Low-Molecular-Weight Hydrolysate from Black Goat Extract Had Antioxidative and Anti-Inflammatory Effects in Macrophage Cells via Inhibition of MAPKs and NF-κ B Pathways

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Introduction
Excessive calorie intake and exposure to stress and pollution can generate harmful radicals in the body, including reactive oxygen species (ROS).Various circumstances such as oxidative stress, tissue damage, pathogen invasion, or exposure to lipopolysaccharides (LPS) resembling endotoxins, activate the immune system, encompassing both innate and adaptive immunity.Tis immune response involves diverse types of immune cells including macrophages, natural killer cells, neutrophils, and T and B lymphocytes.Macrophages play a protective role against oxidative stress by secreting prostaglandin E 2 (PGE 2 ), nitric oxide (NO), and proinfammatory cytokines through the expression of infammatory proteins [1].Te activation of these intracellular signaling pathways initiates infammatory processes [2].While infammation is recognized as a major defense mechanism in the body, excessive macrophage activation from infammation can contribute to the development of serious diseases such as diabetes, cancer, atherosclerosis, and arthritis [3].As this is highly related to oxidative stress, antioxidative agents play a protective role in infammatory conditions [4].
Synthetic antioxidants such as butylated hydroxytoluene and butylated hydroxyanisole are commonly employed to inhibit oxidative reactions and infammation [3].However, their usage is constrained by potential side efects.Natural sources, foods, and ingredients encompass a wide array of functional compounds that have antioxidative properties, thereby contributing to the maintenance of human health [5].Bioactive peptides typically exist in inactive forms within parent proteins, and their active forms are released through enzymatic hydrolysis [6].
Several proteolytic enzymes including trypsin, alcalase, ProteAX, favourzyme, FoodPro ® alkaline protease, pan- creatin, and protease P have been employed to generate antioxidative hydrolysates and peptides from diverse sources.Tese sources encompass a variety of origins, including fsh, milk, marine species, soybean, and plant [7,8], donkey skin [9], and pig skin [10].Especially, the bioactive peptides with low molecular weight are acknowledged for their potent antioxidative activity and several other functionalities, which are attributed to their ability to easily traverse the intestinal barrier [11].Previous studies have reported the diverse functionalities of low-molecular-weight peptides (<3 kDa) on cells [9,10,12].
Black goat meat is recognized as a nutritious animal protein due to its low-fat content and abundance of essential amino acids and minerals, such as Ca and Fe [13].In Korea, not only the meat but also the meat and bones of the black goat are consumed in the form of an extract that is prepared by processing the entire black goat carcass with Korean medicinal herbs under high temperature and pressure.Tis extract is well known to enhance the immune system and anti-infammatory function in young children, pregnant women, and the elderly [13].
Te aim of the current study was to assess the antioxidative and anti-infammatory efects of low-molecularweight hydrolysate (<3 kDa) derived from black goat extract (termed "APT6 < 3 kDa") on macrophage cells and to investigate the underlying mechanism of action.Furthermore, peptides exhibiting substantial antioxidative and antiinfammatory activities were isolated and identifed from APT6 < 3 kDa.

Preparation of Low-Molecular-Weight Hydrolysate from Black Goat Extract (BGE).
To create a low-molecular-weight hydrolysate from black goat extract (BGE), we followed the procedure outlined in Figure S1.We obtained fve female black goats (Capra hircus coreanae) with a carcass weight of 10-11 kg and aged 6-9 months from a local meat market in Chuncheon, Korea.Te entire black goat carcass (about 60% fresh meat percent) was sliced into small pieces measuring 5 × 5 × 5 cm 2 and washed under running water.Next, we extracted 2.5 kg of meat and bone with 7.5 L of distilled water in an autoclave at 121 °C for 10 hours with a slight modifcation of Kim et al. [14].Following extraction, we removed the bone and lyophilized the remaining meat and broth, which we subsequently used for hydrolysis.We hydrolyzed the raw material mixture using 0.67% (w/w) of alcalase (pH 8.0, 50 °C), ProteAX (pH 7.0, 50 °C), and trypsin (pH 8.0, 37 °C) enzymes for 2 h (6 h total).Te enzymes in the hydrolysate were denatured at 95 °C for 10 minutes, resulting in a hydrolysate referred to as "APT6."We separated APT6 under the molecular weight of 3 kDa using an ultrafltration membrane, which we called ATP6 <3 kDa.

