王蓓^1,2, 常化松², 苏松坤¹, 孙丽萍², 王凯^,21 福建农林大学蜂学学院,福州 350002
2 中国农业科学院蜜蜂研究所,北京 100093

Antioxidative and Anti-inflammatory Activities of Ethanol Extract of Geopropolis from Stingless Bees

WANG Bei^1,2, CHANG HuaSong², SU SongKun¹, SUN LiPing², WANG Kai^,2 1 College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou 350002
2 Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093

通讯作者: 王凯,E-mail： kaiwang628@gmail.com

收稿日期:2018-09-28接受日期:2018-11-30网络出版日期:2019-03-01

基金资助:

国家自然科学基金青年科学基金.（31702287） 国家蜂产业技术体系专项.（CARS-44） 国际合作项目“常青蜂场蜂胶有效成分鉴定”.（CARS-44）

Received:2018-09-28Accepted:2018-11-30Online:2019-03-01
作者简介 About authors
王蓓,E-mail： m13121180309@163.com。







摘要
【目的】 无刺蜂(stingless bees)是热带和亚热带地区重要的授粉昆虫之一,以尾部无蛰针为典型特征,其蜂胶采集量较意大利蜜蜂（ Apis mellifera ligustica ）更多,然而其蜂胶活性研究却相对匮乏。本文以来源于马来西亚无刺蜂 ( Heterotrigona itama )采集蜂胶的乙醇提取物(ethanol extract of H. itama propolis, EEHI)为研究对象,旨在探究其体外抗氧化和抗炎活性。【方法】 采用福林酚法和硝酸铝法测定EEHI中总酚酸和总黄酮含量,并采用DPPH和ABTS ^+·自由基清除能力评价EEHI的体外抗氧化能力;在此基础上,采用细菌脂多糖(LPS)诱导小鼠巨噬细胞RAW 264.7炎症模型,通过CCK-8法检测EEHI对细胞相对存活率的影响,在保证EEHI对细胞无细胞毒性作用基础上,分别采用Griess法和实时荧光定量PCR（RT-qPCR）技术评估EEHI对LPS诱导的RAW 264.7细胞中炎症介质一氧化氮（NO）释放量的影响以及其对炎症因子（ IL-1β 、 IL-10 和 INOS ）和抗氧化基因（ HO-1 ）信使RNA表达的影响;进一步利用免疫印迹和免疫荧光方法,以NF-κB炎症信号通路为切入点,研究EEHI对LPS诱导巨噬细胞中p-IκΒ α 和IκΒ α 蛋白表达以及NF-κB-p65蛋白移位的影响,从而探究EEHI潜在的抗炎机理。 【结果】 EEHI中总酚酸和总黄酮含量分别为54.70 mg GAE·g^-1和116.20 mg QE·g^-1;DPPH和ABTS^+·自由基清除能力IC₅₀值分别为275.60和 284.00 μg·mL^-1。EEHI对RAW 264.7细胞的安全浓度为0—40 μg·mL^-1。在LPS诱导的Raw 264.7细胞炎症模型中,相对于LPS刺激组,0—40 μg·mL^-1的EEHI以浓度依赖方式显著地抑制了LPS诱导的RAW 264.7细胞中NO的释放量,且降低了细胞中炎症因子 IL-1β 、 IL-10 和 INOS 的基因表达量,并增强了抗氧化基因 HO-1 的表达。进一步研究发现,0—40 μg·mL^-1的EEHI以浓度依赖方式显著抑制了LPS诱导的RAW 264.7细胞IκΒ α 蛋白的磷酸化,且40 μg·mL^-1的EEHI显著降低了NF-κB-p65蛋白的核移位现象,因此推测EEHI可能是通过抑制LPS诱导的NF-κB信号通路的激活进而发挥了体外抗炎活性。 【结论】 无刺蜂蜂胶乙醇提取物中含有大量多酚类化合物,具有较好的抗炎和抗氧化效果,极具开发利用价值。
关键词： 无刺蜂;无刺蜂蜂胶;抗氧化;抗炎;NF-κB;

Abstract
【Objective】Stingless bees are one of the important pollinators in tropic and subtropical area, differ from Apis mellifera ligustica with typical stingless characteristic. Stingless bee collected more propolis than that of A. m. ligustica . Nevertheless, the study on the geopropolis activity was relatively scarce. The objective of this study is to evaluate the in vitro antioxidant and anti-inflammatory activities of the ethanol extract of geopropolis collected from stingless bee, Heterotrigona itama , which is an indigenous stingless bee species in Malaysia. 【Method】The content of total phenolic acid and total flavonoids in the EEHI (ethanol extract of H. itama propolis) was determined by Folin-phenol method and AlNO₃ colorimetry, respectively. The antioxidant activity was investigated using DPPH and ABTS ^+·free radical scavenging assays. Moreover, the inflammatory model of murine macrophage RAW 264.7 was induced by bacterial endotoxin lipopolysaccharide (LPS) and the effect of EEHI on the relative cell viability was detected via CCK-8 method. On the basis of ensuring that EEHI has no cytotoxic effect on cells, Griess method and RT-qPCR technique were applied to evaluate the effect of EEHI on the release of inflammatory mediator NO, and on the expression of inflammatory factors ( IL-1β , IL-10 and INOS ) as well as antioxidant gene ( HO-1 ) in LPS-activated macrophages, respectively. In order to explore the potential anti-inflammatory mechanisms of EEHI, the effects of EEHI on the expression of p-IκB α and IκΒ α in macrophages induced by LPS and the translocation of NF-κB-p65 protein were further studied by Western blot and immunofluorescence methods. 【Result】The content of total phenolic acid and total flavonoids in the EEHI was 54.70 mg GAE·g ^-1 and 116.20 mg QE·g ^-1, and the IC₅₀ value of the EEHI for scavenging of DPPH and ABTS ^+· free radicals was 275.60 and 284.00 μg·mL ^-1, respectively. The safe concentration of EEHI to RAW 264.7 cells was 0-40 μg·mL ^-1. In LPS-challenged macrophages, EEHI at 0-40 μg·mL ^-1 significantly inhibited the release of NO as well as the expression of pro-inflammatory cytokine genes ( IL-1β , IL-10 and INOS ), and enhanced the expression of antioxidant gene HO-1 in a dose-dependent manner, compared with the LPS-treated control. Furthermore, it was noticed that EEHI at 0-40 μg·mL ^-1 significantly inhibited the LPS-stimulated phosphorylation of IκΒ α protein in a dose-dependent manner, and EEHI at 40 μg·mL ^-1 significantly reduced the nuclear migration of NF-κB-p65 protein. It was suggested that EEHI had potential anti-inflammatory effects by inhibiting the activation of NF-κB inflammatory signaling pathway induced by LPS.【Conclusion】The ethanol extract of geopropolis collected from H. itama contains a large number of polyphenols, which has good antioxidant and anti-inflammatory effects, and is of great value for exploitation and utilization in the future.
Keywords：stingless bee;geopropolis;antioxidant;anti-inflammatory;NF-κB;


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本文引用格式
王蓓, 常化松, 苏松坤, 孙丽萍, 王凯. 无刺蜂蜂胶乙醇提取物的体外抗氧化及抗炎活性[J]. 中国农业科学, 2019, 52(5): 939-948 doi:10.3864/j.issn.0578-1752.2019.05.015
WANG Bei, CHANG HuaSong, SU SongKun, SUN LiPing, WANG Kai. Antioxidative and Anti-inflammatory Activities of Ethanol Extract of Geopropolis from Stingless Bees[J]. Scientia Acricultura Sinica, 2019, 52(5): 939-948 doi:10.3864/j.issn.0578-1752.2019.05.015

