摘要
为了研究陈化对于广陈皮抗氧化活性的影响,以新鲜干燥果皮、贮藏1 a和10 a广陈皮(Pericarpium Citri Reticulatae ‘Chachi’,PCR-C)为研究对象,提取纯化得到广陈皮黄酮类化合物提取物(PCR-CF),以叔丁基过氧化氢(t-BHP)诱导HepG2 细胞作为氧化应激损伤模型,评价陈皮的抗氧化活性,同时利用qRT-PCR和Western blot技术进一步探究其抗氧化活性作用分子机制。结果显示,广陈皮陈化过程中,橙皮苷含量呈现波动,但总体上是下降趋势;而多甲氧基黄酮及其总含量呈增加趋势,其中PCR-C10F中含量最高,PMFs总含量达到(98.66±0.56) mg/g。而且,不同贮藏年份PCR-CF可明显提高SOD、GSH水平和降低MDA含量,且不同年份间差异显著,PCR-C10F抗氧化活性最强,SOD、GSH、MDA水平分别达到(139.38±17.38) U/mg和(117.81±3.22) μmol/g、(0.39±0.03) nmol/mg。相关性分析表明,川陈皮素的积累是PCR-C10F维持氧化还原平衡的主要活性成分。此外,PCR-C10F显著上调Nrf2 mRNA以及其在胞核蛋白中的表达;从而激活抗氧化酶的mRNA和蛋白表达水平,HO-1的mRNA表达水平和蛋白表达水平显著降低。研究表明,陈皮在陈化过程中,多甲氧基黄酮含量呈现增加趋势,抗氧化活性不断增强,PCR-C10F能够通过调控Nrf2-ARE抗氧化信号通路起到氧化应激抵御保护作用。
关键词
广陈皮(Pericarpium Citri Reticulatae ‘Chachi’,PCR-C)来源于茶枝柑(Citri reticulata ‘Chachi’)的干燥成熟果皮,主要产于广东省江门市新会
氧化应激与人类多种慢性疾病的发生和发展密切相
本研究以新鲜干燥果皮以及贮藏1、10 a的广陈皮为研究对象,分析广陈皮在陈化过程中黄酮类化合物的含量变化;以t-BHP诱导氧化应激损伤的人肝癌细胞HepG2细胞为模型,探究PCR-CF的抗氧化活性作用,并通过qRT-PCR和Western blot技术研究PCR-C10F的抗氧化作用分子机制,旨在探究广陈皮在陈化过程中黄酮类化合物含量变化与抗氧化活性的相关性,为陈皮的品质控制及合理使用提供理论参考。
人肝癌细胞(human hepatoma cell,HepG2 cell)购买于中科院上海细胞库干细胞库。新鲜果皮、贮藏1 a和10 a的PCR-C药材,购自广东省江门市新会区双水镇柑泽园,样品经湖北中医药大学药学院鉴定。MEM培养基购于美国Gibco公司;胎牛血清购于南乌拉圭Quacell公司;色谱级的乙腈购于美国Fisher公司;色谱级的甲酸购于天津市科密欧化学试剂有限公司;BCA法蛋白测定试剂盒(BCA02)购于北京鼎国昌盛科技有限公司;GSH(A006-2-1)、MDA(A003-4-1)、T-SOD(A001-1-2)试剂盒购于南京建成生物科技有限公司;RNApure 超纯总RNA快速提取试剂盒购于北京艾德莱生物科技有限公司;荧光定量 PCR 所用引物合成,购于北京擎科生物科技公司;TaKaRa SYBR Premix Ex TaqTM Ⅱ 荧光定量试剂盒和TaKaRa Prime Script RT reagent Kit with gDNA Eraser反转录试剂盒(RR047A),均购于宝生物工程(大连)有限公司;细胞核蛋白与细胞浆蛋白抽提试剂盒(P0028);小鼠单抗β-actin(BM0627)、HRP标记羊抗小鼠二抗(BA1051)、HRP标记羊抗兔二抗(BA1054)均购于武汉博士德生物工程有限公司;兔多抗Nrf2(A0674)购于武汉爱博泰克生物科技有限公司;兔多抗HO-1(10701-1-AP)购于武汉三鹰生物技术有限公司;兔多抗NQO1(DF6437)购于美国Affinity Biosciences公司;兔多抗SOD1(PA5-27240)购于美国Invitrogen公司;兔单抗GSTO1(ab138491)和兔多抗GSH-Px(ab22604)购于Abcam公司;小鼠单抗Histone H3(bsm-33042M)购于北京博奥森生物技术有限公司。
超声破碎仪,VCX750,美国索尼克斯;全波长酶标仪,MULTISKAN GO,美国Thermo公司;高效液相色谱仪,e2695,美国Waters公司;微量核酸测定仪,NanoDrop2000,美国 NanoDrop Technologies 公司;电泳仪,DYY-8C,北京六一仪器厂;凝胶成像系统,Gel Doc XR+,美国LI-COR公司;荧光定量PCR仪,Qtower2.2,德国 Analytik JenaAG公司;垂直电泳槽,DYCZ-24DN,北京六一仪器厂;电转仪,DYCZ-40,北京六一仪器厂。
1)HepG2细胞的培养。细胞培养于含10%胎牛血清、1%双抗及 MEM 基础培养基中,置于37 ℃,5% CO2细胞培养箱中,待其长至瓶壁的80%左右,用胰蛋白酶消化传代,每隔3~4 d传代1次。
2)广陈皮中黄酮类化合物的提取纯化。参照笔者所在实验室前期研究方
3)HPLC分析。参照笔者所在实验室前期研究方
4)SOD、GSH、MDA水平的测定。参照笔者所在实验室前期研究方
5)实时荧光定量PCR(qRT-PCR)检测基因表达水平。将生长状态良好、处于对数生长期的HepG2细胞按照6×1
6)蛋白免疫印迹(Western blot)检测蛋白表达。细胞的处理与分组与RNA提取的细胞处理方式一致。用加入了蛋白酶抑制剂(PMSF)的RIPA细胞裂解液进行细胞总蛋白的提取。按照细胞核蛋白与细胞浆蛋白抽提试剂盒的方法得到细胞核蛋白。4 ℃、10 000 r/min,离心5 min,取上清并用BCA试剂盒测定蛋白浓度。根据目标蛋白分子质量大小选择分离胶的浓度,先恒压80 V电泳至溴酚蓝指示剂在浓缩胶与分离胶交界处成线状,再改为恒压120 V至溴酚蓝到凝胶底部,约需90 min。电泳结束后进行转膜,5% 脱脂奶粉的TBST中封闭2 h,一抗4 ℃孵育过夜,二抗室温孵育2 h,ECL显影并进行拍照。
7)数据统计与分析。试验至少有3个独立的平行试验,数据结果以平均值±标准偏差(Mean ± SD)表示。采用IBM SPSS Statistic Version 25.0统计软件分析数据,多组间比较采用单因素ANOVA方差分析,P<0.05时认为有显著性差异。
PCR-CF中主要黄酮类化合物含量如

