Camellia sinensis source antioxidant peptide, and preparation method and application thereof

Abstract

The application discloses a camellia nitidissima source antioxidant peptide and a preparation method and application thereof, and belongs to the technical field of biology. The amino acid sequence of the antioxidant peptide is Leu-Gly-Leu-Phe. The application extracts protein from camellia nitidissima petals, and carries out enzymolysis by pepsin, ultrafiltration, gel chromatography, ion exchange chromatography and reverse phase high performance liquid chromatography separation and purification, combines with activity tracking and screening, obtains a single active peptide, and identifies the sequence of the single active peptide by mass spectrometry. The antioxidant peptide has good scavenging capacity on ABTS and DPPH free radicals, can inhibit cell lipid peroxidation reaction, and has significant antibacterial activity with a minimum bactericidal concentration of 8 mg / mL. The application firstly obtains the small molecule peptide with antioxidant and antibacterial activity from camellia nitidissima, and the small molecule peptide can be applied to antioxidants, food, health products, cosmetics, medicines or feed additives, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an antioxidant peptide derived from Camellia chrysantha, its preparation method, and its application. Background Technology

[0002] Antioxidant peptides are a class of small-molecule bioactive peptides with free radical scavenging, lipid oxidation inhibition, and metal ion chelation capabilities. They typically consist of 2 to 20 amino acid residues and have a molecular weight of less than 3000 Daltons. Excessive accumulation of reactive oxygen species (ROS) in the body triggers oxidative stress, leading to oxidative damage to proteins, lipids, and DNA, which is closely related to the development of various chronic diseases such as aging, cardiovascular disease, neurodegenerative diseases, diabetes, and cancer. Antioxidant peptides can efficiently scavenge ROS through mechanisms such as hydrogen atom transfer, single electron transfer, or metal ion chelation, blocking the lipid peroxidation chain reaction, thereby preventing and mitigating oxidative damage. This is of great significance for maintaining normal physiological functions and reducing disease incidence. In the food and cosmetics industries, lipid oxidation is a major factor leading to product rancidity, discoloration, flavor deterioration, and decreased nutritional value. Antioxidant peptides can effectively inhibit ROS-induced lipid peroxidation, extend product shelf life, and maintain the clean label attributes of naturally derived products.

[0003] Antioxidant peptides extracted from natural substances have become ideal alternatives to synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) due to their high activity, low toxicity, and good biocompatibility. While synthetic antioxidants are inexpensive and have significant antioxidant effects, long-term use poses potential risks of hepatotoxicity, carcinogenicity, and endocrine disruption, leading many countries and regions to impose strict restrictions on their dosage and application. In contrast, naturally derived antioxidant peptides, obtained through enzymatic release, microbial fermentation, or chemical synthesis, offer greater safety. Currently, numerous studies have extracted and identified various antioxidant peptides from different natural food proteins. In the cereal category, antioxidant peptides from wheat germ, rice, corn, oats, barley, and millet have been widely reported, such as Leu-Pro-Phe and Ala-Tyr-Leu isolated from corn protein. In the legume category, antioxidant peptides from soybeans, mung beans, peas, chickpeas, and black beans have also been extensively studied; Leu-Leu-Pro-His-His and Tyr-Glu-Glu-Leu from soybean protein have been shown to have significant activity. Carnosine and its derivatives, Phe-Phe-Cys, and Leu-Tyr-Pro from meat, eggs, and dairy products have also demonstrated excellent antioxidant properties. In the marine field, salmon skin collagen peptides, oyster peptides, and sea cucumber peptides have become research hotspots in recent years. In addition, nuts, seeds, edible fungi, and traditional Chinese medicine are also emerging sources of antioxidant peptides.

