Isolation, purification, and characterization of antioxidant peptides from fresh mare's milk

Abstract In this study, the whey protein of fresh mare's milk was used as raw material. The antioxidant peptide liquid XMNDT was extracted from fresh mare's milk solution and purified. The peptide had a molecular weight of 1594.89 kDa and was mainly composed of VAPFPQPVVPYPQR. Antioxidant peptide XMNDT could inhibit the proliferation of A549 lung cancer cells in the G1 phase, accelerate cell apoptosis, increase the activity of SOD and the amount of GSH, and reduce the secretion of MDA. It also exhibited certain antioxidant capacity and free radical scavenging. These data can provide a basis for research on new antioxidant properties by reducing the inflammation caused by aging.


| PR EFACE
Fresh mare's milk is a grassland resource with distinctive national characteristics and profound cultural background. The nutritional value of mare's milk is higher than that of other dairy products. From the perspective of nutrition and health care, the effect of mare's milk is better than that of cow's milk. Mare's milk is similar to human milk in terms of chemical composition (Cagali et al., 2014;Claeys et al., 2014;Li et al., 2020). It contains rich nutrients that are involved in human metabolism and functions in adjusting the human body's physiological function, improving immunity, and preventing disease. Unsaturated fatty acid and low-molecular-weight fatty acid can prevent hypercholesterolemia and atherosclerosis and exert therapeutic effects on tuberculosis, emphysema, and chronic gastroenteritis symptoms (Kawuli, 2015). With the development of bioactive peptides in milk and dairy products, an increasing number of bioactive peptides from milk sources have been found. They play many roles in the cardiovascular system, immune system, digestive system, and nervous system. The biological activities of these biologically active polypeptides from milk sources have been gradually exploited to produce healthy or functional food to improve human health.
Oxidative stress occurs when the body is subjected to various harmful stimuli in vivo. During oxidative stress, highly active molecules, such as reactive oxygen free radicals (reactive oxygen species) and reactive nitrogen free radicals (reactive nitrogen species) are produced beyond the oxidation degree of oxide removal.
Therefore, an imbalance of oxidation system and antioxidant system occurs, leading to tissue damage. The most common diseases induced by elevated levels of oxidative stress are heart disease, | 4019 WAILI et AL. cancer, osteoarthritis, rheumatoid arthritis, diabetes, and neurodegenerative problems, such as Alzheimer's disease and Parkinson's disease (Jenner & Olanow, 1996). Antioxidants may slow down the effects of oxidative stress. Milk sources of active peptide have various human metabolic and physiological functions, are easy to digest and absorb, promote immunity, exhibit antibacterial and antiviral activities, and reduce blood pressure and blood lipid levels (Gill et al., 2000;Martínez-Maqueda et al., 2012;Mohanty et al., 2016;Tsuruki et al., 2005). Meanwhile, it's food safety is very high, which is the most popular research topic and a promising functional factor in the international food industry. Most proteins are digested and absorbed in the form of low peptides, which are absorbed in the form of free amino acids. Peptides are digested faster and are absorbed more than free amino acids, indicating that peptides have higher biological and nutritional values than free amino acids.
A bioactive peptide is a 25-amino acid protein with different compositions and arrangement from two different to general linear dipeptides. Its ring structure is complex as it is a multifunctional compound derived from protein. Moreover, it is extracted by membrane separation technology, ion-exchange chromatography or ion gel chromatography, and capillary electrophoresis, reversed-phase high-performance liquid chromatography (RP-HPLC), and time-offlight mass spectrometry were used to further purify and identify the active peptides and to reveal more physiological activities of the peptides. In this study, whey protein of fresh mare's milk was treated with trypsin enzyme solution for 3 hr and then enriched with Sephadex G-75 to separate proteins by molecular weight. In 3-10 kDa ultrafiltration membranes, the proteins were separated into two groups, namely, A and B. Group B showed the best antioxidant activity. In the B component, the proteins were further purified by RP-HPLC and then identified using MALDI-TOF/TOF. The molecular weight of the antioxidant peptide was 1594.89 kDa, and the amino acid composition was VAPFPQPVVPYPQR. Fresh mare's milk antioxidant peptide could inhibit A549 cell proliferation of lung cancer and cause arrest in the G1 phase and increased apoptosis compared with the model group. The antioxidant peptides showed increased glutathione (GSH) content and superoxide dismutase (SOD) activity. The antioxidant activity decreased the secretion of malondialdehyde (MDA), providing the basis for the study of antioxidant peptides in milk sources.

