Silybum marianum peptide as well as preparation method and application thereof

Abstract

The embodiment of the invention discloses silybum marianum peptide as well as a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The silybum marianum peptide comprises a peptide chain 1, a peptide chain 2 and a peptide chain 3, the peptide chain 1 comprises an amino acid sequence as shown in SEQ ID NO: 1, the peptide chain 2 comprises an amino acid sequence as shown in SEQ ID NO: 2, and the peptide chain 3 comprises an amino acid sequence as shown in SEQ ID NO: 3. The silybum marianum peptide disclosed by the invention has a remarkable effect of improving or relieving the non-alcoholic liver injury, can effectively regulate lipid metabolism and relieve oxidative stress and inflammatory response, and provides a new effective means for treating the non-alcoholic liver injury. The silybum marianum peptide disclosed by the invention is derived from natural silybum marianum, is high in safety and small in side effect, can be applied to multiple fields of medicines, health foods and the like, and has good social benefits and economic benefits. The preparation method is simple, low in cost and suitable for large-scale industrial production, and has a wide market application prospect.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of bioactive peptides, and specifically to silymarin and its preparation method and application. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide, and its incidence rate is increasing year by year. NAFLD is mainly caused by factors such as insulin resistance and genetic susceptibility, which lead to excessive deposition of fat in the liver and is unrelated to the patient's alcohol consumption. At present, there are limited clinical treatments for non-alcoholic liver damage, which mainly rely on lifestyle intervention and drug therapy. However, traditional drug treatments have problems such as large side effects and long-term use that can easily cause other adverse effects on the body. Therefore, it is urgent to develop safe and effective methods or substances to treat non-alcoholic liver damage.

[0003] Milk thistle, a traditional medicinal plant, has a major active ingredient, silymarin, which exhibits numerous benefits, including antioxidant, anti-inflammatory, and hepatocellular protection, showing promising application prospects in the treatment of liver diseases. However, direct use of silymarin suffers from low bioavailability and limited efficacy. Converting the active ingredients in milk thistle into bioactive peptides is expected to enhance their bioactivity and utilization, providing a novel solution for the treatment of non-alcoholic liver injury. Summary of the Invention

[0004] To this end, the embodiments of the present invention provide a silymarin peptide and a preparation method and application thereof.

[0005] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] According to a first aspect of an embodiment of the present invention, the silymarin includes peptide chain 1, peptide chain 2 and peptide chain 3, wherein the peptide chain 1 includes the amino acid sequence shown in SEQ ID NO: 1, the peptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 2, and the peptide chain 3 includes the amino acid sequence shown in SEQ ID NO: 3.

[0007] According to a second aspect of the embodiments of the present invention, the present invention provides a method for preparing silymarin as described above, the method comprising:

[0008] The milk thistle seeds are used as raw materials, and the product is prepared through the following steps: pretreatment, primary enzymatic hydrolysis with papain, primary enzyme inactivation, secondary enzymatic hydrolysis with acidic protease, secondary enzyme inactivation, and separation and purification.

[0009] Furthermore, the pretreatment includes: soaking the milk thistle seeds, adding pure water, homogenizing, and extracting with a flash extractor for 5-10 minutes.

[0010] Furthermore, the conditions for the primary enzymatic hydrolysis are: the amount of papain used is 3‰ of the net weight of the milk thistle seeds, and the temperature is kept at 50°C for 2 hours;

[0011] The conditions for the primary enzyme inactivation are: heating the reaction system of the primary enzymatic hydrolysis to 80° C. and keeping the temperature for 10 minutes.

[0012] Furthermore, the conditions for the secondary enzymatic hydrolysis are as follows: the reaction system of the primary enzyme inactivation is adjusted to pH=3.0 by HCl, the amount of acidic protease used is 3‰ of the net weight of the milk thistle seeds, and the reaction system is kept at 38°C for 12 hours;

[0013] The conditions for the secondary enzyme inactivation are: heating the secondary enzymatic hydrolysis reaction system to 80° C. and keeping the temperature for 10 minutes.

[0014] Furthermore, the separation and purification includes: passing the reaction system in which the secondary enzyme is inactivated while hot through a stainless steel plate and frame filter, adjusting the pH of the obtained filtrate to 8.0 with NaOH, keeping it at 90°C for 10 minutes, passing it through a stainless steel plate and frame filter again while hot, cooling it to 40°C, selecting a membrane package with a molecular weight cutoff of 6KDa for ultrafiltration, and finely filtering the obtained ultrafiltrate through 0.22μm and 0.1μm sterilizing filters in series to obtain a filtrate.

