HeLa cells were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China), and the mycoplasma testing method (Young et al., 2010) was used to ensure that no mycoplasma contamination occurs. Methods by both Schantz et al. (2010) and Yang et al. (2013)were used with some modifications to measure the cell viability and intracellular antioxidation. Briefly, HeLa cells were transferred into a 96-well plate with 100 μL per well of DMEM medium at a density of 1 × 10⁵/mL. The Hela cells were then incubated with 5% CO₂ for 24 h at 37°C. The medium was discarded and the cells were carefully washed by autoclaved PBS (pH = 7.4) thrice and followed by the treatment with 100 μL different concentrations of the essential oils (WPEO or BPEO) or their standards (α-pinene, β-pinene, 2-carene, 3-carene, limonene, linalool or caryophyllene) for another 24 h. Different concentrations (0.001–2 μL/mL) of essential oils and standards were dissolved in different concentrations of DMSO, i.e., 0.001, 0.01 and 0.1 in 0% DMSO, 0.2 in 2% DMSO, 0.4 in 4% DMSO, 0.5 in 5% DMSO, 1 in 10% DMSO, 2 in 20% DMSO, respectively. Therefore, the corresponding concentrations of DMSO were used as solvent controls for the cell viability and intracellular antioxidation experiments. The growth medium was used to replace the oils and the standards in the control well. After repeated washing, 5 mg/mL of MTT was added for another 4-h incubation, and 150 μL of DMSO was then added to dissolve the purple crystals. The absorbance at 570 nm was then measured.

In the intracellular antioxidation assay, the cells were transferred and incubated according to the same procedures in the above cell viability assay. An aliquot of 100 μL of 10 μmol/L DCFH-DA was mixed with 100 μL of 0.1 μL/mL oils or the standard solution for 1 h. After the wells were carefully washed by PBS (pH = 7.4), each well was added with 100 μL of 200 μmol/L H₂O₂. The fluorescence intensities were measured at every 5 min during the 1-h incubation period with the excitation and emission wavelengths at 525 and 488 nm, respectively. The integral area of the relative fluorescence intensity of the sample was ∫SA, while the integral area of the relative fluorescence intensity of the control with the only medium was ∫CA. The blank was prepared without both the oils and H₂O₂ solution. The intracellular antioxidant activity CAA unit was calculated according to the following equation: CAA (%) = (1–∫SA/∫CA) × 100% (Wolfe and Liu, 2007).

The phosphomolybdenum complex method was applied to evaluate the total antioxidant activities (Ozkan et al., 2007). Briefly, 0.4 mL WPEO, BPEO, standards or synthetic antioxidants (BHT and PG) solution of different concentrations dissolved in 100% ethanol or Vc dissolved in distilled water was added into 4 mL phosphorus molybdenum reagents (28.0 mmol/L sodium phosphate, 0.6 mmol/L sulfuric acid, and 4.0 mmol/L ammonium molybdate). The reactions were carried out at 95°C for 1.5 h. The absorbance at 695 nm was measured after cooling down.

Superoxide radical (SR), hydroxyl radical (HR), and DPPH radical (DR) scavenging activities of WPEO, BPEO, standards, or synthetic antioxidants (BHT, PG, or Vc) were measured according to our previous work (Wang et al., 2014) and based on the pyrogallol autoxidation method (Gulcin et al., 2010), the benzoic acid fluorescence method (Chen et al., 2002) and the method described by Zhan et al. (2006) with slight modifications, respectively.

The inhibition of lipoprotein lipid peroxidation activity (ILLP) was measured by the method described by Pandey et al. (2007) with slight modifications using homogenates of egg yolk as the lipid-rich media. Egg yolk homogenate (0.4 mL, dissolve in 0.1 mol/L PBS, pH = 7.5, 2%, v/v) was added with 0.1 mL WPEO, BPEO, standards or synthetic antioxidants (BHT, PG or Vc) solution of different concentrations, 0.4 mL FeSO₄ (25.0 mmol/L, dissolved in 1% H₂ SO₄), and then the final volume was made up to 4 mL by PBS. The above mixture was shaken in a water bath at 37 °C for 30 min to induce lipid peroxidation. 1 mL of 20% TCA (w/v) was then added, well mixed, and reacted for 10 min. After centrifugation (4,000 r/min, 12 min), 4 mL of the supernatant was mixed with 2 mL 0.28% TBA (w/v) and reacted and boiled for 15 min. After cooling, the absorbance at 532 nm was measured. The control was a substitution of the sample with 0.1 mL of ethanol or distilled water. ILLP was calculated by the following equation: ILLP (%) = (Ac–As)/Ac × 100% (Ac is the absorbance of the control, while As is the absorbance of the sample).