| Enzymatic hydrolysis of antioxidant peptides from fresh mare's milk
Fresh mare's milk was centrifuged at 11180×g for 15 min for degreasing. Skim milk and 2 mol/L acetic acid sodium acetate buffer were mixed with 10% acetic acid to adjust the pH to 4.6. The sample was incubated in a 40°C water bath for 20 min and centrifuged at 7155×g for 15 min. The supernatant was collected (whey protein), freeze-dried, sealed, and stored in a refrigerator at 4°C. The whey protein powder of fresh mare's milk was accurately weighed to 100 mg, diluted to a 30 mg/ml solution, and placed in a 90°C water bath for 10 min, following a previous method (Oftedal et al., 1983;Pikul et al., 2008) for the determination of antioxidant activity.
Antioxidant activity was determined with enzyme species, enzyme dosage, enzymolysis time, enzyme, and proportion of whey protein powder to investigate the factors of process conditions and to screen fresh mare's milk for antioxidant peptide enzymes.

| Enrichment of antioxidant peptides from fresh mare's milk by ultrafiltration
Ultrafiltration centrifuge tubes with molecular weights of 3, 10, and 30 kDa were selected. Hydrolysis was performed at 1207×g centrifugation for 30 min. Molecular weights of <3, >3, <10, >10, and <30 kDa were used to determine the antioxidant activity of five peptides. The peptides with strong antioxidant activity were frozen at 4°C (Lignitto et al., 2010;Wang et al., 2013).

| Peptide purification
The antioxidant peptide with the strongest antioxidant activity was prepared into 20 mg/ml, and 3 ml of the culture fluid was allowed to pass through a 0.45 µm micropore filter. The filtrate was added to a Sephadex g-75 glycemic gel column (1.5 cm × 60 cm) and then eluted with distilled water at a rate of 0.5 ml/min. The OD value of the eluent was determined at a wavelength of 280 nm. Sephadex G-75 was used to separate components with the strongest antioxidant value.
Approximately 10 mg of the sample was accurately weighed and dissolved in 1 ml of deionized water. The solution was centrifuged at 12880×g for 15 min and filtered through a 0.45 µm membrane.

| Structural analysis
A 0.5 µl sample was first placed on the MALDI target plate, dried naturally, placed on 0.5 µl of 4 mg/ml CHCA solution (50% acetonitrile solution containing 0.1% TFA), and dried naturally at room temperature. The samples were analyzed by mass spectrometry using a 5,800 MALDI-TOF/TOF mass spectrometer. The laser source was 355 nm wavelength Nd: YAG laser, the acceleration voltage was 20 kV, and the data were collected by positive ion mode and automatic data acquisition mode. The enzyme was used to correct the peptide at myoglobin and then corrected by external standard.
The primary mass spectrum of the sample ranged from 300 Da to 3,600 Da. MS was performed using reflector positive model with the following parameters: CID (OFF), mass range (700-5,000 Da), focus mass (1,600 Da), fixed laser intensity (4,500) digitizer, and bin size (0.5 ns). Tandem mass spectrometry was performed using the 2 KV positive model with the following parameters: CID (ON), precursor, mass, windows (relative 100 resolution, FWHM), fixed, laser, intensity (5,000), digitizer: bin size (1 ns). The data obtained were retrieved using the MASCOT (V 2.3) software. The search parameters were as follows: NCBI protein database, trypsin digestion, a leaky cutting site, tolerance level for 100 ppm, two MS tolerance at 0.8 Da, no fixed modification, and modified variable settings for methionine oxidation (Motta et al., 2014).

