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基于脂多糖诱导RAW264.7细胞模型的白茶寡肽分段物抗炎能力评估

In Vitro Anti-inflammatory Activity of White Tea Oligopeptide Fractions

    摘要
  • 摘要:
    目的 探究不同性质（分子量大小、亲疏水性、带电性质）白茶寡肽与抗炎活性的潜在关联，为白茶抗炎肽开发提供基础数据。
    方法 采用Sephadex G-15柱、C18固相萃取柱、强阴离子交换柱、强阳离子交换柱4种分离纯化方式，以分子量大小、亲疏水性、正负带电性将白茶寡肽粗提物进行分段。建立脂多糖诱导的RAW264.7细胞炎症模型，通过测定NO、TNF-α和IL-1β炎症指标来评估各白茶寡肽分段物的抗炎活性，从中筛选具有最强抗炎活性的分段物，并使用超高效液相色谱-质谱联用仪对其进行寡肽序列鉴定和物质构成分析。
    结果 不同性质白茶寡肽的抗炎活性强度为：低分子量分段物＞中、高分子量分段物；疏水性分段物＞亲水性分段物；负电性质分段物＞正电性质分段物，其中低分子量分段物G15-3(MD)抗炎活性最强，与地塞米松（阳性对照药）无显著差异（P＞0.05），对NO、TNF-α和IL-1β的抑制率分别为（34.21±2.53）%、（52.36±6.24）%、（76.43±1.41）%。对最强抗炎活性分段物G15-3(MD)鉴定得到5条寡肽序列。
    结论 白茶寡肽分段物抗炎活性强弱如下：分子量大小＞带电性质＞亲疏水性，低分子量分段物G15-3(MD)抗炎活性最强。N端和C端疏水性氨基酸和碱性氨基酸的存在对抗炎活性有重要贡献，发现两条潜在抗炎活性肽KRW和FGGN。

     

    Abstract:
    Objective Anti-inflammatory activity of oligopeptide fractions in white tea was evaluated by the lipopolysaccharide-induced RAW264.7 cell method.
    Method The oligopeptides extract of Shoumei and Baimudan White Teas were separated and purified in fractions by differential molecular weight, hydrophilicity, and electric charge using Sephadex G-15 chromatography, C18 solid-phase extraction, and strong anion and cation chromatography. Lipopolysaccharide-induced RAW264.7 cells were used in an in vitro test to determine the anti-inflammatory activity of the fractions based on their inhibition rates against NO, TNF-α, and IL-1β. Oligopeptide fractions showing significant anti-inflammatory potentials were sequenced and chemically analyzed by UPLC-Q-Orbitrap-MS.
    Result The oligopeptide fractions of low molecular weight were higher in the anti-inflammatory activity than those of medium or high molecular weight. The hydrophobic fractions were higher in the activity than the hydrophilic ones. And the negatively charged fractions were higher in the activity than the positively charged fractions. Of all the selected fractions, the low molecular weight, hydrophobic G15-3(MD) with basic amino acids at the N- and C-termini showed the strongest anti-inflammatory effect that was not significantly different from what exerted by dexamethasone (P>0.05). It contained 5 bands in the sequence and displayed significant inhibition rates on NO, TNF-α, and IL-1β at (34.21±2.53)%, (52.36±6.24)%, and (76.43±1.41)%, respectively.
    Conclusion Molecular weight of the oligopeptide fractions isolated from the two varieties of white tea seemed to be the most critical factor, followed by electric charge and hydrophilicity/polarity, that correlated with the in vitro anti-inflammatory effect on the RAW264.7 cells. And the low molecular weight, hydrophobic G15-3(MD) was found to be strongly anti-inflammatory like the commonly used drug, dexamethasone, for inflammation. It was postulated that the KRW and FGGN peptides in G15-3(MD) might contribute significantly to the outstanding activity.

     

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