假蒟提取物的体外抗氧化和抗炎效果研究
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摘要
本研究旨在分析假蒟提取物的活性成分并评价其体外抗氧化和抗炎活性。试验通过ABTS法检测3种假蒟提取物(石油醚、正丁醇及乙酸乙酯提取物)的抗氧化能力;采用LPS法建立猪小肠上皮细胞(IPEC-J2)体外炎症模型,用假蒟提取物预处理后,以ELISA法检测细胞上清液中肿瘤坏死因子(TNF-α)、白细胞介素-1(IL-1)和白细胞介素-6(IL-6)的含量。结果表明,3种假蒟提取物总抗氧化活性由大到小依次是假蒟正丁醇提取物、乙酸乙酯提取物、石油醚提取物(P<0.05),其中假蒟正丁醇提取物总生物碱、总酚含量最高,分别为2.81 mEq/100 g、161.82 mg/g。与空白组相比,LPS组IPEC-J2细胞的TNF-α(P<0.05)、IL-1(P>0.05)及IL-6(P<0.05)含量均升高;与LPS组相比,假蒟预处理组的这几种细胞因子含量均显著降低(P<0.05),由高到低依次为乙酸乙酯提取物>石油醚提取物>正丁醇提取物。综合试验结果,假蒟提取物具有一定的抗氧化作用,能影响IPEC-J2细胞炎性因子的分泌,抑制炎症反应。
This study was aimed to investigate the active ingredient and evaluate the antioxidant and anti-inflammatory activities of Piper sarmentosum Roxb. extracts in vitro.The antioxidant activities of three pepper extracts(petroleum ether,n-butanol and ethyl acetate) in vitro were determined by ABTS method.The inflammation model of IPEC-J2 cell was established with LPS in vitro,the levels of tumor necrosis factor(TNF-α),interleukin-1(IL-1) and interleukin-6(IL-6) were detected in cell supernatant by ELISA after pretreatment of Piper sarmentosum Roxb. extract.The results showed that the total antioxidant activity of Piper sarmentosum Roxb. extract in descending order were n-butanol extract,ethyl acetate extract and petroleum ether extract(P<0.05).The contents of total alkaloids and total phenols of Piper sarmentosum Roxb. n-butanol extract were the highest,which were 2.81 mEq/100 g and 161.82 mg/g,respectively.In anti-inflammatory experiments,compared with the blank group,the levels of TNF-α(P<0.05),IL-1(P>0.05) and IL-6(P<0.05) in IPEC-J2 cells increased in LPS group,while compared with the Piper sarmentosum Roxb. pretreatment groups,these indexes in LPS group decreased significantly(P<0.05).The concentrations of cytokines from high to low were ethyl acetate extract,petroleum ether extract and n-butanol extract.In conclusion,the extract of Piper sarmentosum Roxb. had antioxidant activity,and it could affect the secretion of cytokines in IPEC-J2 cells and inhibit the inflammation.
This study was aimed to investigate the active ingredient and evaluate the antioxidant and anti-inflammatory activities of Piper sarmentosum Roxb. extracts in vitro.The antioxidant activities of three pepper extracts(petroleum ether,n-butanol and ethyl acetate) in vitro were determined by ABTS method.The inflammation model of IPEC-J2 cell was established with LPS in vitro,the levels of tumor necrosis factor(TNF-α),interleukin-1(IL-1) and interleukin-6(IL-6) were detected in cell supernatant by ELISA after pretreatment of Piper sarmentosum Roxb. extract.The results showed that the total antioxidant activity of Piper sarmentosum Roxb. extract in descending order were n-butanol extract,ethyl acetate extract and petroleum ether extract(P<0.05).The contents of total alkaloids and total phenols of Piper sarmentosum Roxb. n-butanol extract were the highest,which were 2.81 mEq/100 g and 161.82 mg/g,respectively.In anti-inflammatory experiments,compared with the blank group,the levels of TNF-α(P<0.05),IL-1(P>0.05) and IL-6(P<0.05) in IPEC-J2 cells increased in LPS group,while compared with the Piper sarmentosum Roxb. pretreatment groups,these indexes in LPS group decreased significantly(P<0.05).The concentrations of cytokines from high to low were ethyl acetate extract,petroleum ether extract and n-butanol extract.In conclusion,the extract of Piper sarmentosum Roxb. had antioxidant activity,and it could affect the secretion of cytokines in IPEC-J2 cells and inhibit the inflammation.