[0004] Among numerous preparation methods, enzymatic hydrolysis has become the most commonly used method for extracting bioactive peptides due to its advantages such as mild reaction conditions, high controllability, high safety, and ease of industrial production. Commonly used proteases include pepsin, trypsin, chymotrypsin, papain, bromelain, alkaline protease, neutral protease, and flavor protease. Different proteases have different cleavage site specificities, which can produce a mixture of peptides with different sequence characteristics and molecular weight distributions, thus affecting their antioxidant activity. New strategies such as single-enzymatic hydrolysis and combined enzymatic hydrolysis, stepwise enzymatic hydrolysis and simultaneous enzymatic hydrolysis, ultrasound-assisted enzymatic hydrolysis, and microwave-assisted enzymatic hydrolysis are constantly emerging, improving enzymatic hydrolysis efficiency and peptide yield. Enzymatic hydrolysis products usually need to undergo a series of separation and purification steps, such as desalting, ultrafiltration, gel filtration chromatography, ion exchange chromatography, and reversed-phase high-performance liquid chromatography, combined with mass spectrometry identification techniques such as MALDI-TOF / TOF-MS and LC-MS / MS, to obtain a single active peptide and determine its amino acid sequence.

[0005] Golden camellia (Camellia nitidissima) is a plant belonging to the genus Camellia in the family Theaceae. It is a rare species with exceptionally golden-yellow flowers, earning it the titles of "Queen of Camellias" and "Giant Panda of the Plant Kingdom." Its wild resources are mainly distributed in the Shiwan Mountains area of ​​Fangchenggang City, Guangxi Zhuang Autonomous Region, and the surrounding areas of Nanning City, China. Due to its narrow distribution area and low natural reproduction rate, it is listed as a national second-class protected wild plant. The medicinal history of golden camellia can be traced back to the "Guangxi Medicinal Materials Standard," where it is commonly used in folk medicine as an adjunct treatment for pharyngitis, dysentery, hypertension, and hyperlipidemia. Modern pharmacological studies have shown that golden camellia contains abundant bioactive components, mainly including flavonoids, polyphenols, saponins, polysaccharides, and proteins and polypeptides (accounting for approximately 8% to 12% of dry weight). These active components endow golden camellia with various pharmacological activities, including antioxidant, cholesterol-lowering, anti-tumor, lipid-regulating, blood sugar-lowering, anti-inflammatory, antibacterial, and antiviral effects.

[0006] Based on the aforementioned pharmacological activities, *Camellia chrysantha* has expanded from a rare wild plant into a unique resource with both medicinal and edible uses. Currently, products such as *Camellia chrysantha* flower tea, beverages, oral liquids, facial masks, and skincare essences are available on the market, demonstrating broad prospects for food development, cosmetic applications, and ecological economics. However, in-depth analysis of existing technologies reveals that current research and applications mainly focus on small molecule compounds (flavonoids, polyphenols, saponins) and polysaccharides in *Camellia chrysantha*, with a serious lack of development and utilization of its protein and peptide components. Research on the extraction, compositional analysis, and functional evaluation of *Camellia chrysantha* proteins is extremely limited, with only sporadic reports on total protein content determination and a lack of systematic protein separation and characterization. More importantly, research on the release of bioactive peptides through proteolysis of *Camellia chrysantha* is completely lacking; no technical schemes for preparing antioxidant peptides through enzymatic hydrolysis using *Camellia chrysantha* as a raw material have been publicly disclosed. No reports have been found on small molecule peptides with clearly defined amino acid sequences derived from *Camellia chrysantha*, indicating a significant research gap in this field.

