2.1. Materials and Reagents

TF dried samples were purchased from Guangzhou Qingping Chinese Herbal Medicine Wholesale Market (Guangdong Province, China) and are of Guangxi Province (China) origin. Botanical identification of TF was performed by Professor Gang Hao from College of Life Science, South China Agriculture University. HPD 300 macroporous resin was obtained from Beijing H&E Ltd Corporation (Beijing, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from Sigma-Aldrich (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and Trizol reagent were purchased from GIBCO (Grand Island, NY, USA). The assay kits for catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). And the ROS assay kits were produced by MeilunBio Co., Ltd. (Dalian, Liaoning, China). Human matrix metalloproteinase 1 (MMP-1) and tumor necrosis factor-α (TNF-α) ELISA kits were purchased from Cusabio Biotech Co., Ltd. (Wuhan, China). All other chemical reagents used in this study were of analytical grade.

2.2. Preparation and Determination of TFFs

TF were grounded with a cutting machine and then passed through a 50-mesh sieve to obtain fine powder. The dried powders were soaked with 70% ethyl alcohol, and then extracted by heating and reflux at 80°C for 3 h. After three times of heating reflux extraction, the alcohol liquid was collected and concentrated into stiff paste using a rotary evaporator (YRSHYQ, SH, China), which was dried at 45°C to obtain the alcohol extracts: TF total flavonoids (TFFT). Subsequently, TFFT was redissolved with distilled water, and then partitioned with ethyl acetate to obtain the crude flavonoids (TFFC). Following this, the impurities in TFFC were removed through HPD 300 macroporous resin using distilled water, 50% ethanol and 70% ethanol. The 70% alcohol eluates were condensed to yield TFFP. Using rutin as the standard, the aluminum nitrate method was employed to determine the flavonoid content in TFFT, TFFC, and TFFP. The measured standard curve was y = 15.687x + 0.0006 (R² = 0.9970) (1). The absorbance values measured at 510 nm were substituted into the standard curve to calculate the rutin equivalent, with the results expressed as the percentage of rutin equivalent per milligram of sample (%) [16].

2.3. FT-IR Spectra and LC-MS/MS Analysis

The FT-IR spectra were determined by potassium bromide (KBr) pellet method as reported previously [17]. TFFC and TFFP powder (2 mg) was mixed with KBr powder (200 mg), and then pressed into a pellet. The pellet was subjected to infrared scanning in the range of 4000–500 cm⁻¹ using a Fourier transform infrared spectrophotometer (Bruker, Ettlingen, Germany).

The components of flavonoids were identified through LC-MS/MS. TFFC and TFFP were dissolved in 50% acetonitrile to the final concentration of 50 ppm, and the mixture solution was passed through a 0.22 μm nylon membrane. Chromatographic analysis was performed on an Agilent Technologies 1290 Series System (Agilent, USA). The column was a Waters ACQUIY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm). The binary mobile phase consisted of acetonitrile (phase A) and 0.01% formic acid in water (phase B). The gradient elution program was as follows: 2%–50% phase A at 0–18 min, 50%–95% phase A at 18–21 min, maintain 95% phase A at 21–23 min and 2% phase A at 23–25 min. The flow rate was 0.2 mL/min; 5 μL sample was injected into the system and the eluent was monitored for UV absorption at 210 nm. The MS data was recorded by Bruker maXis impact system (Bruker, Germany) with an ESI interface in negative ion mode. The parameters of ESI-MS were set as follows: sheath gas flow rate of 35 arbitrary units, aux gas flow rate of 10 arb arbitrary units, capillary temperature of 320°C, heater temperature of 350°C, and spray voltage of 3.0 kV. Primary mass spectra were recorded through full MS scan with 70,000 resolution ratio and a scanning range of 70–1050 m/z. The secondary mass spectrometry was detected through MS² with a resolution ratio of 17,500.

2.4. Antioxidant Activity of TFFs In Vitro

The in vitro antioxidant capacity of TFFs was evaluated by measuring the DPPH radical scavenging capacity, ABTS radical scavenging capacity, ferric ion reducing antioxidant power (FRAP), and total reducing power. Vitamin C (V_C) was used as an antioxidant positive control. The TFFs (TFFT, TFFC, and TFFP) sample concentrations were set as: 50, 100, 200, 400, and 800 μg/mL.

