Lemon peel was freeze-dried, crushed, and passed through mesh 60. Fifty grams of the lemon peel powder were mixed with 480 mL of 70% ethanol and extracted in a water bath at 60°C for 4 h. The extracts were filtered, evenly passed through a column filled with AB-8 macroporous resin, collected, and evaporated by rotary evaporation to obtain LPP.

A certain amount of chlorogenic acid standard was weighed and added into deionized water to prepare chlorogenic acid standard solution. Then 1.0 mL chlorogenic acid standard solution with different concentrations was drawn and added into a 25 mL volumetric flask. The 3.0 mL Folin-Ciocalteu reagent was added for mixing. After 5 min of reaction, 4.5 mL of saturated Na₂CO₃ solution was added into the volumetric flask, and the reaction was conducted at 30°C to avoid light for 30 min. The final absorbance was determined at 747 nm and the standard curve of chlorogenic acid was plotted. Lemon peel polyphenols was diluted to 10⁻⁴ times in gradient. The absorbance value of lemon peel polyphenols was determined according to the above method. The polyphenol content of lemon peel polyphenols was calculated according to the standard curve.

Standards of gallic acid, neochlorogenic acid, (+)-catechin, caffeic acid, (−)-Catechin gallate, isochlorogenic acid A, rosmarinic acid, and protocatechuic acid were placed in centrifuge tubes to prepare standard solutions by dissolving in methanol (1.0 mg/mL). Then, the standard solutions were filtered through an organic membrane (0.22 μm) and stored in a 1.5 mL brown vial until use.

Components of LPP were detected using following chromatographic conditions: chromatographic column: Thermo Scientific Accucore C18 (4.6 mm × 150 mm, 2.6 μm); mobile phase A: 100% methanol, B: 0.5% acetic acid solution; flow rate: 0.5 mL/min; column temperature: 30°C; detector: UV-Vis; detection wavelength: 285 nm; injection volume: 10 μL. The content of each component in lemon peel was calculated by external standard method as follows: Mx = Cr × Ax/Ar × C, where, Mx (mg/g): contents of component; Cr (mg/mL): mass concentration of standard; Ax: measured peak area of sample; Ar: measured peak area of standard; and C (1.0 mg/mL): concentration of sample stock solution.

First, 0.01 g DPPH reagent (Phygene Life Sciences Company, Fuzhou, Fujian, China) was dissolved in a 250 mL volumetric flask with anhydrous ethanol to adjust the concentration of DPPH to 0.1 mol/L. Then, various volumes of LPP extract stock solution were added into plugged test tubes, using ultrapure water to a total volume of 0.1 mL. Next, 4.00 mL of 0.1 mol/L DPPH-free radical solution was added, vortexed, and allowed to rest for 30 min in the dark. Anhydrous ethanol was used instead of the sample as the control group. Last, 200 μL of the final reaction solution was collected to measure absorbance at 517 nm with a spectrophotometer. The measured absorbance was used to calculate the scavenging ability of lemon peel extract on DPPH radicals by the following equation: DPPH clearance rate (%) = [1-(A₁-A₂)/A₀] × 100%, where A₀: absorbance of 0.1 mL anhydrous ethanol and 4.00 mL DPPH blank control; A₁: absorbance of 0.1 mL sample solution and 4.00 mL DPPH solution after reaction; A₂: absorbance of 0.1 mL sample solution and 4.00 mL anhydrous ethanol.

ABTS⁺ (Phygene Life Sciences Company, Fuzhou, Fujian, China) free radical working solution was prepared by mixing 5 mL of ABTS⁺ solution (7 mmol/mM) and 88 μL of potassium perphosphate water solution (140 mmol/mL) in the dark for 12 h to stabilize free radical ions. Various volumes of an extract stock solution with the active ingredients from lemon peel were added into plugged test tubes and volume was increased to 0.1 mL with ultrapure water, followed by adding 4.00 mL of the prepared ABTS⁺ radical working solution. After thorough mixing, the reaction was carried out at room temperature for 10 min. An equal volume of ethanol with equal volume was used as the control. Absorbance was measured at 734 nm wavelength (11). The scavenging ability of lemon peel extract on ABTS⁺ radicals was calculated per the following equation: ABTS⁺ clearance rate (%) = [1-(A₁-A₂)/A₀] × 100%, where A₀: absorbance of 0.1 mL anhydrous ethanol and 4.00 mL ABTS⁺ blank control; A₁: absorbance of 0.1 mL sample solution and 4.00 mL ABTS⁺ solution after reaction; A₂: absorbance of 0.1 mL sample solution and 4.00 mL anhydrous ethanol.

