk thistle peptide

[0047] 1. Sample preparation

[0048] 1.1 Materials and reagents

[0049] Trypsin, Promega; acetonitrile, Fisher Chemical; formic acid, Fluka; ammonium bicarbonate (NH4HCO3), Sigma; dithiothreitol, Sigma; iodoacetamide, Sigma.

[0050] 1.2 In-gel digestion

[0051] The gel strips were cut into pieces and the gel pieces were destained using 50% acetonitrile with 50 mM ammonium bicarbonate (NH4HCO3). The gel pieces were dehydrated by incubation with 100% acetonitrile for 5 min, after which the liquid phase was removed and a solution of dithiothreitol (DTT) was added to a final concentration of 10 mM and incubated at 37 °C for 60 min. The gel pieces were dehydrated again by incubation with 100% acetonitrile and after removal of the liquid phase, iodoacetamide was added to a final concentration of 55 mM and incubated at room temperature for 45 min in the dark. The gel pieces were then washed with 50 mM ammonium bicarbonate and dehydrated again by incubation with 100% acetonitrile. Finally, the gel pieces were resuspended in 50 mM ammonium bicarbonate with 10 ng / μΐ of trypsin and incubated on ice for 1 h. After removal of the excess solution from the sample, the gel pieces were digested overnight at 37 °C. The peptides were extracted from the gel pieces using 50% acetonitrile / 5% formic acid and 100% acetonitrile, respectively, and the peptide solutions were stored frozen until use.

[0052] 2. Liquid chromatography-mass spectrometry analysis

[0053] 2.1 Materials and reagents

[0054] Ultrapure water (H2O), Fisher Chemical; acetonitrile, Fisher Chemical; formic acid, Fluka.

[0055] Mass spectrometer: Thermo Scientific™ Q Exactive Plus.

[0056] 2.2 Mass spectrometry parameter settings

[0057] The peptides were separated using an EASY-nLC 1000 ultra-high performance liquid system after dissolution in liquid chromatography mobile phase A. Mobile phase A was a solution of 0.1% formic acid and 2% acetonitrile in water; mobile phase B was a solution of 0.1% formic acid and 90% acetonitrile in water. The liquid chromatography gradient was set as follows: 0-22 min, 6%-35% B; 22-26 min, 35%-80% B; 26-30 min, 80% B, at a flow rate of 550 nL / min.

[0058] The peptide fragments were injected into the NSI ion source for ionization and then into the Thermo Scientific™ Q Exactive Plus mass spectrometer for analysis after separation by an ultra-high performance liquid system. The ion source voltage was set to 2.2 kV, and both the peptide fragment parent ions and their secondary fragments were detected and analyzed using high-resolution Orbitrap. The primary mass spectrometry scan range was set to 350-1800 m / z, and the scan resolution was set to 70,000; the secondary scan resolution was set to 17,500. The data acquisition mode used a data-dependent scan (DDA) program, i.e., after the primary scan, the top 20 peptide fragment parent ions with the highest signal intensity were selected in turn to enter the HCD collision pool for fragmentation using a fragmentation energy of 28%, and secondary mass spectrometry analysis was also performed in turn. In order to improve the effective utilization rate of the mass spectrometer, the automatic gain control (AGC) was set to 5e4, the signal threshold was set to 5e3 ions / s, the maximum injection time was set to 200 ms, and the dynamic exclusion time of tandem mass spectrometry scanning was set to 20.0 s to reduce repeated scanning of the parent ions.

[0059] 2.3 Database search

[0060] Tandem mass spectrometry (MS / MS) data of the peptide fragments: The peptide sequence was deduced from the mass-to-charge ratio (m / z) of the b ions (N-terminal fragments, marked in blue) and the y ions (C-terminal fragments, marked in red). Through mass spectrometry analysis of the silymarin peptides, it was found that the silymarin peptides prepared by the present application include peptide chain 1, peptide chain 2, and peptide chain 3, the peptide chain 1 includes the amino acid sequence shown in SEQ ID NO: 1 (WFNRNADEEEGGEL), the peptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 2 (GPHYNPHGKEHGAPDDEVR), and the peptide chain 3 includes the amino acid sequence shown in SEQ ID NO: 3 (TILPDSEGAIDGHL). Taking the WFNRNADEEEGGEL peptide fragment as an example, the mass spectrometry analysis result is shown in Figure 1

[0061] Example 3

[0062] An acute liver injury model was established in mice treated with carbon tetrachloride (CCl4), and the effects of different doses of silymarin peptides were evaluated. The main observation indicators included measuring liver injury markers (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and performing histopathological analysis on liver tissue. This study also explored the regulatory effects of silymarin peptides on the nuclear factor kappa B (NF-κB) signaling pathway, intestinal microbiota, and metabolite profiles.

[0063] (1) Construction of an acute liver injury animal model

[0064] ​8-week-old adult male C57BL / 6J mice were raised in an environment with indoor temperature of 22±2℃, relative humidity of 50%, and light cycle set to 12 hours of light and 12 hours of darkness. The mice could freely drink clean water and eat special-purpose maintenance feed. An acute liver injury model was prepared by intraperitoneal injection of 2g / mL CCl4 vegetable oil solution (dose of 10mg / kg). The mice were observed 2 hours after injection. If symptoms such as decreased appetite, increased water intake, dull and sparse hair, arched back, prominent ear margin vein, rapid breathing, and wet rales in liver auscultation were observed, it indicated that the modeling was successful.

