2.2.1 Preparation of the plant materials and extracts

Freshly harvested stem of A. macrotera was meticulously separated from its leaf, followed by precise segmentation into small, uniform pieces. The leaf and stem of the A. macrotera were dried in an oven at a temperature of 55 °C and pulverized and sieved (60 mesh) (Zhang, 2013). The resulting plant material was stored in a dry environment.

A total of 1.0 kg of samples were sonicated (Ultrasonic cleaner, 300 w) for 1 h with 70 % methanol at (1:15 w/v) at temperature of 25 °C, and the extracts were filtrated. The obtained extract was subjected to reflux using a rotary evaporator, with methanol being evaporated to yield a concentrated solution. Using the LGJ-10C vacuum freeze dryer (Sihuan Furuikeyi Technology Development Co., Ltd.), the liquid was condensed and frozen dried, to obtain 116.3 g of leaf extract and 111.0 g of stem extract of A. macrotera. Finally, the lyophilized extract was stored at −20 °C for further investigation.

2.4.1 Preparation of standard solutions

A single standard (20 mg/mL) was accurately weighed to prepare a stock solution and dissolved in 1 mg/mL methanol. To conduct further analysis, a mixed standard was prepared by mixing 30 individual standard solutions. The resulting mixed standard was then diluted with methanol to achieve a concentration of 2 mg/mL and stored at −20 °C.

2.4.2 Preparation of sample

To prepare the sample, 1 mg of the leaf and stem extract of A. macrotera was dissolved in 1 mL of methanol and centrifuged at 12,000 r/min for 15 min at 4 °C. The upper solution was filtered using a 0.22 μm millipore filter and stored at −20 °C before injection into the UHPLC-HRMS.

2.4.3 Instrumentation and UHPLC-MS/MS conditions

UHPLC analyses were completed using an Ultimate 3000 (Dionex, Sunnyvale, CA, USA) system. Samples were separated at 40 °C using a Thermo Scientific Hypersil GOLD aQ (100 × 2.1 mm, 1.9 μm). The injection volume of the samples was 2 μL. The flow rate was set at 0.30 mL/min. The mobile phase consisted of 0.1 % of formic acid in water (A) and acetonitrile (B), with optimized gradient elution conditions of: 0–2 min, 5–10 % B; 2–5 min, 10–20 % B; 5–10 min, 20–25 % B; 10–12 min, 25–55 % B; 12–20 min, 55–80 % B; 20–25 min, 80–95 % B; 25–26 min, 95–5 % B; 26–30 min, 5 % B.

The UHPLC system was outfitted with a Q-Exactive Orbitrap MS (Thermo Fisher Scientific, Bremen, Germany), this instrument was equipped with a heated electrospray ionization source (HESI) capable of scanning in negative and positive modes between m/z 120 and 1000. The key parameters were as follows: for (-)-ESI and (+)-ESI, the ion spray voltage was 3.0 and 3.5 kV, respectively, and for sheath and auxiliary gas it was one and 10 arbitrary units, respectively. The temperature of the capillary and the auxiliary gas heaters were maintained at 320 and 350 °C, respectively. The MSⁿ spectra were acquired with full MS mode at a resolution of 35,000 and MS² spectrum with the top 3 ions' MS/MS fragmentation (dd-MS²-TOP3) or parallel reaction monitoring mode at a resolution of 17,500. For MS/MS acquisitions, the NCE (normalized collision energy) was 30 %.

2.4.4 Data processing and analysis

A combination of Xcalibur 4.2 software and Compound Discovery 3.0 software (Thermo Fisher Scientific, California, USA) was used for the acquisition and analysis of the data. In the CD software, the raw LC-MS data were imported by using a TCM workflow, consisting of mzvault, mzcloud, and the Orbitrap Traditional Chinese Medicine Library (OTCML). The TCM workflow template was optimized with the following parameters: MS¹ and MS² with mass tolerances of 10 ppm, the retention time limit of 0.5–28 min, the maximum element counts of C₆₀H₆₀O₆₀N₁₀, the minimum peak intensity of 1,000,000 and the S/N threshold of 10. The data processing software of Compound Discoverer 3.1 was used to perform peak matching and peak area extraction on the original data from the primary mass spectrometry.

