Comparison of the nutritional and phytochemical composition and antioxidant activities of Aralia elata (Miq.) Seem fruits in Northeast China

2.1 Chemicals and abbreviations

Minimal essential medium (MEM), penicillin–streptomycin (PSS) solution, foetal bovine serum (FBS) and the fluorescent probe (DCFH-DA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) assays were purchased from Beyotime (Shanghai, China). Kits used to assay malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities and total antioxidant capacity (T-AOC) were purchased from Nanjing Jiancheng Bioengineering Co. Ltd. (Nanjing, Jiangsu, China).

2.2 Raw materials

AFs obtained from Harbin, Benxi, Raohe, Jiaohe, and Mudangjiang were hand-collected from August 25 to September 10, 2019. We selected the shady slopes of the mixed forests of korean pine and A. elata as the sample collection sites in five regions of Harbin, Benxi, Raohe, Jiaohe, and Mudanjiang. Each sampling site was established with three standard areas of 10 m × 10 m. Ten A. elata plants were randomly selected to collect 20 kg of fruits (including rachises) in each standard area. The collected fruits were all reddish-brown, fully ripened fruits to ensure similar states of the AFs. AFs were placed in a fresh-keeping box containing ice packs and transported back to the laboratory as soon as possible. The location and geographical information of the five zones (Harbin, Benxi, Raohe, Jiaohe, and Mudangjiang) is shown in Fig. 1 and Table 1. The prefrozen AF samples were freeze-dried with a vacuum freeze dryer (LGJ-1A-50, Beijing, China) and then crushed with a disintegrator (JP1000B, Zhejiang, China). The powder was stored at −20 °C until use.

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Fig. 1

Map of the location of harvest of Aralia elata (Miq.) Seem fruits.

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2.3 Extraction procedures

AF extracts (AFEs) were obtained using the method described by Tian et al. (2020). Compared with organic solvents such as methanol and ether, ethanol is nontoxic and safe. Therefore, ethanol was chosen as the extraction solvent in this study. AF powders (500 g per region) from Harbin, Benxi, Raohe, Jiaohe, and Mudangjiang were soaked in an ethanol–water solution (80%) for 30 min and extracted with a MARS-II (1000 W, 2450 MHz) microwave-accelerated reaction system. The extraction parameters were as follows: sample–solvent ratio, 1:30; microwave power, 530 W; microwave time, 40 s; and number of extraction cycles, 3. AFEs were concentrated with a rotary evaporator (RE-5203, Shanghai, China) and freeze-dried with a vacuum freeze dryer. AFEs used in all assays were liquid.

2.4 Analysis of the flavonoid content

The flavonoid content was determined using the method reported by Miliauskas et al. (2004), with some modifications. Five hundred microlitres of AFE, 4.5 mL of distilled water, 3 mL of a 5% NaNO₂ solution and 0.3 mL of a 10% AlCl₃ solution were mixed and reacted for 12 min at room temperature. Then, 2 mL of NaOH were added, and the absorbance was measured at 510 nm using an ultraviolet–visible spectrophotometer (Cary 50 Bio, Victoria, Australia). Rutin (0.2 mg/mL) was used as the standard solution.

2.5 Analysis of the total saponin content

The method described by Hiai et al. (1976) was used to determine the total saponin content. Five hundred microlitres of AFE were evaporated in a water bath at 45 °C. Then, a 5% vanillin–glacial acetic acid solution (0.2 mL) and perchloric acid (0.8 mL) were added. The mixtures were heated in a constant temperature water bath at 60 °C for 15 min. The samples were transferred to an ice bath. After 5 min, 5 mL of glacial acetic acid were added, and the absorbance was measured at 560 nm. Oleanolic acid (1 mg/mL) was used as the standard solution.

2.6 Analysis of the total anthocyanin content

The total anthocyanin content of AFEs was determined using the pH-differential method (Lee et al., 2005). Two millilitres of AFE were diluted 10 times with pH 1 and pH 4.5 buffer and then incubated at room temperature for 60 min. The absorbance was measured at 520 nm and 700 nm, respectively. The total anthocyanin content was calculated using the method described by Lee et al. (2005) with the following equation: $$\text{Anthocyanin content}\left(\text{mg}/\text{100g}\right)=\frac{A\times MW\times DF\times V\times {10}^2}{\epsilon \times 1}$$

where A= (A_{520 nm} – A_{700 nm})_{pH 1.0} – (A_{520 nm} – A_{700 nm})_{pH 4.5}; MW = 449.2 g/mol for the molecular weight of cyanidin-3-glucoside; DF = fold dilution; l = path length in cm; V = sample volume; and ε = 26900 L^.mol/cm for the molar extinction coefficient of cyanidin-3-glucoside.