引文
[1] 张恒慧,尤建嵩,徐永平,等.抗仔猪腹泻产肠毒素大肠杆菌疫苗的研究进展[J].中国畜牧兽医,2015,42(2):347-351. ZHANG H H,YOU J S,XU Y P,et al.Research progress on ETEC vaccines against piglet diarrhea[J].China Animal Husbandry & Veterinary Medicine,2015,42(2):347-351.(in Chinese)
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[4] 龚凌霄,倪勤学,张英.植物抗毒素研究进展及其作为食品功能性成分的应用前景[J].天然产物研究与开发,2012,24(3):421-425. GONG L X,Ni Q X,ZHANG Y.Research on phytoalexins and its application prospect as a food functional component[J].Natural Product Research and Development,2012,24(3):421-425.
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[9] LEI D,CHANN C,WANG Y J,et al.Antioxidative and antiplatelet effects of aqueous inflorescence piper betle extract[J].Journal of Agricultural and Food Chemistry,2003,51(7):2083-2088.
[10] 么春艳.胡椒科植物大叶蒟镇痛、抗抑郁、抗焦虑和毒性研究[D].上海:复旦大学,2009. MO C Y.Antionciceptive,antidepressant,anxiolytic and toxicity studies on Piper laetispicum C.DC[D].Shanghai:Fudan University,2009.(in Chinese)
[11] 潘胜利,解静,钱伏刚,等.大叶蒟中抗抑郁酰胺类化合物[J].药学学报,2005,40(4):355-357. PAN S L,JIE J,QIAN F G,et al.Antidepressant amides from Piper laetispicum C.DC[J].Acta polyphylla,Acta pharmaceutics Sinica,2005,40(4):355-357.(in Chinese)
[12] 王道平,危莉,彭小冰,等.顶空固相微萃取-气质联用法分析新鲜假蒟挥发性化学成分[J].中国实验方剂学杂志,2013,19(10):142-145. WANG D P,WEI L,PENG X B,et al.SPME-GC-MS analysis of volatile constituents from fresh Piper sarmentosum[J].Chinese Journal of Experimental Traditional Medical Formulae,2013,19(10):142-145.(in Chinese)
[13] 周璐丽,王定发,周汉林.假蒟的研究进展[J].热带农业科学,2015,35(5):69-72. ZHOU L L,WANG D F,ZHOU H L.Research advances of Pipe sarmentosum Roxb.[J].Chinese Journal of Tropical Agriculture,2015,35(5):69-72.(in Chinese)
[14] SIREERATAWONG S,VANNASIRI S,SRITIWONG S,et al.Antiinflammatory,anti-nociceptive and antipyretic effects of the ethanol extract from root of Piper sarmentosum Roxb.[J].Journal of the Medical Association of Thailand,2010,93:S1-S6.
[15] 王定发,周璐丽,周雄,等.假蒟提取物对断奶仔猪生长性能、血液指标的影响[J].动物营养学报,2015,27(10):3233-3240. WANG D F,ZHOU L L,ZHOU X,et al.Effects of Piper sarmentosum extracts on growth performance and blood indexes of weaned piglets[J].Chinese Journal of Animal Nutrition,2015,27(10):3233-3240.(in Chinese)
[16] PARK Y K,KOO M H,IKEGAKI M,et al.Comparison of the flavonoid aglycone content of Apis mellifera propolis from various regions of Brazil[J].Arquivos De Biologia E Technologia,1997,40(1):97-106.
[17] SINGLETON V L,ROSSI J A.Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents[J].American Journal of Enology and Viticulture,1965,16:144-158.
[18] 张江涛,王喆之.山茱萸生物碱提取及含量测定的初步研究[A].药物分析论坛[C].2003. ZHANG J T,WANG Z Z.Preliminary study on extraction and content determination of Cornus officinalis alkaloids[A].Pharmaceutical Analysis Forum[C].2003.(in Chinese)
[19] 何军,祝林,奉建芳.附子总生物碱含量测定方法比较[J].现代中药研究与实践,2003,6:20-22. HE J,ZHU L,FENG J F.Comparison on determination of total alkaloids from Radix aconiti lateralis preparata by titration and UV[J].Research and Practice of Chinese Medicines,2003,6:20-22.(in Chinese)
[20] MATEVSKI V,MARIN P D,ALIMPI■ A,et al.Composition and biological effects of Salvia ringens (Lamiaceae) essential oil and extracts[J].Industrial Crops and Products,2015,76:702-709.
