不同生长方式防风的HPLC特征图谱和体外抗炎活性比较研究

HPLC Specific Chromatograms of Saposhnikoviae Radix with Different Growing Methods and Comparison of the in Vitro Anti-inflammatory Activity

目的:建立不同生长方式防风的特征图谱,并比较其体外抗炎活性。方法:采用高效液相色谱法(HPLC),AgilentZORBAXSB-C_(18)色谱柱(250 mm×4.6 mm,5μm),流动相为甲醇(A)-水(B),梯度洗脱,流速为1.0 mL·min^(−1),检测波长为254 nm;对54批防风样品的HPLC结果进行分析;评价不同生长方式防风的水提物对脂多糖(LPS)诱导小鼠单核巨噬细胞RAW264.7分泌一氧化氮(NO)和肿瘤坏死因子-α(TNF-α)2种炎症因子的影响。结果:建立了野生、多年生栽培及1年生栽培3种不同生长方式防风的HPLC特征图谱,确定了7个特征峰的相对保留时间,指认了特征图谱中升麻素苷、升麻素、5-O-甲基维斯阿米醇苷、亥茅酚苷4个特征峰,并对3种不同生长方式防风的特征图谱进行比较。3种不同生长方式防风均可抑制LPS诱导的小鼠RAW264.7细胞分泌NO和TNF-α,抗炎作用强度为野生>多年生栽培>1年生栽培,其中野生防风与多年生栽培防风作用差异无统计学意义,但两者与1年生栽培防风差异有统计学意义(P<0.01)。结论:建立的HPLC特征图谱可通过升麻素的相对峰面积对野生防风与栽培防风进行鉴别;防风发挥抗炎作用不仅与色原酮类成分有关,还与挥发油等其他成分有关,为科学评价防风饮片质量提供参考。

Objective:To establish the high performace liquid chromatography(HPLC)specific chromatograms of Saposhnikoviae Radix with different growing methods and compare their anti-inflammatory effects in vitro.Methods:HPLC conditions are as below:Agilent ZORBAX SB-C_(18) column(250 mm×4.6 mm,5μm),gradient elution with methanol(A)-water(B)at the flow rate of 1.0 mL·min^(−1),detection wavelength of 254 nm.The HPLC results of 54 batches of Saposhnikoviae Radix were analyzed.Furthermore,the effects of water extract of different Saposhnikoviae Radix samples on the secretion of nitric oxide(NO)and tumor necrosis factor-alpha(TNF-α)by lipopolysaccharide(LPS)-stimulated mouse RAW264.7 cells were evaluated.Results:The specific chromatograms of wild,annual,and perennial Saposhnikoviae Radix samples were respectively established and the relative retention time of seven characteristic peaks was determined.Four characteristic peaks were respectively identified as prim-O-glucosylcinmifugin,4'-O-β-D-glucosyl-5-O-methylvisamminol,cinmifugin,and sec-O-glucosylhamaudol.All the samples were verified to inhibit the secretion of NO and TNF-αby LPS-stimulated mouse RAW264.7 cells and the anti-inflammatory activity was in the order of wild Saposhnikoviae Radix>perennial Saposhnikoviae Radix>annual Saposhnikoviae Radix,with insignificant difference between the former two and significant different between the former two and annual Saposhnikoviae Radix.Conclusion:According to the relative peak area of cimicifugin in HPLC specific chromatograms,wild Saposhnikoviae Radix can be discriminated from that derived from cultivated Saposhnikoviae Radix.The anti-inflammatory activity of Saposhnikoviae Radix is related to the chromones,volatile oils,and other components.The result lays a basis for scientific evaluation of the quality of Saposhnikoviae Radix decoction pieces.