2.7 Uhplc-MS/MS analysis

The AFEs were passed through a macroporous resin column and sequentially eluted with deionized water and 20% and 80% ethanol. The solutions eluted with the 80% ethanol solution were collected, concentrated with a rotary evaporator, and freeze-dried to obtain total saponins. The total saponins were dissolved in methanol (1 mg/mL) and filtered with a 0.22 μm filter membrane.

The chromatographic analysis was conducted using an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm) at 40 °C. A volume of 5 μL was injected at a flow rate of 0.4 mL/min. The mobile phase consisted of 0.1% formic acid (A) and pure acetonitrile (B). The elution programme was as follows: 0–3 min, 5–15% B; 3–3.5 min, 15% B; 3.5–6 min, 15–30% B; 6–6.5 min, 30% B; 6.5–12 min, 30–70% B; 12–12.5 min, 70% B; 12.5–18 min, 70–100% B; and 18–20 min, 100% B.

Mass spectrometry was carried out in negative ion detection mode, and the scanning ranges of the primary and secondary mass spectra were 100–1200 m/z and 50–1000 m/z, respectively. Each gas path used nitrogen. The specific parameters were as follows: ion spray floating voltage, 5500 V; source gas 1, 50 psi; source gas 2, 50 psi; temperature, 550 °C; declustering potential, 90 V; and collision energy, 10 V.

2.8 Free amino acid analysis

The method described by Yoo and Chang (2016) was modified slightly to analyse the content of free amino acids. AF powders (50 mg per region) from Harbin, Benxi, Raohe, Jiaohe, and Mudangjiang were hydrolysed with 6 mol/L HCl for 22 h at 110 °C. The suspension was filtered into a 50 mL volumetric flask. One millilitre of the filtrate was dried under a vacuum at 45 °C, dissolved by adding 2 mL of sodium citrate buffer solution, and filtered with a 0.22 μm filter membrane. The amount of amino acids was determined using an amino acid analyser (L-3000, Jiangsu, China). A mixed amino acid standard working solution (100 nmol/mL) was used as the standard solution.

2.9 Cell culture

HEK293 cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in MEM supplemented with 1% PSS and 10% FBS at 37 °C in an incubator with 5% CO₂. HEK293 cells (5–6 × 10³ cells/mL) were seeded into flat-bottomed plates and cultured for 24 h in a CO₂ incubator.

2.10 Analysis of cytotoxicity/proliferation and protective effects

Once all the cells grew to approximately 70–80% confluence, the medium was removed, and the cells were pretreated with AFE (1–200 μg/mL) for 24 h. Normal cells without treatment were regarded as the control group. Cell viability was measured using a CCK-8 assay to evaluate the cytotoxicity or proliferation effect of the AFEs. The cells were pretreated with AFE (1–50 μg/mL) for 24 h. Then, the medium was gently removed, and the cells were exposed to 400 μM H₂O₂ for 4 h. HEK293 cells incubated with H₂O₂ (400 μM) served as the control group, and the protection potential of the control group was designated as 0.0%. Cell viability was measured using a CCK-8 assay to evaluate the protective effects of AFE on H₂O₂-induced oxidative stress.

2.11 Cck-8 assay

Ten microlitres of CCK-8 solution were added to the cells and incubated at 37 °C for 3 h. The 96-well plate was subsequently placed in a multimode microplate reader (Tecan, Infinite M200 Pro) to measure the absorbance at 450 nm. Six parallel experiments with cells in each treatment group were performed, and all the assays were repeated three times. Cell viability was calculated using the following equation: $$C\text{e}llviability\left(\%\right)=\frac{O{D}_{\text{injure group}}-O{D}_{\text{blank group}}}{O{D}_{\text{control group}}-O{D}_{\text{blank group}}}$$

2.12 Measurement of intracellular ROS production

A DCFH-DA fluorescent probe was used to determine intracellular ROS production. Normal cells without treatment were used as the control group, and H₂O₂-treated cells served as the injury group. Incomplete medium containing DCFH-DA (10 μM) was added to the cells, and the cells were then incubated at 37 °C for 20 min. The DCFH-DA fluorescent probe was removed, and the cells were washed three times with phosphate-buffered saline (PBS). The green fluorescence in HEK293 cells was analysed using a fluorescence microscope (BX43 upright microscope, Olympus, Long Island, New York, USA). The fluorescence intensity was immediately detected using a multimode microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 525 nm.