2.5.1 DPPH radical scavenging activity

The DPPH free radical scavenging was determined according to the previously reported method (Zong et al., 2021). Briefly, 1 mL of different concentrations (0.0025, 0.005, 0.01, 0.025, 0.05, 0.1 and 0.15 mg/mL) of the leaf and stem extracts were mixed with 1 mL of a DPPH ethanol solution (0.2 mM) in a test tube. The background group used ethanol instead of the DPPH solution, and the control group used ethanol instead of the sample solution. Ascorbate was used as a positive control. After 30 min of incubation in the dark at 37 °C, a 200 µL aliquot was taken and placed in a 96-microwell detector, and the absorbance was measured at 517 nm using a multifunctional microplate reader (BMG LABTECH, Germany). All determinations were performed in triplicate. The DPPH radical scavenging activity was calculated as follows: $$DPPHradicalscavengingactivity\left(\%\right)=(1-\frac{(\text{Abss}-\text{Absb})}{\text{Absc}})\times 100\%$$

Where Abss, Absc and Absb correspond to the sample, control, and background absorbances, respectively.

2.5.2 ABTS radical scavenging activity

A previously reported method was used to determine the free radical scavenging capability of ABTS (Kuppusamy et al., 2015). Briefly, a total of 1 mL of different concentrations (0.005, 0.01, 0.025, 0.05, 0.1 and 0.15 mg/mL) of the leaf and stem extracts were mixed with 2 mL of an ABTS solution in a test tube. The background group used purified water instead of the ABTS solution, and the control group used purified water instead of the sample solution. Ascorbic acid was used as a positive control. The solutions were incubated for 6–8 min at 37 °C without light, after which a 200 µL aliquot was removed and placed in a 96-microwell detector. The absorbance at 734 nm was determined using a multi-function microplate reader. All determinations were performed in triplicate. The ABTS radical scavenging activity was determined as follows: $$ABTSradicalscavengingactivity\left(\%\right)=(1-\frac{(\text{Abss}-\text{Absb})}{\text{Absc}})\times 100\%$$

Where Abss, Absc and Absb correspond to the sample, control, and background absorbances, respectively.

2.5.3 •OH radical scavenging activity

The hydroxyl radical scavenging activity was evaluated by the salicylate method, with slight modifications to that previously reported (Zong et al., 2021). Briefly, the leaf and stem extract were separately diluted to a concentration (0.0625, 0.125, 0.25, 0.5 and 1.0 mg/mL) using pure water as the solvent. Then 25 µL of ferrous sulfate (9 mmol/L), 0.03 % hydrogen peroxide, and salicylate-ethanol solution (9 mmol/L) were each mixed with a 125 µL aliquot of different sample solution in a 96-microwell detector. The background group used purified water instead of the 0.03 % hydrogen peroxide, and the control group used purified water instead of the sample solution. Positive control was provided by ascorbic acid. Under dark conditions, the mixture was heated to 37 °C for 30 min, and the absorbance at 510 nm was determined using a multi-function microplate reader. All experiments were performed in triplicate and the ABTS radical scavenging activity was calculated as follows: $$·OHradicalscavengingactivity\left(\%\right)=(1-\frac{(Abss-Absb)}{\mathit{Absc}})\times 100\%$$ Where Abss, Absc and Absb correspond to the sample, control, and background absorbances, respectively.

