| 陈海锦,丁美红,史冬玲,等.乳痈方对人乳腺上皮细胞炎症模型的体外抗炎作用及其药效物质含量测定研究[J].浙江中医药大学学报,2023,47(7):712-721. |
| 乳痈方对人乳腺上皮细胞炎症模型的体外抗炎作用及其药效物质含量测定研究 |
| Preliminary Study on Ruyong Formula Aqueous Extract Anti-inflammatory Activity in vitro and Establishment of A Method Determining the Pharmacodynamics Substances |
| DOI：10.16466/j.issn1005-5509.2023.07.002 |
| 中文关键词: 乳痈方 HBL-100 LPS 抗炎作用 LC-MS/MS 含量测定 方法学 细胞样品 |
| 英文关键词: Ruyong Formula HBL-100 LPS anti-inflammatory LC-MS/MS content determination methodology cell samples |
| 基金项目:浙江省基础公益研究计划项目(LGC20B050010) |
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| 中文摘要: |
| [目的] 以脂多糖(lipopolysaccharide，LPS)刺激人乳腺上皮细胞HBL-100建立炎症模型，研究乳痈方的抗炎作用并测定其中的药效成分含量。[方法] 以LPS刺激HBL-100细胞，通过检测一氧化氮(nitric oxide，NO)浓度、白介素-6(interleukin-6，IL-6)水平、细胞计数试剂(cell counting kit-8，CCK-8)细胞活力、肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)及白介素-1β(interleukin-1β，IL-1β)mRNA的表达，确认炎症模型的建立及抗炎活性。以HBL-100细胞为研究对象，采用超高效液相色谱-三重四级杆质谱联用法(ultra performance liquid chromatography tandem-mass spectrometry，UPLC-MS/MS)建立同时测定毛蕊异黄酮等7个成分含量的方法，并对方法的线性、基质效应、回收率、准确度、精密度和稳定性等进行研究。[结果] 成功建立HBL-100细胞炎症模型。研究发现，高剂量(1.0 mg·mL-1)乳痈方水提物对HBL-100细胞具有一定杀伤作用，中、低剂量(0.5、0.25 mg·mL-1)乳痈方水提物能够有效降低NO及IL-6水平，对HBL-100细胞炎症模型的抗炎作用显著。高剂量组中能够检测到阿魏酸、柴胡皂苷a、刺芒柄花素、甘草苷、黄芪甲苷、槲皮素及毛蕊异黄酮7个药效成分，而中、低剂量组可检测到除槲皮素及黄芪甲苷以外的5个药效成分。[结论] 建立了LPS诱导的HBL-100细胞炎症模型，并初步验证了乳痈方的抗炎活性，建立了测定HBL-100细胞中乳痈方中7个成分含量的LC-MS/MS分析方法，为后续乳痈方提取物的细胞药代动力学、乳痈方中主要抗炎成分及乳腺炎的相关细胞信号通路研究奠定了基础。 |
| 英文摘要: |
| [Objective] To establish the inflammation model and study the anti-inflammatory effect of Ruyong Formula(RYF) with lipopolysaccharide(LPS)-induced human mammary epithelial cells HBL-100， and establish a method simultaneously determining the contents of pharmacodynamics substances. [Methods] HBL-100 cells were induced by LPS to establish the inflammation model. The anti-inflammatory activity was confirmed by nitric oxide(NO) and interleukin-6(IL-6) concentration， cell counting kit-8 (CCK-8) cell viability， tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) mRNA expression. Taking HBL-100 cells as the research object， ultra performance liquid chromatography tandem-mass spectrometry(UPLC-MS/MS) was used to establish the simultaneous determination of calycosin， formononetin， ferulic acid， saikoside a ， quercetin， astragaloside Ⅳ and liquiritin methods for the content of seven components of the RYF， including linearity， matrix effect， recovery， accuracy， precision and stability. [Results] The inflammation model was successfully established， and the anti-inflammatory effect of RYF was preliminarily studied. The high concentration of RYF (1.0 mg·mL-1) aqueous extract could injure the cells， and the medium (0.5 mg·mL-1 ) and low concentrations (0.25 mg·mL-1) of RYF aqueous extract could decrease the level of NO and IL-6 efficiently， which meant they had anti-inflammation. A rapid and stable analytical method for the measurement of seven components of RYF was established in this paper. There were seven components of ferulic acid， saikoside a， formononetin， liquiritin， astragaloside Ⅳ， quercetin， calycosin assayed in high concentration of RYF group. However， there were only five components in medium and low concentrations groups except astragaloside Ⅳ and quercetin. [Conclusion] LPS-induced HBL-100 cells were used to establish an inflammation model， and the anti-inflammatory activity of RYF was preliminarily verified. A method for simultaneous determination content of components of RYF using UPLC-MS/MS was established， which could provide a reference for the study of the main anti-inflammatory components of RYF， the cellular pharmacokinetics and the related cell signaling pathway. |
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