The DPPH assay was performed according to the method reported previously with some modifications [18]. Take 20 μL of samples at different concentrations (50, 100, 200, 400, and 800 μg/mL) and add them to a 96-well plate, followed by the addition of 180 μL of 150 μmol/L DPPH solution. After 10 min of reaction time in the dark, the absorbance values at 517 nm were measured on a 96-well plate with 20 μL of various concentrations of samples and 180 μL of 150 μmol/L DPPH solution. Anhydrous ethanol was used as the blank, while a V_C solution was used as the positive control. DPPH was used as the background control instead of anhydrous ethanol. The DPPH radical scavenging activity (%) was calculated using the formula: (1−(A_sample − A_background)/A_blank) × 100%(2). “A” stands for absorbance.

For ABTS radical scavenging capacity assay, the procedure followed the method reported previously with slight modifications [19]. The stock solution was prepared by mixing equal amounts of 7 mmol/L ABTS solution and 4.9 mmol/L potassium persulfate solution. The stock solution was diluted with ethanol to a gradient concentration: 0.15, 0.20, 0.25, 0.30, and 0.35 mmol/L. The working solution was obtained by mixing 180 μL of ABTS solution and 20 μL of water. After 5 min of reaction in the dark, the absorbance values at 734 nm were measured on a 96-well plate with 20 μL of various concentrations of samples and 180 μL of ABTS working solution. Water was used as the blank, while a V_C solution was used as the positive control. ABTS was used as the background control instead of anhydrous ethanol. ABTS radical scavenging activity (%) was calculated using the formula: (1 – (A _sample – A _background)/A _blank) × 100% (3).

The FRAP assay was performed according to the reported method with some modifications [20]; 0.3 mol/L sodium acetate solution, 10 mmol/L TPTZ hydrochloric acid solution, and 20 mmol/L FeCl₃ solution in a 10:1:1 ratio were combined to make the TPTZ working solution. In a 96-well plate, add 20 μL of FeSO₄ solution with different concentrations (0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mmol/L) and 180 μL of TPTZ working solution, and then react for 5 min at room temperature, protected from light, to determine the absorbance value at 593 nm and plot the standard curve. By substituting samples for FeSO₄, the absorbance at 593 nm was calculated. The FRAP of TFFs was determined using the FeSO₄ standard curve. FRAP values were expressed as millimolar FeSO₄ equivalent (mM Fe²⁺ Eq), defined as the concentration of FeSO₄ required to produce the same absorbance as the sample at 593 nm. Water and V_C solution were substituted for the samples as the blank and positive groups, respectively.

For total reducing power assay, the procedure followed reported method with slight modifications [21]. In a centrifuge tube, equal parts 1% potassium ferricyanide, PBS (PH = 6.6), and sample solution were combined. After 20 min of reaction at 50°C, the centrifuge tube was quickly cooled with running water, and the reaction was stopped with 10% trichloroacetic acid. After centrifugation, 90 μL of supernatant, 90 μL of distilled water, and 18 μL of 0.1% ferric chloride were placed on a 96-well plate for 10 min, and absorbance was measured at 700 nm. For the blank and positive controls, the sample solution was replaced with distilled water and V_C, respectively. Total reducing power = A _sample – A _blank(4).