2.5.4 FRAP and CUPRAC/antioxidant capacity assays for the reduction of transition metal ions

The ferric reducing ability of plasma (FRAP) assay was determined according to the previously reported method (Masek et al., 2020) with slight modifications, which involved the reduction of Fe³⁺ to Fe²⁺. The FRAP reagent consisted of acetate buffer (300 mM, pH 3.6), ferric chloride (20 mM), and a solution of 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) in hydrochloric acid (40 mM) at the ratio of 10:1:1 (v/v/v). A total of 20 µL of different concentrations (0.0625, 0.125, 0.25, 0.5, 1.0 and 2.0 mg/mL) of the leaf and stem extracts were mixed with 180 µL of the FRAP reagent in a 96-well plate and incubated for 5 min at 25 °C in the dark. The background group used purified water instead of the sample solution. The absorbance at 593 nm was determined using a multi-function microplate reader. Ferrous sulfate (FeSO₄·7H₂O) was used to develop a 0.05–1.6 µmol/L standard curve. All results were expressed as Fe²⁺ content (Fe²⁺ mmol/L). A triplicate of each test was performed.

The cupric reducing antioxidant capacity (CUPRAC) assay was determined using the previously reported method with slight modifications (Han et al., 2012), which involved the reduction of Cu²⁺ to Cu⁺. A 60 µL aliquot of CuSO₄ (0.01 M) was mixed with 60 µL of an ethanol solution of neocuproine (7.5 mM) and 60 µL of a buffer solution CH₃COONH₄ (1 M) in a 96-microwell detector, followed by the addition of different concentrations (0.125, 0.25, 0.5, 1.0 and 2.0 mg/mL) of the leaf and stem extracts. The background group used purified water instead of the sample solution. After 30 min of incubation at 25 °C in the dark, the absorbance at 450 nm was determined using a multi-function microplate reader. CUPRAC was defined as: $$\mathrm{\Delta }A=A_1-A_0$$

Where A₀ is the absorbance of the reagent test, and A₁ is the absorbance of the sample.

2.7.1 Cell culture

The Cell Bank of Chinese Academy of Sciences (Shanghai) provided the macrophage-like cell line RAW264.7. The culture medium was high-glucose Dulbecco’s modified Eagle medium (DMEM), with 10 % heat-inactivated FBS and 1 % penicillin. Cells were grown at 37 °C in a humidified incubator with 5 % CO₂. Cells in the log growth cycle were selected for the experiments. The cells used for the experiments were all in the fourth to fifteenth passages. Cells were fluid-changed on the day before inoculation.

2.7.2 Cell viability assay

The cells seeded in a 96-well plate (5 × 10⁴ cells/mL) were divided into either the blank, control, or drug groups (0.1, 0.2, 0.4 mg/mL). First, RAW264.7 cells were treated with the respective concentrations of the drug solutions (100 μL) for 24 h. Then, 10 μL of MTT (5 mg/mL) solution was added to each well, except for the blank group. Subsequently, the cells were placed in an incubator for a further 4 h incubation. Finally, the absorbance at 490 nm was measured using a multifunctional microplate reader after 100 μL of DMSO solution was added to each well and mixed for 10 min. The cell viability was calculated as follows: $$Cellviability\left(\%\right)=\frac{(\text{OD sample group - OD blank group})}{\text{(OD control group - OD blank group)}}\times 100\%$$

2.7.3 NO concentration assay

RAW264.7 cells (3 × 10⁵ cells/mL) were placed in a 96-well plate and grown for 24 h until the cells were completely adherent. cells were treated with DEX (1 uM) and the leaf and stem extracts (0.1, 0.2, and 0.4 ug/mL). After 1 h, LPS (0.1 μg/mL) was added to treat the cells for 18 h. The supernatant was collected and mixed with 100 μL of Griess reagent, then the absorbance was measured at 540 nm, according to the requirements of the NO detection kit (Beyotime, Shanghai, China). The absorbance was measured on a multi-function microplate reader at the wavelength of 540 nm. The concentration of NO was then calculated from the standard curve. All of the tests were measured in triplicate.

