Litsea cubeba cake meal antioxidant peptide as well as preparation method and application thereof

Abstract

The invention discloses a litsea cubeba cake meal antioxidant peptide as well as a preparation method and application thereof, and belongs to the technical field of deep processing of oil crop residues. The amino acid sequence of the litsea cubeba cake meal antioxidant peptide is as shown in SEQ ID NO.1, and the molecular weight of the litsea cubeba cake meal antioxidant peptide is 701.50 Da. The litsea cubeba cake meal antioxidant peptide is prepared by taking litsea cubeba cake meal as a raw material and utilizing an enzymolysis process and a chromatographic preparation technology, and the litsea cubeba cake meal antioxidant peptide can be applied to preparation of medicines for preventing or improving the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The invention belongs to the technical field of deep processing of oil crop residues, and particularly relates to a litsea cubeba cake antioxidant peptide and a preparation method and application thereof. Background Art

[0002] The liver plays a crucial role in the human body, playing a central role in the synthesis, breakdown, transport, and transformation of lipids. In patients with non-alcoholic fatty liver disease (NAFLD), disorders in the liver's lipid synthesis, uptake, transport, and oxidation occur. Increased activity of enzymes involved in lipid synthesis leads to increased fatty acid synthesis. Simultaneously, decreased activity of enzymes involved in fatty acid oxidation reduces fatty acid oxidation. If untimely intervention is not promptly implemented, it can progress to non-alcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. However, the pathogenesis of NAFLD is complex and its mechanisms remain largely unresolved. Drugs used to treat NAFLD include metformin and pioglitazone, which improve insulin resistance; resmetirol, a lipid-lowering drug; and vitamin E, an antioxidant. However, these drugs suffer from limitations such as short efficacy and high toxicity. Therefore, the search and development of NAFLD drugs with sustained efficacy, low toxicity, and safety is a key research priority.

[0003] The processing of Litsea cubeba essential oil produces a large amount of waste meal. If not properly handled, this waste meal can rot and cause soil and water pollution. However, this waste meal still contains many valuable components, and discarding it directly would waste these resources. Utilizing the protein in this meal has become a pressing issue. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention uses Litsea cubeba cake as raw material and utilizes enzymatic hydrolysis and chromatographic preparation technology to prepare Litsea cubeba cake antioxidant peptides with the efficacy of improving non-alcoholic fatty liver disease. The Litsea cubeba cake antioxidant peptides can be used in the preparation of drugs for preventing or improving non-alcoholic fatty liver disease, thereby solving the technical problems raised in the background technology.

[0005] Specifically, the technical solution of the present invention includes the following contents: One of the objectives of the present invention is to provide an antioxidant peptide from litsea cubeba cake. The amino acid sequence of the antioxidant peptide from litsea cubeba cake is shown in SEQ ID NO.1, and the molecular weight of the antioxidant peptide from litsea cubeba cake is 701.50 Da.

[0006] A second object of the present invention is to provide a method for preparing antioxidant peptides from Litsea cubeba cake, the preparation method comprising the following steps: mixing defatted Litsea cubeba cake powder and phosphate buffer to obtain a mixed solution; The mixed solution and alkaline protease are mixed and enzymatically hydrolyzed to obtain an enzymatic hydrolysis product, and the enzymatic hydrolysis product is sequentially subjected to enzyme inactivation treatment and centrifugal separation to obtain an enzymatic hydrolyzate of the litsea cubeba cake; The enzymatic hydrolysate of litsea cubeba cake is subjected to ultrafiltration and classification to obtain the ultrafiltration enzymatic hydrolysate of litsea cubeba cake; The ultrafiltration enzymatic hydrolysate of Litsea cubeba cake was purified by gel column chromatography to obtain gel chromatography enzymatic hydrolysate of Litsea cubeba cake; The gel chromatography hydrolysate of Litsea cubeba cake was purified by reverse phase high performance liquid chromatography to obtain antioxidant peptides from Litsea cubeba cake.