0 引言

【研究意义】无刺蜂（stingless bees）是热带和亚热带地区重要的授粉昆虫之一,其区别于意大利蜜蜂（ Apis mellifera ligustica ）的主要特征是尾部无蛰针^[1]。无刺蜂蜂种繁多, Heterotrigona itama 是最具有代表性的一种,该种无刺蜂主要分布于热带和亚热带雨林气候地区,其蜂胶年产量远大于意大利蜜蜂,且胶源植物来源广泛,生物学活性多样,具有很高的生产价值和发展空间^[2,3]。明确无刺蜂蜂胶（geopropolis）的体外抗氧化和抗炎效果,对进一步研究其生物学活性及特色蜂产品开发具有重要意义。【前人研究进展】无刺蜂蜂胶是由无刺蜂采集植物树脂并混合蜂蜡以及泥土等制成的具有不同颜色的固体胶状物,其在蜂箱中主要用来筑巢、填补蜂箱缝隙以及防御病虫害入侵^[1]。无刺蜂蜂胶作为一种民间药物,在热带地区一直用于修复伤口,治疗消化、呼吸、皮肤疾病,并用作抗菌剂和防腐剂等^[4,5],具有丰富的药理学活性,如抗炎^[6]、抗氧化^[4]、抑菌^[7]、抗癌^[8]、抗病毒^[9]、镇痛^[1]等。无刺蜂蜂胶主要含有苯丙酯类、黄酮类、酚酸类、可水解鞣质、三萜类、皂苷类以及生物碱类化合物,其中酚酸类、黄酮类和萜烯类化合物是其主要成分^[10]。通过薄层色谱分析发现, H. itama 蜂胶甲醇提取物中含有萜烯类、黄酮类、酚酸类、类固醇、皂角苷和香豆素类化合物^[8,11]。笔者课题组前期通过UHPLC-Q-TOF/MS联用技术分析 H. itama 蜂胶乙醇提取物,发现含有没食子酸、咖啡酸、香草酸、苯甲酸、短叶松素、山奈酚、倒捻子素等多种酚酸、黄酮类和丰富的萜烯类化合物^[12]。近年来,大量研究证明意大利蜜蜂蜂胶（主要是中国杨树型蜂胶、巴西酒神菊属型蜂胶）的抗氧化、抗炎活性与蜂胶中的酚酸类和黄酮类化合物密切相关,如山奈酚、咖啡酸苯乙酯、槲皮素、阿替匹林-C等^[13]。炎症是机体在受到外界刺激下引起的一系列免疫应激反应,通过调节相关的炎症介质和细胞因子能够有效地缓解炎症反应,如 IL-1β 、 IL-6 、 TNF-α 等^[14]。进一步研究证明蜂胶中的活性物质主要通过调节花生四烯酸代谢、L-精氨酸、抑制NF-κB等炎症相关信号通路实现其抗炎活性,其中核转录因子NF-κB是巨噬细胞炎症过程主要的信号通路,NF-κB被激活会造成促炎因子高表达^[13,15]。笔者课题组前期研究发现富含多酚的中国蜂胶能够通过调节炎症相关因子（ IL-6 、 IL-10 、 IL-1β 等）的表达,抑制脂多糖（LPS）诱导的巨噬细胞IκΒα蛋白的磷酸化进而抑制NF-κB-p65入核,缓解脂多糖诱导的急性炎症反应^[15]。FRANCHIN等^[16]研究发现, Melipona scutellaris 蜂胶的正己烷和水溶性组分能够通过调节 IL-1β 和 TNF-α 炎症因子从而缓解机械性炎症过敏。此外,从 M. scutellaris 无刺蜂蜂胶中分离得到的香豆素类单体成分Cinnamoyloxy-mammeisin（CNM）能够降低肽聚糖诱导的巨噬细胞炎症反应中ERK-1/2、JNK、p38 MAPK和AP-1等蛋白的磷酸化,并且抑制NF-κB信号通路的激活从而减缓巨噬细胞的炎症反应^[17]。【本研究切入点】 H. itama 蜂胶具有抗氧化、NO清除能力、抗糖尿病和抑菌等生物学活性^[6,10],但与其抗炎和抗氧化相关的分子机制研究未见报道。【拟解决的关键问题】通过体外自由基清除能力评估 H. itama 蜂胶乙醇提取物（ethanol extract of H. itama propolis,EEHI）的抗氧化活性,分析EEHI对RAW 264.7小鼠巨噬细胞增殖活力及NO释放量的影响,并测定EEHI对脂多糖诱导的细胞炎症因子和抗氧化基因在mRNA转录水平表达的影响,通过进一步的免疫印迹和免疫荧光技术探究EEHI对NF-κB信号通路的影响。

1 材料与方法

试验于2017年11月至2018年6月份在中国农业科学院蜜蜂研究所蜂产品研究室完成。

1.1 供试材料、试剂与仪器

H. itama 蜂胶样品于2017年10月采集于马来西亚沙捞越州诗巫市常青蜂场,-20℃冰箱储存。

CCK-8试剂盒（Dojindo,日本）;LPS（脂多糖）（Sigma-Aldrich,美国）;DMEM高糖培养基,FBS（Gibco Laboratories,美国）;青链霉素混合液（100×）（索莱宝生物科技有限公司,中国）;1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基、2,2’-联氮-双-（3-乙基苯并噻唑啉-6-磺酸二铵盐）自由基（2,2’-azinobis-(3-ethylbenzthiazoline- 6-sulphonate) radical,ABTS^+·）（Sigma-Aldrich,美国）;PCR引物,Prime Script^TM RT Master Mix试剂盒,TB Green ?Premix Ex Taq^TM（生工生物（上海）股份有限公司）,异硫氰酸胍,BSA（Sigma-Aldrich,美国）;特异性抗体：IkappaB-alpha（Iκ-Β α ）（Cell Signaling Technology,美国）,phosphor-IkappaB-alpha（P-IκΒ α ）（Cell Signaling Technology,美国）, β -actin购自Abcam公司（Cambridge,美国）;NF-κB-p65（Cell Signaling Technology,美国）;BCIP/NBT碱性磷酸酶显色剂（碧云天,中国）;一抗稀释液、二抗稀释液（武汉赛博生物科技有限公司,中国）。

SpectraMax?i3酶标仪（美谷分子仪器有限公司,中国）;NanoDrop 2000超微量分光光度计（赛默飞世尔科技有限公司,美国）;PCR仪（东胜创新生物科技有限公司,中国）;荧光定量PCR仪（杭州博中科技有限公司,中国）;激光共聚焦扫描显微镜（Leica,德国）。

1.2 方法

1.2.1 样品前处理 称取25 g粉碎的蜂胶样品,按物料比1﹕15加入100%无水乙醇,40℃超声3 h,过夜静置后取上清,重复3次。混合3次上清浸提液减压旋蒸至恒重,-20℃储存。

1.2.2 总酚酸和总黄酮含量的测定 EEHI中总酚酸的含量检测采用福林酚法^[18]。取一定浓度的EEHI样品溶液150 μL于1.5 mL离心管中,同时加入等体积的福林酚试剂,振荡混匀后室温避光反应 5 min,然后加入450 μL 2%（W/V）的碳酸钠溶液,摇匀后室温避光反应2 h,取200 μL的反应试剂加入96孔板中,设置2个副孔,同时设置样品空白对照,于 λ =765 nm处测吸光值。以没食子酸当量（gallic acid equivalent,GAE）为标准,计算EEHI中总酚酸的含量。

EEHI中总黄酮的含量的测定采用硝酸铝法^[18]。取一定浓度的EEHI样品溶液300 μL于1.5 mL离心管中,同时加入硝酸铝（100 g·L^-1）溶液和醋酸钾（9.8 g·L^-1）溶液各20 μL,振荡混匀后加入660 μL的蒸馏水,摇匀后室温避光,静置反应1 h后,取200 μL加入96孔板中,设置2个副孔,同时设置样品空白对照,于 λ =415 nm 处测吸光值。以槲皮素（quercetin equivalent,QE）为标准,计算EEHI中总黄酮的含量。

1.2.3 自由基清除能力测定 EEHI的ABTS^+·自由基清除能力参考YANG等^[18]方法并适当调整。于1.5 mL离心管中加入250 μL ABTS^+·工作液和150 μL样品,振荡均匀后避光反应10 min。取150 μL反应液加入96孔板,于 λ =734 nm波长处测定吸光度,记为A1,同时做样品空白A2和试剂空白A0。每组样品同时设置3个平行值。以试样质量浓度和清除率计算线性回归方程,并计算其半抑制浓度。EEHI样品的ABTS^+·自由基清除能力用IC₅₀表示。计算公式：清除率（%）=[1-(A1-A0)/A2]×100。式中,A1为样品上清液吸光值,A0为试剂空白组吸光值,A2为样品空白组吸光值。