2.13 Measurement of MDA levels, antioxidant enzyme activities, and T-AOC

Normal cells without treatment were used as the control group, and H₂O₂-treated cells served as the injury group. Cells digested with trypsin were washed twice with PBS. The remaining cell pellet was lysed in ice-cold RIPA lysis buffer containing 1 mM PMSF for 40 min. The SOD, GSH-Px, and CAT activities and T-AOC and MDA contents were determined according to the instructions provided with the corresponding assay kits. All assays were repeated at least three times.

2.14 Radical scavenging activity measurement

The radical scavenging activity of AFEs (1 g/mL) was determined. The ABTS^.+, DPPH^., FRAP, hydroxyl radical, superoxide anion, H₂O₂ and ferrous ion-chelating abilities were determined using the methods described by Re et al. (1999), Yen and Chen (1995), Benzie and Strain (1996), Halliwell et al. (1987), Chan and Bielski (1974), Jayaprakah et al. (2001), and Singh and Rajini (2004), respectively. The results are presented as mmol of Trolox per kg d.m. All assays were repeated at least three times.

2.15 Statistical analysis

All assays were conducted three times independently, and the data are reported as the mean ± standard deviation. The data were subjected to one-way analysis of variance (ANOVA) using SPSS 20.0 software to analyse the statistical significance. Significance was established at a level of P < 0.05. Principal component analysis (PCA) was performed using SPSS 20.0 software to assess similarity and for the classification of data.

3.1 Contents of flavonoids, anthocyanins and saponins in AFEs from five regions

Fig. 2 presents the variations in flavonoid, anthocyanin and saponin contents in AFEs from five different regions. Significant differences were observed in the AFEs from the five regions (p < 0.05). The flavonoid, anthocyanin, and saponin contents in the AFE from Harbin were higher than those in AFEs from the other four regions. The anthocyanin contents in the AFEs from Jiaohe and Benxi were significantly lower than those in the AFEs from Harbin, Raohe and Mudanjiang. The lowest saponin content was detected in the AFE from Raohe among the five regions (211.20 ± 36.64 mg OA/g). The flavonoid, anthocyanin and saponin contents in the AFE from Mudanjiang were at intermediate levels among the AFEs from five regions.

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Fig. 2

Flavonoid (A), Anthocyanins (B), and Saponin (C) content of Aralia elata (Miq.) Seem fruits. Vertical bars indicate mean values ± SD. The different letters within each column indicate significant differences at p < 0.05.

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3.2 Identification of saponins

Among the complex and diverse active ingredients of A. elata, saponins are the main effective ingredient. Saponin components are important for the functional properties of A. elata. Research scholars also have used araloside as their main research object (Han et al., 2021). The saponins in the AFEs were analysed qualitatively in this experiment to assess the quality of AFs at the monomer compound level. As shown in Fig. 3, approximately 11 visible peaks were determined, 6 of these peaks were identified (Table 2). AFEs presented similar qualitative components in fruits from different regions. Among the components that were identified, araloside A was present at the highest concentration in AFs from all regions except Benxi. The highest araloside A content was detected in the AFE from Harbin. Chikusetsusaponin IV was the second most abundant compound in the AFEs from the five regions among all the identified components. Congmuyenoside X was present at the highest concentration among known ingredients in the AFE from Benxi.

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Fig. 3

Major components of Aralia elata (Miq.) Seem fruits determined using UHPLC-MS/MS.