2.5. Cell Culture and Establishment of UV-Damaged HaCaT Cells

Cell proliferation inhibition was utilized to investigate the toxicity of TFFs on HaCaT cells. Specifically, when the cells entered the logarithmic growth phase, the media was removed and the cells were washed twice with PBS. Trypsin was employed to digest the cells for 2 min. After gentle blowing and mixing, the cells were centrifuged at 34 × g for 5 min. The concentration of cells was adjusted to 8 × 10⁴ cells/mL using Complete culture medium. The complete cell culture medium was prepared by mixing DMEM, FBS, and a double-antibiotic solution (a mixture of penicillin and streptomycin) in the ratio of 9:1:0.1. Samples were dissolved in DMSO to form a 100 mg/mL stock solution, which was then aliquoted and stored at −20°C. Before use, the stock solution was diluted to the desired concentration with complete culture medium, ensuring the final DMSO concentration in the treated cells was below 1%. All sample solutions were filter-sterilized using a 0.22-μm membrane filter before application to the cells. A 100-μL cell suspension was added to 96-well plates and grown in a CO₂ incubator at 37°C for 24 h. The growth media was gradually drawn out, and 100 μL samples were added to each well. Complete culture medium was used as a normal control. After incubated for 24 h at 37°C, the cell viability was tested by MTT method as described in previous literature [22]. Then, the absorbance value was detected at 570 nm. Cell viability (%) = A _sample/A _control × 100%(5).

Prior to analyzing the UV protection of flavonoids, the UV irradiation dose should be determined. When the HaCaT cells reached 80%–90% confluence, 500 μL cells were seeded in 24-well plates at a density of 1.2 × 10⁵ cells/mL. After incubation for 24 h, the culture medium was slowly sucked out, and the cells was washed carefully twice with PBS. Then, PBS (300 μL/well) was added before UV irradiation to avoid cell dehydration. The UV irradiation dose was set at 10, 20, 30, 40, and 50 mJ/cm² using UV irradiation device (HOPE-MED 8140, Tianjin Development Zone Hepu Industry & Trade Co. Ltd., Tianjin, China). After treatment with UV irradiation, the PBS was replaced with culture medium (500 μL/well). After 24 h of incubation, the cell viability was tested by the MTT method and calculated as equation (6). The UV irradiation dose was chosen for further study when the survival rate of HaCaT cells is about 50%.

The experiment was performed in control group, model group, and TFFs groups. After the UV-damaged HaCaT cells model was established, the survival rate of the cells was measured by MTT assay after adding 500 μL sample solution for 24 h, and the cell morphology was observed under a microscope (LIOO, Germany).

2.6. Determination of the Effect of TFFs on Antioxidant System and MMP-1 and TNF-α of UVB-Damaged HaCaT Cells

ROS, CAT, GSH-Px, SOD, and MDA levels were detected following the methods of the previous study with some modifications [23]. After the UV-damaged HaCaT cells model was established, the cells with treatments of TFFs for 24 h were collected. The cell pellet was disrupted using an ultrasonic cell disruptor (Ningbo Scientz Biotechnology Co., Ltd., Zhejiang, China) under ice-water bath conditions. The procedure was performed at 300 W power output, with each cycle lasting 3–5 s followed by a 30-s cooling interval. This process was repeated for three to five cycles. Thereafter, the ROS, CAT, GSH-Px, SOD, and MDA levels were quantified using the BCA protein commercial kits. DCFH-DA was used to detect intracellular ROS detection of HaCaT cells and the levels of MMP-1 and TNF-α were evaluated using the commercial kits (Cusabio Biotech Co., Ltd., Wuhan, China).

2.7. C. elegans Stains and Maintenance

C. elegans N2 (wild type) was used in this study and the age-synchronized C. elegans N2 were collected following the methods of the previous study [24]. Worms were grown on nematode growth media (NMG) plates seeded with E. coli strain OP50 as a food. Then, 100 μM FUdR (5-fluoro-2′-deoxyuridine) was added to NMG to prevent the worm from laying eggs and hatching new worms to interfere with the experiments. Samples were dissolved in DMSO to form a 500 μg/μL stock solution, which was then aliquoted and stored at −20°C. Before use, the stock solution was diluted to the desired concentration with S medium, ensuring the final DMSO concentration in the treated C. elegans was below 0.1%. All sample solutions were filter-sterilized using a 0.22-μm membrane filter before application to the C. elegans. The protocols for preparing NGM, M9 buffer, and S medium used in the experiments are described in previous reference [25].

2.8. Determination of TFFs Concentrations and Stress Resistance Test of C. elegans

The L4-synchronized worms were collected with M9 buffer, and the density was regulated using S medium to contain 5–10 worms per 10 μL. To determine the experimental TFFs concentration, we added 20 μL of different concentrations of TFFs solution, 50 μL of 5-FUdR solution, 10 μL of OP50, and 20 μL of worms into 96-well plates, in which 20 μL of S medium was used in the blank group instead of the sample solution. A minimum of 120 C. elegans should be included in each group, recorded the number of live worms in each well at this time, and then calculated the survival rate of worms after incubation in an incubator at 20°C for 48 h. Survival rate (%) = (Number of live C. elegans/Total number of C. elegans) × 100%(7).