HaCaT cells (Procell Life Science & Technology Co., Ltd, Wuhan, Hubei, China) were cultured with DMEM medium containing 10% fetal bovine serum after resuscitation in 5% CO₂ for 24 h to allow cells to adhere (12). After 24 h, the original medium was discarded. After adherence, HaCaT cells were cultured with DMEM medium (Solarbio Life Sciences, Beijing, China) containing H₂O₂ (adding 31% hydrogen peroxide solution to adjust the concentration, Sigma, St. Louis, MO, USA) at the final concentration of 20, 40, 60, 80, 100, and 120 μmol/L for 4 h. The cell survival rates were measured, and then the appropriate concentration of H₂O₂ was selected for subsequent experiments. Then the HaCaT cells in logarithmic growth phase were divided into five groups: normal group, model group, vitamin C (Vc) group, low-concentration LPP group (LPPL, 50 μg/mL) and high-concentration LPP group (LPPH, 100 μg/mL). HaCaT cells in the normal group were not treated but were cultured with new culture medium to maintain normal growth. The model group included HaCaT cells subjected to oxidative damage. After adherence, model HaCaT cells were cultured with DMEM medium (Solarbio Life Sciences, Beijing, China) containing H₂O₂ at a appropriate concentration for 4 h. HaCaT cells in the Vc group were treated identically with H₂O₂, and then the culture medium was discarded. The Vc cells were then further cultured with the medium containing 100 μg/mL Vc for 12 h. HaCaT cells in the LPPL and LPPH groups were treated with H₂O₂ and the cells were cultured with medium containing 50 and 100 μg/mL LPP, respectively, for 12 h.

After the cells were treated by the procedures described in Section Cell Experiment, 20 μL MTT solution (5 g/L) was added into each well and incubated for 4 h. Then, the medium was discarded, and 150 μL of DMSO was added to each well. Absorbance was measured at 490 nm (Varioskan LUX multifunctional enzyme labeling instrument, Thermo Fisher Scientific, Inc., Waltham, MA, USA) after shaking and in the dark for 20 min. The cell survival rate was calculated as follows: cell survival rate (%) = (OD value in sample group/OD value in normal group) × 100% (13).

After the cells were treated, cell supernatant and cells were collected, and then the cells were lysed in an ice-water bath with an ultrasonic cell disruptor (shocking for 3–5 s with 4 s intervals, repeat 3 times). Then, the LDH level in the cell supernatant was determined according to the kit's instructions, and the levels of LDH, SOD, MDA, GSH, and CAT (Solarbio Life Sciences, Beijing, China) were determined by a multifunctional microplate reader, per manufacturer's instructions.

After treatment, the cells from all 5 groups were collected and lysed in an ice-water bath with an ultrasonic cell disruptor (shocking for 3–5 s with 4 s intervals). RNA was extracted from cells using TRIzol^TM (Thermo Fisher Scientific, Inc.) and diluted to 1 μg/μL. The cDNA template was obtained using 1 μL of diluted RNA solution after reverse transcription. Then, 1 μL cDNA template and 10 μL SYBR Green PCR MasterMix, 1 μL of upstream and downstream primers (Thermo Fisher Scientific, Inc., Table 1), and 7 μL of sterile distilled water were mixed, reacted at 95°C for 60 cycles and 95°C for 15 s in each cycle; 55°C for 30 s; 95°C for 30 s; 55°C for 35 s (Stepone Plus qPCR instrument, Thermo Fisher Scientific, Inc.). Relative gene expression was calculated using 2^−ΔΔCt, with GAPDH as the internal reference (14).