EEHI的DPPH自由基清除能力采用YANG等^[18]方法并适当调整。于1.5 mL离心管中加入125 μL DDPH工作液和125 μL样品,振荡均匀后避光反应30 min。取100 μL反应液加入96孔板,于 λ =517 nm波长处测定吸光度,记为A1,同时做样品空白A2和试剂空白A0。每组样品同时设置3个平行值。以试样质量浓度和清除率计算其线性回归方程,并计算其半抑制浓度。EEHI样品的DDPH自由基清除能力用IC₅₀表示,清除率计算公式同上。

1.2.4 抗炎活性测定 细胞培养及细胞活力测定：RAW 264.7小鼠巨噬细胞系由浙江大学胡福良教授惠赠。用含10%胎牛血清和1%青链霉素混合液（100×）的DMEM高糖培养基培养小鼠巨噬细胞RAW 264.7,37℃、5% CO₂ 培养箱孵育,1 d传代一次。将1.5×10⁵个/mL小鼠巨噬细胞RAW 264.7均匀接种于96孔板中,待细胞长到70% 左右,加入不同浓度的EEHI。处理24 h后,每孔加入10 μL CCK-8反应2 h后,于 λ =450 nm处检测吸光值,以样品空白组为对照计算细胞相对存活率。

Griess法测定样品对细胞中NO含量的影响：将1.5×10⁵个/mL小鼠巨噬细胞RAW 264.7均匀接种于24孔板中,待细胞长到70%左右,加入不同浓度的EEHI孵育1 h后,加入终浓度为1 μg·mL^-1脂多糖诱导细胞炎症反应,继续孵育24 h。收集细胞培养液,5 000 r/min离心10 min,取上清,与等体积Griess A试剂混合均匀后,取100 μL反应液于96孔板中并加入50 μL Griess B,避免气泡产生,摇匀,于 λ =540 nm处检测吸光值。根据NO^2-标准曲线计算NO的含量。

样品对细胞中相关炎症因子在mRNA水平表达影响的检测：将1.5×10⁵个/mL RAW 264.7细胞均匀接种于24孔板中,待细胞长到70%左右,加入不同浓度的EEHI孵育1 h,而后加入终浓度为1 μg·mL^-1脂多糖,孵育 6 h。采用CarryHelix RNA提取试剂盒提取细胞总RNA,采用NanoDrop 2000超微量分光光度计测所提取样品RNA 的浓度和纯度,使用PrimeScript^TM RT Master Mix反转录试剂盒,以1000 ng总RNA反转录合成cDNA模板,反转录产物置于-20℃待用。实时荧光定量PCR采用TB Green ?Premix Ex Taq^TM试剂盒进行,引物序列如表1所示。反应体系（总体积10 μL）：TB Green ?Premix Ex Taq^TM 5.0 μL,上游引物0.2 μL,下游引物0.2 μL,cDNA模板0.2 μL,RNase Free dH₂O 4.4 μL。

Table 1
表1
表1实时荧光定量PCR相关引物序列
Table 1The primer sequence of related genes in RT-qPCR

基因Gene	上游引物序列Upstream primer sequence	下游引物序列Downstream primer sequence
INOS	5′-TTTCCAGAAGCAGAATGTGACC-3′	5′-AACACCACTTTCACCAAGACTC-3′
HO-1	5′-ACATTGAGCTGTTTGAGGAG-3′	5′-TACATGGCATAAATTCCCACTG-3′
IL-1β	5′-CCAACAAGTGATATTCTCCATGAG-3′	5′-ACTCTGCAGACTCAAACTCCA-3′
IL-10	5′-CTATGCTGCCTGCTCTTACTG-3′	5′-CAACCCAAGTAACCCTTAAAGTC-3′
GAPDH	5′-GAGAAACCTGCCAAGTATGATGAC-3′	5′-TAGCCGTATTCATTGTCATACCAG-3′

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免疫印迹（Western blot）：将1.5×10⁵个/mL RAW 264.7细胞均匀接种于6孔板中,待细胞生长至90%融合度,经不同浓度的EEHI孵育1 h,加入终浓度为1 μg·mL^-1的脂多糖,于37℃,5% CO₂培养箱孵育0.5 h后,用含酶抑制剂的NP-40蛋白裂解液收集提取蛋白样品,然后采用BCA测定样品蛋白浓度,按照20 μg的蛋白总上样量对各组细胞样本蛋白进行免疫印迹检测,经过12%的SDS-PAGE凝胶电泳、转印、杂交、碱性磷酸酶显色等步骤,得到IκΒ α 、p-IκΒ α 和 β -actin的杂交条带。

NF-κB-p65的免疫荧光定位：将处于对数生长期的细胞按9×10⁴个/mL接种到爬片共聚焦小皿中,37℃,5% CO₂培养箱过夜孵育,预处理40 μg·mL^-1的EEHI 1 h,加入终浓度为1 μg·mL^-1的脂多糖0.5 h后,用固定液 （甲醇﹕丙酮=1﹕1）固定0.5 h,0.5%的Triton X-100透化0.5 h,10%血清封闭液室温封闭0.5 h,加入1﹕50的一抗（NF-κB-p65）稀释液和1﹕500的二抗羊抗兔（IgG）稀释液分别孵育1 h,DAPI（1﹕2 000稀释）染核处理封片,激光共聚焦扫描显微镜观察,并分析结果。

1.3 数据处理与分析

试验结果均以平均值±标准差（mean±SD）表示,数据采用SPSS软件进行ANOVA分析,Graphpad Prism 5.0作图。 P <0.05表示差异显著, P <0.01表示差异极显著。

2 结果

2.1 总酚酸和总黄酮的含量及体外自由基清除能力

由表2可知,EEHI的总酚酸含量为54.70 mg GAE·g^-1,总黄酮含量为116.20 mg QE·g^-1。EEHI抑制50% DPPH和ABTS^+·自由基的浓度分别为275.60和284.00 μg·mL^-1。

Table 2
表2
表2EEHI总酚酸和总黄酮的含量及体外自由基清除能力
Table 2Total phenolic acid, total flavonoids contents and radical-scavenging activity of EEHI

总酚酸含量 Total phenolic acid content (mg GAE·g-1)	总黄酮含量 Total flavonoids content (mg QE·g-1)	DPPH自由基清除力IC50 DPPH radical-scavenging activity IC50 (μg·mL-1)	ABTS+·自由基清除力IC50 ABTS+· radical-scavenging activity IC50 (μg·mL-1)
54.70±0.65	116.20±0.12	275.60±0.22	284.00±0.01

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2.2 EEHI对巨噬细胞RAW 264.7细胞活力的影响

为保证EEHI对细胞的代谢生长无明显的毒性,采用CCK-8法检测0—50 μg·mL^-1的EEHI对小鼠巨噬细胞 RAW 264.7细胞相对增殖活力的影响,结果如图1所示。与对照组相比,RAW 264.7细胞经过不同浓度EEHI处理后,0—40 μg·mL^-1的EEHI对细胞生长无显著抑制效果,而50 μg·mL^-1的EEHI对细胞的生长有显著的抑制作用。因此,40 μg·mL^-1为EEHI的安全浓度上限。

图1

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图1不同浓度EEHI对RAW 264.7细胞活力的影响

Fig. 1Effects of different concentrations of EEHI on the viability of RAW 264.7 cells

**： P <0.01

2.3 EEHI对小鼠巨噬细胞RAW 264.7中NO释放量的影响

通过典型的Greiss法检测不同浓度EEHI对脂多糖诱导的巨噬细胞炎症反应模型中NO释放量降低的效果。如图2所示,RAW 264.7细胞单独经过脂多糖刺激后,NO含量相对于对照组明显升高。在经过EEHI孵育后再加入脂多糖刺激与脂多糖组相比,NO释放量呈极显著下降趋势,且具有浓度依赖性。因此,EEHI能够抑制炎症过程中NO含量的产生,从而缓解炎症反应。

图2

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图2不同浓度EEHI对RAW 264.7细胞NO释放量的影响

Fig. 2Effects of different concentrations of EEHI on the NO production of RAW 264.7 cells

***： P <0.001

2.4 EEHI对巨噬细胞 RAW 264.7炎症相关因子表达的影响

RT-qPCR结果表明,EEHI能够极显著地抑制脂多糖诱导的 INOS 、 IL-1β 和 IL-10 等基因的表达,其中EEHI对 INOS 、 IL-1β 表达的抑制作用与浓度相关。此外,EEHI在10 μg·mL^-1时能够显著促进 HO-1 表达,在20—50 μg·mL^-1时能极显著增加 HO-1 表达（图3）。因此,在安全浓度范围以内,EEHI具有良好的抗氧化和抗炎能力。