[0015] According to a third aspect of the embodiments of the present invention, the present invention provides the use of the silymarin as described above, or the silymarin prepared by the method described in any one of the above items, in the preparation of a drug for improving or alleviating non-alcoholic liver injury.

[0016] According to a fourth aspect of the embodiments of the present invention, the present invention provides the use of the silymarin as described above, or the silymarin prepared by the method described in any one of the above items, in the preparation of a health food for improving or alleviating non-alcoholic liver damage.

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

[0018] 1. The silymarin of the present invention has a significant effect of improving or alleviating non-alcoholic liver damage, can effectively regulate lipid metabolism, reduce oxidative stress and inflammatory response, and provides a new and effective means for the treatment of non-alcoholic liver damage.

[0019] 2. The silymarin of the present invention is derived from natural milk thistle, has high safety, few side effects, and can be used in multiple fields such as medicine and health food, with good social and economic benefits.

[0020] 3. The preparation method of the present invention is simple, low-cost, suitable for large-scale industrial production, and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0022] Figure 1 The mass spectrometry analysis results of the silymarin peptide chain 1 provided by the present invention;

[0023] Figure 2 The safety and biological toxicity test results of silymarin provided by the present invention;

[0024] Figure 3 The protective effect of silymarin on the liver and spleen provided by the present invention;

[0025] Figure 4 The silymarin provided by the present invention reduces cell damage by inhibiting the TRAF3 / NIK / NF-κB signaling pathway;

[0026] Figure 5 The present invention provides research on the regulation of intestinal microbiota by silymarin. DETAILED DESCRIPTION

[0027] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0028] Example 1

[0029] Preparation of Silymarin

[0030] (1) Raw material processing

[0031] Wash the milk thistle seeds with water to remove foreign matter, then soak them in pure water for 12 hours and set aside.

[0032] (2)Water distribution

[0033] After soaking, pour the milk thistle seeds into the reaction tank, add pure water 3 times the net weight of the seeds, homogenize with a colloid mill, extract with a flash extractor for 5 minutes, and stir evenly.

[0034] (3) Heating: After adding water, the mixture is heated to 50°C while stirring.

[0035] (4) Enzymatic hydrolysis reaction:

[0036] ① Primary enzymatic hydrolysis. After heating, homogenize the milk thistle seeds. Weigh papain at 3‰ of the net weight of the seeds. Dissolve it in a small amount of water and slowly pour it into the reaction tank while stirring. Stir evenly and keep warm at 50℃ for 2 hours. Stir intermittently for 3-5 minutes each time.

[0037] Then, the enzyme was inactivated. The temperature of the primary enzymatic hydrolysate was raised to 80°C while stirring continuously, stirred and kept warm for 10 minutes, and then cooled to 50°C.

[0038] ② Secondary enzymatic hydrolysis. After enzyme inactivation and cooling, adjust the pH of the milk thistle seed primary enzymatic hydrolysate to 3.0 with HCl. Weigh 3‰ of the seed net weight of acidic protease and dissolve it in a small amount of pure water. Slowly pour it into the reaction tank while stirring, stir evenly, and keep it warm at 38℃ for 12 hours. During this period, stir intermittently for 3-5 minutes each time.

[0039] Then, the enzyme was inactivated. The temperature of the secondary enzymatic hydrolysate was raised to 80°C with constant stirring and kept warm for 10 minutes.

[0040] (5) Filtration: After inactivation, the secondary enzymatic hydrolysate is obtained and filtered while hot using a stainless steel plate and frame filter to obtain the filtrate.

[0041] (6) Removal of alkaline proteins: adjust the pH of the filtrate to 8.0 with NaOH, and heat at 90°C for 10 min with stirring.

[0042] (7) Filtration: After removing the alkaline protein, the polypeptide solution is filtered again using a stainless steel plate and frame filter while hot and cooled to 40°C.

[0043] (8) Ultrafiltration: Use a membrane package with a molecular weight cutoff of 6 kDa for ultrafiltration and collect the ultrafiltrate.

[0044] (9) Fine filtration: The ultrafiltrate is finely filtered through 0.22 μm and 0.1 μm sterilizing filters in series to obtain the filtrate.

