Kaempferia galanga rhizomes were purchased from Qingping Chinese medicinal herbs market (Guangdong Province, China), and were identified by deputy chief pharmacist Junbiao Wu (The Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, China). Prior to use, the rhizomes were ground into powder.

The reference standard of n-alkanes (C₈−C₂₀) was received from ANPEL Laboratory Technologies (Shanghai) Inc. (Shanghai, China). 2,2-diphenyl-1-picrylhydrazyl (DPPH, 98%), 2,2′-amino-di(2-ethyl-benzothiazoline sulfonic acid-6) ammonium salt (ABTS, 98%), ascorbic acid (≥ 99%), 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), and acridine orange were acquired from Sigma-Aldrich (Shanghai, China). Malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were obtained from Nanjing Jiancheng Biotechnology Co., Ltd (Nanjing, China). Dimethyl sulfoxide (DMSO) was obtained from MP Biomedicals (CA, USA). Diphenyl-1-pyrenylphosphine (DPPP, catalog no. DJ799) was obtained from Macklin (Shanghai, China). Other chemicals used in this study were of analytical grade.

The DPPH assay was used to determine the DPPH radical scavenging activity of KGEO according to the described method (16–17) after making the changes. One milligram of DPPH was dissolved in ethanol (5 ml) to prepare a fresh DPPH working solution. Fifty microlitres of different concentrations of KGEO sample solutions (1, 5, 10, 20, 30, and 40 mg/ml) were mixed well with 50 μL of 0.5 mM DPPH solution to react in the dark at room temperature for 30 min as a sample group. For the blank group, the sample was replaced with an equal volume (50 μL) of ethanol, and ascorbic acid was used as a positive control. A microplate reader (M1000pro, Tecan, Männedorf, Switzerland) was used to determine the absorbance at 517 nm. Three replicates of the experiment were carried out. The IC₅₀ (mg/ml) value was determined by the following Eq. 1:

where A₀ represents the absorbance values of the control and A₁ represents the absorbance values of KGEO. The scavenging rate and IC₅₀ of ascorbic acid were calculated under the same conditions.

The ABTS assay was carried out based on a previously reported method (16) with some modifications. To prepare the ABTS radical stock solution, ABTS (7.4 mM) solution and K₂S₂O₈ (2.45 mM) solution were mixed with an equal volume and reacted for 12−16 h in the dark. Before use, the ABTS radical stock solution was diluted withphosphate buffered saline (PBS) until the absorbance was 0.70 ± 0.02 at 734 nm. Fifty microlitres of different concentrations of sample solutions (0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2.0 mg/ml) were mixed with 150 μL of ABTS radical working solution to react for 6 min in the dark at room temperature, which was used as the experimental group. For the blank group, the sample solutions were replaced by the same volume of ethanol (50 μL). The absorbance of each group was measured at 734 nm using a microplate reader. The results are presented as the IC₅₀ value (mg/ml), and the scavenging rate was determined according to Eq. 1. The inhibition percentage and IC₅₀ of ascorbic acid were calculated under the same conditions.

The ability of KGEO to inhibit hydroxyl radicals was evaluated according to a reported method (18) with some modifications. KGEO samples (1 ml) with a range of concentrations (1, 5, 10, 20, and 30 mg/ml) were combined with 1 ml of 1 mM salicylic acid ethanol solution, 1 ml of FeSO₄ (1 mM), and 1 ml of 0.01% H₂O₂ (v/v), which were mixed in order. The mixture was maintained at 37°C for 30 min, and then the absorbance (A_i) was measured at 510 nm. Ascorbic acid was used for the positive control group. An equal volume of distilled water replaced the 0.01% H₂O₂ to prepare A_j. A₀ was prepared with an ethanol solution instead of KGEO samples. The ability of KGEO to inhibit hydroxyl radicals was determined using Eq. 2:

The total reduction ability of KGEO was evaluated by using the method reported in the literature (19–20) with some modifications. KGEO samples (0.5 ml) of a range of concentrations (0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 g/ml) were mixed with 0.2 M phosphate buffer (pH 6.6) solution (2.5 ml) and 1% potassium ferricyanide (2.5 ml) in order. Then, the mixture was maintained at 50°C for 20 min. Afterward, 10% trichloroacetic acid (2.5 ml) was added, followed by incubation for 10 min at room temperature. After incubation, 0.10% ferric chloride (0.5 ml) and distilled water (2.5 ml) were added to the mixture (2.5 ml), and then the absorbance was measured at 700 nm. The EC₅₀ value was the value at which the absorbance was 0.5. Ascorbic acid was used as the positive control.