图3

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图3不同浓度EEHI对RAW 264.7细胞炎症相关基因表达的影响

A：诱导型一氧化氮合酶 INOS ;B：白介素-10 IL-10 ;C：白介素-1 β IL-1β ;D：血红素氧合酶1 HO-1 。*： P <0.05;***： P <0.001
Fig. 3Effects of different concentrations of EEHI on the related inflammatory genes expression in RAW 264.7 cells

2.5 EEHI对脂多糖诱导的IκΒ α 活化的抑制作用

采用Western blot方法检测细菌脂多糖诱导的巨噬细胞中IκΒ α 和磷酸化IκΒ α 的蛋白相对表达水平,结果发现脂多糖刺激组IκΒ α 的磷酸化明显增加,而经过不同浓度EEHI孵育后处理组IκΒ α 的磷酸化水平被抑制,且随着EEHI浓度的增加,p-IκΒ α 显著降低（图4）。

图4

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图4不同浓度EEHI对细菌脂多糖诱导的IκB α 活化的抑制作用

Fig. 4Inhibitory effects of different concentrations of EEHI on the activation of IκΒ α induced by LPS

2.6 EEHI对巨噬细胞p65(NF-κB)核定位的影响

为进一步探究EEHI对NF-κB激活的抑制作用,采用激光共聚焦扫描显微镜观察40 μg·mL^-1的EEHI对脂多糖诱导的炎症信号通路NF-κB-p65入核定位的影响,结果表明脂多糖刺激组显著激活了NF-κB-p65入核,而经过40 μg·mL^-1的EEHI预处理后,NF-κB-p65入核受到了明显的抑制（图5）。

图5

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图5EEHI对NF-κB-p65激活的影响

Fig. 5Effects of EEHI on NF-κB-p65 activation

3 讨论

无刺蜂蜂胶是由无刺蜂采集的用来维持蜂群健康、防止病敌害的树脂混合无刺蜂分泌物以及泥土的一种天然特色蜂产品。国内的研究主要集中在意大利蜜蜂采集的蜂胶,由于地理和环境优势,国外对无刺蜂蜂产品研究较多,但关于无刺蜂蜂胶的药理学活性研究目前尚不全面。本研究采用的蜂胶采自马来西亚本土无刺蜂蜂种 H. itama ,是典型的无刺蜂蜂胶,具有一定的代表性和研究价值。

酚酸类化合物被报道具有抗氧化、抑菌、消炎、抗过敏和抑菌等生物学活性。同时,一些黄酮类化合物被报道具有抗炎、抗氧化、抗癌、保肝、肠道保护作用^[19,20]。越来越多的研究结果证明,蜂胶的体外抗氧化能力与其含有丰富的总酚酸和总黄酮息息相关^[21]。蜂胶的DPPH和ABTS^+·自由基清除能力是评估蜂胶体外抗氧化水平的两种方法,且这两种方法具备重现性、稳定性良好的特点。蜂胶中的总酚酸、总黄酮含量是评估蜂胶质量的重要指标。通过检测蜂胶的总酚酸和总黄酮含量,笔者发现EEHI的总酚酸含量与文献报道结果一致,且比 M. fasciculata 和 Geniotrigona thoracica 蜂胶中的总酚酸含量高（分别为47.78和29.10 μg·mL^-1）。总黄酮含量高于 G. thoracica 蜂胶（61.5 μg·mL^-1）^[4],却低于文献^[7]报道的163.9 μg·mL^-1,推测可能是由于样品采集地点^[4,22]和所采用的总黄酮当量的标准品^[23,24]不同而造成。通过DPPH和ABTS^+·自由基清除能力试验发现,该蜂胶虽然富含相对于 G. thoracica 蜂胶高的酚酸和黄酮,但是自由基清除能力却与文献报道存在明显差异,由此证明虽然多酚类化合物对蜂胶抗氧化能力起主要作用,但不一定高含量的酚酸和黄酮类化合物就具有最高的抗氧化能力,也可能与EEHI中酚酸黄酮类化合物的分子结构基团有关^[3,25]。

炎症发生时会产生超氧自由基、过氧亚硝酸盐、过氧化氢、次氯酸和NO等高活性物质。NO是一种具有血管舒张、神经传导和炎症反应生物功能的小分子。巨噬细胞在脂多糖刺激下会促进 iNOS 催化精氨酸产生NO和促炎细胞因子,而在巨噬细胞中NO的过量产生会导致一系列的生理反应,如炎症、细胞毒性和自身免疫失调^[26],所以天然毒副作用小的NO抑制剂对缓解炎症反应是一个很好的选择。 INOS （诱导型一氧化氮合酶）作为一氧化氮合酶之一,是炎症条件下表达的关键酶,能够通过脂多糖诱导产生高水平含量的NO,从而影响细胞的氧化还原状态并诱导蛋白质、脂类和DNA的氧化,加速炎症反应的发生^[27]; IL-1β （白介素-1 β ）是一种细胞促炎因子,在释放前列腺素中扮演着重要的角色,并且与中性粒细胞在炎症反应中的迁移直接相关^[16]; IL-10 （白介素-10）是一种具有释放免疫介质、呈递抗原、抑制单核巨噬细胞释放炎症因子等多功能的细胞因子; HO-1 （血红素氧合酶1）能被过氧化氢、紫外线、NO等多种引起氧化应激的反应诱导,催化血红素降解产生一系列具有抗氧化、抗炎、抗凋亡等分子^[28]。通过Griess和RT-qPCR方法研究发现,EEHI能够极显著抑制脂多糖诱导的巨噬细胞的NO释放量,并抑制 INOS 的表达,与前人报道一致^[11],EEHI能够依赖浓度关系清除NO含量,在采用脂多糖诱导的小鼠巨噬细胞Raw 264.7细胞炎症反应模型中,EEHI具有与中国蜂胶和巴西绿胶相近的NO清除能力^[16]。此外,相对于脂多糖刺激组,EEHI极显著抑制了细胞炎症因子基因 IL-1β 、 IL-10 的表达量,并显著上调细胞中抗氧化蛋白基因 HO-1 的表达,表明EEHI能够通过降低细胞中炎症介质的表达,增强对其氧化应激的保护作用,从而减缓巨噬细胞的炎症反应,与文献报道相一致^[10],EEHI具有NO清除能力,且与意大利蜜蜂蜂胶（主要是中国杨树型蜂胶、巴西酒神菊属型蜂胶）及巴西无刺蜂 M. scutellaris 蜂胶相同,能够通过调节炎症介质的表达缓解炎症反应的发生,如 IL-1β 、 INOS 、 IL-10 、 HO-1 等。值得注意的是,EEHI与中国杨树型蜂胶对抗炎症因子 IL-10 的调节作用相同,均抑制 IL-10 的表达,但与巴西酒神菊属型蜂胶的调节作用不一致,巴西酒神菊属型蜂胶在低浓度时促进炎症因子 IL-10 的表达,高浓度时表现出抑制作用^[14-15,29]。

IκΒ α 是一种抑制NF-kB活性的抑制蛋白,正常情况下,NF-kB抑制蛋白IκΒ α 与P65/P50异二聚体在细胞质中相互作用从而掩盖NF-kB家族中的核定位序列。在脂多糖刺激下,IκΒ α 迅速磷酸化后被蛋白酶降解,导致游离的NF-kB自由进入细胞核^[30]。NF-κB是具有激活转录基因功能的一组蛋白质,参与调节许多与免疫和炎症相关的基因。细胞正常状态时,NF-κB-IκΒs处于动态平衡,在受到外界刺激下,IκΒs被降解蛋白酶降解后,NF-κB家族中的核定位序列与IκΒs相互作用并携带定位信号,使被激活的NF-κB由细胞质迅速向细胞核移动。NF-κB-p65被激活后暴露出核定位位点,快速转移到细胞核中并大量分泌炎症因子,对机体产生伤害^[31]。通过Western blot和免疫荧光进一步探究EEHI缓解脂多糖诱导的巨噬细胞炎症反应的作用机制,发现EEHI能够显著抑制IκΒ α 的磷酸化和炎症信号通路NF-κB-p65的激活,由此推测EEHI与中国杨树型蜂胶^[15]、巴西无刺蜂 M. scutellaris 蜂胶中的CNM^[17]在NF-κB信号通路作用机理上可能存在一致性,主要是通过降低IκΒ α 的磷酸化并抑制NF-κB-p65的激活从而抑制NF-κB的激活,缓解炎症因子在细胞核内的分泌和转录,从而缓解炎症反应。