RNA Extraction and Quantitative Reverse-Transcription
Polymerase Chain Reaction (RT-qPCR).mRNA levels of iNOS, COX-2, TNF-α, IL-1β, and IL-6 in RAW 264.7 cells were assessed using RT-qPCR.To conduct the assay, RAW 264.7 cells were treated with APT6 < 3 kDa for 1 hour prior to activation with LPS for 6 h [16].Total RNA was extracted from the cells, and cDNA was synthesized using a commercially available kit (Qiagen, Hilden, Germany).Te ratio of 260 : 280 nm in all samples was matched in the range of 1.8-2.0.Te RT-qPCR reaction was performed using targeted primers (Table S1) and SYBR Green master mix (BioFact, Daejeon, Korea), and the relative mRNA expression was calculated using the 2 −ΔΔCt method and normalized to the mRNA expression of the housekeeping gene (β-actin) using a LightCycler 96 instrument (Roche Diagnostics, Penzberg, Germany).

Separation and Identifcation of Peptides Having Antioxidative and Anti-Infammatory Activities in APT6 < 3 kDa.
Te protocol for separating and identifying peptides having high antioxidative and anti-infammatory activities in APT6 < 3 kDa involved several steps (Figure S1).First, lyophilized APT6 <3 kDa was dissolved in distilled water and separated using chromatography (NGC Quest 10 Plus; Bio-Rad, CA, USA) equipped with a Superdex peptide column (GE Healthcare, Piscataway, NJ, USA).Five fractions (F1, F2, F3, F4, and F5) were obtained at 215 nm.Tese fve fractions were then lyophilized and evaluated for antiinfammatory activity using the ORAC assay and tested for their ability to inhibit NO and PGE 2 levels in LPSstimulated RAW 264.7 cells at 250 μg/mL.
Te amino acid sequences of the peptides with the highest antioxidative and anti-infammatory activities were analyzed by a Triple TOF 5600 system (ABSciex, Foster City, CA, USA) with a YMC-Pack C8 column [11].A linear gradient method with a fow rate of 0.25 mL/min was employed, where mobile phases A and B consisted of 0.1% formic acid in water and acetonitrile, respectively.Te mass spectra were obtained through electrospray ionization in positive mode.Te full-scan mass spectra ranged from 360 to 1,800 m/z, and the data was analyzed using Analyst TF 1.7 software and ProteinPilot 4.5.

Statistical Analysis.
Te experiments were conducted in triplicate, and the statistical analysis was carried out using SAS software v.9.4 (SAS Institute Inc., Cary, NC, USA).Te data analysis in the study involved performing a one-way analysis of variance followed by Tukey tests to compare signifcant diferences among the means at the level of p < 0.05.Te results were presented as means and standard errors of the mean (SEM).