To conduct heat stress and oxidative stress experiments, 20 μL of TFFs solution, 50 μL of 5-FudR mixture, 10 μL of OP50, and 20 μL of L4-synchronized worms were added to 96-well plates at 20°C for 72 h. A blank group of 20-μL S medium was used instead of the sample solution. For the heat stress experiment, the 96-well plate (including both blank group and sample groups) was sealed with a sealing membrane, and the sealing membrane was removed after 5 h of incubation at 35°C, and the survival rate of worms was counted after 12 h of incubation in a 20°C incubator. For the oxidative stress experiment, a total of 100 μL of paraquat solution at a concentration of 20 mM was supplied to each well, and worm survival was counted after 10 h of incubation. For the heat stress experiment and the oxidative stress experiment, a minimum of 120 C. elegans should be included in each group.

2.9. Determination of the Effect of TFFs on Antioxidant System of C. elegans

The M9 buffer was used to gather the L4-synchronized worms, and the density was set at 60 worms/well, and add 200 μg/mL TFFs solution (400 μL), 5-FUdR combination (1 mL), and inactivated OP50 (200 μL) to a 6-well plate. The plate was then incubated at 20°C for 5 days. The DCFH-DA technique was used to treat the worms′ ROS levels, and an inverted fluorescence microscope was used to take pictures. After worms were gathered in 1.5 mL centrifuge tubes and mashed with liquid nitrogen to create worm homogenate, the commercial kits were used to measure the protein content, SOD, CAT, and GSH-Px enzyme activities (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.10. Gene Expression Assay by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from 5-day-old adult worms using the total RNA extraction kit and reverse-transcribed into cDNA with the Color Reverse Transcription Kit and stored at −80°C for qRT-PCR. The detection was carried out on the Quant Studio 7 Flex using 2 × Color SYBR Green qPCR Master Mix (ROX2 plus) as the detection method. The gene expression data were analyzed using the comparative 2^−△△CT method, with act-1 taken as the internal control. Primer sequences are listed in Table S1 of Supporting Information.

2.11. Statistical Analysis

The data (from at least three independent experiments) were shown as mean ± standard deviation (SD). Variances among the groups were analyzed by one-way analysis of variance (ANOVA). All statistical analyses were performed by using SPSS 20.0 (SPSS Inc., CA, USA). Variances were considered significant at p < 0.05. Figures were drawn by Origin 2020 and Adobe Illustrator 2022.

3.1. Component Identification of TFFs

The extraction yield and flavonoid content of TFFT, TFFC, and TFFP are presented in Table 1. The higher flavonoid concentration during the isolation operations suggested that the separation and purification of flavonoids was effective.

Then, FT-IR scanning spectrum was evaluated. The FT-IR of TFFC and TFFP revealed that several absorption bands are typical characteristic peaks at 500–4000 cm⁻¹ (Figure 1). The peak form and most recognizable peaks of the TFFC (Figure 1(a)) and TFFP (Figure 1(b)) infrared spectra maps were identical; however, the peak position and intensity differ. The hydroxyl group caused the absorption peaks of TFFC at 3440 cm⁻¹ and TFFP at 3432 cm⁻¹, while the C–H absorption peaks of TFFC and TFFP around 2930 and 2850 cm⁻¹ were induced by benzene ring alternations. The aryl carbonyl stretching vibration maxima of TFFC and TFFP were at wavenumbers 1627 and 1616 cm⁻¹, respectively. There were typical absorption peaks of C=C vibration in the benzene ring between 1516 and 1447 cm⁻¹. The hydroxyl group’s bending vibration peak was discovered around 1320–1158 cm⁻¹. At the wave range 890–700 cm⁻¹, there was an absorption peak generated by substituents on the benzene ring. Furthermore, the ether bond on the epoxy ring in the C₆–C₃–C₆ structure absorbed the TFFC signal at 1063 cm⁻¹. The C–O stretching vibration of the hydroxyl group caused an absorption peak in TFFP at 1200–1050 cm⁻¹. There is no absorption in the range of 1700–1900 cm⁻¹, indicating that there were no carboxyl groups and ester bonds; there was no absorption in the range of 2600–3000 cm⁻¹, indicating that there were no long-chain alkanes and conjugated double bonds, and methyl and methylene are relatively small.