3.2.1 Identification of flavonoids and derivatives

A total of 48 flavonoids were preliminarily identified in A. macrotera. The peaks obtained from mass spectrum at 56, 94, 96, 97, 98, 102, 105, 121, 125, 130, and 132 were accurately identified as catechin, rutin, quercetin, isoquercitrin, kaempferol-3-O-neohesperidoside, luteoloside, kaempferol-7-O-glucoside, eriodictyol, luteolin, kaempferol, and isorhamnetin based on the retention time and MS² pattern of the corresponding reference standard.

The peak 45, with [M−H]^- ion at m/z 451.1246, could be characterized as catechin-glucopyranoside, which produced some characteristic fragment ions at m/z 289.0714 [M−H−C₆H₁₀O₅]^-, 205.0497, and 137.0231. Peak 67 showed a precursor ion at m/z 625.1410 [M−H]^- and was suggested to be quercetin-dihexoside based on the product ions at m/z 463.0885 [M−H−C₆H₁₀O₅]^- and 301.0348 [M−H−2C₆H₁₀O₅]^-, in line with the literature characterization (Sánchez-Salcedo et al., 2016). Peaks 81 and 88 possessed the same parent ion at m/z 755.2040 and produced product ions at m/z 301.0334, 178.9977 and 151.0029, therefore, they were attributed to quercetin rutin rhamnoside. Peak 84 displayed a [M−H]^- ion at m/z 595.1305 and yielded a fragment ion at m/z 301.0347 [M−H−C₆H₁₀O₅−C₅H₈O₄]^-, which corresponded to the loss of a glucose and an arabinose moiety. Thus, peak 84 was tentatively identified as quercetin-arabinosyglucoside based on literature research (Zeng et al., 2017). Peak 106, with a parent ion [M−H]^- at m/z 433.0776, was identified as quercetin-pentoside due to the fragment ion at m/z 301.0338 [M−H−C₅H₈O₄]^- and 271.0237 [M−H−C₅H₈O₄−CO−2H]^-, which were related to the loss of xylan.

Peaks 71, 79, 83, 86, 110, 112, 117, 137 and 140 were respectively characterized as vicenin II, schaftoside, isoschaftoside, eriocitrin, rhoifolin, narcissoside, neodiosmin, demethylnobiletin and demethylnobiletin isomer, when compared with the mzVault.

Peaks 70, 80, and 90 possessed the same precursor ion [M−H]^- at m/z 449.1089, which loses a molecule of rhamnose to produce m/z 285.0404 [M−H−C₆H₁₀O₅−2H]^-, and loses another molecule to produce the quercetin characteristic ion at m/z 125.0232 [M−H−2C₆H₁₀O₅]^-, thus they were identified as the taxifolin-7-rhamnoside isomer. Compared with the literature, peak 114 and peak 126 were identified as ashesperetin or the hesperetin isomer, peak 123 was characterized as calycosin, peak 131 and peak 133 were identified as chrysoeriol or the chrysoeriol isomer, and peak 134 was identified as genkwanin (Vieira de Morais et al., 2021).

Among the 48 kinds of flavonoids and their derivatives in the characterization, 48 were found in the leaf, while only 32 compounds were found in the stem. The leaf-specific compounds included taxifolin-7-rhamnoside, quercetin-rhamnosyl rutin, isorhamnetin-3-O- nehesperidine, and rhoifolin, which indicates that the biological activity of the leaf may be better than that of the stem. Thus, A. macrotera proved to be a promising source of flavonoids that may have valuable applications in the medical, healthcare, food, and pharmaceutical industries.

