FLS（重庆汇达柠檬科技集团有限公司，重庆，中国）的萃取液，干燥，保持柠檬种子恒重，在研钵中研磨，过筛获得柠檬籽粉。The powder was degreased with n-hexane for 90 min (degreasing temperature, 50°C; material liquid ratio, 1 : 30; and power, 200 W). After filtration and drying, flavonoids were extracted from 8 g of degreased lemon seed powder with 300 mL of 95% ethanol for 60 min (temperature, 50°C; power, 200 W). Finally, the sample solution was obtained by filtration and the dry FLS were evaporated using a rotary evaporator (Great Wall R-1050, Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd., Zhengzhou, Henan, China).
２.２.细胞培养
胡man embryonic kidney (HEK) 293T cells (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China) were resuscitated from liquid nitrogen and seeded in Dulbecco’s Modified Eagle’s Medium (DMEM) (high sugar, containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody solution) (Solarbio Life Sciences, Beijing, China). The medium was changed two or three times a week in a saturated humid environment at 37°C and 5% CO2．当细胞融合达到90%时，用胰蛋白酶(0.25%)(Solarbio Life Sciences)消化传代。所有的实验都使用对数期的细胞。
２.３.FLS对hek293t细胞的毒性研究
HEK 293T细胞悬浮液（
1
×
10
4细胞/mL)接种到96孔细胞培养板(
60
μ
l
细胞
+
One hundred.
μ
l
文化
媒介)， 37℃培养24 h至贴壁，20
μ不同浓度的FLS溶液(正常组，50
μ克/毫升，100
μ克/毫升和150
μg/mL)。
２.４.MTT法检测细胞存活率
采用MTT法测定细胞存活率:HEK 293T细胞先在细胞壁上培养24 h, 20
μ升FLS溶液中，用不同浓度（正常组，50
μ克/毫升，100
μ克/毫升和150
μg/mL);20
μ加入L的MTT (5 mg/mL) (Solarbio Life Sciences)，混合培养4 h。丢弃上培养液，加入150
μlof dimethyl sulfoxide (DMSO) (Solarbio Life Sciences), shaking for 30 min in dark period and at 37°C. Optical density (OD) was measured at 490 nm. The experiment was performed in triplicate.
细胞
生存
速度
％
＝
基于“增大化现实”技术
/
作为
×
One hundred.
％，其中Ar为FLS治疗组的OD, As为正常组的OD (Evolution™350,Thermo Fisher Scientific, New York, USA)。
2．5．细胞增殖的观察
HEK 293T细胞悬浮液（
1
×
10
4细胞/mL)接种到96孔细胞培养板(
60
μ
l
细胞
+
One hundred.
μ
l
文化
媒介), cultured at 37°C for 24 h, adhered to the wall, and cultured with 20
μ的H L2O2(0.3 mmol/L)
μ升用不同浓度的溶液FLS（正常组，40
μ克/毫升，100
μ克/毫升和160
μg/mL) was added to the culture for 24 h, and the cell survival rate was measured using the MTT method (Figure
1(a))。
HEK 293T细胞悬浮液（
1
×
10
4细胞/mL)接种到96孔细胞培养板(
60
μ
l
细胞
+
One hundred.