[0007] To obtain antioxidant peptides with well-defined sequences from Camellia chrysantha protein, several technical hurdles need to be overcome. The protein content in Camellia chrysantha petals is relatively low, only about 8% to 12% by dry weight, and it is tightly bound to a large amount of cellulose (about 20% to 25%), polyphenols (about 5% to 8%), and pigments. Polyphenols readily form hydrogen bonds or covalent cross-links with proteins, forming insoluble protein-polyphenol complexes, severely reducing protein extraction rates. While conventional alkali-soluble acid precipitation methods can partially extract proteins, the co-extracted polyphenols and pigments interfere with subsequent enzymatic hydrolysis and chromatographic separation, and alkali treatment may lead to protein denaturation, Maillard reactions, and amino acid racemization. Therefore, a comprehensive extraction scheme combining dephenolization, decolorization, and high extraction rates is needed. Furthermore, the amino acid composition and sequence information of Camellia chrysantha protein are unknown, lacking a basis for targeted protease selection. Different proteases have different cleavage specificities, resulting in diverse peptide mixtures with significantly varying antioxidant activities. To obtain peptides with high free radical scavenging activity, it is necessary to systematically compare the enzymatic hydrolysis effects of various proteases or their combinations, and establish structure-activity relationships between hydrolysis parameters, peptide spectra, and activity. This involves extensive orthogonal experiments and activity screening. Enzymatic hydrolysis products are typically complex mixtures containing dozens to hundreds of different peptides, each with subtle differences in molecular weight, charge, polarity, and hydrophobicity. To separate a single active peptide from this complex matrix, a multi-stage chromatographic system is required, including size exclusion-based gel chromatography, charge difference-based ion exchange chromatography, and hydrophobicity-based reversed-phase high-performance liquid chromatography. Activity tracking screening is required after each stage of separation to confirm the location of the active component and avoid losing low-abundance, highly active peptides. The entire process is multi-step, time-consuming, and results in significant sample loss, demanding extremely high operational precision. After obtaining a single peptide peak, sequence analysis using mass spectrometry is necessary, requiring sufficient sample purity and good ionization efficiency. Furthermore, the evaluation of antioxidant activity cannot be limited to single chemical free radical scavenging experiments, as chemical experimental results may not reflect the true antioxidant effect under physiological conditions. It is also necessary to establish a cellular-level oxidative stress model to verify whether peptides can enter cells, reduce reactive oxygen species levels, and inhibit lipid peroxidation damage. As a national second-class protected plant, Camellia chrysantha has limited wild resources, and artificial cultivation has not yet been widely promoted. Obtaining raw materials is difficult and costly, which further necessitates the design of efficient and low-loss separation processes within the limited resources available. Summary of the Invention

[0008] To address the aforementioned technological gaps and challenges, this invention aims to provide an antioxidant peptide derived from Camellia chrysantha, its preparation method, and its application.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] This invention provides an antioxidant peptide derived from Camellia chrysantha, with the amino acid sequence Leu-Gly-Leu-Phe, i.e., LGLF.

[0011] Furthermore, the antioxidant peptide has free radical scavenging activity, including ABTS free radicals and / or DPPH free radicals.

[0012] Furthermore, the antioxidant peptide can inhibit oxidative stress-mediated cellular lipid peroxidation.

[0013] Furthermore, the antioxidant peptide has the activity of inhibiting the growth of Escherichia coli.

[0014] The present invention also provides a method for preparing the antioxidant peptide, comprising the following steps:

[0015] (a) Protein was extracted from the petals of Camellia chrysantha to obtain protein extraction raw material;

[0016] (b) The protein extraction raw material obtained in step (a) was enzymatically hydrolyzed with protease, inactivated, centrifuged, and the supernatant was concentrated to obtain a concentrated solution.

[0017] (c) Ultrafiltration is performed on the concentrate obtained in step (b), and the filtrate is collected;

[0018] (d) The filtrate obtained in step (c) is subjected to gel chromatography and ion exchange chromatography in sequence to collect the chromatographic fraction with antioxidant activity;

[0019] (e) The chromatographic components obtained in step (d) are separated and purified by reversed-phase high-performance liquid chromatography, and the eluent corresponding to the chromatographic peak with antioxidant activity is collected to obtain the antioxidant peptide.

[0020] To further explain, in step (b), the protease is pepsin, and the amount of pepsin used is 4-6% of the mass of the protein extraction raw material; the enzymatic hydrolysis conditions are pH 1.5-2.5, temperature 35-40℃, and time 3-5 hours; the inactivation conditions are water bath treatment at 90-100℃ for 8-12 minutes.

[0021] To further explain, in step (c), the ultrafiltration is performed sequentially using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa and 3 kDa.

[0022] To further clarify, in step (d), the gel chromatography uses Sephadex G-15 as the separation medium, water as the mobile phase, a flow rate of 0.8-1.2 mL / min, and a detection wavelength of 280 nm; the ion exchange chromatography is anion exchange chromatography, with mobile phase A being 20 mmol / L Tris-HCl buffer (pH 8.0) and mobile phase B being Tris-HCl buffer (pH 8.0) containing 0.8-1.2 mol / L NaCl.