3.2.2 Identification of organic acids

Peaks 1, 9, 10, 13, 15, 16, 21, 33, 60, 62, 64, 76, 78, 85, 93, 101, 107, 109, and 120 were accurately identified as quinic acid, malic acid, α-ketoglutaric acid, citric acid, citraconic acid, cis-aconitic acid, gallic acid, protocatechuic acid, cryptochlorogenic acid, chlorogenic acid, caffeic acid, 4-coumaric acid, suberic acid, ferulic acid, salicylic acid, isochlorogenic acid B, azelaic acid, isoferulic acid, and abscisic acid. This identification was based on the retention time and MS² pattern of the corresponding reference standard.

Peak 44 contained a [M−H]^- ion at m/z 299.0772, and a fragment ion at m/z 137.0231 [M−H−C₆H₁₀O₅]^-, corresponding to the loss of a hexose, and then by the neutral loss of a CO₂, a fragment ion of m/z 93.0332 [M−H−C₆H₁₀O₅−CO₂]^- was identified. Thus, peak 44 was considered to be salicylic acid-hexoside. By comparing with databases and retention time behavior, peaks 48 and 59 showed similar [M−H]^- ions at m/z 341.08780 and produced fragment ions at m/z 179.0339 [M−H−C₆H₁₀O₅]^- and 135.0438 [M−H−C₆H₁₀O₅−CO₂]^-, respectively. Thus, they were characterized as caffeic acid-hexoside (Tang et al., 2022). Peaks 66, 68 and 73 possessed the deprotonation ion [M−H]^- at m/z 355.1035, and displayed fragment ions at m/z 193.0497 [M−H−C₆H₁₀O₅]^-, 175.0390 [M−H−C₆H₁₀O₅−H₂O]^-, and 149.0596 [M−H−C₆H₁₀O₅−CO₂]^-. By comparison with the literature, these peaks were assigned as ferulic acid-hexoside (Mena et al., 2018).

Peaks 11, 24, 27, 35, 41, 42, 43, 54, and 69 were respectively characterized as trans-aconitic acid, geniposidic acid, danshensu, (r)-mandelic acid, 4-hydroxybenzoic acid, 2-isopropylmalic acid, gentisic acid, 2-coumaric acid and 3-coumaric acid by comparison with mzVault.

Peak 12 exhibited a [M−H]^- ion at m/z 175.0248, which was characterized as ascorbic acid, and yielded fragment ions at m/z 146.9599 and 130.8732, in accordance with the previously reported data (Luo et al., 2020). According to the MS² data and retention time behavior reported in the literature (Li et al., 2020), peaks 14, 19 were identified as tartaric acid, mevalonic acid. Peaks 29, 34, 53, and 72 were characterized as vanillic acid, orsellinic acid, isovanillic acid and 4-methoxysalicylic acid (Wang et al., 2021). Peak 26, with a [M−H]^- ion at m/z 218.1034, produced a fragment ion at m/z 146.0810, which was identified as pantothenic acid when comparing with previously reported data (Shi et al., 2022). Coincidentally, the peaks 51 and 61 displayed a molecular ion with a mass of 131.0714 [M−H]^- and generated a product ion at m/z 87.0437 [M−H−CO₂]^- and 85.0644 [M−H−CO₂−2H]^-, which corresponds to a loss of CO₂. Thus, they were proposed to be 2-hydroxyhexanoic acid or its isomer according to published data (Li et al., 2020). Peaks 31 and 55 showed a similar [M−H]^- ion at m/z 151.0401 and produced a fragment ion at m/z 108.0441 [M−H−CO₂]^-. By comparing with databases and considering their retention time behavior, they were respectively characterized as isovanillin and vanillin (Koprivica et al., 2018). Peak 65 gave a [M−H]^- ion at m/z 517.1563 and a major fragment ion at m/z 193.0497 and was identified as ferulic acid-dihexoside, in agreement with the literature (Koprivica et al., 2018). Peaks 37 and 58 gave the same [M−H]^- ion at m/z 285.0616, as well as ions at m/z 152.0103, 153.0183, 108.0202 and 109.0282, and were thus identified as dihydroxybenzoic acid pentoside or its isomer, in accordance with the literature (Peixoto Araujo et al., 2020). Peak 122, with a [M−H]^- ion at m/z 201.1132, produced fragment ions at m/z 139.1116 and 183.1016, which was identified as 3-tert-butylpropionic acid by comparing with public data (Yu et al., 2019).