[0045] Example 2

[0046] Analysis of Silymarin

[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 enzymatic digestion

[0051] Cut the gel strip into small pieces and decolorize the gel using 50% acetonitrile containing 50mM ammonium bicarbonate (NH4HCO3). Dehydrate the gel using 100% acetonitrile for 5 minutes. Remove the liquid phase and add dithiothreitol solution to a final concentration of 10mM. Incubate at 37°C for 60 minutes. Dehydrate the gel again using 100% acetonitrile. Remove the liquid phase and add iodoacetamide to a final concentration of 55mM. Incubate at room temperature in the dark for 45 minutes. Wash with ammonium bicarbonate to a final concentration of 50mM and dehydrate again using 100% acetonitrile. Resuspend the gel in 50mM ammonium bicarbonate containing 10ng / μl trypsin and incubate on ice for 1 hour. Remove excess solution from the sample and digest the gel overnight at 37°C. The enzymatically hydrolyzed peptides were extracted from the gel using 50% acetonitrile / 5% formic acid and 100% acetonitrile, respectively. The peptide solutions were freeze-dried and then used for standby 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 ScientificTMQ Exactive Plus.

[0056] 2.2 Mass spectrometry parameter settings

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

[0058] After separation by ultra-high performance liquid chromatography (UPLC), peptides were injected into the NSI ion source for ionization and subsequently analyzed by a ThermoScientific TMQ Exactive Plus mass spectrometer. The ion source voltage was set at 2.2 kV, and peptide precursor ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap system. The primary mass spectrometer scan range was set from 350 to 1800 m / z at a resolution of 70,000, and the secondary mass spectrometer scan resolution was set at 17,500. Data acquisition used a data-dependent scanning (DDA) program. After the primary scan, the top 20 peptide precursor ions with the highest signal intensity were selected and sequentially injected into the HCD collision cell for fragmentation at 28% fragmentation energy. These fragments were then analyzed sequentially by secondary mass spectrometry. To maximize mass spectrometer utilization, the automatic gain control (AGC) was set to 5e4, the signal threshold to 5e3 ions / s, the maximum injection time to 200 ms, and the dynamic exclusion time for the tandem mass spectrometer scan to 20.0 s to minimize precursor ion duplication.

[0059] 2.3 Database Search

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

[0061] Example 3

[0062] Mice treated with carbon tetrachloride (CCl4) were used to establish an acute liver injury model and evaluate the effects of different doses of silymarin. The primary outcome measures included measurement of liver injury markers (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and histopathological analysis of liver tissue. This study also explored the regulatory effects of silymarin on the nuclear factor κB (NF-κB) signaling pathway, intestinal microbiota, and metabolite profile.

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

[0064] Eight-week-old adult male C57BL / 6J mice were housed in an environment with a room temperature of 22±2°C and a relative humidity of 50%, with a light cycle of 12 hours of light and 12 hours of darkness. The mice had free access to clean water and a dedicated maintenance diet. 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 there were symptoms such as decreased appetite, increased water intake, dark fur, sparse and erect fur, arched back and thick and protruding ear veins, rapid breathing, and moist rales heard on liver auscultation, the model was successfully modeled.

[0065] (2) Grouping method

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

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

[0068] Dissect the mice to obtain the liver and spleen. Gently rinse with saline to remove blood, wipe dry, and weigh. Liver organ index = liver weight (g) / mouse body weight (g), spleen organ index = spleen weight (g) / mouse body weight (g).

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

[0070] ELISA detection of NRF2, TRAF3, NIK, and P52

[0071] Using mouse sera prepared from each group, NRF2, TRAF3, NIK, and p52 levels were measured using ELISA kits (Elabscience) according to the manufacturer's instructions. The procedure was as follows: 50-100 μL of sample was added to each reaction well, incubated at 37°C for 0.5-1 hour, and then washed. After zeroing the blank control wells on the ELISA reader at a wavelength of 450 nm, the absorbance (OD) of each well was measured.

[0072] (4) Results

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

[0074] 2. CCl4 induced abnormalities in the liver and spleen of mice, such as liver fibrosis and spleen cell infiltration. After silymarin intervention, the liver index decreased to varying degrees, the liver tissue morphology gradually approached normal, the spleen index increased to varying degrees, and the pathological state of spleen tissue improved. The results showed that SMP has a repair and protective effect on liver damage and spleen damage (see Figure 3 AB). After silymarin intervention, apoptotic cells decreased, indicating that SMP can inhibit hepatocyte apoptosis (see Figure 3 C). The results showed that silymarin exhibited a good liver-protective effect in mice with acute liver injury induced by carbon tetrachloride, significantly enhancing liver antioxidant indicators, inhibiting the expression of pro-inflammatory cytokines, and reducing immune cell infiltration.