[0023] To further explain, in step (e), the conditions for the reversed-phase high-performance liquid chromatography are as follows: the chromatographic column is a C18 semi-preparative column, mobile phase A is a 0.1% trifluoroacetic acid aqueous solution, mobile phase B is a 0.1% trifluoroacetic acid acetonitrile solution, and gradient elution is used.

[0024] To further explain, the gradient elution procedure is as follows: 0-8 min, 5% B; 8-40 min, 5% B linearly increases to 45% B; 40-43 min, 45% B linearly increases to 95% B; 43-45 min, 95% B; 45-50 min, 95% B decreases to 5% B.

[0025] The present invention also provides the application of the above-described antioxidant peptides in the preparation of antioxidants, food, health products, cosmetics, pharmaceuticals or feed additives.

[0026] The beneficial effects of this invention are:

[0027] 1. This invention is the first to isolate and identify a novel antioxidant peptide from the petals of Camellia chrysantha, with the amino acid sequence Leu-Gly-Leu-Phe (LGLF), filling a technological gap in antioxidant peptides derived from Camellia chrysantha. This peptide consists of four amino acid residues, has a small molecular weight, is easily absorbed and utilized by the human body, and, being derived from a natural plant with both medicinal and edible properties, exhibits high safety, avoiding the potential toxic side effects of synthetic antioxidants (such as BHA and BHT). Furthermore, the antioxidant peptide of this invention demonstrates excellent in vitro free radical scavenging ability, showing dose-dependent scavenging effects against both ABTS and DPPH free radicals. Experiments confirmed that its EC50 values ​​reached 0.95 mg / mL and 0.26 mg / mL, respectively. Although slightly lower than the positive control glutathione, it still falls within the category of highly active natural antioxidant peptides. This peptide is not only effective in chemical systems, but also significantly inhibits hydrogen peroxide-induced lipid peroxidation in HEK293T cells at the cellular level, reducing intracellular malondialdehyde (MDA) levels. This demonstrates its antioxidant function through a dual mechanism of scavenging free radicals and blocking the lipid peroxidation chain reaction, exhibiting a clear cytoprotective effect. Notably, this antioxidant peptide also possesses excellent antibacterial activity, showing significant bactericidal effects against Gram-negative Escherichia coli, with a minimum bactericidal concentration as low as 8 mg / mL. This indicates that the peptide has multifunctional bioactivity, providing experimental evidence for its expanded applications in food preservation and anti-infection.

[0028] 2. The preparation method provided by this invention is scientifically sound, reasonable, and highly operable. Addressing the technical bottleneck of protein extraction from Camellia chrysantha petals, this invention designs a combined extraction process integrating alcohol extraction for impurity removal, alkali dissolution and acid precipitation, and cellulase assistance. This effectively removes interfering substances such as polyphenols and pigments, improving protein purity and yield. Utilizing pepsin-specific enzymatic hydrolysis, the process is mild, highly controllable, and preserves the natural activity of the peptides. Furthermore, through a multi-stage separation system combining ultrafiltration, gel chromatography, ion exchange chromatography, and reversed-phase high-performance liquid chromatography, combined with an activity-tracking screening strategy, a single active peptide was successfully obtained. This method is stable, reproducible, and easily scaled up for industrial production. Based on these advantages, the antioxidant peptides of this invention can be widely applied in antioxidants, food, health products, cosmetics, pharmaceuticals, and feed additives, especially suitable for developing anti-aging skincare products, functional foods, and drugs to assist in the treatment of oxidative stress-related diseases. As a unique plant resource of Guangxi, Camellia chrysantha opens up new avenues for its high-value utilization, possessing significant ecological and economic benefits and promising industrialization prospects. Attached Figure Description

[0029] Figure 1The graph shows the in vitro free radical scavenging activity results of the antioxidant peptide Leu-Gly-Leu-Phe (LGLF) of this invention. The left graph shows the change curve of ABTS free radical scavenging rate with concentration, and the right graph shows the change curve of DPPH free radical scavenging rate with concentration.

[0030] Figure 2 The graph shows the in vitro free radical scavenging activity results of the positive control glutathione. The left graph shows the change in ABTS free radical scavenging rate with concentration, and the right graph shows the change in DPPH free radical scavenging rate with concentration.