According to the results of LC-MS/MS data (Figures 1(c) and 1(d)), the compositions of TFFC and TFFP differ somewhat. TFFC was found to be composed of at least 15 compounds, whereas TFFP was composed of at least 13 compounds based on their total ion chromatograms. The flavonoid components of TFFC and TFFP were determined by their first-level mass spectrometry (MS1) and second-level mass spectrometry (MS2), which are shown in Figures S1–S18 of Supporting Information. And nine flavonoid components were identified and are shown in Table 2. TFFC and TFFP both contained at least seven flavonoid components, including 2′,4,4′-trimethoxychalcone, 8-aldehyde-catechin, Amomutsaokin A, Amomutsaokin G, Tsaokoflavanol A, Tsaokoflavanol B, and Tsaokoflavanol F. Additionally, (−)-catechin and (2R,3R,4R)-3′,5′-dimethoxy-3,4,7,4′-tetrahydroxy-flavan are unique components found only in ATC. It is worth noting that (2R,3R,4R)-3′,5′-dimethoxy-3,4,7,4′-tetrahydroxy-flavan, Tsaokoflavanol A, Tsaokoflavanol B, Tsaokoflavanol F, Amomutsaokin A, and Amomutsaokin G are all new substances that have been isolated from A. tsaoko for the first time.

3.2. Antioxidant Activity of TFFs In Vitro

The results of the antioxidant activity of TFFs are summarized in Table 3. As demonstrated in Figure 2(a), as concentration increased, the DPPH radical scavenging activity in the sample groups correspondingly increased. The scavenging capability of DPPH radical in the sample concentration range of 50–800 μg/mL was comparable to that of V_C. At the concentration of 50 μg/mL, the DPPH radical scavenging activity of TFFC was approximately 50% that of the V_C. TFFC’s DPPH radical scavenging activity was 70.51% at 800 μg/mL, which was 8.39 times that of the low concentration (50 μg/mL). When TFFP was at the concentration of 800 μg/mL, the DPPH radical scavenging rate reached 50.72%. The capacity of TFFs to scavenge ABTS radical increased with concentration (Figure 2(b)). However, when the concentration of ABTS radical increased, the scavenging capacity increasing range of V_C and TFFC declined, showing that the scavenging ability of V_C and TFFC at high concentrations had achieved saturation. At 50–800 μg/mL, TFFC had the greatest ABTS radical scavenging capacity, while the scavenging rate of TFFC at 200–800 μg/mL was comparable to that of V_C, and the scavenging activity of TFFP at 400 μg/mL was 86.07%. The scavenging capacity of TFFT on ABTS radical was the lowest, although at 200 μg/mL, it topped 40%. In the experiment to investigate the reducing ability of TFFs to Fe³⁺, we found that the reducing ability of each sample to Fe³⁺ increased with concentration, indicating that the ability of TFFs and V_C to promote Fe³⁺ reduction was dose dependent (Figure 2(c)). The antioxidant capacities of TFFT, TFFC, and TFFP are comparable at 50 μg/mL. When the concentration reached 100–800 μg/mL, the FRAP value of TFFC was the greatest, even topping the V_C group at 100–400 μg/mL. Finally, we examined the total reducing capacity of flavonoids and found that it increased in a concentration-dependent way in the range of 50–800 μg/mL (Figure 2(d)). The total reducing power of TFFP at 800 μg/mL was 1.58, 1.74 times that of TFFC (0.90) and 1.78 times that of TFFT (0.89), which was comparable to 80% of the total reducing power of the V_C group at the same dose.