Means and standard deviations (SD) of the triplicates for all samples were calculated. All statistical analyses were done by SPSS (Statistical Package for Social Science) Version 16.0. The antioxidant activities and free radical scavenging potential of WPEO, BPEO, their standards, and synthetic antioxidants at the same concentration were compared by one-way ANOVA. Probit analysis was applied to calculate the median effective concentration (EC₅₀) values of the essential oils and the synthetic antioxidants. The higher the EC₅₀ value, the lower the antioxidant and free radical scavenging activities (Gulcin et al., 2010). The ratios of seven standards to essential oils in terms of different antioxidant and scavenging activities were subject to principal component analysis (PCA), which was calculated using PRIMER 6 software package version 6.1.5 (Plymouth Marine Laboratory, United Kingdom). The PCA analysis was also carried based on DPPH radical scavenging activities and superoxide radical scavenging activities of WPEO and BPEO in the present study and five different habitats reported by Li et al. (2020).

MTT, a yellow tetrazolium salt, could be reduced by succinodehydrogenase in the living cells to a purple formazan, which can be dissolved by DMSO and absorbance at 570 nm reflects indirectly the number of the living cells (Adach et al., 2016; Sakulnarmrat et al., 2013). The effects of WPEO, BPEO, and seven standards with different concentrations on cell viability in HeLa cells are shown in Figure 1. No significant difference was observed among WPEO, BPEO, and standards with the concentration ranged from 0.001 to 0.2 μL/mL. They did not cause a significant effect on the cell viability at concentrations lower than 0.2 μL/mL, while the cell viability was reduced when the concentration increased to 0.4 μL/mL. The obvious inhibition of the essential oils and the standards on HeLa cells then occurred when the concentration further increased from 0.4 to 2 μL/mL. The inhibitory effect of WPEO seems slightly lower than that of BPEO on HeLa cells, but both WPEO and BPEO inhibited the cell viability more severely than all the standards. Caryophyllene exhibited the lowest inhibition effects on HeLa cells. The growth of HeLa cells is inhibited by WPEO or BPEO, indicating the pepper essential oils can play an anticancer role in inhibiting HeLa cells when their concentrations are up to 0.4 μL/mL. Previous research has also shown that black pepper extracts have potential immunomodulatory, anti-tumor, and anti-cancer activities, indicating pepper that pepper exhibits the potential to prevent or attenuate carcinogenesis as therapeutic tools (Majdalawieh and Carr, 2010). The essential oils from piper klotzschianum, P. hispidum, and P. arboreum all showed the best cytotoxic activity, inhibiting murine melanoma cell lines and human hepatocellular carcinoma and promyelocytic leukemia (Lima et al., 2019). The essential oils of other plants such as Juniperus oxycedrus L. presented anti-tumor properties in breast cancer cells due to the high level of α-pinene, β-pinene, limonene, and 3-carene (El-Abid et al., 2019). α- and β-pinenes are inhibitors in breast cancer, and in vitro present cytotoxic activity against human cancer cells, instead of healthy cells including red blood cells or even whole organisms (Mercier et al., 2008). Although exhibiting the lowest inhibition effects on HeLa cells in the present study, caryophyllene still showed anti-proliferative properties in cancer cells (Mboge et al., 2019). However, although Lampronti et al. (2006) did not find an anti-tumor effect of the individual α or β-pinenes, they believed that possible synergistic effects with other monoterpenes and sesquiterpenes such as caryophyllene could exist. All these studies provide further insight into the potential applications of terpenes and a mixture of terpenes, i.e., the essential oils as potential anti-tumor agents. Besides the anticancer role of WPEO and BPEO in killing HeLa cells, we focused on the intracellular antioxidant activities.