[0031] Figure 3 The figure shows the effect of the antioxidant peptide Leu-Gly-Leu-Phe (LGLF) of this invention on the malondialdehyde (MDA) content in HEK293T cells under oxidative stress. The figure compares the MDA content of the blank group, control group, damaged group (H2O2 treatment) and protection group (LGLF pretreatment + H2O2 treatment).

[0032] Figure 4 The images show Escherichia coli colonies treated with different concentrations of LGLF, with concentrations of 0, 1, 2, 4, and 8 mg / mL, respectively.

[0033] Figure 5 The graph shows the survival rate of E. coli treated with different concentrations of LGLF. The horizontal axis represents the LGLF concentration (mg / mL), and the vertical axis represents the survival rate (%). Detailed Implementation

[0034] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0035] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0036] Example 1: Protein extraction and enzymatic hydrolysis from Camellia chrysantha petals

[0037] Take dried Camellia chrysantha petals, pulverize them, and pass them through a 60-mesh sieve. Weigh 100 g of the powder and add 2000 mL of 95% ethanol at a material-to-liquid ratio of 1:20 (w / v). Sonicate for 30 min, filter under reduced pressure, and collect the residue. Air-dry the ethanol-extracted powder at room temperature, then add deionized water at a material-to-liquid ratio of 1:20 (w / v). Adjust the pH to 9.0 with 1 mol / L NaOH, sonicate for 30 min, and then magnetically extract for 180 min. Centrifuge the extract at 5000 r / min for 20 min and collect the supernatant. Add 5% (w / v) cellulase to the supernatant and enzymatically hydrolyze for 2 hours at pH 4.8 and 50℃ to obtain the protein extraction raw material.

[0038] Add 4-6% (w / v) pepsin, preferably 5% (w / v), to the above-mentioned protein extraction raw materials. Adjust the pH to 1.5-2.5, preferably 2.0, with 1 mol / L HCl. Incubate the mixture in a constant temperature water bath at 35-40℃, preferably 37℃, for 3-5 hours, preferably 4 hours. After incubation, inactivate the enzyme in a water bath at 90-100℃ for 8-12 minutes, preferably 95℃ for 10 minutes. After cooling to room temperature, centrifuge at 8000 r / min for 20 minutes. Collect the supernatant and vacuum dry to concentrate the solution. Perform ultrafiltration sequentially through ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa and 3 kDa, collecting the final filtrate for later use.

[0039] Example 2: Isolation, purification, and mass spectrometry identification of antioxidant peptides

[0040] The ultrafiltration filtrate obtained in Example 1 was loaded onto a Sephadex G-15 gel chromatography column (1.6 × 60 cm), using deionized water as the mobile phase at a flow rate of 0.8–1.2 mL / min, preferably 1.0 mL / min. The elution curve was monitored at 280 nm. Each component was manually collected based on the retention time of the chromatographic peaks. The antioxidant activity of each component was assessed using ABTS and DPPH radical scavenging assays (see Example 3 for details). The component with the highest activity was selected for further purification.

[0041] The above-mentioned active components were loaded onto a Hisprep Q FF 16 / 10 anion exchange chromatography column. Mobile phase A was 20 mmol / L Tris-HCl buffer (pH 8.0), and mobile phase B was Tris-HCl buffer (pH 8.0) containing 0.8-1.2 mol / L, preferably 1 mol / L NaCl. The flow rate was 4 mL / min, and the detection wavelength was 220 nm. Gradient elution was used, with mobile phase B linearly increasing from 0 to 100%. The eluent corresponding to each elution peak was collected, and the antioxidant activity was tested again. The component with the best activity was freeze-dried to obtain a lyophilized powder.

[0042] The lyophilized powder was reconstituted in a 0.1% trifluoroacetic acid (TFA) aqueous solution, desalted using a C18 solid-phase extraction column, and then separated by reversed-phase high-performance liquid chromatography (RP-HPLC) using an XBridge BEH C18 semi-preparative column (10 × 250 mm, 5 μm). Mobile phase A was a 0.1% TFA aqueous solution, and mobile phase B was a 0.1% TFA acetonitrile solution. The flow rate was 2.5 mL / min, and the detection wavelength was 220 nm. The gradient elution program was set as follows: 0–8 min, 5% B; 8–40 min, 5% B linearly increasing to 45% B; 40–43 min, 45% B linearly increasing to 95% B; 43–45 min, maintaining 95% B; 45–50 min, 95% B decreasing to 5% B. The eluent corresponding to the main chromatographic peaks was collected, lyophilized, and the antioxidant activity was measured again. The fraction with the highest activity was taken as the final purified antioxidant peptide.