[0007] Furthermore, the preparation method of the defatted Litsea cubeba cake powder comprises the following steps: The litsea cubeba cake was pulverized to obtain a crushed material, which was then mixed with n-butanol at a material-liquid ratio of 1 g:8 mL. The crushed material was then ultrasonically treated for 30 min and allowed to stand for defatting. The defatted litsea cubeba cake powder was then obtained by drying and pulverizing.

[0008] Furthermore, the static degreasing process includes a static temperature of 25° C., a static time of 24 h, and replacement of n-butanol every 12 h.

[0009] Furthermore, the molar concentration of the phosphate buffer is 0.05 mol / L.

[0010] Furthermore, the material-liquid ratio of the defatted Litsea cubeba cake powder to phosphate buffer is 1 g to 15 mL.

[0011] Furthermore, the amount of alkaline protease used is 2.0% of the mass of the defatted Litsea cubeba cake powder.

[0012] Furthermore, the conditions of the enzymatic hydrolysis treatment include an enzymatic hydrolysis temperature of 35° C., an enzymatic hydrolysis pH of 9.5, and an enzymatic hydrolysis time of 3 h.

[0013] Furthermore, the process of inactivating the enzyme in the enzymatic hydrolysis product includes keeping the product in a 95° C. water bath for 10 minutes to inactivate the enzyme, and then cooling the product to room temperature.

[0014] Furthermore, the centrifugal separation conditions include a centrifugal speed of 10,000 rpm and a centrifugal time of 20 min.

[0015] Furthermore, the ultrafiltration classification treatment step includes: classifying the litsea cubeba cake hydrolysate through ultrafiltration membranes with molecular weight cutoffs of 1 kDa, 5 kDa and 10 kDa, and screening the component with the strongest scavenging ability for DPPH free radicals and hydroxyl free radicals as the litsea cubeba cake ultrafiltration hydrolysate.

[0016] Furthermore, the gel column chromatography purification process includes the following steps: The ultrafiltration hydrolysate of Litsea cubeba cake was dissolved in double-distilled water to prepare a solution with a concentration of 20 mg / mL, and then separated by Sephadex G-25 column chromatography and eluted with double-distilled water at a flow rate of 1.2 mL / min. A gel chromatography chromatogram was prepared based on the absorbance value at 220 nm. The chromatographic peaks were collected, and the scavenging ability of the components in each chromatographic peak for DPPH free radicals and hydroxyl free radicals was determined. The component with the strongest scavenging ability for DPPH free radicals and hydroxyl free radicals was selected and freeze-dried to obtain the gel chromatography hydrolysate of Litsea cubeba cake.

[0017] Furthermore, the reverse-phase high performance liquid chromatography purification process comprises the following steps: The gel chromatography hydrolysate of Litsea cubeba cake was prepared into a 20 μg / mL solution with double distilled water and purified by RP-HPLC to obtain the antioxidant peptides from Litsea cubeba cake.

[0018] Furthermore, the RP-HPLC purification conditions include an injection volume of 20 μL, a chromatographic column model of Zorbax SBC-18 (4.6×250 mm, 5 μm), a mobile phase acetonitrile concentration uniformly increased from 0 to 30% within 30 minutes, an elution rate of 1.0 mL / min, and an ultraviolet detection wavelength of 220 nm.

[0019] The third object of the present invention is to provide a use of an antioxidant peptide from Litsea cubeba cake in the preparation of a drug for preventing or improving non-alcoholic fatty liver disease.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses Litsea cubeba cake as raw material, and obtains Litsea cubeba cake antioxidant peptides with liver protection efficacy through pretreatment, alkaline protease hydrolysis, membrane ultrafiltration, and chromatographic separation and purification. The molecular weight is determined to be 701.50 Da by ESI-MS. The Litsea cubeba cake antioxidant peptides prepared by the present invention can significantly scavenge DPPH free radicals and hydroxyl free radicals, reduce the liver weight, liver lipid accumulation level, liver collagen fibrosis level and serum AST and ALT activities of mice with non-alcoholic fatty liver disease, increase the activity of antioxidant enzymes (SOD, CAT, GSH-Px) in liver tissue homogenate, and reduce the content of oxidation product malondialdehyde (MDA). Therefore, the Litsea cubeba cake antioxidant peptides prepared by the present invention have the function of improving non-alcoholic fatty liver disease.