| Cell proliferation was detected by MTT assay
The logarithmic growth of tumor cells was evaluated by digesting the cells with 0.25% trypsin and adding 100 µl per hole of a 96-well cell culture plate, with each hole containing approximately 5 × 10 4 cells.
The plate was incubated at 37°C and 5% CO 2 humidity. The cancer cells were observed for adherent growth. The supernatant was added with 5, 50, 500, 1,000, and 2,000 µg/ml antioxidant peptide in each hole. The cell culture medium without samples was used as the control group, and each group had five holes. After culturing for 12, 48, and 72 hr, 5 mg/ml of MTT solution was added to each hole, and 20 µl/holes were cultured for 4 hr. The supernatant was discarded, added with 100 µl of DMSO per hole, and gently shaken for 10 min. At 490 nm wavelength, the absorption value of each hole was measured using an enzyme marker, and the average inhibition rate was calculated.
2.6.3 | Cell cycle was detected by flow cytometry A549 cells were inoculated in six holes, with each hole containing approximately 1 × 10 6 cells. The cells were cultured to logarithmic phase and then washed with culture medium and PBS. Antioxidant peptide was added to the low-, middle-, and high-dose groups. Antioxidant peptide was also added to the control group. The cells were allowed to grow after 48 hr of trypsin treatment. Approximately 0.5 ml of cells were collected per hole, digested for 1-2 min, and added with 2 ml of medium. The single-cell suspension was added into a centrifuge tube, centrifuged at 134×g for Cell inhibition rate = (Control group OD − Test group OD) Control group OD × 100 % 5 min. The supernatant was discarded, and the cells were washed twice with 1 ml of PBS. The supernatant was removed, and 1 ml of PBS was added to the heavy suspension and uniformly mixed. Approximately 2 ml of 70% ethanol precooled at 4°C overnight was added to the cells and centrifuged. The supernatant was discarded, and the cells were washed with PBS and then centrifuged. Approximately 500 µl of PI solution was added to stain for 30 min at room temperature. The cell suspension was subjected to flow cytometry. Cell cycle was measured, and the percentage of cells in the G1, S, and G2 phases was observed.

| Apoptosis was detected by flow cytometry
The A549  then evaluated for apoptosis using flow cytometry (Coyle et al., 2008).
The experiment was repeated three times.

| Antioxidant peptide of fresh mare's milk relieves oxidative stress
2.7.1 | Method for establishing cell hypoxia reoxygenation model The physical model of oxygen (mixed gas, preculture method) was constructed to develop good cell culture dishes in a sterile airtight container. The cells were subjected to air inlet Walter 95%N 2 + 5%CO 2 mixed gas outlet. The outflow of the container was sealed to maintain oxygen for 6 hr. The cells were then placed back into the 37°C and 5% CO 2 incubator, reoxygenated, and cultured for 3 hr.

| Experimental grouping
The good growth of lung cancer cells with 2 × 10 5 cell density were seeded in 60 mm dishes, cultured for 24 hr, and then randomly divided into five groups: blank control group, model group, low-dose peptide group, medium-dose peptide group, and high-dose peptide group. The model and blank control groups were only added with 5% basal culture medium. After 48 hr, the blank control group was allowed to continuously grow normally. The low-dose peptide group, middle-dose peptide group, high-dose peptide group, and model group were treated with hypoxia for 6 hr and reoxygenated for 3 hr. The centrifuged supernatant was used for the experiment.
The contents of GSH, SOD, and MDA were calculated according to the formula given by each reagent box. In each experiment, three holes in the experimental group were used, and the experiment was repeated three times.

| GSH detection
Approximately 2 × 10 5 cells were cultured in 60 mm Petri dish. The were then centrifuged for 1640×g at 4°C for 10 min. Approximately 1 ml of the supernatant was used for color reaction. Absorbance was detected at 420 nm with an enzyme analyzer (Cai et al., 2002). GSH content was calculated according to the following formula:

| SOD testing
Approximately 2 × 10 5 cells were cultured in a 60 mm Petri dish. The adherent cells were allowed to grow until 80% confluency. The blank model group, low-dose group, middle-dose group, and high-dose group of antioxidant peptides were cultured for 48 hr. Antioxidant activity of peptide intervention was determined after 48 hr. Hypoxia was induced for 6 hr, and the cells were reoxygenated for 3 hr. The liquid was absorbed, and 0.25% of pancreatic enzymes was used for digestion for 2-3 min. DMEM medium was added to terminate the digestion. The cell suspension was then loaded into 2 ml EP tube and GSH concentration = (test OD − blank OD) standard OD − blank OD × Standard concentration × Dilution factor ÷ Proteincon centration centrifuged at 1,000 rpm for 10 min. The supernatant was discarded, and the precipitated cells were added with 1 ml of PBS, gently blown, and then centrifuged at 1,000 rpm for 10 min. The supernatant was discarded, and the cells were manually ground ultrasonically for measurement. SOD was tested according to the manufacturer's instructions. WST-1 method was used to detect absorbance at 450 nm with an enzyme analyzer . SOD activity was calculated according to the following formula:

| MDA testing
Approximately 2 × 10 5 of cells were cultured in a 60 mm Petri dish.
The adherent cells were allowed to grow until 80% confluency. Blank model group, low-dose group, middle-dose group, and high-dose group of antioxidant peptides were cultured for 48 hr. The antioxidant activity of peptide intervention was determined after 48 hr. Hypoxia was induced for 6 hr, and the cells were reoxygenated for 3 hr. The cells were collected, added with 0.5 ml of agent five extract, and mixed for 2 min.
The cells were broken and added to 0.1 and 1.5 ml centrifuge tubes.
ELISA was performed according to the kit instructions. Absorbance at 532 nm was then determined (Xingxing et al., 2016). Sample MDA was calculated according to the following formula:

| Statistical analysis
The SPPSS19.0 statistical software was used to analyze the measurement data to represent the group and perform the t test. When p < .05, the difference was statistically significant. All tests were repeated three times.

| Ultrafiltration enrichment of antioxidant peptides from fresh mare's milk
The whey protein of fresh mare's milk was digested with trypsin enzyme for 3 hr and then subjected to high-speed centrifugation to remove insoluble enzyme solution. The ultrafiltration membrane was then enriched for 5 peptides with different molecular weight ranges, namely, <3, >3, <10, >10, and <30 kDa. Different molecular weight peptides were simultaneously determined. Reduction capacity was determined to assess hydroxyl radical scavenging ability. DPPH free radical scavenging ability was used as the index. Antioxidant peptides with a molecular weight of >10 kDa showed the strongest antioxidant activity. Therefore, they were selected for the following experiment. The results are shown in Table 1.

| Separation and purification of antioxidant peptides from fresh mare's milk by Sephadex G-75 gel chromatography
The molecular weight of antioxidant peptide >10 kDa was accurately

| RP-HPLC isolation and purification
Component B was subjected to RP-HPLC separation and purification ( Figure 2). For the gradient collection, a total of 63 pipe components were freeze dried and detected for antioxidant activity.
Among them, two components had strong antioxidant activity, and the activity of component 2 was higher than that of component 1.
The results are shown in Figure 2 and Table 2.

| MALDI-TOF/TOF analysis
The two components were analyzed by MALDI-TOF/TOF. Figure 3 shows the spectrum, combined with the amino acid composition analysis results. The two components with relative peptide molecu-  Figure 3.

TA B L E 2
The antioxidant activity of 1, 2 fraction was obtained by semi -Preparation of RP-HPLC (X ± S, n = 3)

| Effects of antioxidant peptide on the cell cycle of A549 lung cancer cells
As shown in Table 3, compared with the control group, the antioxidant peptide group showed significantly increased ratio of G1 phase cells (p < .05). However, the antioxidant peptide group showed significantly decreased S phase cells compared with the control group (p < .05). Antioxidant peptides could block cell proliferation in the S phase (Table 4, Figure 41-4).

| Apoptosis of A549 lung cancer cells induced by antioxidant peptide
The results showed that with the prolongation of treatment time,

| Oxidative stress index results
Compared with the blank control group, the model group showed significantly decreased secretion of GSH and SOD and significantly The results are shown in Table 6.

| D ISCUSS I ON
Antioxidant peptide is a kind of peptide with antioxidant activity.
There are naturally occurring antioxidant peptides in organisms, mainly carnosine and glutathione (Fraternale et al., 2016;Olivares et al., 2012). However, most antioxidant peptides are isolated from various proteins (Wang et al., 2014). Antioxidant peptides have be- ity. This is similar to the previous report (Verma et al., 2017;Verma et al., 2018). Two components were separated by Sephadex G-75.  In this study, we detected GSH, SOD, and MDA and found that antioxidant peptide induced lung cancer cells growth at 48 hr. Each drug group content in the culture supernatant of GSH cells was significantly increased. SOD activity was significantly increased, whereas MDA content was decreased significantly. The protective effect of antioxidant peptides in whey protein of fresh mare's milk on oxidative stress injury was preliminarily determined. These results show that the antioxidant peptide can regulate the intracellular antioxidant system, inhibit the free radicals produced during oxidative stress, and restore the oxidative damage to a certain extent. However, the effect of antioxidant peptides on the signaling pathway in lung cancer cells and the mechanism of apoptosis induced by lung cancer have not been fully elucidated. Thus, further research is needed.

ACK N OWLED G M ENTS
The authors would like to express their sincere gratitude to the Key

Laboratory of Active Components of Xinjiang Natural Medicine and
Drug Release Technology (XJDX1713). This study was supported by the Study on the characteristic nutrient composition and health function of mare's milk (2017A01002-4-2).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.