图1 PCR-CF中主要黄酮类化合物的含量
Fig.1 The contents of main flavonoids in PCR-CF
1.橙皮苷Hesperidin;2.异橙黄酮Isosinensetin;3.甜橙黄酮Sinensetin;4.5,7,8,4'-四甲氧基黄酮5,7,8,4'-Tetramethoxyflavone;5.川陈皮素Nobiletin;6.3,5,6,7,8,3',4'-七甲氧基黄酮3,5,6,7,8,3',4'-Heptamethoxyflavone;7.橘皮素Tangeretin;8.PMFs.
细胞内SOD、GSH和MDA水平可以反映抗氧化活性水平。如
注 Note:同列字母不同代表差异显著(P<0.05)。Different letters in the same column represent significant differences (P<0.05).
PCR-CF中所含的黄酮类化合物可能是其发挥抗氧化活性的关键成分,为了进一步探究黄酮类化合物与抗氧化活性之间是否具有相关性,对PCR-CF中黄酮类化合物的含量与SOD、GSH和MDA水平进行Pearson相关性分析(
注 Note:*相关性在0.05水平上显著(双尾)。**相关性在0.01水平上显著(双尾)。*Correlation is significant at the 0.05 level (two-tailed). ** Correlation is significant at the 0.01 level (two-tailed).
PCR-C10F抗氧化活性较强,为了进一步探究PCR-C10F对t-BHP诱导的HepG2细胞氧化损伤的保护作用机制,使用qRT-PCR考察了PCR-C10F对氧化应激状态下Nrf2基因水平的影响。如

图2 PCR-C10F对t-BHP诱导HepG2细胞Nrf2 mRNA表达的影响
Fig.2 Effect of PCR-C10F on mRNA expression of Nrf2 in HepG2 cells induced by t-BHP
GADPH作为内参基因。结果以平均值±标准差表示,n=3。*,P<0.05,**,P<0.01,***,P<0.001,下同。GADPH was used as the reference gene. Results were presented as mean ± SD,n=3. *,P< 0.05,** ,P< 0.01,*** ,P< 0.001. The same as below.
为了进一步确定PCR-C10F能否促进细胞内Nrf2转移入核,测定Nrf2 蛋白的表达情况。如