[0021] (2) The treatment method of the present invention not only solves the pollution problem of the by-product of the Litsea cubeba cake, but also realizes the resource utilization of the Litsea cubeba cake, thereby improving the utilization value of the by-product of the Litsea cubeba cake. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The scavenging ability of the litsea cubeba cake enzymatic hydrolysate (LCP) and ultrafiltration fractions (LCP-I to LCP-IV) of the present invention on DPPH free radicals and hydroxyl free radicals at a concentration of 1.0 mg / mL; Figure 2 This is a chromatogram of the Sephadex G-25 glucan gel of the present invention; Figure 3 The scavenging ability of DPPH and hydroxyl radicals of ultrafiltration hydrolysates of Litsea cubeba cake (GLCP-I to GLCP-III) prepared on Sephadex G-25 at a concentration of 1.0 mg / mL was investigated. Figure 4 RP-HPLC analysis of Sephadex G-25 hydrolysate (GLCP-II); Figure 5 This is the mass spectrum of antioxidant peptides from Litsea cubeba cake; Figure 6 This is the structural diagram of the antioxidant peptide from Litsea cubeba cake; Figure 7 Representative images of Oil Red O staining of liver tissues in NAFLD mice; Figure 8 Representative images of MASSON staining of liver tissues in NAFLD mice. DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions of the present invention through the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0024] Unless otherwise specified, the raw materials and reagents used in the present invention are commercially available or can be prepared by known methods.

[0025] Experimental reagents: Total cholesterol (TC), triglyceride (TG), SOD, GSH-Px, MDA, ALT, and AST detection kits were purchased from Nanjing Jiancheng Bioengineering Institute; Experimental animals: Male C57BL / 6J mice (6 weeks old, weighing 20 ± 2 g) were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. High-fat feed and ordinary feed were obtained from Suzhou Shuangshi Experimental Animal Feed Technology Co., Ltd.

[0026] Embodiment 1:

[0027] A preparation method of litsea cubeba pomace antioxidant peptides for improving non-alcoholic fatty liver disease, specifically including the following processes: Remove the branches and leaves from the litsea cubeba pomace, then perform tissue crushing to obtain crushed material. According to the material-liquid ratio of 1 g∶8 mL, add 1 g of the crushed material to 8 mL of n-butanol. Subsequently, after 30 min of ultrasonic treatment, let it stand for defatting at 25 °C for 24 h. During defatting, replace the n-butanol every 12 h. After defatting, perform drying and crushing treatments in sequence to obtain defatted litsea cubeba pomace powder; According to the material-liquid ratio of 1 g∶15 mL, weigh 1 g of defatted litsea cubeba pomace powder and add it to 15 mL of phosphate buffer solution with a molar concentration of 0.05 mol / L, mix and stir to obtain a mixed solution. Adjust the temperature of the mixed solution to 35 °C, then adjust the pH value of the mixed solution to 9.5. At this time, add alkaline protease accounting for 2.0% of the mass of the defatted litsea cubeba pomace powder, and perform enzymatic hydrolysis for 3 h; After enzymatic hydrolysis, place the enzymatic hydrolysis product in a water bath and heat it to 95 °C for heat sterilization treatment for 10 min, then cool it to room temperature. Finally, put it into a centrifuge and centrifuge at a speed of 10000 rpm for 20 min, and collect the supernatant to obtain litsea cubeba pomace enzymatic hydrolysate (LCP); Perform fractionation on the litsea cubeba pomace enzymatic hydrolysate (LCP) through ultrafiltration membranes with a molecular weight cut-off of 1 kDa, 5 kDa, and 10 kDa, and collect the fractionation components LCP-I (MW>10 kDa), LCP-II (5.0 kDa<MW<10 kDa), LCP-III (1.0 kDa<MW<5.0 kDa), and LCP-IV (MW<1.0 kDa). Measure the scavenging abilities of these four fractionation components against DPPH free radicals and hydroxyl free radicals. The results are shown in Figure 1 , and it is found that LCP-IV has the strongest scavenging abilities against DPPH free radicals and hydroxyl free radicals. Take LCP-IV as the litsea cubeba pomace ultrafiltration enzymatic hydrolysate; Dissolve the above-mentioned litsea cubeba pomace ultrafiltration enzymatic hydrolysate (LCP-IV) in double-distilled water to prepare a solution with a concentration of 20 mg / mL, perform separation by Sephadex G-25 column chromatography of dextran gel, elute with double-distilled water, control the flow rate at 1.2 mL / min, and make a gel chromatography chromatogram according to the absorbance value at 220 nm. The results are shown in Figure 2 , collect the chromatographic peaks GLCP-I, GLCP-II, and GLCP-III, measure the scavenging abilities of these three chromatographic peak components against DPPH free radicals and hydroxyl free radicals. The results are shown in Figure 3 , and it is found that GLCP-III has the strongest scavenging abilities against DPPH free radicals and hydroxyl free radicals. Lyophilize GLCP-III to obtain litsea cubeba pomace gel chromatography enzymatic hydrolysate (GLCP-III); The gel chromatography hydrolysate of the above-mentioned litsea cubeba cake (GLCP-III) was prepared into a 20 μg / mL solution with double distilled water and purified by gradient elution using reversed-phase high performance liquid chromatography (RP-HPLC) (injection volume: 20 μL; column model: ZorbaxSB C-18 (4.6×250 mm, 5 μm); the mass percentage concentration of acetonitrile in the mobile phase was increased from 0 to 30% at a constant rate within 30 min; the elution rate was controlled at 1.0 mL / min; the UV detection wavelength was 220 nm) to obtain the litsea cubeba cake antioxidant peptide (GLCP-III-7). The chromatogram of the litsea cubeba cake antioxidant peptide is shown in Figure 4 The molecular weight was determined to be 701.50 Da by ESI-MS. Figure 5 The amino acid sequence was determined by protein / peptide sequence analyzer to be Ala-Lau-Val-Val-Asp-Gly-Lys (abbreviated as ALVVDGK). The specific structure of the antioxidant peptide from Litsea cubeba cake is shown in Figure 6 .

[0028] Mice were divided into two experimental groups. One group was fed a normal diet (NCD group, 10 mice); the other group was fed a high-fat diet (HFD group, 30 mice). After 4 weeks of feeding, the mice in the HFD group were randomly divided into three groups for another 4 weeks: (1) HFD group (10 mice); (2) HFD+PPc group (positive control group of polyene phosphatidylcholine capsule, mice were given HFD and gavage with 142.5 mg / kg / d PPc, 10 mice); (3) HFD+Litsea cubeba cake antioxidant peptide group (mice were given HFD and gavage with 50 mg / kg / d Litsea cubeba cake antioxidant peptide, 10 mice). The mice in the NCD group continued to be fed the corresponding diet for 4 weeks. The body weight of the mice was measured and recorded weekly. The experiment lasted for 8 weeks.

[0029] Effect verification: (1) Evaluation of antioxidant capacity: According to the method described in the literature (Bin Wang, Li Li., Chang-Feng Chi, Jia-Hui Ma, Hong-Yu Luo, Yin-Feng Xu. Purification and characterization of a novel antioxidant peptide derived from blue mussel (Mytilus edulis) protein hydrolysate [J]. Food Chemistry, 2013, 138 (2): 1713-1719), the half-scavenging efficiency (EC50) of the antioxidant peptide from the sorghum cake obtained in Example 1 for DPPH free radicals and hydroxyl free radicals was 1.23 mg / mL and 0.96 mg / mL, respectively, showing significant antioxidant capacity. Compared with the HFD group of mice, the antioxidant peptide from the sorghum cake can significantly increase the activities of SOD, CAT, and GSH-Px in the liver tissue homogenate of NAFLD mice and significantly reduce the MDA content (see Table 1 below), indicating that the antioxidant peptide from the sorghum cake prepared by the present invention has an enhancing effect on the antioxidant capacity of NAFLD mice.