[21] 谭榀新,叶涛,刘湘新,等.植物提取物抗氧化成分及机理研究进展[J].食品科学,2010,31(15):288-292. TAN P X,YE T,LIU X X,et al.Research advances in antioxidant composition of botanical extracts and their action mechanisms[J].Food Science,2010,31(15):288-292.(in Chinese)
[22] MAI T T,FUMIE N,CHUYEN N V.Antioxidant activities and hypolipidemic effects of an aqueous extract from flower buds of Cleistocalyx operculatus (Roxb.) merr.and perry[J].Journal of Food Biochemistry,2009,33(6):790-807.
[23] 吴桂苹,黄菲菲,李恒,等.胡椒总多酚的提取及其抗氧化活性研究[J].农学学报,2016,6(11):67-74. WU G H,HUANG F F,LI H,et al.Extraction and antioxidant activity of total polyphenol from black pepper (Piper nigrum L.)[J]. Journal of Agriculture,2016,6 (11):67-74.(in Chinese)
[24] 黄艳,周璐丽,王定发,等.番石榴叶提取物体外抑菌和抗氧化作用的研究[J].黑龙江畜牧兽医,2017,21:192-194. HUANG Y,ZHOU L L,WANG D F,et al.Antibacterial and antioxidant activity of the Psidium guaijava L.leaf extracts in vitro[J].Heilongjiang Animal Science and Veterinary Medicine,2017,21:192-194.(in Chinese)
[25] HUSSAIN K,ISMAIL Z,SADIKUN A,et al.Antioxidant,anti-TB activities,phenolic and amide contents of standardised extracts of Piper sarmentosum Roxb.[J].Natural Product Research,2009,23(3):238.
[26] FERNANDES M R V,DIAS A L T,CARVALHO R R,et al.Antioxidant and antimicrobial activities of Psidium guajava L.spray dried extracts[J].Industrial Crops and Products,2014,60(1):39-44.
[27] ASADI S,AHMADIANI A,ESMAEILI M A,et al.In vitro antioxidant activities and an investigation of neuroprotection by six salvia species from Iran:Comparative study[J]. Food and Chemical Toxicology,2010,48(5):1341.
[28] STAGOS D,PORTESIS N,SPANOU C,et al.Correlation of total polyphenolic content with antioxidant and antibacterial activity of 24 extracts from Greek domestic Lamiaceae species[J].Food and Chemical Toxicology,2012,50(11):4115.
[29] WIBOOL R,PEERATI R.Studies of the anti-inflammatory and antipyretic activities of the methanolic extract of Piper sarmentosum Roxb.leaves in rats[J]. Songklanakarin Journal of Science and Technology,2007,29(6):1519-1526.
[30] 王梦远,郭晓,刘晓婷,等.中药生物碱的提取及抗炎作用研究[J].动物医学进展,2017,38(4):80-83. WANG M Y,GUO X,LIU X T,et al.Study on the alkaloid extract from traditional chinese medicine and anti-inflammatory effect[J].Progress in Veterinary Medicine,2017,38(4):80-83.(in Chinese)
[31] 张明发,沈雅琴.苦参碱类生物碱抗菌药理作用的研究进展[J].抗感染药学,2018,15(3):369-374. ZHANG M F,SHEN Y Q.Research advances on anti-bacteria pharmacological effects of matrine-type alkaloids[J].Anti-infection Pharmacy,2018,15 (3):369-374.(in Chinese)
[32] 梁馨文,李强强,高景林,等.海南无刺蜂蜂蜜中多酚类物质成分分析及其抗氧化、抗炎活性评价[J].食品科学,2018,39(8):141-148. LIANG X W,LI Q Q,GAO J L,et al.Polyphenic constituents and antioxidant and anti-inflammatory activities of stingless bee honey from Hainan[J].Food Science,2018,39(8):141-148.(in Chinese)
[2] 蒋磊,周明旭,夏芃芃,等.产肠毒素大肠杆菌感染的分子致病机制[J].中国兽医学报,2014,34(9):1551-1560. JIANG L,ZHOU M X,XIA P P,et al.Molecular pathogenic mechanisms of enterotoxigenic Escherichia coli infection[J].Chinese Journal of Veterinary Science,2014,34(9):1551-1560.(in Chinese)
[3] 曹承华,贺雅静,高荧苒,等.LPS介导的炎症反应过程及作用机制[J].河南大学学报(医学版),2017,36(1):70-76. CAO C H,HE Y J,GAO Y R,et al.LPS Mediated inflammatory response and mechanism[J].Journal of Henan University(Medical Science),2017,36(1):70-76.(in Chinese)
[4] 龚凌霄,倪勤学,张英.植物抗毒素研究进展及其作为食品功能性成分的应用前景[J].天然产物研究与开发,2012,24(3):421-425. GONG L X,Ni Q X,ZHANG Y.Research on phytoalexins and its application prospect as a food functional component[J].Natural Product Research and Development,2012,24(3):421-425.
