(A) HPLC chromatogram of methanol extract (MX). Eight fractions were collected, 1 min per fraction. The blue trace was the chromatogram at 254 nm while the red one was at 320 nm. (B) TYR inhibitory activity of MXs and fractions in cell lysate hsTYR assays. Cell lysate containing 3 × 10⁶ human MM418C1 melanoma cells were incubated with MX/the fractions (1 mg/mL) for 1 h. The tyrosinase activity was presented as a percentage of untreated group. Kojic acid (1 mg/mL) was used as positive control. Mean ± SEM (n = 3) of three independent experiments, each performed in triplicate were shown. One-way ANOVA was employed followed by Dunnett’s test. **** p < 0.0001.

¹H NMR spectra (800 MHz, DMSO-d₆ or CD₃OD-d₄) for (A) F1, F2 and F3; (B) F1, 4-hydroxybenzoic acid (1), uridine (2), thymidine (3), cytidine (4); (C) F2, caffeoyl choline (5), 3-caffeoylquinic acid (6), and 5-caffeoylquinic acid (7); (D) F3, indole-3-carboxaldehyde (8), xanthiside (9), 1, 5-dicaffeoylquinic acid (10) and 1, 3-dicaffeoylquinic acid (11).

Chemical structures of compounds 1–11 isolated from the methanol extract of Xanthium strumarium L.

(A) Anti-hsTYR activity of compounds 1–11 using cell lysate assays. Cell lysates prepared from 3 × 10⁶ MM148C1 human melanoma cells were incubated with 1 or 0.5 mg/mL of compounds 1–11 in 5% DMSO for 1 h. Kojic acid was used as a positive control. TYR activity is presented as a percentage of 5% DMSO group (negative control). The cutoff line represents the threshold value for **** p < 0.0001 (threshold: 85.0198%), ns: not statistically significant. (B) Dose–response curve of 4HB against hsTYR. Cell lysate prepared from 3 × 10⁶ MM148C1 human melanoma cells were incubated with 1000.0, 500.0, 250.0, 125.0, 62.5, 31.3, 15.6, 7.8 and 3.9 μg/mL 4HB solution for 1 h. Mean ± SEM (n = 3) of three independent experiments, each performed in triplicate are shown. Two-way ANOVA was employed followed by Šídák’s multiple comparisons test.

Cell-based hsTYR assay for 4HB. (A) Cell viability test (resazurin assay). Human MM418C1 melanoma cells were treated with 1000.0, 500.0, 250.0 and 125.0 μg/mL 4HB/MX. The growth of the cells is expressed as a percentage of the untreated group. Human MM418C1 melanoma cells were treated with 500 μg/mL 4HB (500 4HB) or MX (500 MX) and 0.1 μM α-MSH for 5 days to assess its effect on (B) melanin content and (C) cellular TYR activity. Cells with growth media (0−) were used for comparison with the α-MSH treated cells (0+). 500 μg/mL kojic acid (500 KA) was utilized as a positive control in both assays. The melanin content was determined using the percentage of the untreated group. The unit of TYR activity was determined by following the change in Aλ490/min/mg protein. Mean ± SEM (n = 3) of three independent experiments, each performed in triplicate, are shown. One-way ANOVA was employed followed by Dunnett’s test. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Zebrafish pigmentation assays for MX and 4HB. Synchronized embryos were treated with 4HB and kojic acid at a concentration of 15.63, 7.81 and 3.91 μg/mL. Kojic acid (KA) at 4 mg/mL was used as a positive control and standard E3 medium was used as a negative control. Tested compounds were dissolved in embryo E3 medium. Zebrafish pigmentations were assessed using a stereomicroscope coupled with digital camera for in silico processing/quantification of the pigmented areas. (A–D) lateral view and magnified caudal fin view of embryos at 48 h post-fertilization (hpf). (E) statistical analysis of pigmentation displayed as percentage to untreated control group in zebrafish (n = 9). Image and contrast-based analysis were conducted in Fiji ImageJ 2.9.0 software; One-way ANOVA was employed followed by Dunnett’s test. **** p < 0.0001 versus control. Scale bar = 100 μm.