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3.3 Amino acid contents

The contents of 17 amino acids in AFs obtained from the five origins were analysed, including 8 essential amino acids and 9 nonessential amino acids (Table 3). Significant differences in the contents of amino acids were observed in AFs from the five different regions (p < 0.05). The sum of the contents of nonessential amino acids and essential amino acids in AFs ranged from 2.22 ± 0.25% to 3.60 ± 0.32% and 3.68 ± 0.27% to 5.41 ± 0.41%, respectively. Total amino acid contents of 7.75 ± 0.34%, 6.93 ± 0.21%, 5.89 ± 0.44%, 8.17 ± 0.29%, and 9.01 ± 0.36% were observed in the AFEs from Harbin, Jiaohe, Raohe, Mudanjiang, and Benxi, respectively. According to the experimental results, L-glutamic acid was dominant in the AFs, while L-methionine and L-tyrosine were the least abundant amino acids.

3.4 Cytotoxicity/proliferation impact of AFEs in HEK293 cells

The cytotoxicity and effect of AFEs from different origins on the proliferation of HEK293 cells were tested using a CCK-8 assay (Fig. 4A). In this study, cell viability between 95% and 105% was considered to indicate no cytotoxicity or effect on proliferation. Experimental data showed that the cell viability presented concentration-dependent increases after an incubation with increasing concentrations of AFEs from Jiaohe, Harbin and Benxi. Concentration-dependent decreases in viability were observed after an incubation with increasing concentrations of AFEs from Raohe and Mudanjiang. In particular, the cell viability reached 116.76 ± 1.83% when the concentration of AFE from Harbin was 200 μg/mL. Moreover, when the concentration exceeded 50 μg/mL, AFEs from Jiaohe, Harbin and Benxi altered the proliferation of HEK293 cells, while AFEs from Benxi and Raohe exerted cytotoxic effects.

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Fig. 4

The effect of Aralia elata (Miq.) Seem fruit extract on HEK293 cells. (A) Cytotoxicity effect of Aralia elata (Miq.) Seem fruit extract on HEK293 cells. Normal cells without treatment were regarded as the control group. (B) Protective effect of Aralia elata (Miq.) Seem fruit extract on H₂O₂-induced oxidative damage in HEK293 cells. HEK293 cells incubated with H₂O₂ (400 μM) were used as the control group, and the protection potential of the control group was designated as 0.0%. Vertical bars indicate mean values ± SD. # statistically significant difference versus control group (p < 0.001).

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3.5 Protective effects of AFEs on H₂O₂-induced oxidative stress

An oxidative stress model was established by incubating cells with 400 μM H₂O₂ to evaluate the protective effects of AFE on oxidative injury. A concentration-dependent increase in the potential protective effect of AFEs from the five regions on oxidative stress-induced damage was observed (Fig. 4B). The protective potentials of the AFEs from Jiaohe and Harbin were 18.31 ± 0.20% and 19.28 ± 0.15% (12.5 μg/mL), respectively, which were significantly higher than those of Raohe (10.15 ± 0.20%), Benxi (4.37 ± 0.18%), and Mudanjiang (9.14 ± 0.27%). The protective effects of AFEs from Raohe and Harbin were 23.3 and 17.1 times higher than those of AFEs from Jiaohe (1 μg/mL), respectively. Based on the results, AFEs inhibited H₂O₂-induced oxidative stress injury, but the degree of inhibition of AFEs from different regions was significantly different (p < 0.05).

3.6 Effects of AFEs on ROS generation and MDA levels

Fig. 5 reflects the relative ROS production in HEK293 cells and the green fluorescence intensity. The relative ROS production increased rapidly after HEK293 cells were incubated with H₂O₂, while AFEs reversed this increase. When the concentration of an AFE from any of the five studied regions exceeded 25 μg/mL, it effectively reduced the relative intracellular ROS production compared with that of the injured group to a level that was even lower than the ROS level of normal cells. When the concentration was 12.5 μg/mL, AFEs from Harbin, Raohe and Jiaohe significantly reduced the ROS levels induced by H₂O₂. The AFE obtained from Raohe significantly inhibited ROS production at any concentration (1–50 μg/mL). The fluorescence intensity in photos of cells treated with 50 μg/mL extracts were obtained using laser scanning confocal microscopy. The trend for the fluorescence intensity was consistent with that of relative ROS production. MDA is one of the end products of lipid peroxidation induced by ROS. The MDA level in the injured cells increased substantially compared with that in the control cells. However, the MDA level in the cells pretreated with increasing concentrations of an AFE gradually decreased (Fig. 6F). Overall, the AFE from Harbin reduced the MDA levels to the greatest extent and the AFE from Mudanjiang reduced the levels to the lowest extent among AFEs from the five regions. When the AFE concentration was 25 μg/mL, the order of reduction in MDA levels was Harbin > Benxi > Raohe > Jiaohe > Mudanjiang. Therefore, AFEs resulted in increased resistance to H₂O₂-induced oxidative stress, mainly by reducing intracellular ROS production and MDA levels.