[0043] A small amount of the purified peptide was dissolved in a matrix solution for matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / TOF-MS) and analyzed by mass spectrometry. Fragmentation analysis by primary and secondary mass spectrometry confirmed the amino acid sequence of the antioxidant peptide to be Leu-Gly-Leu-Phe (LGLF), with a molecular weight of approximately 450.5 Da.

[0044] Example 3: In vitro detection of ABTS and DPPH free radical scavenging activities

[0045] 1. ABTS Free Radical Scavenging Assay: A 1:1 (v / v) mixture of 7.4 mmol / L ABTS solution and 2.6 mmol / L potassium persulfate solution was incubated at room temperature in the dark for 12-16 hours to form the ABTS working stock solution. Before use, the solution was diluted 30-40 times with anhydrous ethanol to obtain an absorbance of 0.7 ± 0.02 at 734 nm, yielding the ABTS working solution. 50 μL of LGLF sample solution (prepared in Example 2) at different concentrations (0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 mg / mL) and 150 μL of ABTS working solution were added to each well of a 96-well plate. The plates were incubated in the dark for 10 min, and the absorbance was measured at 734 nm, denoted as A. X Deionized water was used as a control group instead of the sample, and the absorbance value was recorded as A0. According to the formula "ABTS free radical scavenging rate = (1 - A0) / 2", the ABTS free radical scavenging rate is calculated as follows: X The clearance rate at each concentration was calculated using " / A0) × 100%". Glutathione was used as a positive control, and a series of concentrations were prepared for parallel determination.

[0046] 2. DPPH free radical scavenging experiment: Add 100 μL of the above-mentioned series of concentrations of LGLF sample solution and 100 μL of 0.2 mmol / L DPPH ethanol solution to a 96-well plate, respectively. Incubate in the dark for 30 min, and measure the absorbance at a wavelength of 517 nm, denoted as A. X Deionized water was used as the control group instead of the sample, and the absorbance value was recorded as A0; anhydrous ethanol was used as the blank group instead of the DPPH solution, and the absorbance value was recorded as A0. b According to the formula "DPPH free radical scavenging rate = [1 - (A) / 2]", the DPPH free radical scavenging rate is calculated as follows: X - A b The clearance rate at each concentration was calculated using the formula: [A0] × 100%. Glutathione was also used as a positive control in parallel assays.

[0047] The results are as follows Figure 1 , Figure 2 As shown in Table 1, the scavenging rates of LGLF peptide against ABTS and DPPH free radicals at a concentration of 1.6 mg / mL were 78.38% and 92.76%, respectively. The calculated half-maximum effective concentration (Cmax) was... 50The values ​​were 0.95 mg / mL and 0.26 mg / mL, respectively. The positive control glutathione, at 1.6 mg / mL, showed scavenging rates of 93.49% and 90.30% for ABTS and DPPH free radicals, respectively, with EC50 values ​​of 0.079 mg / mL and 0.076 mg / mL, respectively. These results indicate that although the LGLF peptide prepared in this invention has slightly lower activity than glutathione, it still exhibits good in vitro free radical scavenging ability in a clear dose-dependent manner.

[0048] Table 1. Scavenging rates of ABTS and DPPH free radicals at different concentrations of LGLF and glutathione

[0049]

[0050] Example 4: Detection of cellular lipid peroxidation inhibitory activity (MDA content determination)

[0051] Logarithmic growth phase human embryonic kidney cells (HEK293T) were harvested, digested with trypsin, counted, and after adjusting the cell density, seeded into 6-well plates at a density of 1.0 × 10⁶ cells per well. 6 Cells were collected. Four experimental groups were set up: blank group (no cells, only culture medium), control group (normal cell culture), damage group (cells treated with H2O2), and protection group (cells pretreated with LGLF and then treated with H2O2). The cells were incubated in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere.