[0030] Table 1 Effects of antioxidant peptides from Litsea cubeba cake on SOD, CAT, GSH-Px activities and MDA content in liver tissue of NAFLD mice (n=3)

[0031] (2) Improvement of lipid accumulation: Referring to the method of the literature (Li Li, Wang Yu-Mei, Zeng Xiao-Yan, Hu Ying, Zhang Ji, WangBin, Chen Shang-Xing. Bioactive proteins and antioxidant peptides from Litseacubeba fruit meal: Preparation, characterization and ameliorating function onhigh-fat diet-induced NAFLD through regulating lipid metabolism, oxidative stress and inflammatory response[J]. International Journal of BiologicalMacromolecules, 2024, 280:136186), the NAFLD model of male C57BL / 6J mice (weighing 20 g~22 g) was first established: the experimental period was 8 weeks, the HFD group was fed with an appropriate amount of high-fat feed every day, the sample group (HFD+Litsea cubeba cake antioxidant peptide group) and the positive drug group (HFD+PPc group) were fed with a high-fat feed and supplemented with 100 mg / kg / d (Litsea cubeba cake antioxidant peptide obtained in Example 1) and 142.5 mg / kg / d, respectively. The NCD group was administered with a dose of (PPc) once daily by oral gavage. The mice in the NCD group were fed an appropriate amount of normal chow. After the final oral gavage experiment, the mice were fasted for 16 hours and then sacrificed. Liver weights were measured, and eye blood was collected and centrifuged for 15 minutes (4°C, 4000 rpm) to collect serum. The livers were also harvested. The efficacy of antioxidant peptides from Litsea cubeba cake in ameliorating hepatic lipid accumulation in NAFLD mice was evaluated by Oil Red O staining of liver tissue and determination of TC and TG levels in liver tissue. The results are shown in Tables 2 and 3 below.

[0032] Table 2 Effects of antioxidant peptides from Litsea cubeba cake on liver weight in NAFLD mice (number of mice n = 10)

[0033] Table 3 Effects of antioxidant peptides from Litsea cubeba cake on TC and TG levels in liver tissue of NAFLD mice (number of mice n = 10)

[0034] The results showed that compared with the HFD group, the antioxidant peptides from Litsea cubeba cake could significantly reduce the liver weight of NAFLD mice. The results of Oil Red O staining of liver sections showed that the boundaries between liver cells in the sample group gradually became clearer and the number of red lipid droplets decreased compared with the HFD group (see Figure 7 ), the TC and TG contents in the liver tissue were reduced, indicating that the antioxidant peptides from Litsea cubeba cake can reduce the lipid accumulation in the liver cells of NAFLD mice.

[0035] (3) Improvement of liver function: Table 4 Effects of antioxidant peptides from Litsea cubeba cake on serum ALT and AST activities in NAFLD mice (number of mice n = 10)

[0036] The results showed that compared with the HFD group mice, the antioxidant peptides from Litsea cubeba cake could significantly reduce the AST and ALT activities in the serum of NAFLD mice and improve the liver function of NAFLD mice.

[0037] (4) Improvement of liver collagen fibrosis in NAFLD mice: The results showed that compared with the NCD group, the high-fat diet group had a large number of vacuoles in the liver cells and a large amount of blue collagen deposition, showing obvious liver fibrosis. Compared with the high-fat diet group, the area of ​​stained fibrosis area was significantly reduced after intervention with the antioxidant peptide of Litsea cubeba cake (see Figure 8 ), indicating that the antioxidant peptides from Litsea cubeba cake can inhibit liver collagen deposition in NAFLD mice induced by high-fat diet and prevent liver fibrosis.

[0038] The above embodiments and accompanying drawings provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. An antioxidant peptide from Litsea cubeba cake, characterized in that: The amino acid sequence of the antioxidant peptide from litsea cubeba cake is shown in SEQ ID NO. 1, and the molecular weight of the antioxidant peptide from litsea cubeba cake is 701.50 Da.

