Higenamine (HM), an alkaloid found in various plant species, is obtained when norcoclaurine synthase selectively condenses dopamine and 4-hydroxypheny lacetaldehyde to give (S)-higenamine ((S)-HM). The World Anti-doping Agency has listed HM as a prohibited agent in athletics. As a result, many commercial, academic, and regulatory bodies across the globe are invested in finding a rapid method for (S)-HM detection. In the current study, a lateral flow immunoassay (LFA) was developed in which the relevant biosensor was generated as a conjugate of the monoclonal antibody against (S)-HM (namely, MAb E8) and colloidal gold nanoparticles. The HM-γ-globulin conjugates and rabbit anti-mouse IgG antibodies were placed in the test and control zones, respectively. The free (S)-HM molecules in the samples and the immobilized HM-γ-globulin conjugates competitively reacted with the developed biosensor in the LFA. An inverse relationship existed between the biosensors' visible response, which was noted by the variation in the intensity of a pinkish spot in the test zone, and the content of the free (S)-HM. The limit of detection of the developed LFA was 156 ng/mL. Various validation methods confirmed that the LFA exhibited sufficient sensitivity, selectivity, repeatability, and reliability, making it ideal for (S)-HM detection in plant samples and plant-containing products. The developed system required only a small sample volume (20 μL) and a concise sample preparation time compared with conventional LFAs. Thus, the LFA reported in this study could serve as a rapid response kit for the detection of (S)-HM in plant samples. K E Y W O R D S colloidal gold nanoparticles, higenamine, immunochromatographic test strips, lateral flow immunoassay, monoclonal antibody 1 | INTRODUCTION Higenamine (HM; 1-[(4-hydroxyphenyl)methyl]-1,2,3,4-tetrahydroiso quinoline-6,7-diol, Figure 1) is a benzyl tetrahydroisoquinoline alkaloid derived from various plant species, including Aconitum carmichaelii Debeaux. (Ranunculaceae), Nandina domestica Thunb., (Berberidaceae), Nelumbo nucifera Gaertn. (Nelumbonaceae), and Asarum heterotrophies F. Schmidt (Aristolochiaceae). 1,2 Given its chiral character, studies have revealed that the norcoclaurine synthase conformational selectively condenses dopamine and 4-hydroxyphenylacetaldehyde to form the specific enantiomer, (S)-HM. 3-5 From a pharmacological perspective, HM is a partial

Background Quassinoids and canthin-6-one alkaloids are bioactive markers of Eurycoma longifolia (EL) and E. harmandiana (EH) and have been commercially utilized to treat inflammation and male infertility. Objectives This study aims to reveal the contents of bioactive compounds and to compare anti-inflammatory activities of these two species. Methods High-performance liquid chromatography (HPLC) methods coupled with UV-Vis detection were developed and validated for the simultaneous analysis of the chemical profiles and their contents in EL and EH. The anti-inflammatory activities of both species were investigated using RAW 264.7 cell line. Results The HPLC methods provided a sensitivity (LOD) of 0.02–0.05 µg/mL for the eight bioactive compounds (canthin-6-one alkaloids, quassinoids, and scopoletin) with high precision (% relative standard deviation (RSD) ≤6.48) and recoveries between 80.0–120%. The chaparrinone: eurycomanone ratio was high in EH, whereas EL had a higher ratio of eurycomanone: chaparrinone than EH. The contents of total canthin-6-one alkaloids, quassinoids, and scopoletin were 0.01–0.75, 0.19–1.54, and 0.01–0.28 mg/g, respectively, in EL roots and 0.12–1.80, 7.05–9.26, and 0.02 mg/g, respectively, in EH roots. The anti-inflammatory effects of EL and EH extracts varied among the samples due to the variation in their chemical constituents. Conclusions In summary, our study indicated that chaparrinone was the major compound in EH. EH exhibited anti-inflammatory activity the same extent as EL. Highlights EH and EL extracts were analyzed using developed HPLC-UV methods, revealing a high concentration of chaparrinone in EH, and an anti-inflammatory assay indicated that EH had a potency comparable to that of EL.

Eurycoma longifolia (EL) and Eurycoma harmandiana (EH) are natural medicinal plants belonging to the Simaroubaceae family, and are well‐known for their ability to enhance male sexual performance. The present study investigated the phosphodiesterase‐5 (PDE‐5) inhibitory activity of intact roots of EL and EH. Additionally, canthin‐6‐one alkaloids, β‐carboline alkaloids, and quassinoids were also screened for PDE‐5 inhibitory activity. We developed in vitro root and callus cultures of EL and EH to determine their PDE‐5 inhibitory activity. Our results indicated that canthin‐6‐one alkaloids, which include canthin‐6‐one‐9‐O‐β‐D‐glucopyranoside, 9‐methoxycanthin‐6‐one, canthin‐6‐one, and 9‐hydroxycanthin‐6‐one, exhibited PDE‐5 enzymatic inhibitory activity, with IC50 values of 2.86±0.23, 3.30±1.03, 4.31±0.52, and 4.66±1.13 μM, respectively. The ethanolic extract of the intact roots of EL and EH, and the in vitro root culture of EH had large amounts of canthin‐6‐one alkaloids (1.50±0.04, 2.12±0.03, and 3.48±0.08 mg/g dry weight, respectively), and showed potent PDE‐5 inhibition. Our findings indicate that in vitro root cultures of EH may be used to replace intact plants, and canthin‐6‐one‐9‐O‐β‐D‐glucopyranoside should be further investigated for development as a health supplement.

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