图3 PCR-C10F对t-BHP诱导HepG2细胞Nrf2 蛋白表达的影响
Fig.3 Effect of PCR-C10F on protein levels of Nrf2 in HepG2 cells induced by t-BHP
A:分别提取总蛋白和胞核蛋白,通过Western blot 技术测定HepG2 细胞中 Nrf2 蛋白表达情况,Histone H3作为胞核蛋白内参,β-actin作为总蛋白内参The total protein and nuclear protein were extracted to measure the expression level of Nrf2 protein in HepG2 cells via Western blot. Histone H3 was used as the reference protein for nuclear protein and β-actin was used as the reference protein for total protein. B~D分别为Nrf2蛋白在胞核蛋白、总蛋白以及两者之比中表达水平的统计分析结果。结果以平均值±标准差表示,n=3。The statistical analysis results of the expression levels of Nrf2 protein in nucleus protein,total protein and the ratio of nucleus Nrf2 to total Nrf2. Results were presented as mean ± SD,n=3.
测定细胞内NQO1、 GSTO1、SOD1、GSH-Px、HO-1基因的mRNA表达水平,探究PCR-C10F能否通过促进Nrf2入核上调细胞内抗氧化相关基因的表达。如

图4 PCR-C10F对t-BHP诱导HepG2细胞中NQO1、GSTO1、SOD1、GSH-Px和HO-1 mRNA表达水平的影响
Fig.4 Effect of PCR-C10F on mRNA expression of NQO1,GSTO1,SOD1,GSH-Px and HO-1 in HepG2 cells induced by t-BHP
提取分离细胞总RNA后,通过qRT-PCR技术测定HepG2 细胞中NQO1(A)、SOD1(B)、GSTO1(C)、GSH-Px(D)、HO-1(E) 基因mRNA表达水平。GADPH作为内参基因,结果以平均值±标准差表示,n=3。The total RNA were extracted to measure mRNA expression of NQO1 (A),SOD1 (B),GSTO1 (C),GSH-Px (D) and HO-1 (E) in HepG2 cells via qRT-PCR. GADPH was used as the reference gene.Results were presented as mean ± SD,n=3.
进一步分析t-BHP刺激诱导下HepG2细胞中NQO1、GSTO1、SOD1、GSH-Px抗氧化酶蛋白和HO-1 应激反应蛋白的表达情况,结果如

图5 PCR-C10F对t-BHP诱导HepG2细胞中NQO1、GSTO1、SOD1、GSH-Px和HO-1 蛋白表达水平的影响
Fig.5 Effect of PCR-C10F on protein levels of NQO1,GSTO1,SOD1,GSH-Px and HO-1 in HepG2 cells induced by t-BHP
A:提取细胞中总蛋白,通过Western blot技术测定HepG2 细胞中NQO1、GSTO1、SOD1、GSH-Px、HO-1蛋白的表达水平,β-actin作为内参The total protein were extracted to measure the expression levels of NQO1,GSTO1,SOD1,GSH-Px and HO-1 protein in HepG2 cells via western blot. β-actin was used as the reference protein for total protein; B~F分别为NQO1、GSTO1、SOD1、GSH-Px、HO-1 蛋白表达水平的统计分析结果,结果以平均值±标准差表示,n=3。The statistical analysis results of the expression levels of NQO1,GSTO1,SOD1,GSH-Px and HO-1 protein. Results were presented as mean ± SD,n=3.
黄酮类化合物是陈皮发挥生物活性的重要物质基础,中国药典以橙皮苷含量作为陈皮质量评价指标,“陈久者良”在陈皮使用和市场定价中被广泛认同,然而橙皮苷含量变化与陈皮的陈化年限并不一
t-BHP是一种氧化损伤诱导剂,可以与细胞膜上的脂质、蛋白质等生物大分子反应引起氧化损
在正常生理状态下,Nrf2与Keap1以二聚体的形式定位于细胞质中;而一旦受到外界刺激,Nrf2就会与Keap1发生解离,从细胞质转移进至细胞核中,并与ARE作用,激活下游一系列抗氧化酶的表
综上所述,陈皮在陈化过程中多甲氧基黄酮类化合物含量呈现增加的趋势,而且其抗氧化活性也随之增强,而且川陈皮素的积累可能是主要的因素。PCR-C10F表现出较强的抗氧化活性,能够通过调控Nrf2-ARE抗氧化通路下游抗氧化酶mRNA和蛋白的表达减轻t-BHP对HepG2细胞的氧化应激损伤,从而发挥抗氧化活性,为陈皮发挥间接抗氧化活性作用机制的研究提供了理论基础,进而为陈皮的合理贮藏及使用提供一定的理论依据。本研究由于只选取了同一产地同一果园的样本,而且不同年份的样本量有限,还需进一步扩充样品的品种来源及年份深入开展研究。
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