基于化学成分与肠道菌群探讨消癌解毒方中黄连-乌梅药对的配伍机制
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摘要
目的探讨黄连-乌梅药对与消癌解毒方水煎液中部分生物碱类成分的含量差异,并比较药对与全方对小鼠肠道菌群的影响。方法采用Extend-C18(100mm×2.1mm,1.8μm)色谱柱,以0.1%甲酸水(A)-乙腈(B)进行梯度洗脱,流速0.3mL/min,柱温35℃,进样量2μL;离子化模式为电喷雾离子化(ESI),以正离子模式检测,开展方法学验证和含量测定研究。按照给药不同将小鼠分为正常组、全方高浓度组、全方低浓度组、药对高浓度组和药对低浓度组,连续灌胃给药7d,每组分别收集粪便样本,进行16S rRNA基因测序。结果建立的含量测定方法在一定浓度范围内线性关系良好,方法学验证结果均符合相关要求,木兰花碱与非洲防己碱在黄连、药对以及全方中的含量分别为:(4.433±0.133)(8.905±0.154)mg/g,(3.545±0.033)(9.170±0.051)mg/g,(5.287±0.038)(13.861±0.690)mg/g。全方组和药对组中拟杆菌门Bacteroides的丰度均高于正常组,而全方组和药对组中厚壁菌门Firmicutes的丰度均低于正常组。药对高浓度组中的疣微菌门Verrucomicrobia和变形菌门Proteobacteria的丰度高于其他组。结论液质联用方法和相关参数简便、可靠,可用于有关成分的含量检测。与单味药中含量相比,经过药对配伍后,非洲防己碱含量增加,而木兰花碱含量则略有降低;经过全方配伍后,2种成分的含量均增加。此外,肠道菌群实验表明无论是药对还是全方在给药后,会不同程度地影响肠道菌群内部占比改变,其可能对调节肠道系统平衡,具有一定帮助作用,从而为进一步阐释黄连-乌梅在消癌解毒方中配伍机制提供了参考和依据。
OBJECTIVE To explore the difference of some alkaloids in Coptidis Rhizoma-Mume Fructus decoction and Xiaoai Jiedu Prescription,and compare their effects on the intestinal flora of mice.METHODS The separation was performed on Extend-C18 column(100 mm×2.1 mm,1.8μm)with 0.1% aqueous formic acid(A)-acetonitrile solution(B)as the mobile phase for gradient elution.The flow rate was 0.3 mL/min,column temperature was set at 35℃,and injection volume was 2μL.Electrospray ionization source was applied for the analysis under positive ion model.The method was validated and the samples were determined under this condition.50 mice were divided into ZC group,QFH group,QFL group,YDH group and YDL group according to different dosages.After 7 days of oral administration,fecal samples from each group were collected for16 S rRNA gene sequencing.RESULTS The established detection method showed good linearity within certain concentration ranges and the method validation results meet the relevant requirements.The contents of magnoflorine and columbamine in the single herb,herb pair and formula decoction were(4.433±0.133)and(8.905±0.154)mg/g,(3.545±0.033)and(9.170±0.051)mg/g,(5.287±0.038)and(13.861±0.690)mg/g,respectively.In addition,the abundance of Bacteroides in QF and YD were higher than that in the normal group,but the abundance of Firmicutes was opposite.Interestingly,we found that the abundance of Verrucomicrobia and Proteobacteriain YDH were higher than other groups.CONCLUSION The method and related parameters were simple and reliable,which could be used to detect the content of related components.Compared with single herb,the content of columbamine was increased both in herb pair and Xiaoai Jiedu Prescription;the content of magnoflorine was decreased slightly in herb pair,while increased in Xiaoai Jiedu Prescription.After administering herb decoction,the change of intestinal flora diversity revealed Coptidis Rhizoma-Mume Fructus decoction and Xiaoai Jiedu Prescription could regulate the balance of intestinal system.The results further explain the compatibility mechanism of the two drugs in this prescription.
OBJECTIVE To explore the difference of some alkaloids in Coptidis Rhizoma-Mume Fructus decoction and Xiaoai Jiedu Prescription,and compare their effects on the intestinal flora of mice.METHODS The separation was performed on Extend-C18 column(100 mm×2.1 mm,1.8μm)with 0.1% aqueous formic acid(A)-acetonitrile solution(B)as the mobile phase for gradient elution.The flow rate was 0.3 mL/min,column temperature was set at 35℃,and injection volume was 2μL.Electrospray ionization source was applied for the analysis under positive ion model.The method was validated and the samples were determined under this condition.50 mice were divided into ZC group,QFH group,QFL group,YDH group and YDL group according to different dosages.After 7 days of oral administration,fecal samples from each group were collected for16 S rRNA gene sequencing.RESULTS The established detection method showed good linearity within certain concentration ranges and the method validation results meet the relevant requirements.The contents of magnoflorine and columbamine in the single herb,herb pair and formula decoction were(4.433±0.133)and(8.905±0.154)mg/g,(3.545±0.033)and(9.170±0.051)mg/g,(5.287±0.038)and(13.861±0.690)mg/g,respectively.In addition,the abundance of Bacteroides in QF and YD were higher than that in the normal group,but the abundance of Firmicutes was opposite.Interestingly,we found that the abundance of Verrucomicrobia and Proteobacteriain YDH were higher than other groups.CONCLUSION The method and related parameters were simple and reliable,which could be used to detect the content of related components.Compared with single herb,the content of columbamine was increased both in herb pair and Xiaoai Jiedu Prescription;the content of magnoflorine was decreased slightly in herb pair,while increased in Xiaoai Jiedu Prescription.After administering herb decoction,the change of intestinal flora diversity revealed Coptidis Rhizoma-Mume Fructus decoction and Xiaoai Jiedu Prescription could regulate the balance of intestinal system.The results further explain the compatibility mechanism of the two drugs in this prescription.
