Compound
1 was isolated as a white amorphous powder. The molecular formula was deduced to be C
21H
29N
2O
+ from the molecular ion peak [M]
+ at
m/
z 325.2282 (calculated for C
21H
29N
2O
+, 325.2274) in the positive-ion mode of HRESIMS (
Figure S1). The
1H NMR spectrum of
1 (
Table 1,
Figure S2) displayed the presence of the characteristic signals of two aromatic proton sets of a monosubstituted aromatic ring at
δH 7.64 (2H, t,
J = 7.5 Hz),
δH 7.62 (1H, t,
J = 7.5 Hz), and
δH 7.58 (2H, t,
J = 7.5 Hz); 1,2,6-trisubstituted aromatic ring at
δH 7.18 (1H, t,
J = 7.0 Hz) and
δH 7.17 (2H, d,
J = 7.0 Hz); two pairs of relatively deshielded methylene groups at
δH 4.94 (2H, s) and
δH 4.16 (2H, s); two symmetric methyl groups at
δH 2.30 (6H, s); and another two pairs of ethyl groups at
δH 3.67 (4H, m) and
δH 1.56 (6H, t,
J = 7.5 Hz). The
13C NMR data of
1 (
Table 1,
Figure S3), assigned with the aid of the HSQC (
Figure S5) and HMBC experiments (
Figure S6)confirmed 21 carbon signals composed of four methyl groups at
δC 8.4 (2 × C) and
δC 18.7 (2 × C); four methylene carbons at
δC 54.9 (2 × C),
δC 55.7, and
δC 63.4; 12 aromatic carbons (
δC 128.7, 129.1, 129.5 (2 × C), 130.7 (2 × C), 132.3, 134.1 (2 × C), 134.2, 136.7 (2 × C)); and a carbonyl carbon at
δC 164.1. The partial structures of
1 were determined by 2D NMR experiments (
1H-
1H COSY and HMBC). The gross structure of
1 was finally elucidated by the characteristic NMR signals, and its molecular formula (C
21H
29N
2O
+) was confirmed by HRESIMS. The
1H-
1H COSY correlations (
Figure S4) between H-2/H-3/H-4/H-5/H-6 as well as the HMBC correlations of H-2(H-6)/C-7 (
δC 63.4) and H
2-7/C-1, C-2, and C-3 verified the presence of benzyl functionality (
Figure 3). Furthermore, the methylene of H
2-7 showed HMBC correlations with three other carbons: C-8 (
δC 55.7), C-1′ (
δC 54.9), and C-1″ (
δC 54.9) (
Figure 3), providing evidence that a quaternary atom linking C-7, C-8, C-1′, and C-1″ is present. Based on the
1H-
1H COSY spectrum of H
2-1′/H
2-2′ and H
2-1″/H
2-2″ and the HMBC correlations of H
2-1′/H
2-1″ with C-2′/C-2″, along with their symmetric NMR signals, the two ethyl units were assigned and confirmed to be attached to the quaternary atom by the HMBC correlations of H
2-1′/H
2-1″ with C-7 and C-8. The relatively deshielded methylene carbon NMR signals of C-7, C-8, C-1′, and C-1″ and the markedly diminished intensity of the carbon NMR signals observed for C-1′, and C-1″ at
δC 54.9 confirmed that the quaternary atom linking them could be a quaternary ammonium cation, which finally led to the partial structure of A (
Figure 3). Another spin system was observed as a cross-peak between H-12/H-13/H-14 in the
1H-
1H COSY spectrum, representing the 1,2,6-trisubstituted aromatic ring, which was assigned to the 2,6-dimethylated benzene as the partial structure of B by the HMBC correlations of H-12(H-14)/C-10, H-13/C-11(C-15), C-16(C-17)/C-10, C-11, and C-12 (
Figure 3). Finally, the connectivity through the amide bond between the two partial structures of A and B was suggested by the C=O and NH moieties remaining from the molecular formula (C
21H
29N
2O
+) of
1, the detected HMBC correlation of H
2-8/C-9 (
δC 164.1), and the characteristic
13C chemical shifts of C-8 (
δC 55.7) and C-10 (
δC 134.2), although the key HMBC correlation between C-9 and C-10 was missing in
1 due to the absence of protons. Accordingly, the complete structure of
1 was established, as shown in
Figure 1, and it was identified to be denatonium.
The structures of the known compounds (
Figure 1) were determined to be
trans-ferulic acid ethyl ester (
2) [
20], eugenin (
3) [
21], and α-L-glutamyl-L-Leucine (
4) [
22] by comparing their NMR spectroscopic data with those previously reported in the literature and MS data obtained from the LC/MS analysis. To the best of our knowledge, denatonium (
1) was identified from a natural source for the first time, and compounds
2–4 were reported for the first time from
P. multiflorus var.
albus in this study. In natural product chemistry, it is important to take the necessary precautions during the isolation work in order to minimize the possibility of unexpected artifact isolation [
23]. To verify whether compounds
1–4 were genuine natural compounds or artifacts,
P. multiflorus var.
albus was extracted with 80% methanol (
v/
v) for 10 h, and the resultant methanolic extract was subjected to LC/MS analysis. As a result, there was no peak with a molecular ion corresponding to compound
2 in the methanolic extract, whereas compounds
1,
3, and
4 were detected, suggesting that compound
2 was an artifact produced by the extraction with ethanol. In addition, to confirm that the isolated denatonium (
1) is a natural compound, the methanol used for extraction was analyzed using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight (Q-TOF) high-resolution (HR)-MS because methanol can possess denatonium as a component. As a result, there was no detected peak for denatonium in the methanol solvent that we used for extraction (
Figure S7), suggesting that the methanol used does not contain denatonium and the isolated denatonium can be a genuine natural compound.