Although the traditional chemical methods to evaluate antioxidant activities in vitro are quick, simple, cheap, and convenient, they neither truly simulate the internal physiological environment of the human body, nor accurately reflect the antioxidant capacity of the antioxidants in vivo. Besides, the animal models and human experiments are expensive and time-consuming, while the cell model method provides an economical and rapid method for better antioxidant research. Compared with the chemical methods, the cell model method has a better biological correlation and might predict the antioxidant activity of the antioxidants in the human body more accurately. Therefore, within the non-cytotoxic range lower than 0.2 μL/mL on HeLa cells, HeLa cells were chosen to simulate an in vivo environment to investigate the intracellular antioxidant activities of the essential oils and their standards with concentration at 0.1 μL/mL. Non-fluorescent DCFH-DA could enter cells freely and then be hydrolyzed by intracellular esterases to form DCFH (Labieniec and Gabryelak, 2007). Abundant cellular ROS could be simulated by H₂O₂, and then the produced ROS could oxidize the non-fluorescent DCFH to fluorescent DCF, therefore, the measurement of fluorescence intensity reflects the cellular ROS directly (Qian et al., 2012; Xu and Chang, 2012). When antioxidants possess the scavenging ability of ROS, the oxidation of DCFH could be inhibited or even eliminated, and thus the fluorescence intensity of DCF would decrease.

The integral areas of the relative fluorescence intensity of WPEO, BPEO, and seven standards in HeLa cells are shown in Figure 2A. After 1-h cultivation, the relative fluorescence intensity in the control group stimulated by H₂O₂ was nearly eight times that of the blank group without the H₂O₂ stimulation. The relative fluorescence intensities of WPEO, BPEO, and seven standards were all significantly lower than the control, indicating that all of them had the intracellular ROS scavenging activity in HeLa cells. The intracellular antioxidant activity (CAA) of WPEO, BPEO, and seven standards are also shown in Figure 2B. BPEO showed significantly higher CAA than WPEO, while for the standards the order was limonene = 2-carene > α-pinene > β-pinene > 3-carene = caryophyllene = linalool. Limonene, α-pinene, β-pinene, and 3-carene all have higher contents in BPEO than WPEO, and they account for about 75% in BPEO and play a role in scavenging intracellular ROS, leading to the higher CAA of BPEO (Supplementary Table S1). Although 2-carene shows high CAA but lower content in BPEO than WPEO, it only accounts for 1.1 and 0.1% in WPEO and BPEO, respectively, (Supplementary Table S1). The essential oils rich in monoterpenes, oxygenated monoterpenes, and/or sesquiterpenes generally show greater and higher antioxidative potential (Tepe et al., 2004; Deba et al., 2008). In particular, the monoterpenes found in these essential oils may act as radical scavenging agents, as the monoterpenes are very reactive because of the presence of C=C, a double bond between two atoms of carbon (Mercier et al., 2009). In addition, cyclic monoterpenes will preferentially separate from an aqueous phase and thus easily access cell membrane structures due to their lipophilic character (Sikkema et al., 1994). On the other hand, as sesquiterpene hydrocarbons, caryophyllene subjected to antioxidant study always shows a low antioxidant effect (Ruberto and Baratta, 2000).

The activities of synthetic antioxidants have only been examined by chemical methods, while they have not been examined by cell model. Compared with animal models and human experiments, the cell model method is cheap and rapid. However, compared with the chemical methods, the cell model method is relatively expensive and time-consuming. The results from chemical methods, to some extent, could reflect the comparisons between peppers and synthetic antioxidants. In addition, the activities of the pepper essential oils and their components are what we are more interested in. Therefore, the activities of synthetic antioxidants have only been evaluated with chemical methods and without cell model methods.

The capacities in reducing Mo⁶⁺ to Mo⁵⁺ of the antioxidants stand for TAA, which reaction produces a blue-green phosphomolybdenum complex with the maximum absorption wavelength at 695 nm (Merugu et al., 2021). The TAA results of WPEO, BPEO, and synthetic antioxidants (BHT, PG, and Vc) are shown in Figure 3A. In all antioxidants, TAA followed the same trend, i.e., increased rapidly with the sample concentrations; however, the increasing rates varied among different antioxidants. Although the TAA of both pepper essential oils was lower than those of the three synthesized antioxidants, WPEO and BPEO still showed significant and concentration-dependent TAA. The TAA of WPEO, BPEO, and seven standards are also shown in Figure 3B. BPEO showed a significantly higher TAA than WPEO, and the TAA declined in the trend of 2-carene > limonene = linalool > α-pinene = β-pinene = 3-carene > caryophyllene. Limonene, α-pinene, β-pinene, and 3-carene with relatively higher TAA exhibiting higher contents in BPEO than WPEO, while caryophyllene, with the lowest TAA, is significantly richer in WPEO than BPEO, which leads to the higher TAA of BPEO than WPEO (Figure 3). This trend of TAA among essential oil and standards is similar to the trends of CAA, which is BPEO > WPEO and caryophyllene has the lowest values (Figure 2). As a sesquiterpene, caryophyllene always has the lowest CAA or TAA likely due to its structure with higher molecule weight and more steric hindrance than those small molecule monoterpenes, which might work more effectively as reactive and intercellular ROS scavenging antioxidants (Mukai et al., 2018).