In the characterization, a total of 65 types of organic acids and their derivatives were identified in the leaf, whereas only 45 types of compounds were detected in the stem. For example, substances such as gallic acid, geniposidic acid, gentisic acid, and chlorogenic acid, were only found in leaf. Thus, the antioxidant activity of the leaf may be more effective than the stem.

3.2.3 Identification of phenylpropanoids and their derivatives

The majority of phenylethanol glycosides (phgs) have a class of phgs with a caffeoyl group, therefore, the MS² of most compounds will show the same fragment pattern attributed to the caffeoyl group at m/z 161.0232, 135.0438 and 179.0340 (Han et al., 2012), which can therefore be used as diagnosis ions of phenylethanol glycosides. Peak 100 showed the [M−H]^- ion at m/z 477.1402 and produced a fragment ion at m/z 315.1084 [M−H−C₉H₆O₃]^- and 161.0234 [M−H−C₈H₁₀O₃−C₆H₁₀O₅]^-, which indicated the presence of phenethyl glycosides. Thus, it was speculated as calceolarioside B. Analogously, peaks 89 and 95, displayed secondary fragment ions with roughly the same m/z of 161.0232, 179.0340 and 135.0438, which are typical of phenylethanol glycosides. Therefore, peaks 89 and 95 were identified as verbascoside, and isoverbascoside, respectively. (Huang et al., 2010).

Peak 32 was detected at m/z 153.0557, with a fragment ion at m/z 123.0438, corresponding to the loss of a CH₂OH group, and was thus identified as hydroxytyrosol (Peralbo-Molina et al., 2012). Peak 30 carried the precursor [M−H]^- ion at m/z 315.1085 and yielded the main ions at m/z 153.0545 [M−H−C₆H₁₀O₅]^-, 123.0437 and 135.0438, which suggested the presence of hydroxytyrosol. Therefore peak 30 was characterized as hydroxytyrosol-glucoside (Peralbo-Molina et al., 2012).

Peak 40 exhibited the [M−H]^- at m/z 299.1136, which gave its product ion at m/z 137.0233 and 119.0487, affirmed as salidroside according to published data (Li et al., 2017). Peak 46 was characterized as forsythiaside E by comparison with the databases. Peak 50 was detected at m/z 431.1559, with the main characteristic ion at m/z 229.1136, corresponding to the neutral loss of the furanosyl group in the precursor [M−H]^- ion, which is consistent with the fragmentation of osmanthoside H (Huang et al., 2010).

Peaks 89, 95, and 103 were identified as β-hydroxyverbascoside, verbascoside and isoverbascoside, respectively. Peaks 75 and 77 possessed an identical precursor ion at m/z 415.1610 [M−H]^- and produced the same fragment ions at m/z 161.0442 and 101.0230. Thus, these peaks were identified as phenylethylrutinoside by comparing with public data (Zeng et al., 2017). Peak 108 showed a deprotonated ion [M−H]^- at m/z 579.2083 and produced a major fragment ion at m/z 417.1549 [M−H−C₆H₁₀O₅]^- corresponding to the loss of a glucose, hence it was identified as syringaresinol 4-O-β-D-glucopyranoside on the basis of literature (Liu et al., 2022).

A total of 12 substances were found and characterized as phenylpropanoid and its derivatives, of which forsythiaside E was only detected in the leaf. Forsythiaside E may be the main characteristic component of the leaf.