μ
l
文化
媒介), cultured at 37°C for 24 h, adhered to the wall, and cultured with 20
μ的H L2O2(0.3 mmol/L)处理4 h，制备氧化损伤模型。丢弃上介质后，20
μ升用不同浓度的溶液FLS（正常组，40
μ克/毫升，100
μ克/毫升和160
μg/mL) was added to the culture for 24 h, and the cell survival rate was measured using the MTT method (Figure
1(b))。
2.6。细胞凋亡的观察流式细胞仪
根据细胞增殖实验对HEK 293T细胞进行处理，收集并形成单细胞悬液。固定和染色后，用流式细胞仪检测细胞凋亡(Accuri C6, BD Biosciences, San Jose, CA, USA)。
HEK 293T细胞处于对数生长期，用0.25％胰蛋白酶消化，在六孔细胞培养板接种（
1
×
10
5细胞/mL)， 2 mL DMEM在37°C和5% CO的饱和潮湿环境中培养2保温24 h，贴壁，加200
μ的H L2O2（0.3 mmol/L), mixed well, and cultured for 4 h to prepare the oxidative damage model. FLS aqueous extract (200
μL)不同浓度(0
μ50 g / mL,
μ克/毫升，100
μ克/毫升和150
μg/mL)，加入HEK 293T细胞氧化损伤模型每孔，并加入0.1 mol/L的PBS溶液维持平衡。细胞在37°C和5% CO条件下进一步培养224 h。将经FLS处理的HEK 293T细胞从旧培养基中取出，用预冷的PBS洗涤，用200 mol / l与细胞壁分离
μL，取1.5 mL离心管，离心弃上清。再次用预冷PBS洗涤细胞，4000r /min离心15 min，弃上清液，加入800
μL生理盐水，均质。按照试剂盒说明书测定匀浆中MDA、SOD、GSH、GSH- px、CAT的含量(南京建城生物工程研究所，中国南京)。
2.8。单独分析
按照本节细胞培养方法培养hek293t细胞
2.2;the HEK 293T cells in the logarithmic growth stage were digested by 2.5 g/L trypsin to form a single cell suspension. The HEK 293T cells were inoculated into 24-well plates, with 50 cells per well. Each group of cells was inoculated with six wells, five groups in total (normal, control, FLSL, FSLM, and FLSH groups). HEK 293T cells in the normal group were cultured for 1 week without any other treatment. HEK 293T cells in the control group were cultured using 900
μ大号DMEM培养基和100
μL培养液含H2O2(0.3更易/ L);FLSL组、FSLM组、FLSH组采用800
μL DMEM培养基，100
μL培养液含H2O2（0.3 mmol/L), and 100
μ含有FLS L培养介质溶液（40
μg / mL, 80
μ克/毫升和160
μg/mL)。丢弃培养基，用PBS洗涤培养皿孔，用1 mL纯甲醇固定15 min，使用Giemsa染色液(Solarbio Life Sciences)染色。将培养板置于低倍数显微镜下(IX73, Olympus, Tokyo, Japan)，计数50个以上细胞的克隆数，按以下公式计算克隆形成率:
克隆
形成
速度
％
＝
克隆
数量
/
接种
数量
×
One hundred.
％［
13］．
2.9。定量聚合酶链反应(qPCR)
将HEK 293T细胞培养到六孔板(
1
×
10
5细胞/mL)，用不同浓度的FLS处理。用TRIzol Reagent (Thermo Fisher Scientific)提取HEK 293T细胞总RNA。我们添加了1
μL Oligo (dT) 18引物(500 ng)和1.0
μL的总RNA（1.0
μg）至10.0
μL非核酸酶水在65°C梯度PCR仪上加热5分钟。然后，混合物含有4.0
μL的5x反应缓冲液，1.0
μRiboLock RNase Inhibitor (20 U)， 2.0
μL的10毫米dNTP混合，和1.0
μlof RevertAid Reverse Transcriptase (200 U/
μl) (Thermo Fisher Scientific) was added to the total RNA system, which was transcribed into cDNA at 42°C for 60 min and 70°C for 5 min. Amplification conditions were as follows: denaturation at 95°C for 3 min, annealing at 60°C for 30 s, extension at 95°C for 1 min, and a total of 40 cycles. mRNA expressions of SOD, CAT, GSH, and GSH-Px were detected using qPCR (Table
1)．每个基因的cDNA平行扩增3次，得到Ct均值。以管家基因GAPDH为内参，按
2
−
∆
∆
Ct(StepOnePlus, Thermo Fisher Scientific) [
14］．
如图所示
6，FLS含有六种化合物，包括没食子儿茶素，咖啡酸，表儿茶素，牡荆素，槲皮素，橙皮苷和。Their peak retention time on the liquid chromatogram was 5.664 min, 9.715 min, 10.611 min, 20.442 min, 25.017 min, and 31.010 min, respectively. The contents of the six components in the FLS were 18.304 mg/g, 37.294 mg/g, 25.080 mg/g, 108.539 mg/g, 334.585 mg/g, and 145.983 mg/g, respectively.