Wild-type AB zebrafish were provided by Guangdong Laboratory Animals Monitoring Institute. The zebrafish were kept at 28.5 ± 0.5°C with a 14:10 h light/dark cycle and fed flake food three times daily. Embryos were obtained from natural spawning, collected after 45 min of light, and then incubated in E3 medium (21) (CaCl₂ 0.33 mM, NaCl 5 mM, KCl 0.18 mM, MgSO₄ 0.34 mM).

We evaluated the effect of KGEO against H₂O₂-induced embryotoxicity. KGEO was dissolved in DMSO. The 48 h post-fertilization (hpf) embryos were treated with different concentrations of KGEO (0.4, 0.8, and 1.6 μg/ml) for 1 h. After 1 h, H₂O₂ (2 mM) was added as a stimulant. DMSO (0.32%) was used as the solvent control. The embryos were rinsed in fresh medium, and the survival rate was examined after 24 h.

The heartbeat rates of zebrafish embryos were measured after 24 h of H₂O₂ (2 mM) stimulation at 72 hpf. The average heart rate of zebrafish embryos was measured by manual counting with a 10 s period for 15 live zebrafish embryos under a microscope (Nikon Ci-E, Japan). The heartbeat rates were expressed as a percentage.

Intracellular ROS, cell death, and lipid peroxidation were measured by DCF-DA, acridine orange, and DPPP assays to assess the protective effect of KGEO against H₂O₂-induced oxidative stress (22). The zebrafish embryos were treated with phenylthiourea (2 mg/ml) to inhibit pigment pattern formation in zebrafish for better observation of fluorescence images starting at 5 hpf. The 48 hpf zebrafish embryos were treated with or without KGEO (0.4, 0.8, and 1.6 μg/ml) for 1 h. Then, H₂O₂ (2 mM) was added, followed by incubation for 24 h. At 72 hpf, the embryos were washed with E3 medium and transferred into 24-well plates. DCF-DA is an oxidation-sensitive fluorescent probe dye used to detect ROS levels. The zebrafish embryos were incubated with DCF-DA solution (2.5 μM) for 50 min at 28°C in the dark. Apoptotic cells were detected in embryos by using acridine orange (7 μg/ml), a nucleic acid-selective fluorescent dye, and the embryos were incubated for 30 min in the dark at 28°C. When DPPP is oxidized, it becomes fluorescent. DPPP is a fluorescent probe used to measure lipid peroxidation for the detection of membrane damage. The embryos were treated with DPPP solution (20 μg/ml) and incubated for 40 min in the dark at 28.5°C. After incubation, the embryos were washed with E3 medium at least three times and anesthetized with MS222 (150 μg/ml) before visualization. Fluorescence images of embryos were captured using a fluorescence microscope (Nikon Ci-E, Japan), and the fluorescence intensity of individual embryos was quantified using ImageJ software (Fiji version) (National Institutes of Health, Bethesda, USA).

The 48 hpf zebrafish embryos were treated with or without KGEO (0.4, 0.8, and 1.6 μg/ml) for 1 h and then incubated with H₂O₂ (2 mM) for 24 h. At 72 hpf, the embryos from each group were collected and homogenized with buffer (pH 7.2) containing EDTA-2Na (0.0001 M), Tris−HCl (0.01 M), and NaCl (0.65%) using a tissue homogenizer (1,700 r/min, 30 s × 3). After centrifugation (3,000 r/min, 15 min), the supernatants were collected immediately to determine the activity of SOD, CAT and GSH-Px and MDA levels according to the manufacturer’s directions (A003−1, A001−1, A007−1, and A005, Jiancheng, Nanjing, China).

DPPH is often used in antioxidant experiments as a stable radical (23). The ability of KGEO to scavenge DPPH radicals was studied. In the oxidation reaction, KGEO showed a potential DPPH radical scavenging effect with an IC₅₀ value of 19.77 ± 1.28 mg/ml (Table 2), with an increase in DPPH radical scavenging with increasing KGEO concentration. The scavenging effect of KGEO at a concentration of 10 mg/ml was 52.70%, and the activity appeared to be dose-dependent (24). This may be related to the plant source and processing procedure based on the content of its main components. DPPH radicals can be scavenged in the form of reduced DPPH-H, when a hydrogen radical combines with a DPPH radical (25). An EO with DPPH radical scavenging activity is beneficial for inhibiting damage to biomolecules (protein, sugars, PUFA, DNA) caused by reactive radical species in susceptible food and biological systems (24).

The ABTS radical scavenging assay is also usually employed to determine the in vitro antioxidant activity of different substrates. The scavenging activity of KGEO at different concentrations for ABTS radicals was determined. The IC₅₀ of the EO was determined to be 1.41 ± 0.01 mg/ml (Table 2). The EO exhibited higher activity toward the ABTS radical compared to the DPPH radical. After proton dissociation, the ABTS radical (ABTS+) accepts a single electron and combines with hydrogen (H+) to form stable ABTS, which plays a significant role in free radical scavenging. The main mechanisms in the process of free radical scavenging are proton priority loss and single electron transfer (26). The DPPH radical scavenging assay can only be used in hydrophobic antioxidant systems, while the ABTS radical scavenging assay can be used in hydrophobic and lipophilic systems, which are more common (27).

