Identification of phosphodiesterase type-5 (PDE-5) inhibitors in herbal supplements using a tiered approach and associated consumer risk

The use of herbal supplements for improved sexual performance is a common practice amongst the youth and some senior citizens in Ghana. These products are considered ‘natural’ and greatly preferred over synthetic alternatives due to the assurance of little to no adverse effects by producers. However, the high rate of adulteration often compromises their safety. Forty herbal supplements, of which 25 were previously shown to result in medium to high intake of phosphodiesterase type-5 (PDE-5) inhibitors using a PDE-Glo bioassay, were further investigated using liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis to examine the reliability of the bioassay and whether the observed higher responses could be ascribed to inherent plant constituents or adulterants. Results showed significant amounts of vardenafil, tadalafil and especially sildenafil, in 2, 1 and 10 samples, respectively, with total concentration levels resulting in estimated daily intakes (EDIs) above 25 mg sildenafil equivalents with six supplements even having EDIs above 100 mg sildenafil equivalents. Only one sample contained a natural ingredient (icariin), but its concentration (0.013 mg g−1) was too low to explain the observed potency in the bioassay. The estimated concentrations of PDE-5 inhibitors in 35 supplements, according to the bioassay, were in line with those of the LC–MS/MS analysis. However, discrepancies were observed for five supplements. Further examination of one of the latter supplements using the PDE-Glo bioassay to select the positive fraction and further examination with LC–MS/MS and 1H-NMR revealed the presence of hydroxythiohomosildenafil, a sildenafil analogue not yet included in the liquid chromatography–mass spectrometry reference library. This study demonstrates the significance of applying a tiered approach, where the use of a bioassay is followed by chemical analysis of bioactive samples in order to identify unknown bioactive compounds.

在加纳的年轻人与部分老年群体中，通过服用草本补充剂以改善性功能是一种常见做法。此类产品因生产商宣称几乎无不良反应，被视为“天然”产品，远较合成类替代品更受青睐。然而，极高的掺假率往往会损害其安全性。本研究针对40款草本补充剂展开分析：此前通过PDE-Glo生物测定法检测发现，其中25款摄入的5型磷酸二酯酶（PDE-5）抑制剂水平处于中高水平。为验证该生物测定法的可靠性，并探究检测到的高响应究竟源于草本固有成分还是掺假物质，研究采用液相色谱-串联质谱（LC–MS/MS）对样本进行进一步检测。结果显示，分别有2份、1份与10份样本中检出足量的伐地那非、他达拉非与西地那非；所有阳性样本的总浓度折算为西地那非当量后，其每日预估摄入量（EDIs）均超过25mg，其中6款补充剂的EDIs甚至高于100mg西地那非当量。仅1份样本检出天然成分淫羊藿苷，但其浓度仅为0.013 mg·g⁻¹，过低而无法解释生物测定中观察到的活性。根据生物测定法估算，35款补充剂中的PDE-5抑制剂浓度与LC–MS/MS检测结果相符，但另有5款样本出现检测结果不一致的情况。针对其中1款不一致的样本，研究先通过PDE-Glo生物测定法筛选出活性组分，再结合LC–MS/MS与氢核磁共振（1H-NMR）展开检测，最终检出羟基硫代同型西地那非——一种尚未被纳入液相色谱-质谱参考库的西地那非类似物。本研究证实了采用分层检测策略的重要性：先通过生物测定法筛选活性样本，再对其进行化学分析，以此识别未知的活性化合物。