UF-HPLC–MS Screening of Histamine Inhibitors in Traditional Chinese Medicines Reduces False Positives and False Negatives

With allergies weighing on increasingly foggy minds this spring, research financed by the National Natural Science Foundation of China and published in the Journal of Chromatography A, by Jin Qi and a team of researchers from the China Pharmaceutical University in Nanjing, PR China, attempted to find answers in Radix Paeoniae alba (RPA), also known as Chinese peony, which is used in select traditional Chinese herbal formulas as an allergy treatment (1).

Flower of pink peony with yellow stamens | Image Credit: © ArtCookStudio - stock.adobe.com

In the study, the combination of ultrafiltration with high performance liquid chromatography–mass spectrometry (UF-HPLC–MS), along with enzyme channel blocking and directional enrichment, was found effective for screening natural histidine decarboxylase (HDC) inhibitors in RPA, while reducing both false-positive and false-negative results.

The combined methods utilized in the research yielded several viable compounds for the inhibition of HDC, a unique enzyme that serves as a catalyst for histamine production, and for which traditional Chinese medicines (TCM) like Dang Gui Yin Zi and Fang Feng Tong Sheng San are considered important sources. Up to now, natural HDC inhibitors–including ones found in cinnamon bark and strawberry cultivars–have been detected mostly via a time-consuming and complicated extraction-separation-identification process.

Researchers are now finding that UF-HPLC–MS is a faster and simpler method, and an effective one. UF-HPLC-MS combines ultrafiltration, which separates molecules based on their size, with HPLC, which separates molecules based on their chemical properties, followed by mass spectrometry detection. This technique allows for the efficient separation, identification, and quantification of complex mixtures of compounds, providing valuable insights into sample composition and aiding in the discovery and characterization of unknown substances.

However, UF-HPLC–MS can produce both false-positive and false-negative readings as a result of non-specific binding of molecules and target proteins. To combat the false positives using this method, the researchers introduced enzyme channel blocking (ECB), by applying a known inhibitor to the TCMs prior to the incubation of their extracts and proteins. However, because the content values of compounds may vary during screening, directional enrichment (DE) was also applied to categorize high- and low-content compounds and reduce false negatives.

These contemporaneous methods, concluding with DE, identified three compounds among low-content components of RPA that showed HDC inhibitory activity. ECB eliminated two of the three, leaving catechin as a significant result (IC50 0.52 mM). Looking at high-content components, gallic acid (IC50 1.8 mM) and paeoniflorin (IC50 > 2 mM) were also identified as inhibitors within RPA.

Whether these compounds are put to effective use to tamp down histamine overproduction remains to be seen, but this research at least suggests the possibility that natural inhibitors may one day help to alleviate annoying allergy symptoms.

Reference

(1) Hu, G.; Wang, L.; Li, X.; Qi, J. Rapidly and accurately screening histidine decarboxylase inhibitors from Radix Paeoniae alba using ultrafiltration-high performance liquid chromatography/mass spectrometry combined with enzyme channel blocking and directional enrichment technique. J. Chromatogr. A 2023, 1693, 463859. DOI: 10.1016/j.chroma.2023.463859