无刺蜂蜂胶乙醇提取物的体外抗氧化及抗炎活性
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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-IκΒα和IκΒα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284.00μg·mL-1。EEHI对RAW 264.7细胞的安全浓度为0—40μg·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—40μg·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—40μg·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IκΒα蛋白的磷酸化,且40μg·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。
【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-IκBα and IκΒα 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 284.00μg·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 IκΒα 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.
【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-IκBα and IκΒα 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 284.00μg·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 IκΒα 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.
引文
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[2]ISMAIL M M.The potential of heterotrigona farming for a high income agro-entrepreneur project in Malaysia.OPTIONS,2016,20(1):12-17.
[3]SANCHES M A,PEREIRA A M S,SERR?O J E.Pharmacological actions of extracts of propolis of stingless bees(Meliponini).Journal of Apicultural Research,2017,56(1):1-8.DOI:10.1080/00218839.2016.1260856.
[4]DUTRA R P,DE BARROS ABREU B V,CUNHA M S,BATISTA MC A,TORRES L M B,NASCIMENTO F R F,RIBIRO M N S,GUERRA R N M.Phenolic acids,hydrolyzable tannins,and antioxidant activity of geopropolis from the stingless bee Melipona fasciculata Smith.Journal of Agricultural and Food Chemistry,2014,62(12):2549-2557.DOI:10.1021/jf404875v.
[5]ARAúJO M J A M,BOSCO S D M G,SFORCIN J M.Pythium insidiosum:inhibitory effects of propolis and geopropolis on hyphal growth.Brazilian Journal of Microbiology,2016,47(4):863-869.DOI:10.1016/j.bjm.2016.06.008.
[6]LIBERIO S A,PEREIRA A L A,DUTRA R P,REIS A S,ARAúJO MJ A M,Mattar N S,Silva L A,Ribeiro M N S,Nascimento F R F,GUERRA R N M,MONTEIRO-NETO V.Antimicrobial activity against oral pathogens and immunomodulatory effects and toxicity of geopropolis produced by the stingless bee Melipona fasciculata Smith.BMC Complementary and Alternative Medicine,2011,11:108.DOI:10.1186/1472-6882-11-108.
[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.http://www.researchgate.net/publication/297046720.
[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.
[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 ID296086.DOI:10.1155/2015/296086.
[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.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)
[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.
[12]ZHAO L L,YU M J,SUN M H,XUE X F,WANG T T,CAO W,SUNL 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.
[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.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)
[14]WANG K,HU L,JIN X L,MA Q X,MARCUCCI M C,NETTO A AL,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.
[15]BANG S Y,KIM J H,KIM H Y,LEE Y J,PARK S Y,LEE S J,KIMY H.Achyranthes japonica exhibits anti-inflammatory effect via NF-κΒsuppression and HO-1 induction in macrophages.Journal of Ethnopharmacology,2012,144(1):109-117.DOI:10.1016/j.jep.2012.08.037.
[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-1βand TNF-α.Journal of Ethnopharmacology,2012,143(2):709-715.DOI:10.1016/j.jep.2012.07.040.
[17]FRANCHIN M,ROSALEN P L,DA CUNHA M G,SILVA R L,COLóN 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.
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