Research Center for Traceability and Standardization of TCMs, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, PR China; Jiangsu Key Laboratory of TCM Evaluation and Translational Research, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, PR China.
Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing, 210012, PR China.
J Chromatogr A. 2023 Mar 29;1693:463859. doi: 10.1016/j.chroma.2023.463859. Epub 2023 Feb 15.
Histidine Decarboxylase (HDC), an unique enzyme responsible for the synthesis of histamine, which is an important mediator in allergy. Inhibition of HDC activity to decrease histamine production is one way to alleviate allergic symptoms. Traditional Chinese medicines (TCMs) with reported anti-allergy effect is one of important source to search for natural HDC inhibitor. Ultrafiltration combined with high-performance liquid chromatography/mass spectrometry (UF-HPLC/MS) is an effective method for screening HDC inhibitor from TCMs. Nevertheless, false-positive and false-negative results caused by the non-specific binding and the neglection of the trace active compounds are major problems in this method. In this study, an integrated strategy that combined UF-HPLC/MS with enzyme channel blocking (ECB) technique and directional enrichment (DE) technique was developed to seek natural HDC inhibitors from Radix Paeoniae alba (RPA), and at the same time, to reduce false-positive and false-negative results. HDC activity was detected to determine the validity of the screened compounds by RP-HPLC-FD in vitro. Molecular docking was applied to assay the binding affinity and binding sites. As a result, three compounds were screened from low content components of RPA after the DE. Among them, two non-specific compounds were eliminated by ECB, and the specific compound was identified as catechin, which has obvious HDC inhibition activity with IC 0.52 mM. Furthermore, gallic acid (IC 1.8 mM) and paeoniflorin (IC>2 mM) from high content components of RPA were determined having HDC inhibitory activity. In conclusion, the integrated strategy of UF-HPLC/MS combined with ECB and DE technique is an effective mode for rapid and accurate screening and identification of natural HDC inhibitors from TCMs.
组氨酸脱羧酶（HDC）是一种独特的酶，负责合成组胺，组胺是过敏反应中的重要介质。抑制 HDC 活性以减少组胺的产生是缓解过敏症状的一种方法。具有报道的抗过敏作用的中药是寻找天然 HDC 抑制剂的重要来源之一。超滤结合高效液相色谱/质谱联用（UF-HPLC/MS）是从中药中筛选 HDC 抑制剂的有效方法。然而，由于非特异性结合和忽略痕量活性化合物，该方法存在假阳性和假阴性结果的主要问题。在这项研究中，我们开发了一种将超滤结合高效液相色谱/质谱联用（UF-HPLC/MS）与酶通道阻断（ECB）技术和定向富集（DE）技术相结合的综合策略，从白芍（RPA）中寻找天然 HDC 抑制剂，同时减少假阳性和假阴性结果。通过体外反相高效液相色谱-荧光检测（RP-HPLC-FD）检测 HDC 活性来确定筛选化合物的有效性。应用分子对接测定结合亲和力和结合位点。结果，从 RPA 的低含量成分中筛选出三种化合物。其中，两种非特异性化合物通过 ECB 被消除，特异性化合物被鉴定为儿茶素，其具有明显的 HDC 抑制活性，IC 0.52 mM。此外，从 RPA 的高含量成分中鉴定出没食子酸（IC 1.8 mM）和芍药苷（IC>2 mM）具有 HDC 抑制活性。总之，超滤结合高效液相色谱/质谱联用（UF-HPLC/MS）与 ECB 和 DE 技术相结合的综合策略是一种从中药中快速、准确筛选和鉴定天然 HDC 抑制剂的有效模式。