Amino Acid Composition of APT6 and APT6 < 3 kDa.
Te amino acid composition of APT6 and its low-molecularweight peptide (APT6 < 3 kDa) is shown in Table S2.Glutamic acid and glycine were identifed as the two major amino acids in both APT6 and APT6 < 3 kDa, with concentrations ranging from 12.57 to 13.23 g/100 g and 9.94 to 11.18 g/100 g, respectively.Tis fnding is consistent with previous studies that have reported glutamic acid and glycine as the predominant amino acids in other BGEs [14,17].Aspartic acid, proline, alanine, leucine, lysine, and arginine were also abundant in APT6 and APT6 < 3 kDa.Following ultrafltration with a 3 kDa cutof, threonine, leucine, tyrosine, and phenylalanine were increased, whereas aspartic acid, glycine, and cysteine were decreased.Te essential amino acid (EAA) content of APT6 and APT6 < 3 kDa was found to be 32.96% and 35%, respectively, which surpassed the EAA content of black goat meat extract (23.9%) [17] and veal meat and bone hydrolysate (21.82%) [18].However, the reported EAA content of chicken meat hydrolysate (44.5%) [19] exceeds that of APT6 and APT6 < 3 kDa.
Animal sources hold great potential as raw materials for generating protein hydrolysates that possess high antioxidative properties [20].Te contents of antioxidative amino acids (AAA) and hydrophobic amino acids (HAA) in APT6 and APT6 < 3 kDa were determined to be 42.55% and 50.39%, and 41.24% and 49.55%, respectively (Table S2).Te AAA and HAA composition of hydrolysates varies depending on the protein sources and enzymes used.Veal meat and bone hydrolysate produced using nutrase and trypsin had 43.7% AAA and 36.01%HAA [18].AAA (51.7%) and HAA (43.5%) contents of oyster hydrolysate generated with alcalase were higher and lower, respectively, than those of APT6 and APT6 < 3 kDa [21].Rapeseed protein hydrolysate produced with alcalase consisted of 37.35% AAA and 38.65% HAA [22], which were lower than the contents in APT6 and APT6 < 3 kDa.

Antioxidative Activities of BGE and Its Hydrolysates.
Te antioxidative activities (FRAP, ABTS, and ORAC) of BGE and hydrolysates are presented in Table 1.Te FRAP, ABTS, and ORAC activities of BGE were measured to be 5, 64.28, and 45.08 μmol TE/g dry matter, respectively.However, all the antioxidative activities of the hydrolysate (APT6) were found to be signifcantly higher than those of BGE (p < 0.05).Low-molecular-weight hydrolysate (APT6 < 3 kDa) exhibited the highest FRAP, ABTS, and ORAC activities (6.64, 206.98, and 247.44 μmol TE/g dry matter, respectively).In comparison, the ABTS activity of donkey-hide gelatin hydrolysate (<3 kDa) hydrolyzed with protease P and blue mussel hydrolysate (<1 kDa) prepared with pepsin was reported to be 51.59 μmol TE/g dry matter [9] and 55.25 μmol TE/g dry matter [3], respectively, which were substantially lower than the ABTS activity of APT6 < 3 kDa.Additionally, APT6 < 3 kDa exhibited higher ORAC activity than hydrolysates from pig skin (141.39 μmol TE/g dry matter) [23] and catfsh protein (16 μmol TE/g) [20], but lower activity than hydrolysate from donkey hide gelatin (307.26μmol TE/g dry matter) [9].Generally, enzymatic hydrolysis enhances the antioxidative activities compared to those of the original material [9,23,24].It has been reported that antioxidative peptides isolated from various food sources typically range in size from 500 to 1,800 Da.Low-molecular-weight peptides demonstrate greater biological efects due to their ease of intestinal wall penetration [12].Several studies have highlighted the anti-infammatory efect of protein hydrolysates exhibiting antioxidative activities [3,24].Collectively, our fndings suggest that APT6 and APT6 < 3 kDa could serve as natural antioxidants and potentially possess antiinfammatory properties.