Hydroxyl radicals are powerful oxidants that can react with many redox-sensitive elements and organics non-selectively in the environment and damage various biomolecules (28). As a main ROS, hydroxyl radicals can lead to lipid peroxidation or other oxidative damage (29). Different concentrations of KGEO were evaluated. KGEO had hydroxyl radical scavenging activity, with an IC₅₀ of 3.09 ± 0.34 mg/ml, as shown in Table 2. KGEO exhibited higher activity toward the DPPH radical and lower activity toward the ABTS radical. Similar to DPPH and ABTS radicals, hydroxyl radical species are neutralized by a hydrogen atom.

The ferricyanide reduction method was used to determine the reducing power. The electron-donating activity is a significant mechanism of antioxidant activity, which can be indicated by Fe³⁺ reduction (19). The reducing power was assessed on KGEO. As presented in Table 2, the EC₅₀ value for Fe³⁺ reduction of KGEO was 389.38 ± 4.07 mg/ml. This result suggested that KGEO has weak potency to donate electrons to reactive free radicals. During the ferric reducing assay, reducing agents reduced the Fe³⁺−ferricyanide complex to the ferrous form. Thus, Fe²⁺ can be assessed by the density of blue color in the reaction medium at 700 nm (30).

The protective effect of KGEO on oxidative stress in vivo induced by H₂O₂ was established using a zebrafish model. The survival rate of zebrafish embryos treated with different concentrations of KGEO is shown in Figure 1A. After exposure to H₂O₂, the survival rate dropped to 66.67% compared to 100.00% of the control group. However, the survival rate increased after preadministration of 0.4, 0.8, and 1.6 μg/ml KGEO, and the rate rose to 73.33, 86.67, and 93.33%, respectively. DMSO (0.32%) as a solvent control had no effect on the survival rate. The heartbeat rate of embryos treated with H₂O₂ (2 mM) was decreased to 80.56%, while pretreatment with different concentrations of KGEO (0.4, 0.8, and 1.6 μg/ml) recovered the heart rate to 91.16, 93.94, and 98.48%, respectively (Figure 1B). These results suggested that KGEO alleviated oxidative damage caused by H₂O₂.

The fluorescence images of intracellular ROS generation are shown in Figure 2A. After treatment with H₂O₂, the DCF fluorescence intensity was enhanced to 170.63%, with brighter fluorescence compared to the control group, while the fluorescence intensity was reduced up to 103.31% by cotreatment with KGEO.

Acridine orange preferentially stains necrotic and late apoptotic cells, as it is able to permeate cells with disrupted plasma membranes. Fluorescence images showing cell death in zebrafish embryos as assessed by acridine orange staining are shown in Figure 2B. In the H₂O₂-treated group, the fluorescence intensity was significantly enhanced to 157.01% compared with the control group. After KGEO was administered, the fluorescence expression was significantly decreased, and the fluorescence value decreased to 105.04% with increasing KGEO concentration.

The fluorescence images for DPPP are shown in Figure 2C, and the fluorescence intensity indicated the degree of lipid peroxidation. H₂O₂ significantly induced lipid peroxidation, with the fluorescence intensity increasing to 170.55%, while lipid peroxidation was reduced to 108.00% after KGEO treatment.

The fluorescence intensities of images of ROS generation, cell death, and lipid peroxidation were measured using ImageJ software. The results suggested that KGEO showed protective activity in zebrafish embryos damaged by H₂O₂-induced oxidative damage via a decrease in ROS and lipid peroxidation and by alleviating cell death.

The antioxidant enzyme activities (SOD, CAT, and GSH-Px) and MDA levels were evaluated in H₂O₂-induced zebrafish embryos. The results are shown in Figure 3. In the H₂O₂-treated group compared to the control group, the activities of CAT, GSH-Px, and SOD in zebrafish embryos were decreased by 3.62−, 2.26−, and 1.56−fold, respectively, and MDA levels were increased by 2.27−fold. However, after treatment with different concentrations of KGEO (0.4, 0.8, and 1.6 μg/ml), the oxidative damage was alleviated in a dose-dependent manner. When treated with KGEO (1.6 μg/ml), the activities of CAT, GSH-Px, and SOD were increased by 3.78−, 2.5−, and 1.80−fold, respectively, and the MDA level showed a decrease of 63.00% compared to the H₂O₂-treated group. The results indicated that H₂O₂ caused oxidative damage and lipid peroxidation, which were due to the decrease in antioxidant enzyme activities and the increase in the formation of MDA, while KGEO alleviated this damage.