3.2.4 Identification of other classes of compounds

A total of 19 other classes of compounds were detected in the leaf and stem extracts of the A. macrotera. This includes amino acids, sugars, alkaloids, and unsaturated fatty acids. For example, by comparing peak 18 with a reference public data set, peak 18 was determined to be l-tyrosine, (Jin et al., 2022) and amino acid of interest. It produced a precursor ion at m/z 182.0812 [M + H]⁺ and diagnostic ions at m/z 136.0756[M + H-CO₂]⁺ and 119.0491[M + H-CO₂-OH]⁺ in the positive-ion mode. Analogously, peaks 2, 7, 25, and 38 were assigned to arginine, l-glutamic acid, l-phenylalanine, and l-tryptophan, respectively.

3.3.1 DPPH free radical scavenging assay

The antioxidant activity of the leaf and stem extract were first determined by measuring the DPPH scavenging capacity, which showed that these extracts had a good scavenging activity against DPPH radicals. As shown in Fig. 3(a), the extracts of leaf and stem, at concentrations of 0.0025–0.15 mg/mL, showed dose-dependent effects of DPPH radical clearance with an the biochemical half maximal inhibitory concentration (IC₅₀) value of 0.019 and 0.067 mg/mL, respectively. Notably, the leaf extract at 0.1 mg/mL concentration showed a 91.53 ± 1.85 % clearance of DPPH radicals, with a better clearance of DPPH radicals than the stem extract.

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Fig. 3

Effect of DPPH radical scavenging of the A. macrotera extracts. Among them, VC represents vitamin C, EAM-L represents leaf extract, and EAM-S represents stem extract. Different concentrations of the extracts and Vc (final concentrations of 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1 and 0.15 mg/mL). a. Effect of ABTS radical scavenging of the A. Macrotera extracts. Different concentrations of the extract and Vc (final concentrations of 0.005, 0.01, 0.025, 0.05, 0.1 and 0.15 mg/mL, respectively). b. Hydroxyl radical scavenging action of the A. Macrotera extracts. Different concentrations of the extracts and Vc (final concentrations of 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/mL, respectively). c. Data are presented as the means ± SD of three independent experiments, * is compared to the Vc group, *P＜0.05, **P＜0.01.***P＜0.005.

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3.3.2 ABTS free radical scavenging assay

The clearance ability of ABTS radicals by the leaf and stem extract is shown in Fig. 3(b). The results showed that the extracts of leaf and stem, at concentrations of 0.005–0.15 mg/mL, showed dose-dependent effects of ABTS radical clearance with an IC₅₀ value of 0.03 and 0.05 mg/mL, respectively. In particular, the leaf extract at 0.1 mg/mL concentration showed a 95.4 ± 2.15 % clearance of ABTS radicals, with a better ABTS radical clearance than the stem extract.

3.3.3 Hydroxyl free radical scavenging assay

As shown in Fig. 3(c), the leaf and stem extract at a concentration of 0.5 mg/mL showed a 54.84 ± 8.25 % and 45.64 ± 3.04 % clearance, respectively, to the hydroxyl radicals, with an IC₅₀ value of 0.347 and 0.558 mg/mL, respectively. The experimental results indicate that both the leaf and stem extract had a good scavenging effect on the hydroxyl free radicals.

3.3.4 FRAP and CUPRAC/antioxidant capacity assays for the reduction of transition metal ions

The FRAP assay and the CUPRAC assay were conducted to measure the antioxidant potential of the plant extracts to reduce iron and copper ions. As shown in Fig. 4, the FRAP determines the ability to reduce Fe³⁺ to Fe²⁺, while CUPRAC determines the ability to reduce Cu²⁺ to Cu⁺. A. macrotera extracts were found to be more effective in reducing iron and copper ions with increasing concentrations. Extraction of A. macrotera exhibited a good ability to reduce transition metal ions. For example, at the concentration of 1 mg/mL, the iron ion reduction capacity of the leaf and stem extract was found to be 1.785 ± 0.089 and 0.67 ± 0.022 mmol/L, respectively. A high level of active compounds, such as polyphenolic compounds, may make the extracts capable of reducing transition metal ions.