[0052] After incubation, each well of the protection group was added with LGLF peptide solution (prepared in Example 2, using sterile culture medium) to a final concentration of 500 μmol / L, while the remaining groups were added with an equal volume of sterile culture medium. After incubation for another 24 hours, each well of the protection and damage groups was added with H2O2 solution to a final concentration of 400 μmol / L, while the control and blank groups were added with an equal volume of culture medium. Incubation was continued for another 12 hours to induce oxidative stress damage.

[0053] Cells from each well were collected into centrifuge tubes and washed twice with pre-cooled phosphate buffer. Following the instructions of the malondialdehyde (MDA) assay kit (TBA method), cells were lysed with lysis buffer, centrifuged, and the supernatant was mixed with thiobarbituric acid reaction solution. The mixture was incubated in a boiling water bath for color development. After cooling, the absorbance was measured at 532 nm. The intracellular MDA content was calculated based on the standard curve. Each experimental group had three replicates.

[0054] The results are as follows Figure 3As shown in Table 2, compared with the control group, the intracellular MDA content in the H2O2-damaged group was significantly increased (P<0.05), indicating that oxidative stress successfully induced cellular lipid peroxidation. Compared with the damaged group, the intracellular MDA content in the protected group pretreated with LGLF peptide was significantly decreased (P<0.05), indicating that LGLF peptide can effectively inhibit oxidative stress-mediated cellular lipid peroxidation, reduce the generation of MDA, the end product of lipid peroxidation, and thus protect cells from oxidative damage.

[0055] Table 2. Results of MDA content detection under oxidative stress.

[0056]

[0057] Example 5: Antibacterial activity detection

[0058] Gram-negative Escherichia coli (E. coli) was selected as the test strain, and the antibacterial properties of different concentrations of Leu-Gly-Leu-Phe were evaluated using the plate count method. The bacterial strain was inoculated into tryptic soy peptone broth and cultured for 12 h until the logarithmic growth phase. The bacterial culture was then diluted to 10⁻¹⁰ with PBS solution. 8 CFU / mL.

[0059] LGLF peptide was prepared into different final concentrations of 1, 2, 4, and 8 mg / mL using sterile PBS solution.

[0060] The samples were incubated at 37 °C with shaking at 150 r / min for 12 h. Each reaction solution was appropriately diluted and spread onto tryptic soy agar plates. Afterward, the plates were incubated upside down at 37 °C for 16 h, and the bacterial colony count was recorded. Three replicates were set for each concentration group, and the results are expressed as averages. PBS solution was used instead of LGLF solution as a blank control group, and the colony count was recorded as B0. The colony counts of samples at different concentrations were recorded as B1. The survival rate of *E. coli* at each concentration was calculated using the formula: "Survival rate = B1 / B0 × 100%".

[0061] The results are as follows Figure 4 , Figure 5 As shown in Table 3, the number of E. coli colonies gradually decreased with increasing Leu-Gly-Leu-Phe concentration. When the concentration increased to 8 mg / mL, the survival rate of E. coli decreased from 100% to 0%, indicating that no viable colonies were detected at this concentration. The minimum bactericidal concentration (MBC) was 8 mg / mL, demonstrating that the antioxidant peptide LGLF of this invention has significant bactericidal activity against E. coli.

[0062] Table 3. Survival rate of Escherichia coli treated with different concentrations of LGLF

[0063]