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Fig. 5

The effect of Aralia elata (Miq.) Seem. fruit extract on reactive oxygen species (ROS) generation. (A) Production of intracellular ROS was measured using the fluorescence probe DCFH-DA. (B) Fluorescence images in HEK293 cells were collected by laser scanning confocal microscopy (200 × ), 12.5 μg/mL Aralia elata (Miq.) Seem. fruit extract + 400 mM H₂O₂. (a) control group; (b) injure group; (c) Jiaohe; (d) Harbin; (e) Raohe; (f) Mudanjiang; (g) Benxi. Vertical bars indicate mean values ± SD. *p < 0.001 versus the injury group.

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Fig. 6

The effect of Aralia elata (Miq.) Seem. fruit extract on antioxidant status of H₂O₂-exposed HEK293 cells. (A) Superoxide dismutase (SOD) activity; (B) Catalase (CAT) activity; (C) Glutathione peroxidase (GSH-Px) activity; (D) Total antioxidant capacity (T-AOC) activity; (E) Malondialdehyde (MDA) level. Vertical bars indicate mean values ± SD. * statistically significant differerces versus injure group (p < 0.001).

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3.7 Regulatory effects of AFEs on antioxidant enzymes and T-AOC

Relevant kits were used to determine the antioxidant enzyme and total antioxidant activities in HEK293 cells. The results are shown in Fig. 6. CAT activity, SOD activity, GSH-Px activity, and T-AOC were significantly decreased in HEK293 cells damaged by H₂O₂ compared with those in the control group. Nevertheless, CAT activity, SOD activity, GSH-Px activity, and T-AOC were significantly increased in HEK293 cells pretreated with AFE. Moreover, the antioxidant enzyme activity and T-AOC levels remained similar. The SOD activity and T-AOC of the cells treated with the AFE obtained from Raohe were 12.38 ± 0.84 U/mg protein and 0.65 ± 0.01 mM TE/g FM (50 μg/mL), respectively, which were significantly lower than those of cells treated with the AFEs obtained from the other four regions. The highest CAT activity was observed in cells treated with the AFE (50 μg/mL) obtained from Harbin, followed by the AFEs obtained from Jiaohe, Benxi, Mudanjiang and Raohe. The GSH-Px activity in cells treated with the AFEs obtained from the five regions was in the following order: Mudanjiang > Benxi > Harbin > Raohe > Jiaohe (50 μg/mL). Notably, the T-AOCs of cells treated with AFEs obtained from all regions except Raohe were not significantly different, but the differences were significant compared with the injury group.

3.8 Effects of AFEs on the free radical scavenging capacity

Seven assays were used as supplementary methods to evaluate the chemical antioxidant potential of AFEs (Fig. 7). Significant differences were observed in the antioxidant activities of AFEs from different regions. Based on descriptive statistics, the ferrous ion-chelating ability and hydroxyl radical and superoxide anion scavenging activities were normally distributed, and the values of the remaining indicators were positively skewed. The highest DPPH^. free radical scavenging ability was observed for the AFE from Raohe among the AFEs, followed by those from Harbin, Mudanjiang, Benxi, and Jiaohe. The ABTS^.+ radical scavenging activity of the AFEs ranged from 1.69 to 1.86 mmol TE/100 g dm, among which the AFE from Raohe showed the highest activity. The FRAP of AFEs ranged from 7.70 to 4.52 mmol TE/100 g dm in the order of Harbin > Mudanjiang > Raohe > Jiaohe > Benxi. The AFE from Jiaohe had the strongest ability to remove hydroxyl radicals among the AFEs from the five regions. Meanwhile, the AFE from Raohe had the strongest ability to remove superoxide anions. The ferrous ion-chelating ability of the AFEs from the five regions was significantly different, with the largest value observed for the Jiaohe AFE and the smallest value observed for the Harbin AFE. The H₂O₂ scavenging ability of the Harbin extracts was the strongest, and the difference between the AFEs of the remaining four regions was not significant.