Among all the ROS, superoxide anion radical is one of the strongest free radicals. SR results, measured by the autoxidation of the pyrogallol method of WPEO, BPEO, and synthetic antioxidants (BHT, PG, and Vc) are shown in Supplementary Figure S1A. For all antioxidants, SR showed the same trend, i.e., it increased rapidly with the sample concentrations. However, the rates of increase differed among the antioxidants. WPEO and BPEO showed significant and concentration-dependent SR, which are similar, a little higher and 2-fold as that of Vc, BHT, and PG, respectively. The SR of WPEO, BPEO, and seven standards are also displayed in Supplementary Figure S1B. The EC₅₀ values (mg/mL) of all samples and standards are shown in Table 1. High EC₅₀ values indicate low scavenging activities, and thus the order of SR was BPEO > Vc > WPEO > BHT > PG, and SR among the oils and standards declined in the trend of limonene > BPEO > 3-carene > WPEO > 2-carene > β-pinene > α-pinene > linalool > caryophyllene (Table 1).

Hydroxyl radical scavengers play a significant role in biological systems due to the high activity of hydroxyl radicals. All samples studied here exhibited HR in a dose-dependent manner to varying degrees (Supplementary Figure S2A). BPEO and WPEO presented stronger HR than the synthetic antioxidants, BHT, and even several-folds as Vc. HR of WPEO, BPEO, and seven standards are also illustrated in Supplementary Figure S2B. The EC₅₀ results showed that HR declined in the trend of BPEO > BHT > WPEO > PG > Vc, and the HR order among the oils and standards was BPEO > WPEO > linalool > 3-carene > caryophyllene > α-pinene > β-pinene > 2-carene > limonene (Table 1). Compared with the synthetic antioxidants and single standard, both WPEO and BPEO are the most effective in removing OH.

Antioxidants always result in a decrease in absorbance at 517 nm by the reaction of DPPH radicals scavenging. DPPH assays are simply chemical assays and may only be used to define the chemical profile of a preparation. However, the application of this quick and easy chemical assay combined with other pharmacological experiments, to some extent, could help reflect the differences between peppers and synthetic antioxidants. Supplementary Figure S3A exhibits the DR results of WPEO, BPEO, and synthetic antioxidants (BHT, PG, and Vc). The scavenging activity increased with the concentration from 1 to 5 mg/mL of WPEO and BPEO, though they showed lower DR than the synthetic antioxidants. The EC₅₀ results also showed PG > Vc > BHT > BPEO > WPEO (Table 1). On the other hand, the EC₅₀ results showing the DR order of oils and standards followed the decreasing trend of BPEO > WPEO > limonene > 3-carene > α-pinene > β-pinene > 2-carene > linalool > caryophyllene (Table 1). The most abundant component in BPEO, 3-carene, was significantly stronger than caryophyllene, the major component in WPEO, indicating that BPEO was more DR active than WPEO. Furthermore, DR of both WPEO and BPEO were better than those of the single standards in removing DPPH radicals.

Lipid peroxidation could lead to cell damage and different oxidant reactions in organisms. Among the two essential oils and the three synthetic antioxidants, the ILLP followed the declining order of BHT > PG > BPEO > WPEO > Vc, and they were all concentration-dependent (Supplementary Figure S4A; Table 1). The EC₅₀ results of oil samples and standards showed that ILLP followed the decreasing trend of linalool > BPEO > WPEO > 3-carene > caryophyllene > α-pinene > limonene > β-pinene > 2-carene (Supplementary Figure S4B; Table 1).

All the above orders, although based on different activities, followed a similar trend, with BPEO always having higher activities than WPEO, and sometimes the essential oils had higher capacities than the synthetic antioxidants, which showed good evidence for future research on natural antioxidants. BPEO and WPEO were generally better than those of the single standards in different aspects.