3.3. Establishment of UVB-Damaged HaCaT Cells and TFFs Anti-UVB Damage Effect

The survival rate of normal HaCaT cells revealed that TFFs samples had no harmful impact on HaCaT cells at concentrations ranging from 25 to 400 μg/mL, and showed a certain proliferation promotion effect as concentration increased (Figure 3(a)). The survival percentage of HaCaT cells was more than 120% when the concentration of TFFs was greater than 50 μg/mL. To avoid the sample effect on promoting proliferation impact on activity evaluation, the following TFFs experiment concentrations were chosen: 12.5, 25, and 50 μg/mL.

We used different UVB radiation energies to irradiate HaCaT cells, and the results of cell survival rate after 24 h of culture are presented in Figure 3(b). The cell survival rate dropped as the irradiation energy increased. Cell survival was 48.12% when the UV intensity was 30 mJ/cm². There is currently no common standard for radiation dosage in both domestic and international studies. However, investigations have indicated that when the survival rate of HaCaT cells is about 50%, the levels of intracellular ROS and free radicals rise, causing damage to the antioxidant enzyme system [29]; the optimal radiation dose for establishing injured cell model was decided based on the median lethal radiation intensity. As a result, the irradiation energy of 30 mJ/cm² was used for the following tests in this study.

The survival rate of HaCaT cells injured by UVB at 30 mJ/cm² varied with concentration. The model group’s HaCaT cells survival rate was 47.33%, demonstrating that the UV damage model was successfully established. TFFC and TFFP might considerably increase cell viability at low concentrations (^#p < 0.05). At medium concentrations, TFFT, TFFC, and TFFP all demonstrated substantial protective effects on HaCaT cells. HaCaT cells viability rose to more than 80% at high concentrations of TFFC, suggesting that TFFC had a potent anti-UVB damaging impact at high concentration. The uneven rhomboid shape of normal HaCaT cells is seen in the image (Figure 3(d)). Following irradiation, the model group’s adhering living cells decreased in number, their volume shrank, and their form took on a rounder form. Following 50 μg/mL TFFT, TFFC, and TFFP treatment, the number of living HaCaT cells increased in comparison to the model group. Additionally, the morphology and size of the cells did not significantly differ from those of the control group, suggesting that all of the flavonoids found in TF protected HaCaT cells from UVB irradiation.

3.4. Determination of the Antioxidant Activity of TFFs in HaCaT Cells

Long-term UV radiation exposure to the skin can cause the body’s antioxidant system to be destroyed by the fast buildup of intracellular ROS, which can hinder normal cell metabolism and cause a cascade of oxidative stress events [30]. After UV irradiation, the ROS level of HaCaT cells treated with 50 μg/mL TFFs was measured using flow cytometry in conjunction with the DCFH-DA fluorescent probe method to assess the protective impact on UV-damaged HaCaT cells. The mean fluorescence intensity of the cells in the model group was considerably higher (^∗p < 0.05) than that of the control group (Figures 3(e) and 3(f)), suggesting that UV irradiation may raise the ROS levels in HaCaT cells. The amount of intracellular ROS was reduced by TFFs to varied degrees when compared to the model group. TFFC exhibited the highest level of inhibition, with ROS levels just 40.8% of the model control group, whilst TFFT and TFFP groups had levels 65.33% and 48.57% of the model group, respectively. As a result, HaCaT cells′ capacity to reduce intracellular ROS levels was graded as follows: TFFC > TFFP > TFFT.

The antioxidant system is responsible for maintaining the homeostatic balance of intracellular redox levels. To further investigate the protective effects of TFFs on UVB-induced oxidative stress in HaCaT cells, the activities of CAT, SOD, GSH-Px, and MDA were analyzed. The CAT concentration of HaCaT cells in the model group was 0.18 U/mL, which was significantly lower than that of the control group (4.24 U/mL) (Figure 3(g)). The TFFs in each sample increase the level of CAT enzyme in HaCaT cells, including TFFP with the strongest protection and TFFT playing the least role. Figure 3(h) illustrates that TFFP had the strongest ability to improve the GSH-Px level in HaCaT cells, whereas TFFC and TFFT could both considerably increase the GSH-Px level in HaCaT cells at concentrations of 25 μg/mL and 50 μg/mL, respectively. SOD activity was most strongly influenced by TFFC, although SOD levels were also markedly raised by TFFT and TFFP (Figure 3(i)). In UVB-damaged HaCaT cells, TFFT was unable to suppress MDA formation, while TFFC had a more potent inhibitory effect on MDA than TFFP (Figure 3(j)).