引文
[1]王琼,刘沈林,严士海,等.乌梅黄连复方对人结肠癌细胞HT29增殖和迁移的影响[J].南京中医药大学学报,2014,30(6):538-541.
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[11]王雪,赵龙玉,赵凤春,等.应用Illumina高通量测序技术探究长根菇多糖对小鼠肠道菌群的影响[J].食品科学,2015,36(19):222-226.
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[13]邹忠杰,龚梦鹃,谢媛媛,等.黄连总生物碱对乙醇致大鼠胃黏膜损伤保护作用的代谢组学研究[J].中华中医药杂志,2013,28(3):807-810.
[14]郑进保.药用植物提取物在控制酒精发酵染菌和抑制肠道有害菌中的初步研究[D].厦门:厦门大学,2008.
[15]GUO S,JIANG K,WU H,et al.Magnoflorine ameliorates lipopolysaccharide-induced acute lung injury via suppressing NF-kappaB and MAPK activation[J].Front Pharmacol,2018,9:982.
[16]BAO M,CAO Z,YU D,et al.Columbamine suppresses the proliferation and neovascularization of metastatic osteosarcoma U2OS cells with low cytotoxicity[J].Toxicol Lett,2012,215(3):174-180.
[17]魏晓,刘威,袁静,等.人类肠道菌群与疾病关系的元基因组学研究进展[J].中国微生态学杂志,2011,23(1):75-80.
[18]李亚丹,任宏伟,吴彦彬,等.拟杆菌与肠道微生态[J].微生物学通报,2008,35(2):281-285.
[19]WANG T,CAI G,QIU Y,et al.Structural segregation of gut microbiota between colorectal cancer patients and healthy volunteers[J].ISME J,2012,6(2):320-329.
[2]朱萱萱,王琼,刘沈林,等.乌梅黄连复方对人结肠癌Lovo和HT29细胞株抗增殖作用的实验研究[J].中华中医药学刊,2013,31(11):2346-2348.
[3]江晓,李沙沙,张斌,等.黄连乌梅药对不同配伍比例的指纹图谱研究[J].辽宁中医药大学学报,2012,14(10):195-197.
[4]谭佳妮,石文静,沈卫星,等.消癌解毒方对结肠癌的抑制作用及对Treg细胞的影响[J].南京中医药大学学报,2017,33(5):515-518.
[5]石文静,谭佳妮,沈卫星,等.消癌解毒方对结肠癌的抑制作用及机制探讨[J].中华中医药杂志,2018,34(1):69-72.
[6]石文静,谭佳妮,沈卫星,等.消癌解毒方含药血清对人结肠癌细胞增殖及糖酵解过程的影响[J].中国实验方剂学杂志,2017,23(20):120-125.
[7]NICHOLSON JK,HOLMES E,KINROSS J,et al.Host-gut microbiota metabolic interactions[J].Science,2012,336(6086):1262-1267.
[8]KARLSSON FH,TREMAROLI V,NOOKAEW I,et al.Gut metagenome in European women with normal,impaired and diabetic glucose control[J].Nature,2013,498(7452):99-103.
[9]CANI PD.Gut microbiota and obesity:lessons from the microbiome[J].Brief Funct Genomics,2013,12(4):381-387.
[10]GEVERS D,KUGATHASAN S,DENSON LA,et al.The treatment-naive microbiome in new-onset Crohn's disease[J].Cell Host Microbe,2014,15(3):382-392.
[11]王雪,赵龙玉,赵凤春,等.应用Illumina高通量测序技术探究长根菇多糖对小鼠肠道菌群的影响[J].食品科学,2015,36(19):222-226.
[12]贾盛佼,王莉莉,于新娟,等.肠道菌群参与结直肠癌发生的模式[J].世界华人消化杂志,2013,21(9):779-784.
[13]邹忠杰,龚梦鹃,谢媛媛,等.黄连总生物碱对乙醇致大鼠胃黏膜损伤保护作用的代谢组学研究[J].中华中医药杂志,2013,28(3):807-810.
[14]郑进保.药用植物提取物在控制酒精发酵染菌和抑制肠道有害菌中的初步研究[D].厦门:厦门大学,2008.
[15]GUO S,JIANG K,WU H,et al.Magnoflorine ameliorates lipopolysaccharide-induced acute lung injury via suppressing NF-kappaB and MAPK activation[J].Front Pharmacol,2018,9:982.
[16]BAO M,CAO Z,YU D,et al.Columbamine suppresses the proliferation and neovascularization of metastatic osteosarcoma U2OS cells with low cytotoxicity[J].Toxicol Lett,2012,215(3):174-180.
[17]魏晓,刘威,袁静,等.人类肠道菌群与疾病关系的元基因组学研究进展[J].中国微生态学杂志,2011,23(1):75-80.
[18]李亚丹,任宏伟,吴彦彬,等.拟杆菌与肠道微生态[J].微生物学通报,2008,35(2):281-285.
[19]WANG T,CAI G,QIU Y,et al.Structural segregation of gut microbiota between colorectal cancer patients and healthy volunteers[J].ISME J,2012,6(2):320-329.
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