After the cells were lysed with RIPA cell lysate, supernatant was separated, and protein concentration was determined by a protein assay kit. A total of 30–50 μg protein was loaded for SDS-PAGE separation (Thermo Fisher Scientific, Inc.) followed by electroblot transfer onto a nitrocellulose (NC) membrane. The membrane was sequentially blocked, washed, and labeled with primary antibodies against SOD, CAT, GSH and GSH-Px and secondary antibodies (Thermo Fisher Scientific, Inc.). Bound antibodies were detected using the chemiluminescence method [iBright FL1000 (Thermo Fisher Scientific, Inc.)] (15).

All experiments were conducted in triplicate to obtain an average value. Data were analyzed by SPSS 23 statistical software. One-way ANOVA was used to compare between groups. P < 0.05 was considered statistically significant.

According to the experimental method, the standard curve of chlorogenic acid standard solution was drawn. The regression equation of the standard curve was y = 0.226x-0.002 (R²= 0.997), y was the concentration of chlorogenic acid, and x was the absorbance value. According to the calculation of standard curve, the content of LPP (chlorogenic acid) reached 79.8%.

As illustrated in Figure 1, LPP scavenged DPPH and ABTS⁺ free radicals in a dose-dependent manner. Within the concentration range of 0-120 μg/mL, the scavenging ability increased with LPP concentration. The DPPH and ABTS⁺ free radical scavenging activities were expressed as 77.19 and 93.74 μg/mL LPP by IC₅₀. The ability to scavenge DPPH and ABTS⁺ radicals in the Vc positive control group was lower than that of LPP.

By observing the effects of different concentrations of H₂O₂ on HaCaT cells survival rate, it was found that the concentration of 20 μg/mL had almost no effect on the cell survival rate, and the cell survival rate decreased slightly at the concentration of 40 and 60 μg/mL, while the cell survival rates were significantly decreased at the concentration of 80 and 100 μg/mL, when the concentration reached 120 μg/mL, almost all the cells died (Figure 2). Therefore, the concentration of 100 μg/mL was selected as the further experimental concentration to observe the inhibitory effect of LPP on H₂O₂ induced oxidative damage of cells.

As expected, survival rate of HaCaT cells in the model group was significantly lower than that in the normal group (Figure 3, P < 0.05). However, the survival rates of HaCaT cells treated with Vc (100 μg/mL) and LPP (50 and 100 μg/mL) were improved compared with the model group (P < 0.05). The protective effects of LPP were dose-dependent and significantly stronger than Vc at same concentration (100 μg/mL, P < 0.05).

As shown in Figure 4, the level of LDH was the lowest in the normal group (120.70 ± 9.24 U/L) and highest in the model (623.55 ± 15.91 U/L). However, LPP reduced the LDH levels in cells with oxidative damage. Higher concentrations of LPP (100 μg/mL) were associated with lower LDH levels. LPP could reduce the level of LDH, regulate the LDH level of oxidative damage cells gradually return to the normal state, and the regulation effect of LPP was stronger than Vc at the same concentration of 100 μg/mL.

Compared with the normal group, levels of SOD, GSH and CAT in HaCaT cells in the model group were significantly decreased, as expected, and the level of MDA was significantly increased (P < 0.05, Table 2). Compared with the model group, the levels of SOD, GSH, and CAT were increased after treatment with Vc (100 μg/mL) or LPP (50 and 100 μg/mL) treatment, and the level of MDA was decreased. This effect was also enhanced with increasing LPP concentration.

As shown in Figure 5, compared with the normal group, the expression of Bax, Caspase-3, Nrf 2, and HO-1 in the HaCaT cells in the model group was significantly increased, and Bcl-2 was significantly decreased (P < 0.05). However, compared with the model group, the expression of Bcl-2 in skin cells was increased after LPP (50 and 100 μg/mL) treatment, and Bax, Caspase-3, Nrf 2, and HO-1 were decreased. These effects were strongly affected by changes in LPP concentration, and the effect of LPP (100 μg/mL) appeared to be stronger than that of Vc (100 μg/mL), in good agreement with the results from the prior experiments.

As shown in Figure 6 and Table 3, the main active components in LPP were found to be protocatechic acid, caffeic acid, neochlorogenic acid, (+)-catechin, gallic acid, (−)-catechin gallate, isochlorogenic acid A and rosmarinic acid with contents of 20.625, 102.795, 25.575, 50.82, 17.49, 110.715, 69.96, and 255.915 mg/g, respectively.