2. The method for preparing antioxidant peptides from Litsea cubeba cake according to claim 1, wherein: The preparation method comprises the following steps: mixing defatted Litsea cubeba cake powder and phosphate buffer to obtain a mixed solution; The mixed solution and alkaline protease are mixed and enzymatically hydrolyzed to obtain an enzymatic hydrolysis product, and the enzymatic hydrolysis product is subjected to enzyme inactivation and centrifugal separation to obtain a litsea cubeba cake enzymatic hydrolyzate; The enzymatic hydrolysate of litsea cubeba cake is subjected to ultrafiltration and classification to obtain the ultrafiltration enzymatic hydrolysate of litsea cubeba cake; The ultrafiltration enzymatic hydrolysate of Litsea cubeba cake was purified by gel column chromatography to obtain gel chromatography enzymatic hydrolysate of Litsea cubeba cake; The gel chromatography hydrolysate of Litsea cubeba cake was purified by reverse phase high performance liquid chromatography to obtain antioxidant peptides from Litsea cubeba cake.

3. The method for preparing antioxidant peptides from Litsea cubeba cake according to claim 2, wherein: The preparation method of the defatted Litsea cubeba cake powder comprises the following steps: The litsea cubeba cake was pulverized to obtain litsea cubeba cake powder, which was then mixed with n-butanol at a material-liquid ratio of 1 g:8 mL. The mixture was then ultrasonically treated for 30 min and allowed to stand for defatting. The defatted litsea cubeba cake powder was then obtained by drying and pulverizing.

4. The method for preparing antioxidant peptides from Litsea cubeba cake according to claim 2, wherein: The material-liquid ratio of the defatted Litsea cubeba cake powder to phosphate buffer is 1 g to 15 mL.

5. The method for preparing antioxidant peptides from Litsea cubeba cake according to claim 2, characterized in that: The amount of alkaline protease used is 2.0% of the mass of the defatted Litsea cubeba cake powder.

6. The method for preparing antioxidant peptides from Litsea cubeba cake according to claim 2, characterized in that: The enzymatic hydrolysis treatment conditions include an enzymatic hydrolysis temperature of 35° C., an enzymatic hydrolysis pH of 9.5, and an enzymatic hydrolysis time of 3 h.

7. The method for preparing antioxidant peptides from Litsea cubeba cake according to claim 2, characterized in that: The ultrafiltration and classification treatment step comprises: subjecting the litsea cubeba cake hydrolysate to ultrafiltration classification through ultrafiltration membranes with molecular weight cutoffs of 1 kDa, 5 kDa and 10 kDa, and screening the component with the strongest scavenging ability for DPPH free radicals and hydroxyl free radicals as the litsea cubeba cake ultrafiltration hydrolysate.

8. The method for preparing antioxidant peptides from Litsea cubeba cake according to claim 2, characterized in that: The process of gel column chromatography purification comprises the following steps: The ultrafiltration hydrolysate of Litsea cubeba cake was dissolved in double-distilled water to prepare a solution with a concentration of 20 mg / mL, and then separated by Sephadex G-25 column chromatography and eluted with double-distilled water at a flow rate of 1.2 mL / min. A gel chromatography chromatogram was prepared based on the absorbance value at 220 nm. The chromatographic peaks were collected, and the scavenging ability of the components in each chromatographic peak for DPPH free radicals and hydroxyl free radicals was determined. The component with the strongest scavenging ability for DPPH free radicals and hydroxyl free radicals was selected and freeze-dried to obtain the gel chromatography hydrolysate of Litsea cubeba cake.

9. The method for preparing the antioxidant peptide from Litsea cubeba cake for improving non-alcoholic fatty liver disease according to claim 2, characterized in that: The process of reversed-phase high performance liquid chromatography purification comprises the following steps: The gel chromatography hydrolysate fractions of Litsea cubeba cake were prepared into a 20 μg / mL solution with double distilled water and purified by RP-HPLC to obtain Litsea cubeba cake antioxidant peptides.

10. Use of the litsea cubeba cake antioxidant peptide according to claim 1 or the litsea cubeba cake antioxidant peptide prepared by the method for preparing the litsea cubeba cake antioxidant peptide according to any one of claims 2 to 9 in the preparation of a medicament for preventing or improving non-alcoholic fatty liver disease.

Images