[5] 金立志.植物提取物添加剂在动物营养中的应用及其机制的研究进展[J].动物营养学报,2010,22(5):1154-1164. JIN L Z.Phytogenic feed additives:Nutritional functions and mechanism of action in monogastric animals[J].Chinese Journal of Animal Nutrition, 2010,22 (5):1154-1164.(in Chinese)
[6] 国家中医药管理局.中华本草第3卷.[M].上海:上海科学技术出版社,1999. NATIONAL EDITORIAL BOARD OF CHINESE MATERIA MEDICA.The Third Volume of Chinese Materia Medica[M].Shanghai:Shanghai Scientific and Technical Publishers,1999.(in Chinese)
[7] 冯岗,袁恩林,张静,等.假蒟中胡椒碱的分离鉴定及杀虫活性研究[J].热带作物学报,2013,34(11):2246-2250. FENG G,YUAN E L,ZHANG J,et al.Isolation and identification of piperine from Piper sarmentosum and its insecticidal activity[J].Chinese Journal of Tropical Crops,2013,34 (11):2246-2250.(in Chinese)
[8] HAFIZAH A H,ZAITON Z,ZULKHAIRI A,et al.Piper sarmentosum as an antioxidant on oxidative stress in human umbilical vein endothelial cells induced by hydrogen peroxide[J].Journal of Zhejiang University Science B,2010,11:357-365.
[9] LEI D,CHANN C,WANG Y J,et al.Antioxidative and antiplatelet effects of aqueous inflorescence piper betle extract[J].Journal of Agricultural and Food Chemistry,2003,51(7):2083-2088.
[10] 么春艳.胡椒科植物大叶蒟镇痛、抗抑郁、抗焦虑和毒性研究[D].上海:复旦大学,2009. MO C Y.Antionciceptive,antidepressant,anxiolytic and toxicity studies on Piper laetispicum C.DC[D].Shanghai:Fudan University,2009.(in Chinese)
[11] 潘胜利,解静,钱伏刚,等.大叶蒟中抗抑郁酰胺类化合物[J].药学学报,2005,40(4):355-357. PAN S L,JIE J,QIAN F G,et al.Antidepressant amides from Piper laetispicum C.DC[J].Acta polyphylla,Acta pharmaceutics Sinica,2005,40(4):355-357.(in Chinese)
[12] 王道平,危莉,彭小冰,等.顶空固相微萃取-气质联用法分析新鲜假蒟挥发性化学成分[J].中国实验方剂学杂志,2013,19(10):142-145. WANG D P,WEI L,PENG X B,et al.SPME-GC-MS analysis of volatile constituents from fresh Piper sarmentosum[J].Chinese Journal of Experimental Traditional Medical Formulae,2013,19(10):142-145.(in Chinese)
[13] 周璐丽,王定发,周汉林.假蒟的研究进展[J].热带农业科学,2015,35(5):69-72. ZHOU L L,WANG D F,ZHOU H L.Research advances of Pipe sarmentosum Roxb.[J].Chinese Journal of Tropical Agriculture,2015,35(5):69-72.(in Chinese)
[14] SIREERATAWONG S,VANNASIRI S,SRITIWONG S,et al.Antiinflammatory,anti-nociceptive and antipyretic effects of the ethanol extract from root of Piper sarmentosum Roxb.[J].Journal of the Medical Association of Thailand,2010,93:S1-S6.
[15] 王定发,周璐丽,周雄,等.假蒟提取物对断奶仔猪生长性能、血液指标的影响[J].动物营养学报,2015,27(10):3233-3240. WANG D F,ZHOU L L,ZHOU X,et al.Effects of Piper sarmentosum extracts on growth performance and blood indexes of weaned piglets[J].Chinese Journal of Animal Nutrition,2015,27(10):3233-3240.(in Chinese)
[16] PARK Y K,KOO M H,IKEGAKI M,et al.Comparison of the flavonoid aglycone content of Apis mellifera propolis from various regions of Brazil[J].Arquivos De Biologia E Technologia,1997,40(1):97-106.
[17] SINGLETON V L,ROSSI J A.Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents[J].American Journal of Enology and Viticulture,1965,16:144-158.