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Fig. 7

Analysis of the radical scavenging activity of the extracts of Aralia elata (Miq.) Seem fruits. (A) DPPH^. free radical scavenging ability; (B) ABTS^.+ radical scavenging activity; (C) Hydroxyl radicals scavenging ability; (D) superoxide anionradical scavenging ability; (E) Ferric reducing antioxidant power; (F) Ferrous ion-chelating ability; (G) H₂O₂ levels.

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3.9 Correlation coefficient

The correlation coefficient was calculated to clarify the relationship between the phytochemical components and the antioxidant activity in AFs. A positive and significant correlation was observed between saponin contents and cellular antioxidant activity (r = 0.908, CAT; r = 0.768, T-AOC; r = 0.521, SOD; r = 0.618, GSH-Px). In addition, the data showed a clear linear relationship between anthocyanin contents and the free radical scavenging capacity (r = 0.681, DPPH; r = 0.865, ABTS; r = 0.942, FRAP; r = 0.917, H₂O₂). However, the contents of flavonoids and amino acids exerted a minor effect on the cellular antioxidant capacity and the free radical scavenging capacity (Fig. 8). Thus, saponins had a substantial contribution to the cellular antioxidant capacity, and anthocyanins were important compounds affecting the free radical scavenging capacity.

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Fig. 8

Correlation coefficients of phytochemical compounds and antioxidant-related index. CAT: the activity of catalase; SOD: the activity of superoxide dismutase; GSH-Px: the activity of glutathione peroxidase; T-AOC: the total antioxidant capacity; MDA: the malondialdehyde levels; ROS: the production of reactive oxygen species; DPPH: the DPPH^. free radical scavenging ability; ABTS: the ABTS^.+ radical scavenging activity; FRAP: the ferric reducing antioxidant power; Fe²⁺ : the ferrous ion-chelating ability; H₂O₂: the H₂O₂ levels. * The correlation is significant at the 0.05 level.

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3.10 Principal component analysis

PCA was used as an unsupervised method to examine the similarity among AFs from Harbin, Benxi, Raohe, Jiaohe, and Mudangjiang. The phytochemical compounds and antioxidant capacities of AFEs from five regions were analysed using PCA. As shown in Fig. 9A, principal component 1 (PC1), the first component of phytochemical compounds, accounted for 39.84% of the total variance, and PC2 represented 32.84% of the total variance. Harbin data were near the X-axis and separated by essential amino acids, nonessential amino acids, total free amino acids and P. The Jiaohe data were also close to the X-axis but had a large negative score on the PC1 axis. Benxi and Raohe data had large positive scores on PC2 and were clearly separated from other regions. They were mainly affected by anthocyanins and flavonoids. Mudanjiang data had a negative score on the PC2 axis, which was opposite to the Benxi and Raohe data. The antioxidant capacity was evaluated using PCA, and the classification of regions was different from the classification obtained from the phytochemical compound PCA. PC1 and PC2 accounted for 74.88% of the total variance, of which PC1 explained 44.54% and PC2 explained 30.34% (Fig. 9B). Benxi, Mudanjiang and Jiaohe data were grouped into one cluster. Harbin and Raohe data were located at relatively opposite ends of the PC1 axis and had large negative scores on PC2. Raohe data were mainly affected by the ABTS^.+ and DPPH^. radical scavenging activities. Meanwhile, Harbin data were separated according to the FRAP assay, CAT activity, protective effect on oxidative stress, and H₂O₂ scavenging ability.

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Fig. 9

Principal component analysis characterization (PC1 vs PC2) of Aralia elata (Miq.) Seem. fruit origin using the phytochemical compounds (A, 16 variables) and antioxidant-related index (B, 15 variables). EAA: Essential amino acids; NEAA: Non-essential amino acid ; TAA: Total free amino acids; Cell Viability: Cytotoxicity of AE fruits extracts in HEK293 cells; Protection: Protection against H₂O₂-oxidative stress; CAT: the activity of catalase; SOD: the activity of superoxide dismutase; GSH-Px: the activity of glutathione peroxidase; T-AOC: the total antioxidant capacity; MDA: the malondialdehyde levels; ROS: the production of reactive oxygen species; DPPH: the DPPH^. free radical scavenging ability; ABTS: the ABTS^.+ radical scavenging activity; FRAP: the ferric reducing antioxidant power; Fe²⁺ : the ferrous ion-chelating ability; H₂O₂: the H₂O₂ levels.

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