[0064] In summary, this invention uses dried petals of *Camellia chrysantha* as raw material. A protein extract was obtained through alcohol extraction to remove impurities, followed by alkali dissolution and acid precipitation combined with cellulase-assisted extraction. Further controlled enzymatic hydrolysis with pepsin was performed. Utilizing multi-stage separation techniques including ultrafiltration, gel chromatography, ion exchange chromatography, and reversed-phase high-performance liquid chromatography, combined with stepwise screening for ABTS and DPPH free radical scavenging activity, a single antioxidant peptide was successfully isolated and purified from the *Camellia chrysantha* protein hydrolysate. Its amino acid sequence was identified as Leu-Gly-Leu-Phe (LGLF) by MALDI-TOF / TOF-MS mass spectrometry. In vitro activity evaluation results showed that this peptide exhibited good scavenging ability against both ABTS and DPPH free radicals, with EC50... 50 The values ​​were 0.95 mg / mL and 0.26 mg / mL, respectively. Cellular experiments further confirmed that LGLF peptides significantly reduced malondialdehyde (MDA) levels in hydrogen peroxide-induced HEK293T cells, effectively inhibiting oxidative stress-mediated lipid peroxidation. Antibacterial experiments showed that LGLF peptides exhibited significant bactericidal activity against *Escherichia coli*; at a concentration of 8 mg / mL, the survival rate of *E. coli* decreased to 0, with a minimum bactericidal concentration (MBC) of 8 mg / mL. This invention fills the technological gap in antioxidant peptides derived from *Camellia chrysantha*. The provided preparation method is reasonable, reproducible, and easy to scale up for production. The obtained LGLF peptides have a clear structure, excellent activity, and high safety, and can be widely used as active ingredients in antioxidants, food, health products, cosmetics, pharmaceuticals, and feed additives, opening up new avenues for the high-value utilization of this unique plant resource.

[0065] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. An antioxidant peptide derived from Camellia chrysantha, characterized in that, Its amino acid sequence is Leu-Gly-Leu-Phe, i.e., LGLF.

2. The antioxidant peptide according to claim 1, characterized in that, The antioxidant peptide has free radical scavenging activity, including ABTS free radicals and / or DPPH free radicals.

3. The antioxidant peptide according to claim 1 or 2, characterized in that, The antioxidant peptides can inhibit oxidative stress-mediated cellular lipid peroxidation.

4. The antioxidant peptide according to claim 1, characterized in that, The antioxidant peptide has the activity of inhibiting the growth of Escherichia coli.

5. A method for preparing the antioxidant peptide according to any one of claims 1-4, characterized in that, Includes the following steps: (a) Protein was extracted from the petals of Camellia chrysantha to obtain protein extraction raw material; (b) The protein extraction raw material obtained in step (a) was enzymatically hydrolyzed with protease, inactivated, centrifuged, and the supernatant was concentrated to obtain a concentrated solution. (c) Ultrafiltration is performed on the concentrate obtained in step (b), and the filtrate is collected; (d) The filtrate obtained in step (c) is subjected to gel chromatography and ion exchange chromatography in sequence to collect the chromatographic fraction with antioxidant activity; (e) The chromatographic components obtained in step (d) are separated and purified by reversed-phase high-performance liquid chromatography, and the eluent corresponding to the chromatographic peak with antioxidant activity is collected to obtain the antioxidant peptide.

6. The preparation method according to claim 5, characterized in that, In step (b), the protease is pepsin, and the amount of pepsin used is 4-6% of the mass of the protein extraction raw material; the enzymatic hydrolysis conditions are pH 1.5-2.5, temperature 35-40℃, and time 3-5 hours; the inactivation conditions are water bath treatment at 90-100℃ for 8-12 minutes.

7. The preparation method according to claim 5, characterized in that, In step (c), the ultrafiltration is performed sequentially using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa and 3 kDa.

8. The preparation method according to claim 5, characterized in that, In step (d), the gel chromatography uses Sephadex G-15 as the separation medium, water as the mobile phase, a flow rate of 0.8-1.2 mL / min, and a detection wavelength of 280 nm; the ion exchange chromatography is anion exchange chromatography, with mobile phase A being 20 mmol / L Tris-HCl buffer (pH 8.0) and mobile phase B being Tris-HCl buffer (pH 8.0) containing 0.8-1.2 mol / L NaCl.

9. The preparation method according to claim 5, characterized in that, In step (e), the conditions for the reversed-phase high-performance liquid chromatography are as follows: the chromatographic column is a C18 semi-preparative column, the mobile phase A is a 0.1% trifluoroacetic acid aqueous solution, the mobile phase B is a 0.1% trifluoroacetic acid acetonitrile solution, and gradient elution is used.

10. The use of the antioxidant peptide according to any one of claims 1-4 in the preparation of antioxidants, food, health products, cosmetics, pharmaceuticals or feed additives.

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