Efects of APT6 < 3 kDa on RAW 264.7 Cell Viability.
Te efects of APT6 < 3 kDa on RAW 264.7 cell viability are shown in Figure 1.APT6 < 3 kDa at 10-500 μg/mL increased RAW 264.7 cell viability (Figure 1(a)).Following stimulation with LPS for 24 h, the cell viability decreased to 67.94% compared to the control (p < 0.05).LPS decreases the viability of RAW 264.7 cells by releasing infammatory substances acting as cytotoxic agents [25].Te stimulation in this study appeared to induce a lower cell state in RAW264.7 cells, which could afect the impact of agents on the result.However, treatment with 500 μg/mL of APT6 < 3 kDa signifcantly elevated the cell viability to 86.03% (Figure 1(b)).
In this study, APT6 < 3 kDa at concentrations ranging from 10 to 500 μg/mL did not exhibit any cytotoxic efects on RAW 264.7 cells, and treatment with 500 μg/mL of APT6 < 3 kDa efectively restored the reduced cell viability induced by LPS.Terefore, the concentration range of 10-500 μg/mL was selected to evaluate the antiinfammatory activity of APT6 < 3 kDa in this study.

Efect of APT6 < 3 kDa on NO, PGE 2 , and ROS Production.
In the infammatory condition, LPS stimulation leads to the upregulation of iNOS and COX-2 expression, resulting in the excessive production of PGE 2 and NO in macrophages [6].NO is a free radical produced by iNOS, while PGE 2 is a product of arachidonic acid metabolism by COX-2 [26].Consequently, NO and PGE 2 serve as representative indicators of an infammatory response.Consistent with expectations, treatment of RAW 264.7 cells with LPS at a concentration of 1 μg/mL for 24 hours increased the production of NO and PGE 2 production (Figures 1(c) and 1(d)).However, treatment with 100 μg/mL of APT6 < 3 kDa signifcantly reduced the level of NO from 37.24 μM (LPS only; 100%) to 34.33 μM (92.18%) (p < 0.05), and the maximum reduction to 33.27 μM (89.87%) was achieved with 500 μg/mL of APT6 < 3 kDa (Figure 1(c)).Moreover, the production of PGE 2 induced by LPS (100%) was reduced to 91.68% and 70.14% following treatment with 250 μg/mL and 500 μg/mL APT6 < 3 kDa, respectively (p < 0.05; Figure 1(d)).Tese results demonstrated that APT6 < 3 kDa at concentrations of 250 and 500 μg/mL can efectively downregulate the production of NO as well as PGE 2 production in LPS-stimulated RAW 264.7 cells.
Oxidative stress plays a critical role in the development of chronic infammation and the induction of cellular damage.ROS is generated under imbalanced oxidant conditions in the body [24].In this study, LPS stimulation resulted in oxidative stress, as evidenced by an increase in ROS production (141.88%) in RAW 264.7 cells.However, treatment with 250 μg/mL of APT6 < 3 kDa signifcantly suppressed ROS production to 133.13% (Figure 1(e)).Tis indicates that the antioxidative properties of APT6 < 3 kDa efectively mitigated oxidative stress in LPS-stimulated RAW 264.7 cells.Terefore, the consumption of these dietary antioxidants may confer protection against oxidative stress and prevent the progression of infammation.

Efects of APT6 < 3 kDa on iNOS and COX-2 mRNA and Protein Expression.
To explain the mechanisms underlying the inhibition of NO and PGE 2 levels in RAW 264.7 cells by APT6 < 3 kDa, the mRNA and protein expression levels of iNOS and COX-2 were evaluated.As shown in Figures 2(a) and 2(b), the mRNA levels of iNOS and COX-2 increased following stimulation with LPS (1 μg/mL) for 6 h compared to RAW 264.7 cells without APT6 < 3 kDa and LPS (p < 0.05).However, treatment with APT6 < 3 kDa at concentrations of 100-500 μg/mL signifcantly inhibited the expression of iNOS, reducing it from LPS-only treatment (100%) to 81.33%-66.77%(Figure 2(a)).Conversely, APT6 < 3 kDa did not exert a signifcant inhibitory efect on  ).In addition, treatment with 500 μg/mL of APT6 < 3 kDa resulted in a signifcant suppression of TNF-α levels to 87.33% compared to the LPS-only treatment (p < 0.05; Figure 3(f )).Tese results suggest that the reduction in IL-1β and TNF-α production observed following treatment with APT6 < 3 kDa may be associated with the downregulation of IL-1β and TNF-α transcription.However, the inhibition of IL-6 transcription did not result in a substantial reduction of IL-6 levels in RAW 264.7 cells.