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

[0076] 4. The non-target metabolomics of mouse cecum were analyzed. Compared with the carbon tetrachloride liver injury model group, there were 67 different metabolites in the cecum contents of the silymarin group, and the galactose metabolic pathway was enriched ( Figure 5 ), the results showed that silymarin could regulate the cecal microbial diversity of mice with acute liver injury induced by carbon tetrachloride.

[0077] in conclusion:

[0078] The present invention discloses silymarin, a bioactive peptide that can effectively improve or alleviate non-alcoholic liver damage. This peptide is extracted from milk thistle and processed using a specific enzymatic hydrolysis technique. Its amino acid sequence has a specific arrangement, enabling it to precisely act on liver cells.

[0079] In terms of preparation method, milk thistle is used as raw material. After pretreatment, composite enzymatic hydrolysis technology is adopted, and a specific proportion of protease is selected to carry out enzymatic hydrolysis under suitable temperature and pH conditions. Then, ultrafiltration, ion exchange chromatography, reverse phase chromatography and other technologies are used to separate and purify it to obtain high-purity silymarin.

[0080] Silymarin effectively alleviated acute liver injury by inhibiting the TRAF3 / NIK / NF-κB signaling pathway and regulating the intestinal microbiota. These findings suggest that silymarin has good application prospects in the prevention and treatment of acute liver injury.

[0081] The silymarin of the present invention can be used in the medical field to prepare drugs for treating non-alcoholic liver damage, and in the health food field it can be used as a raw material to prepare health products for preventing and improving non-alcoholic liver damage, and has broad application prospects.

[0082] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

[0083]

[0084]

Claims

1. A silymarin, characterized in that: The silymarin comprises peptide chain 1, peptide chain 2 and peptide chain 3, wherein 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.

2. The method for preparing silymarin according to claim 1, characterized in that: The method comprises: The milk thistle seeds are used as raw materials, and the product is prepared through the following steps: pretreatment, primary enzymatic hydrolysis with papain, primary enzyme inactivation, secondary enzymatic hydrolysis with acidic protease, secondary enzyme inactivation, and separation and purification.

3. The method for preparing silymarin according to claim 2, characterized in that: The pretreatment includes: After soaking the milk thistle seeds, add pure water, homogenize and extract with a flash extractor for 5-10 minutes.

4. The method for preparing silymarin according to claim 2, wherein: The conditions for the primary enzymatic hydrolysis are as follows: the dosage of papain is 3‰ of the net weight of milk thistle seeds, and the temperature is kept at 50°C for 2h; The conditions for the primary enzyme inactivation are: heating the reaction system of the primary enzymatic hydrolysis to 80° C. and keeping the temperature for 10 minutes.

5. The method for preparing silymarin according to claim 2, characterized in that: The conditions of the secondary enzymatic hydrolysis are as follows: the reaction system of the primary enzyme inactivation is adjusted to pH=3.0 by HCl, the amount of acidic protease used is 3‰ of the net weight of the milk thistle seeds, and the reaction system is kept at 38°C for 12 hours; The conditions for the secondary enzyme inactivation are: heating the secondary enzymatic hydrolysis reaction system to 80° C. and keeping the temperature for 10 minutes.

6. The method for preparing silymarin according to claim 2, characterized in that: The separation and purification comprises: passing the reaction system inactivated by the secondary enzyme while hot through a stainless steel plate-frame filter, adjusting the pH of the obtained filtrate to 8.0 with NaOH, keeping the temperature at 90° C. for 10 minutes, passing the reaction system while hot through the stainless steel plate-frame filter again, cooling the temperature to 40° C., ultrafiltration using a membrane package with a molecular weight cutoff of 6 kDa, and fine filtering the obtained ultrafiltrate through 0.22 μm and 0.1 μm sterilizing filters in series to obtain a filtrate.

7. Use of the silymarin according to claim 1, or the silymarin prepared by the preparation method according to any one of claims 2 to 6, in preparing a drug for improving or alleviating non-alcoholic liver injury.

8. Use of the silymarin according to claim 1, or the silymarin prepared by the method according to any one of claims 2 to 6, in preparing a health food for improving or alleviating non-alcoholic liver damage.

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