近年来,大量的研究证明植物以及蜂胶中的酚酸、黄酮具有良好的抗炎及抗氧化效果,包括山奈酚、阿替匹林-C、倒捻子素等。黄酮类化合物不仅具有清除巨噬细胞中NO的含量和降低 INOS 表达的效果,更能够通过调节NF-κB以及多种蛋白信号通路缓解机体炎症的发生^[13]。同时,植物中的萜烯类化合物具有潜在的自由基清除能力,并能够降低细胞中NO含量以及炎症相关因子（ IL-1α 、 TNF-α ）的表达,同时抑制NF-κB的活性^[32]。由此,笔者推测EEHI的抗炎和抗氧化活性,不仅与其丰富的多酚类化合物密切相关,丰富的萜烯类化合物也可能为良好的抗氧化和抗炎活性提供了一定的辅助作用,而关于萜烯类化合物的活性效果需要进一步验证。

4 结论

无刺蜂蜂胶含有丰富的多酚类化合物,且具有良好的体外自由基清除能力。在细菌脂多糖诱导小鼠巨噬细胞炎症反应试验中, H. itama 蜂胶乙醇提取物（EEHI）显著抑制了细胞炎症因子 IL-1β 、 IL-10 的表达,上调细胞中抗氧化基因 HO-1 的表达,并通过降低细胞中 INOS 的表达从而抑制细胞中NO的释放;另外,EEHI可通过抑制IκΒ α 蛋白的磷酸化和NF-κB-p65的激活从而抑制NF-κB转录因子的活性,缓解炎症反应。研究结果可为无刺蜂相关特色蜂产品开发提供理论依据。

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BMC Complementary and Alternative Medicine, 2011,11:108. DOI: 10.1186/1472-6882-11-108.
DOI:10.1186/1472-6882-11-108URLPMID:3225302 [本文引用: 2]
Background Native bees of the tribe Meliponini produce a distinct kind of propolis called geopropolis. Although many pharmacological activities of propolis have already been demonstrated, little is known about geopropolis, particularly regarding its antimicrobial activity against oral pathogens. The present study aimed at investigating the antimicrobial activity of M. fasciculata geopropolis against oral pathogens, its effects on S. mutans biofilms, and the chemical contents of the extracts. A gel prepared with a geopropolis extract was also analyzed for its activity on S. mutans and its immunotoxicological potential. Methods Antimicrobial activities of three hydroalcoholic extracts (HAEs) of geopropolis, and hexane and chloroform fractions of one extract, were evaluated using the agar diffusion method and the broth dilution technique. Ethanol (70%, v/v) and chlorhexidine (0.12%, w/w) were used as negative and positive controls, respectively. Total phenol and flavonoid concentrations were assayed by spectrophotometry. Immunotoxicity was evaluated in mice by topical application in the oral cavity followed by quantification of biochemical and immunological parameters, and macro-microscopic analysis of animal organs. Results Two extracts, HAE-2 and HAE-3, showed inhibition zones ranging from 9 to 13 mm in diameter for S. mutans and C. albicans, but presented no activity against L. acidophilus. The MBCs for HAE-2 and HAE-3 against S. mutans were 6.25 mg/mL and 12.5 mg/mL, respectively. HAE-2 was fractionated, and its chloroform fraction had an MBC of 14.57 mg/mL. HAE-2 also exhibited bactericidal effects on S. mutans biofilms after 3 h of treatment. Significant differences (p < 0.05) in total phenol and flavonoid concentrations were observed among the samples. Signs toxic effects were not observed after application of the geopropolis-based gel, but an increase in the production of IL-4 and IL-10, anti-inflammatory cytokines, was detected. Conclusions In summary, geopropolis produced by M. fasciculata can exert antimicrobial action against S. mutans and C. albicans, with significant inhibitory activity against S. mutans biofilms. The extract with the highest flavonoid concentration, HAE-2, presented the highest antimicrobial activity. In addition, a geopropolis-based gel is not toxic in an animal model and displays anti-inflammatory effect.

[7] IBRAHIM N, ZAKARIA A J, ISMAIL Z, MOHD K S. Antibacterial and phenolic content of propolis produced by two Malaysian stingless bees, Heterotrigona itama and Geniotrigona thoracica
International Journal of Pharmacognosy and Phytochemical Research, 2016, 8(1): 156-161..
URL [本文引用: 2]

[8] CINEGAGLIA N C, BERSANO P R O, ARAúJO M J A M, BúFALO M C, SFORCIN J M, .Anticancer effects of geopropolis produced by stingless bees on canine osteosarcoma cells in vitro
. Evidence-Based Complementray and Alternative Medicine 2013, 2013: Article ID 737386. DOI: 10.1155/2013/737386.
DOI:10.1155/2013/737386URLPMID:3652194 [本文引用: 2]
Geopropolis is produced by indigenous stingless bees from the resinous material of plants, adding soil or clay. Its biological properties have not been investigated, such as propolis, and herein its cytotoxic action on canine osteosarcoma (OSA) cells was evaluated. OSA is a primary bone neoplasm diagnosed in dogs being an excellent model in vivo to study human OSA. spOS-2 primary cultures were isolated from the tumor of a dog with osteosarcoma and incubated with geopropolis, 70% ethanol (geopropolis solvent), and carboplatin after 6, 24, 48, and 72 hours. Cell viability was analyzed by the crystal violet method. Geopropolis was efficient against canine OSA cells in a dose- and time-dependent way, leading to a distinct morphology compared to control. Geopropolis cytotoxic action was exclusively due to its constituents since 70% ethanol (its solvent) had no effect on cell viability. Carboplatin had no effect on OSA cells. Geopropolis exerted a cytotoxic effect on canine osteosarcoma, and its introduction as a possible therapeutic agent in vivo could be investigated, providing a new contribution to OSA treatment.

[9] COELHO G R, MENDONCA R Z, DE SENNA VILAR K, FIGUEIREDO C A, BADARI J C, TANIWAKI N, NAMIYAMA G, DE OLIVEIRA M I, CURTI S P, SILVA P E, NEGRI G. Antiviral action of hydromethanolic extract of geopropolis from Scaptotrigona postica against Antiherpes simplex virus(HSV-1)
Evidence-Based Complementray and Alternative Medicine 2015, 2015: Article ID 296086. DOI: 10.1155/2015/296086.
[本文引用: 1]

[10] 陈佳玮, 申小阁, 胡福良 . 无刺蜂蜂胶化学成分及生物学活性的研究进展
天然产物研究与开发, 2016,28:2021-2029.
URL [本文引用: 3]
无刺蜂蜂胶化学组成复杂,具有丰富而突出的生物学活性,但目前有关其研究相对较少。本文以国内外研究文献为基础,系统归纳了无刺蜂蜂胶已探明的包括35种黄酮类、13种酚酸类和53种萜烯类化合物在内的主要化学成分,并阐述了其在抗菌、抗氧化、抗炎、抗癌等方面的生物学活性,以期为无刺蜂蜂胶化学标准化及质量控制的研究提供依据,并为全面评价无刺蜂蜂胶的药用价值及其开发利用提供参考。
CHEN J W, SHEN X G, HU F L . Review on chemical composition and biological activities of geopropolis
Natural Product Research and Development , 2016,28:2021-2029. (in Chinese)
URL [本文引用: 3]
无刺蜂蜂胶化学组成复杂,具有丰富而突出的生物学活性,但目前有关其研究相对较少。本文以国内外研究文献为基础,系统归纳了无刺蜂蜂胶已探明的包括35种黄酮类、13种酚酸类和53种萜烯类化合物在内的主要化学成分,并阐述了其在抗菌、抗氧化、抗炎、抗癌等方面的生物学活性,以期为无刺蜂蜂胶化学标准化及质量控制的研究提供依据,并为全面评价无刺蜂蜂胶的药用价值及其开发利用提供参考。

[11] IBRAHIM N ,NIZA N F S M, RODI M M M, ZAKARIA A J, ISMAIL Z, MOHD K S. Chemical and biological analyses of Malaysian stingless bee propolis extracts
Malaysian Journal of Analytical Science, 2016,20(2):413-422. DOI: 10.17576/mjas-2016- 2002-26.
DOI:10.17576/mjasURL [本文引用: 2]