3.5. Effect of TFFs on TNF-α and MMP-1 Level in UV-Damaged HaCaT Cells

When UV irradiation of HaCaT cells occurred, the creation of ROS was tightly correlated with the activation of TNF-α and MMP-1 expression [23]. Based on the previous experimental findings, TFFs may have an anti-UVB damage impact by inhibiting the generation of ROS in UV-damaged HaCaT cells, boosting the activity of antioxidant enzymes like SOD, and lowering MDA peroxide formation.

We explored the effect of 50 μg/mL TFFT, TFFC, and TFFP on TNF-α and MMP-1 levels in UV-damaged HaCaT cells. Figure 3(k) demonstrates that TFFC most strongly suppressed TNF-α production, with its release being only 7.74 U/mL greater than that of the control group. Figure 3(l) demonstrates that TFFC had the most pronounced downregulation effect on MMP-1. After being treated with TFFC, the MMP-1 content in HaCaT cells dropped to 231.4 U/mL, and just 30.57% of the content in the model group. As a result, HaCaT cells′ capacity to suppress TNF-α and MMP-1 production was graded as follows: TFFC > TFFP > TFFT.

3.6. Effects of TFFs on the Antistress Ability of C. elegans

The survival rates of C. elegans treated with different concentrations of TFFs for 48 h were almost unaffected (Figure 4(a)), and the survival rates were all above 90%, indicating that TFFs had no toxic effect on C. elegans in the concentration range of 50–200 μg/mL, with 200 μg/mL being the highest experimental concentration of TFFT, TFFC, and TFFP.

Figure 4(b) demonstrates the findings of the influence of TFFs on the capacity of C. elegans to tolerate heat stress after 5 h at 35°C. When compared to the control group, the model group’s C. elegans survival rate was 48.4%, which was considerably lower than the control group (p < 0.05), demonstrating that the model was established successfully. The findings revealed that, as compared to the model group, each experimental group improved C. elegans survival rates. The higher the concentration of flavonoids, the greater the C. elegans survival rate. In the paraquat-induced C. elegans oxidative damage model, all concentrations of TFFs significantly enhanced C. elegans survival (p < 0.05), and the survival rate of worms rose with concentration (Figure 4(c)). At a concentration of 200 μg/mL, the TFFs significantly increased C. elegans resistance to paraquat, increasing C. elegans survival rate by 48.31% (TFFT), 49.86% (TFFC), and 46.49% (TFFP), respectively, compared to the model group, but the protective effects of the three TFFs on oxidative stress C. elegans differed little.

3.7. Effects of TFFs on the Antioxidant System of C. elegans

In Figure 4(d), the ROS fluorescence intensity of the control group was significantly higher than that of the TFFs treatment groups, indicating that TFFs could effectively alleviate the accumulation of ROS in the body of C. elegans. Figures 4(e), 4(f), 4(g) depict the effects of TFFs on SOD, CAT, and GSH-Px in C. elegans. TFFs-treated groups considerably increased the vigor of SOD, CAT, and GSH-Px enzymes as compared to the control group. Among them, TFFC had the greatest effect on enhancing SOD and CAT enzyme activity, whereas TFFP had the greatest potential to enhance GSH-Px levels.

3.8. Effects of TFFs on the Antioxidant Genes of C. elegans

qRT-PCR was used to determine the effects of TFFs on the mRNA expression of gcs-1, gst-4, gst-10, sod-3, and ctl-1 (Figure 5). TFFs significantly raised the transcript levels of gst-10, sod-3, and ctl-1 in C. elegans. Only TFFP raised the expression of gst-10 substantially. TFFT and TFFC also had a tendency to influence the expression of gst-10; however, this was not statistically significant. When compared to the control group, all three flavonoid samples enhanced the expression of sod-3 and ctl-1 to varying degrees, but had no effect on the expression of gcs-1 and gst-4 genes.