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Fig. 4

The ability of the A. macrotera extracts to reduce ferric ions (Fe³⁺) to ferrous ions (Fe²⁺). EAM-L represents leaf extract, and EAM-S represents stem extract. Different concentrations of the extracts. (final concentrations of 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL). b. The ability of the A. macrotera extracts to reduce Copper ions (Cu²⁺) to Cuprous ions (Cu⁺). EAM-L represents leaf extract, and EAM-S represents stem extract. Different concentrations of the extracts. (final concentrations of 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL). * is compared to the EAM-L. *P＜0.05, **P＜0.01, ***P＜0.005.

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3.3.5 Relationship between the identified compounds and antioxidant

The above antioxidant experiments indicate that the overall antioxidant activity of the leaf extract is better than that of the stem extract. This finding aligns with our prior component characterization of the extracts. Specifically, during the component identification experiment, a total of 65 organic acids were identified in the leaf extract, whereas only 45 were detected in the stem extract. Furthermore, substances such as gallic acid were only detected in the leaf extract, indicating that organic acid compounds have excellent antioxidant capabilities. Compounds such as gallic acid have been empirically demonstrated to possess commendable antioxidant properties (Badhani et al., 2015), which corroborates why the leaf appears to have better antioxidant capacity compared to the stem.

3.5.1 Effects of the A. macrotera extracts on the viability of RAW264.7 cells

To test the potential cytotoxicity of the leaf and stem extract against RAW264.7 cells, the cell viability was determined by the MTT assay. A variety of concentrations of leaf and stem extract (0.1, 0.2, and 0.4 mg/mL) were applied to cells for 24 h. As shown in Fig. 6(a), none of the cells treated with these extracts showed any cytotoxicity.

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Fig. 6

Protective effects of EAM-L and EAM-S against the LPS-induced oxidative stress in RAW264.7 cells. EAM-L represents leaf extract, and EAM-S represents stem extract. a. RAW264.7 cells were treated with different concentrations(final concentrations of 100, 200 and 400 μg/mL) of EAM-L and EAM-S for 24 h, and the cell viability was determined by the MTT assay. b. RAW264.7 cells were pre-treated with different concentrations of EAM-L and EAM-S for 1 h before treatment with LPS(100 ng/mL), the concentration of dexamethasone is 1 μM and then were treated with 100 ng/mL of LPS for 18 h to detect the amount of no release. Data are presented as the mean ± SD of three independent experiments, # is compared to the con group, * is compared to the LPS group,###P＜0.05, *P＜0.05, **P＜0.01.***P＜0.005.

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3.5.2 NO assay

To investigate the anti-inflammatory potential of the leaf and stem extract, we determined the levels of NO (a key proinflammatory cell mediator) in the supernatant of the cells treated with LPS using the Griess reagent. NO production was inhibited in the LPS-treated cells by pretreatment with leaf and stem extract in RAW264.7 cells, as shown in Fig. 6(b). As a result, pretreatment of the cells showed significant and dose-dependent inhibition with leaf and stem extract. Therefore, our results indicate that leaf and stem extract can inhibit the generation of inflammatory mediators of macrophages treated with LPS. This may be a source of a novel anti-inflammatory agent.

3.5.3 Relationship between the identified compounds and anti-inflammatory

The above cell experiments indicate that the overall anti-inflammatory activity of leaf extract is better than that of stem extract. This finding aligns with our prior component characterization of the two extracts. Specifically, during the component identification experiment, a total of 48 flavonoids were identified in the leaf extract, whereas only 32 were detected in the stem extract. Furthermore, in the quantitative experiments, the leaf extract consisted of 22 % of flavonoids while the stem extract contained only 9 %. The significant anti-inflammatory capacity of flavonoids has previously been confirmed in the literature (Maleki et al., 2019).