[0065] (2) Grouping method

[0066] The control group was given physiological saline by gavage daily from day 1 to day 14, and a single intraperitoneal injection of olive oil on day 14. The CCl4 group was also given physiological saline by gavage daily, and additionally injected with 10μL / g body weight of 10% CCl4 (dissolved in olive oil) intraperitoneally on the last day. The treatment groups were given different doses of polypeptides on the basis of CCl4 modeling: the low-dose group was given 50mg / kg SMP by gavage, the medium-dose group was given 100mg / kg, and the high-dose group was given 200mg / kg. All mice were given daily gavage. These treatment groups were also injected with 10μL / g body weight of 10% CCl4 (dissolved in olive oil) intraperitoneally on day 14.

[0067] (3) Liver wet weight, spleen wet weight, liver index and spleen index determination and blood glucose monitoring

[0068] The mice were dissected to obtain the liver and spleen, which were gently rinsed with physiological saline to remove blood stains, and then weighed after drying. Among them, the liver organ index = liver weight (g) / mouse body weight (g), and the spleen organ index = spleen weight (g) / mouse body weight (g).

[0069] About 5μL of blood was collected from the tip of the mouse tail every day using a portable blood glucose meter (Bayer, Leverkusen, Germany) to monitor the fasting blood glucose concentration.

[0070] ELISA method for detecting NRF2, TRAF3, NIK, P52

[0071] Using the mouse serum prepared in each group, the levels of NRF2, TRAF3, NIK, and P52 were determined according to the instructions of the ELISA kit (Elabscience). The operation steps are as follows: 50-100μL of sample was added to each reaction well, and after incubation at 37℃ for 0.5-1 hour, it was washed. On the ELISA detector, the blank control well was adjusted to zero at 450nm wavelength, and the absorbance OD value of each well was detected.

[0072] (4) Results

[0073] 1、8-week-old adult male C57BL / 6J mice were given a maximum dose of peptide per day: 200 mg / kg SMP, for 14 consecutive days. The control group was given an equal amount of normal saline by gavage daily. There was no difference in body weight, fasting blood glucose value, liver and spleen wet weight between the control group and the SMP group (p>0.05, Figure 2 A-C), SMP treatment had no effect on serum ALT activity (p>0.05, Figure 2 D). The results showed that the milk thistle peptide had no toxicity to mice.

[0074] 2、CCl4 induces abnormal liver and spleen in mice, such as liver fibrosis, spleen cell infiltration, etc. After the intervention of milk thistle peptide, the liver index decreased to different degrees, the liver tissue morphology gradually approached normal, the spleen index increased to different degrees, and the spleen tissue pathological state improved. The results showed that SMP had repair and protection effects on liver damage and spleen damage (see Figure 3 A-B). After the intervention of milk thistle peptide, the number of apoptotic cells decreased, indicating that SMP could inhibit hepatocyte apoptosis (see Figure 3 C). The results showed that in the acute liver injury mice induced by carbon tetrachloride, milk thistle peptide showed good liver protection effect, significantly enhanced the antioxidant index of liver, inhibited the expression of pro-inflammatory cytokines, and reduced immune cell infiltration.

[0075] 3、SMP reversed the decrease of Nrf2 and TRAF3 protein expression and the increase of NIK and p52 protein expression in the liver of CCl4-induced mice (see Figure 4 D-F). The results showed that milk thistle peptide regulated the expression of key proteins in the NIK / NF-κB signaling pathway in mice, and increased the expression of antioxidant marker protein nuclear factor E2-related factor 2 (Nrf2).

[0076] 4、Analysis of the cecum non-target metabolome of mice, compared with the carbon tetrachloride liver injury model group, there were 67 kinds of differential metabolites in the cecal contents of the milk thistle peptide group, which enriched the galactose metabolism pathway (see Figure 5 ), the results showed that milk thistle peptide could regulate the diversity of cecal microorganisms of acute liver injury mice induced by carbon tetrachloride.

[0077] Conclusion:

[0078] The application discloses milk thistle peptide which can effectively improve or relieve non-alcoholic liver damage. The bioactive peptide is extracted from milk thistle and treated by specific enzymolysis technology, has specific arrangement of amino acid sequence, and can accurately act on liver cells.

[0079] In the preparation method, the S. mooreana is used as raw material, pretreated, and then subjected to complex enzymatic hydrolysis by using specific proportion of protease under suitable temperature and pH value, and then subjected to separation and purification by ultrafiltration, ion exchange chromatography and reverse phase chromatography to obtain high-purity S. mooreana peptide.

[0080] The S. mooreana peptide effectively relieves acute liver injury by inhibiting TRAF3 / NIK / NF-κB signal pathway and regulating intestinal microbiota.

[0081] The S. mooreana peptide of the present application can be used for preparing medicine for treating non-alcoholic liver injury in the medical field, and can be used as raw material for preparing health products for preventing and improving non-alcoholic liver injury in the health food field, and has wide application prospect.

[0082] Although the present application has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application are within the scope of the present application.

[0083]

[0084]

Claims

1. A silymarin peptide, characterized in that, The SOD peptide consists of peptide chain 1, peptide chain 2 and peptide chain 3, the amino acid sequence of the peptide chain 1 is SEQ ID NO: 1, the amino acid sequence of the peptide chain 2 is SEQ ID NO: 2, and the amino acid sequence of the peptide chain 3 is SEQ ID NO:

3.

2. The use of the SOD peptide of claim 1 in the preparation of a drug for improving or relieving non-alcoholic liver injury.

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