Isolation and Identifcation of Antioxidative and Anti-
Infammatory Peptides in APT6 < 3 kDa.Te APT6 < 3 kDa was separated into fve fractions (F1-F5) by fast protein liquid chromatography using a Superdex peptide 30/100 GL column (Figure 5(a)).Te ORAC values of these fractions are presented in Figure 5(b).Among the fractions, F5 showed the highest ORAC activity (p < 0.05) of 979.88 μmol TE/g dry matter, which was 3.6-fold higher than that of APT6 < 3 kDa.F2 and F5 signifcantly reduced the production of NO and PGE 2 in LPS-stimulated RAW 264.7 cells compared to APT6 < 3 kDa and LPS-only treatment (p < 0.05; Figures 5(c  F5 exhibited both the highest antioxidative and antiinfammatory efects among the fve fractions, and therefore, it was selected for further analysis of its constituent peptides.In total, 43 peptides were identifed in F5, and the sequences of representative peptides are listed in Table 2. Notably, glycine (G), proline (P), hydroxyproline (X), and alanine (A) were the predominant amino acids in F5.Especially, G-P-X and G-P sequences were frequently detected.Te antioxidative and immunomodulatory properties of peptides can be infuenced by their structure, amino acid composition, length, and hydrophobicity [7].For instance, collagen contains a G-P1-P2 triplet sequence, where P1 is predominantly proline (20-30%) and P2 is typically hydroxyproline [29].Te exposure of the glycine residue is essential for Fe (II)-binding activity [30].Te G-P-X sequence, found in peptides derived from marine sources or animal skin, is known to exhibit high antioxidative activity [31].According to Jin et al. [32], G-P displays potent dipeptidyl peptidase IV-inhibitory activity, suggesting its potential application in pharmacological agents for infammatory diseases.In addition, HAA contributes to the antioxidative and anti-infammatory properties of peptides [7].In the case of the 43 peptides identifed in F5, they exhibited HAA content ranging from 50.00 to 83.33%.Tese results suggest that the high antioxidative and antiinfammatory efects observed in F5 and APT6 < 3 kDa may be attributed to the amino acid composition and characteristics of these peptides.However, further investigations are required to explore the specifc antioxidative and anti-infammatory activities of each peptide within F5.

Conclusions
Te present study provides evidence for the antioxidative and anti-infammatory properties of APT6 < 3 kDa, a lowmolecular-weight hydrolysate of BGE.APT6 < 3 kDa exhibited potent antioxidative activity, reducing oxidative stress in LPS-stimulated RAW 264.7 cells.Moreover, it demonstrated anti-infammatory efects by inhibiting the NF-κB and MAPK pathways, resulting in a decrease in the production of proinfammatory cytokines and downregulation of infammatory protein expression.Notably, a specifc fraction of APT6 < 3 kDa (F5), which contained 43 short peptides, showed high antioxidative and antiinfammatory activities, likely attributed to its high proportion of HAA.Tese fndings highlight the potential application of APT6 < 3 kDa and its peptides in the functional food industry.Furthermore, from a broader perspective in the future, it will be worthwhile to explore the immune regulatory efects of APT6 < 3 kDa.However, future investigations will be warranted to evaluate the antiinfammatory efects and absorption profle of APT6 < 3 kDa and its purifed peptides in other cell lines and animal models.

Table 1 :
Antioxidative activities of BGE and its hydrolysates.

Table 2 :
Amino acid sequences of peptides in F5 and their HAA composition (%).