[12] ZHAO L L, YU M J, SUN M H, XUE X F, WANG T T, CAO W, SUN L P . Rapid determination of major compounds in the ethanol extract of geopropolis from Malaysian stingless bees, Heterotrigona itama , by UHPLC-Q-TOF/MS and NMR
. Molecules , 2017,22(11):1935. DOI: 10.3390/molecules22111935.
DOI:10.3390/molecules22111935URLPMID:29125569 [本文引用: 1]
Abstract A reliable, rapid analytical method was established for the characterization of constituents of the ethanol extract of geopropolis (EEGP) produced by Malaysian stingless bees- Heterotrigona itama -by combining ultra-high-performance liquid chromatography with quadruple time-of-flight mass spectrometry (UHPLC-Q-TOF/MS). Based on known standards, the online METLIN database, and published literature, 28 compounds were confirmed. Phenolic acids, flavones, triterpenes and phytosterol were identified or tentatively identified using characteristic diagnostic fragment ions. The results indicated that terpenoids were the main components of EEGP, accompanied by low levels of phenolic acids, flavonoids, and phytosterol. Two major components were further purified by preparative high-performance liquid chromatography (PHPLC) and identified by nuclear magnetic resonance (NMR) as 24( E )-cycloart-24-ene-26-ol-3-one and 20-hydroxy-24-dammaren-3-one. These two triterpenes, confirmed in this geopropolis for the first time, are potential chemical markers for the identification of geopropolis from Malaysian stingless bees, H. itama .

[13] 王凯, 张江临, 胡福良 . 蜂胶抗炎活性及其分子机制研究进展
中草药, 2013,44(16):2321-2329.
DOI:10.7501/j.issn.0253-2670.2013.16.025URL [本文引用: 3]
蜂胶是一种具有广泛药理学活性的蜂产品.虽然不同地区、不同来源的蜂胶成分差异很大,但各种蜂胶所具有的生物学活性基本类似.就国内外蜂胶及其单体活性成分的抗炎作用及其分子机制进行综述,旨在为蜂胶生物学活性的基础性研究提供思路,并为蜂胶产品的标准化提供参考.
WANG K, ZHANG J L, HU F L . Research progress in anti-inflammatory activity of propolis and its molecular mechanism
Chinese Traditional and Herbal Drugs , 2013,44(16):2321-2329. (in Chinese)
DOI:10.7501/j.issn.0253-2670.2013.16.025URL [本文引用: 3]
蜂胶是一种具有广泛药理学活性的蜂产品.虽然不同地区、不同来源的蜂胶成分差异很大,但各种蜂胶所具有的生物学活性基本类似.就国内外蜂胶及其单体活性成分的抗炎作用及其分子机制进行综述,旨在为蜂胶生物学活性的基础性研究提供思路,并为蜂胶产品的标准化提供参考.

[14] WANG K, HU L, JIN X L, MA Q X, MARCUCCI M C ,NETTO A A L, SAWAYA A C H F, HUANG S, REN W K, CONLON M A, TOPPING D L, HU F L. Polyphenol-rich propolis extracts from China and Brazil exert anti-inflammatory effects by modulating ubiquitination of TRAF6 during the activation of NF-κB
Journal of Functional Foods, 2015,19:464-478. DOI: 10.1016/j.jff.2015.09.009.
DOI:10.1016/j.jff.2015.09.009URL [本文引用: 2]
Propolis has documented anti-inflammatory properties, although its mechanisms of action are poorly understood. In this study, the anti-inflammatory effects of polyphenol-rich propolis extracts (PPE) from China (CPPE) and Brazil (BPPE) were examined. Oral administration of PPE to lipopolysaccharide (LPS)-challenged mice decreased serum proinflammatory cytokine concentrations and inhibited pulmonary nuclear factor (NF)-κB activation. Both PPE types modulated LPS-induced key inflammatory mediators production in RAW 264.7 macrophages. They also suppressed NF-κB activation in HEK 293T cells, correlating well with their inhibitory effects on IκB phosphorylation and p65 nuclear translocation in LPS-activated macrophages. We found PPE suppressed NF-κB activation through delaying the ubiquitination of TRAF6 in HeLa-T6RZC stable cells and by directly disrupting the polyubiquitin synthesis in anin vitrokinase assay system. Overall, analysis showed substantial compositional differences between CPPE and BPPE; nevertheless, they both displayed similar anti-inflammatory properties through NF-κB-responsive inflammatory gene expressions by inhibiting TRAF6 dependent canonical NF-κB pathway.

[15] BANG S Y, KIM J H, KIM H Y, LEE Y J, PARK S Y, LEE S J, KIM Y H .Achyranthes japonica exhibits anti-inflammatory effect via NF-κΒ suppression and HO-1 induction in macrophages
Journal of Ethnopharmacology , 2012,144(1):109-117. DOI: 10.1016/j.jep.2012. 08.037.
[本文引用: 4]

[16] FRANCHIN M ,DA CUNHA M G,DENNY C,NAPIMOGA M H,CUNHA T M,KOO H,DE ALENCAR S M,IKEGAKI M, ROSALEN P L. Geopropolis from Melipona scutellaris decreases the mechanical inflammatory hypernociception by inhibiting the production of IL-1 β and TNF- α .
Journal of Ethnopharmacology , 2012,143(2):709-715. DOI: 10.1016/j.jep.2012.07.040.
DOI:10.1016/j.jep.2012.07.040PMID:22885134 [本文引用: 3]
The pharmacological activity of geopropolis collected by stingless bees (important and threatened pollinators), a product widely used in folk medicine by several communities in Brazil, especially in the Northeast Region, needs to be studied.The aim of this study was to evaluate the antinociceptive activity of Melipona scutellaris geopropolis (stingless bee) using different models of nociception.The antinociceptive activity of the ethanolic extract of geopropolis (EEGP) and fractions was evaluated using writhing induced by acetic acid, formalin test, carrageenan-induced hypernociception, and quantification of IL-1 and TNF- . The chemical composition was assessed by quantification of total flavonoids and phenolic compounds.EEGP and its hexane and aqueous fractions showed antinociceptive activity. Both EEGP and its aqueous fraction presented activity in the mechanical inflammatory hypernociception induced by the carrageenan model, an effect mediated by the inhibition of IL-1 and TNF- . The chemical composition of EEGP and its hexane and aqueous fractions showed a significant presence of phenolic compounds and absence of flavonoids.Our data indicate that geopropolis is a natural source of bioactive substances with promising antinociceptive activity.

[17] FRANCHIN M, ROSALEN P L ,DA CUNHA M G, SILVA R L, COLO?N D F, BASSI G S, DE ALENCAR S M, IKEGAKI M, ALVES-FILHO J C, CUNHA F Q, BEUTLER J A, CUNHA T M. Cinnamoyloxy-mammeisin isolated from geopropolis attenuates inflammatory process by inhibiting cytokine production: Involvement of MAPK, AP-1, and NF-κB
Journal of Natural Products, 2016,79(7):1828-1833. DOI: 10.1021/acs.jnatprod.6b00263.
DOI:10.1021/acs.jnatprod.6b00263URLPMID:27367493 [本文引用: 2]
Abstract Chemical compounds belonging to the class of coumarins have promising anti-inflammatory potential. Cinnamoyloxy-mammeisin (CNM) is a 4-phenylcoumarin that can be isolated from Brazilian geopropolis. To our knowledge, its anti-inflammatory activity has never been studied. Therefore, the present study investigated the anti-inflammatory activity of CNM and elucidated its mechanism of action on isolated macrophages. Pretreatment with CNM reduced neutrophil migration into the peritoneal and joint cavity of mice. Likewise, CNM reduced the in vitro and in vivo release of TNF-α and CXCL2/MIP-2. Regarding the possible molecular mechanism of action, CNM reduced the phosphorylation of proteins ERK 1/2, JNK, p38 MAPK, and AP-1 (subunit c-jun) in PG-stimulated macrophages. Pretreatment with CNM also reduced NF-κB activation in RAW 264.7 macrophages stably expressing the NF-κB-luciferase reporter gene. On the other hand, it did not alter IκBα degradation or nuclear translocation of p65. Thus, the results of this study demonstrate promising anti-inflammatory activity of CNM and provide an explanation of its mechanism of action in macrophages via inhibition of MAPK signaling, AP-1, and NF-κB.