[18] 张江涛,王喆之.山茱萸生物碱提取及含量测定的初步研究[A].药物分析论坛[C].2003. ZHANG J T,WANG Z Z.Preliminary study on extraction and content determination of Cornus officinalis alkaloids[A].Pharmaceutical Analysis Forum[C].2003.(in Chinese)
[19] 何军,祝林,奉建芳.附子总生物碱含量测定方法比较[J].现代中药研究与实践,2003,6:20-22. HE J,ZHU L,FENG J F.Comparison on determination of total alkaloids from Radix aconiti lateralis preparata by titration and UV[J].Research and Practice of Chinese Medicines,2003,6:20-22.(in Chinese)
[20] MATEVSKI V,MARIN P D,ALIMPI■ A,et al.Composition and biological effects of Salvia ringens (Lamiaceae) essential oil and extracts[J].Industrial Crops and Products,2015,76:702-709.
[21] 谭榀新,叶涛,刘湘新,等.植物提取物抗氧化成分及机理研究进展[J].食品科学,2010,31(15):288-292. TAN P X,YE T,LIU X X,et al.Research advances in antioxidant composition of botanical extracts and their action mechanisms[J].Food Science,2010,31(15):288-292.(in Chinese)
[22] MAI T T,FUMIE N,CHUYEN N V.Antioxidant activities and hypolipidemic effects of an aqueous extract from flower buds of Cleistocalyx operculatus (Roxb.) merr.and perry[J].Journal of Food Biochemistry,2009,33(6):790-807.
[23] 吴桂苹,黄菲菲,李恒,等.胡椒总多酚的提取及其抗氧化活性研究[J].农学学报,2016,6(11):67-74. WU G H,HUANG F F,LI H,et al.Extraction and antioxidant activity of total polyphenol from black pepper (Piper nigrum L.)[J]. Journal of Agriculture,2016,6 (11):67-74.(in Chinese)
[24] 黄艳,周璐丽,王定发,等.番石榴叶提取物体外抑菌和抗氧化作用的研究[J].黑龙江畜牧兽医,2017,21:192-194. HUANG Y,ZHOU L L,WANG D F,et al.Antibacterial and antioxidant activity of the Psidium guaijava L.leaf extracts in vitro[J].Heilongjiang Animal Science and Veterinary Medicine,2017,21:192-194.(in Chinese)
[25] HUSSAIN K,ISMAIL Z,SADIKUN A,et al.Antioxidant,anti-TB activities,phenolic and amide contents of standardised extracts of Piper sarmentosum Roxb.[J].Natural Product Research,2009,23(3):238.
[26] FERNANDES M R V,DIAS A L T,CARVALHO R R,et al.Antioxidant and antimicrobial activities of Psidium guajava L.spray dried extracts[J].Industrial Crops and Products,2014,60(1):39-44.
[27] ASADI S,AHMADIANI A,ESMAEILI M A,et al.In vitro antioxidant activities and an investigation of neuroprotection by six salvia species from Iran:Comparative study[J]. Food and Chemical Toxicology,2010,48(5):1341.
[28] STAGOS D,PORTESIS N,SPANOU C,et al.Correlation of total polyphenolic content with antioxidant and antibacterial activity of 24 extracts from Greek domestic Lamiaceae species[J].Food and Chemical Toxicology,2012,50(11):4115.
[29] WIBOOL R,PEERATI R.Studies of the anti-inflammatory and antipyretic activities of the methanolic extract of Piper sarmentosum Roxb.leaves in rats[J]. Songklanakarin Journal of Science and Technology,2007,29(6):1519-1526.
[30] 王梦远,郭晓,刘晓婷,等.中药生物碱的提取及抗炎作用研究[J].动物医学进展,2017,38(4):80-83. WANG M Y,GUO X,LIU X T,et al.Study on the alkaloid extract from traditional chinese medicine and anti-inflammatory effect[J].Progress in Veterinary Medicine,2017,38(4):80-83.(in Chinese)
[31] 张明发,沈雅琴.苦参碱类生物碱抗菌药理作用的研究进展[J].抗感染药学,2018,15(3):369-374. ZHANG M F,SHEN Y Q.Research advances on anti-bacteria pharmacological effects of matrine-type alkaloids[J].Anti-infection Pharmacy,2018,15 (3):369-374.(in Chinese)
[32] 梁馨文,李强强,高景林,等.海南无刺蜂蜂蜜中多酚类物质成分分析及其抗氧化、抗炎活性评价[J].食品科学,2018,39(8):141-148. LIANG X W,LI Q Q,GAO J L,et al.Polyphenic constituents and antioxidant and anti-inflammatory activities of stingless bee honey from Hainan[J].Food Science,2018,39(8):141-148.(in Chinese)