[18] YANG H S, DONG Y Q, DU H J, SHI H M, PENG Y H, LI X B . Antioxidant compounds from propolis collected in Anhui, China
Molecules, 2011,16(4):3444-3455. DOI: 10.3390/molecules16043444.
DOI:10.3390/molecules16043444URLPMID:21512452 [本文引用: 4]
The antioxidant activities of the chloroform, ethyl acetate and n-butanol extract fractions from propolis collected in Anhui, China were evaluated in this study. The ethyl acetate fraction contained the highest amount of total phenolics and total flavonoids, and showed the greatest 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging capacities and Ferric Reducing/Antioxidant Power (FRAP). The antioxidant activity of twenty-two compounds isolated from the ethyl acetate fraction was also evaluated using the above-mentioned three assays. The results indicated that phenolics contributed to the antioxidant activity of propolis collected in Anhui, China. Therefore, propolis collected in Anhui, China and its phenolics might be used as a natural antioxidant.

[19] HELENO S A, MARTINS A, QUEIROZ M J, FERREIRA I C . Bioactivity of phenolic acids: Metabolites versus parent compounds: A review
Food Chemistry, 2015,173:501-513. DOI: 10.1016/j. foodchem.2014.10.057.
DOI:10.1016/j.foodchem.2014.10.057URLPMID:25466052 [本文引用: 1]
Phenolic acids are present in our diet in different foods, for example mushrooms. Due to their bioactive properties, phenolic acids are extensively studied and there is evidence of their role in disease prevention. Nevertheless, in vivo, these compounds are metabolized and circulate in the organism as glucuronated, sulphated and methylated metabolites, displaying higher or lower bioactivities. To clarify the importance of the metabolism of phenolic acids, knowledge about the bioactivity of metabolites is extremely important. In this review, chemical features, biosynthesis and bioavailability of phenolic acids are discussed, as well as the chemical and enzymatic synthesis of their metabolites. Finally, metabolite bioactive properties are compared with that of the corresponding parental compounds.

[20] OTEIZA P I, FRAGA C G, MILLS D A, TAFT D H . Flavonoids and the gastrointestinal tract: Local and systemic effects
Molecular Aspects of Medicine, 2018,61(1):41-49. DOI: 10.1016/j.mam.2018. 01.001.
DOI:10.1016/j.mam.2018.01.001URLPMID:29317252 [本文引用: 1]
Abstract The gastrointestinal (GI) tract plays a central role in the absorption, distribution, metabolism, and excretion of flavonoids, which ultimately define the health effects of these bioactives. These aspects are modulated by the interactions of flavonoids with other dietary components, environmental factors, the host, and the GI microbiota. Flavonoid can target molecules in the luminal content, the different GI tract cell types, and the microbiota. Importantly, flavonoid actions at the GI tract can have an impact systemically, e.g. on glucose homeostasis, lipid and energy metabolism, or cardiovascular risk factors. The beneficial actions of flavonoids at the GI include their capacity to: i) protect the intestinal epithelium against pharmacological insults and food toxins; ii) modulate the activity of enzymes involved in lipid and carbohydrate absorption; iii) maintain the intestinal barrier integrity; iv) modulate the secretion of gut hormones; v) modulate the GI tract immune system; vi) exert potential anti-colorectal cancer activity; and vii) shape microbiota composition and function. Further understanding of the mechanisms mediating the effects of flavonoids on the intestine (and its microbiota) is of critical importance given the relevance of the GI tract on sustaining overall health and of the widespread recommendations of increasing the intake of plant bioactives.

[21] 张江临, 王凯, 胡福良 . 蜂胶的抗氧化活性及其分子机制研究进展
中国中药杂志, 2013,38(16):2645-2652. DOI: 10.4268/ cjcmm20131621.
DOI:10.4268/cjcmm20131621URL [本文引用: 1]
蜂胶是蜜蜂工蜂采集的植物树脂与其上颚腺、蜡腺等的分泌物混合而成的胶黏性物质,具有广泛的药理活性及保健功能。蜂胶的抗氧化活性一直被认为是蜂胶最重要的生物活性之一。该文针对不同地理来源、不同提取方法获得的蜂胶提取物以及蜂胶中几种重要单体活性成分的抗氧化活性方面的研究进展进行了综述,并总结了蜂胶及其单体成分发挥抗氧化活性的可能存在的分子机制,旨在为更深入地研究蜂胶的药理活性提供思路,同时也为蜂胶产品的深度开发提供参考。
ZHANG J L, WANG K, HU F L . Advance in studies on antioxidant activity of propolis and its molecular mechanism
China Journal of Chinese Materia Medica , 2013,38(16):2645-2652. DOI: 10.4268/ cjcmm20131621. (in Chinese)
DOI:10.4268/cjcmm20131621URL [本文引用: 1]
蜂胶是蜜蜂工蜂采集的植物树脂与其上颚腺、蜡腺等的分泌物混合而成的胶黏性物质,具有广泛的药理活性及保健功能。蜂胶的抗氧化活性一直被认为是蜂胶最重要的生物活性之一。该文针对不同地理来源、不同提取方法获得的蜂胶提取物以及蜂胶中几种重要单体活性成分的抗氧化活性方面的研究进展进行了综述,并总结了蜂胶及其单体成分发挥抗氧化活性的可能存在的分子机制,旨在为更深入地研究蜂胶的药理活性提供思路,同时也为蜂胶产品的深度开发提供参考。

[22] ARAúJO M J A, BúFALO M C, CONTI B J, FERNANDES JR A, TRUSHEVA B, BANKOVA V, SFORCIN J M . The chemical composition and pharmacological activities of geopropolis produced by Melipona fasciculata Smith in northeast Brazil
. Journal of Molecular Pathophysiology , 2015,4(1):12-20. DOI: 10.5455/jmp. 20150204115607.
[本文引用: 1]

[23] 董捷, 张红城, 李浩, 李慧 . 八种蜂花粉醇提物中总多酚和总黄酮含量测定
食品工业科技, 2008,29(11):80-83.
DOI:10.3321/j.issn:1002-6630.2008.12.053URL [本文引用: 1]
为测定蜂花粉的功能性成分,用不同浓度乙醇（0%、30%、50%、70%、80%、 100%）对杏花、茶花、荷花、油菜、荞麦、向日葵、玫瑰、五味子蜂花粉进行提取。结果表明：八种蜂花粉中多酚和黄酮类物质最适宜用80%乙醇提取,荷花 花粉水提多酚提取量高于醇提物。五味子花粉的总多酚含量最高,为19.74±0.20mg/g,荷花花粉的总多酚含量最低,为1.50±0.02mg /g;油菜花粉的总黄酮含量最高,为24.00±0.17mg/g,荷花花粉的总黄酮含量最低,为0.90±0.01mg/g。
DONG J, ZHANG H C, LI J, LI H . The contents of total polyphenols and flavonoids in ethanolic extracts from eight kinds of bee pollens
Science and Technology of Food Industry , 2008,29(11):80-83. (in Chinese)
DOI:10.3321/j.issn:1002-6630.2008.12.053URL [本文引用: 1]
为测定蜂花粉的功能性成分,用不同浓度乙醇（0%、30%、50%、70%、80%、 100%）对杏花、茶花、荷花、油菜、荞麦、向日葵、玫瑰、五味子蜂花粉进行提取。结果表明：八种蜂花粉中多酚和黄酮类物质最适宜用80%乙醇提取,荷花 花粉水提多酚提取量高于醇提物。五味子花粉的总多酚含量最高,为19.74±0.20mg/g,荷花花粉的总多酚含量最低,为1.50±0.02mg /g;油菜花粉的总黄酮含量最高,为24.00±0.17mg/g,荷花花粉的总黄酮含量最低,为0.90±0.01mg/g。

[24] 李红兵, 米佳, 张林锁, 罗青, 李晓莺, 曹有龙, 李越鲲, 戴国礼, 冉林武, 闫亚美 . 不同花粉多酚类物质组成比较
食品研究与开发, 2015,36(20):111-114. DOI: 10.3969/j.issn.1005-6521.2015.20.030.
DOI:10.3969/j.issn.1005-6521.2015.20.030URL [本文引用: 1]
采用分光光度计法、HPLC、HPLC-MS法分析测定、鉴定枸杞花粉等多酚类物质含量及主要组成。结果表明,枸杞花粉等6种不同多酚类物质来源的植物材料总多酚类物质含量的变化幅度为0.82 g/100 g~3.92 g/100 g。其中油菜花粉总多酚类物质含量最高,其次是枸杞花粉。各种花粉中的主要多酚类物质是黄酮类。枸杞花粉黄酮类物质含量分别是荷花花粉、油菜花粉、荞麦花粉、向日葵花粉和茶花粉的1.42、0.72、2.78、1.20、2.37倍。初步分离鉴定出枸杞花粉多酚类物质10个,其中主要组分有3个,含量均大于其它所测市售花粉。结果表明,较其它5种蜂花粉材料,枸杞花粉中多酚类物质主要组分与之差异较大,是较好的开发功能性食品的资源。
LI H B, MI J, ZHANG L S, LUO Q, LI X Y, CAO Y L, LI Y K, DAI G L, RAN L W, YAN Y M . The difference of the compositions of polyphenols from different bee pollen
Food Research and Development , 2015,36(20):111-114. DOI: 10.3969/j.issn.1005-6521.2015.20.030. (in Chinese)
DOI:10.3969/j.issn.1005-6521.2015.20.030URL [本文引用: 1]
采用分光光度计法、HPLC、HPLC-MS法分析测定、鉴定枸杞花粉等多酚类物质含量及主要组成。结果表明,枸杞花粉等6种不同多酚类物质来源的植物材料总多酚类物质含量的变化幅度为0.82 g/100 g~3.92 g/100 g。其中油菜花粉总多酚类物质含量最高,其次是枸杞花粉。各种花粉中的主要多酚类物质是黄酮类。枸杞花粉黄酮类物质含量分别是荷花花粉、油菜花粉、荞麦花粉、向日葵花粉和茶花粉的1.42、0.72、2.78、1.20、2.37倍。初步分离鉴定出枸杞花粉多酚类物质10个,其中主要组分有3个,含量均大于其它所测市售花粉。结果表明,较其它5种蜂花粉材料,枸杞花粉中多酚类物质主要组分与之差异较大,是较好的开发功能性食品的资源。

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[27] PARK S Y, KIM J H, LEE S J, KIM Y H . Involvement of PKA and HO-1 signaling in anti-inflammatory effects of surfactin in BV-2 microglial cells
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78 Surfactin inhibits proinflammatory mediator synthesis in LTA-activated BV-2 cells. 78 Surfactin suppresses NF-κB and STAT-1, but potentiates phosphorylation of STAT-3. 78 Surfactin induces HO-1 expression and Nrf-2 activation. 78 Surfactin induces cAMP and CREB phosphorylation. 78 PKA inhibitor blocks HO-1 induction and surfactin’s antiinflammatory effects.

[28] WANG K, JIN X L, LI Q Q ,SAWAYA A C H F,LE LEU R K,CONLON M A,WU L M, HU F L. Propolis from different geographic origins decreases intestinal inflammation and Bacteroides spp. populations in a model of DSS-induced colitis
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[29] DOS SANTOS H F, CAMPOS J F, DOS SANTOS C M, BALESTIERI J B P, SILVA D B, CAROLLO C A, DE PICOLI SOUZA K, ESTEVINHO L M, DOS SANTOS E L . Chemical profile and antioxidant, anti-inflammatory, antimutagenic and antimicrobial activities of geopropolis from the stingless bee Melipona orbignyi .
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Geopropolis is a resin mixed with mud, produced only by stingless bees. Despite being popularly known for its medicinal properties, few scientific studies have proven its biological activities. In this context, the objective of this study was to determine the chemical composition and antioxidant, anti-inflammatory, antimutagenic and antimicrobial activities of theMelipona orbignyigeopropolis. The hydroalcoholic extract of geopropolis (HEGP) was prepared and its chemical composition determined by high performance liquid chromatography coupled to diode array detector and mass spectrometry (HPLC-DAD-MS). The antioxidant activity was determined by the capture of free radicals and inhibition of lipid peroxidation in human erythrocytes. The anti-inflammatory activity was evaluated by the inhibition of the hyaluronidase enzyme and the antimutagenic action was investigated inSaccharomyces cerevisiaecolonies. The antimicrobial activities were determined against bacteria and yeasts, isolated from reference strains and hospital origin. The chemical composition of HEGP included flavonoids, derivatives of glycosylated phenolic acids and terpenoids. HEGP showed high antioxidant activity, it inhibited the activity of the inflammatory enzyme hyaluronidase and reduced the mutagenic effects inS. cerevisiae. In relation to the antimicrobial activity, it promoted the death of all microorganisms evaluated. In conclusion, this study reveals for the first time the chemical composition of the HEGP ofM. orbignyiand demonstrates its pharmacological properties.

[30] WANG K, PING S, HUANG S, HU L, XUAN H Z, ZHANG C P, HU F L. Molecular mechanisms underlying the in vitro anti-inflammatory effects of a flavonoid-rich ethanol extract from Chinese propolis(poplar type)
Evidence-Based Complementary Alternative Medicine 2013, 2013 : Article ID127672. DOI: 10.1155/2013/127672.
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China produces the greatest amount of propolis but there is still lack of basic studies on its pharmacological mechanisms. Our previous study found that ethanol extract from Chinese propolis (EECP) exerted excellent anti-inflammatory effects in vivo but mechanisms of action were elusive. To further clarify the possible mechanisms underlying the anti-inflammatory effects of Chinese propolis (poplar type), we utilized EECP to analyze its chemical composition and evaluated its potential anti-inflammatory effects in vitro. High-performance liquid chromatography (HPLC) profile indicated that EECP contained abundant flavonoids, including rutin, myricetin, quercetin, kaempferol, apigenin, pinocembrin, chrysin, and galangin. Next we found that EECP could significantly inhibit the production of NO, IL-1??, and IL-6 in lipopolysaccharide- (LPS-) stimulated RAW 264.7 cells and suppress mRNA expression of iNOS, IL-1??, and IL-6 in a time- and dose-dependent manner. Furthermore, we found that EECP could suppress the phosphorylation of I??B?? and AP-1 but did not affect I??B?????s degradation. In addition, using a reporter assay, we found that EECP could block the activation of NF-??B in TNF-??-stimulated HEK 293T cells. Our findings give new insights for understanding the mechanisms involved in the anti-inflammatory effects by Chinese propolis and provide additional references for using propolis in alternative and complementary therapies.

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[32] MARQUES F M, FIGUEIRA M M, SCHMITT E F P, KONDRATYUK T P, ENDRINGER D C, SCHERER R, FRONZA M. In vitro anti-inflammatory activity of terpenes via suppression of superoxide and nitric oxide generation and the NF-κB signalling pathway
Inflammopharmacology , 2018, DOI: 10.1007/s10787-018- 0483-z.
DOI:10.1007/s10787-018-0483-zURLPMID:29675712 [本文引用: 1]
Abstract BACKGROUND AND AIMS: Terpenes are considered the main components of essential oils and an important source for the identification of novel lead molecules. This study aimed to investigate the in vitro anti-inflammatory activity of L-carveol, L-carvone, and m-cimene (monoterpenes) and of valencene and guaiene (sesquiterpenes). METHODS: The influence on intracellular nitric oxide (NO) and pro- and anti-inflammatory cytokine (TNF-02±, IL-102± and IL-10) production and on nuclear factor kappa B (NF-0202B) activity was determined using Griess reagent, immunoenzymatic assay kits (ELISA) and chemiluminescence measurements in cell-based assays, respectively. Antioxidant activity was assayed through the protective effect against cellular oxidative damage produced by superoxide anion production (O 2 00·- ) and hydrogen peroxide on macrophages and by the quenching activity of the NO radical. RESULTS AND DISCUSSION: Terpenes reduced the pro-inflammatory cytokines TNF-02± and IL-102± and increased the production of IL-10. In addition, the terpenes, especially guaiene (53.309000900±0900092.4%) and m-cymene (38.109000900±0900090.6%), significantly inhibited NO production in a macrophage cell culture-based assay, whereas no effect was observed in the scavenging activity of this radical. L-carveol and m-cymene significantly inhibited O 2 00·- production with reductions of approximately 68.609000900±0900092.2% and 48.209000900±0900094.2%, respectively, at a concentration of 1000020204M. Moreover, these terpenes were verified to suppress NF-0202B activity. The results indicate that these terpenes have therapeutic potential and may be used to suppress inflammatory diseases or as a leading compounds.