In the HPLC chromatograms of the ether extract of three agarwood samples (S1, S2, S3) detected at UV 254 nm, a total of fifty-six compounds (four types of chromones) were detected (Figure 3
). By comparing their retention time and MS spectra with reference compounds, twenty-six of them were identified, and thirty compounds were tentative identified. The identified or characterized results of THPECs, EPECs and DEPECs were listed in Table 2
, and the retention time of THPECs, EPECs and DEPECs were in the range of 12~30 min, 18~34 min, 21~43 min, respectively. The results of FTPECs were showed in Table 3
, and the retention time of FTPECs were in the range of 34~80 min, except for compound 20
2.3.1. Structural Analysis of THPECs
Taking compounds 1 and 3 for example, the characteristic fragment ions at m/z 331([M + H–18]+) and 313 ([M + H–18–18]+), indicated both were THPECs. The molecular weight (MW) of these two compounds were determined as 348 based on the protonated precursor ion at m/z 349. According to formula (1) (MW − (30a + 16b +34c) = 254), a, b and c were calculated as 1, 4 and 0, respectively, which meant compounds 1 and 3 had four hydroxyl and one methoxyl substituent groups on the whole compound. Furthermore, the benzyl ion at m/z 121 was found in compound 1, which meant one methoxy substitution occurred on its benzyl moiety according to Formula (2), thus, the other four hydroxyl substitutions occurred on its chromone moiety. Neither benzyl ion nor chromone moiety ions were observed in compound 3, thus the positions of substituent groups were uncertain. By searching the literature, we found six THPECs with the MW = 348 were reported, including four of them possessing one methoxy substitution occurring on the benzyl moiety, and four hydroxyl substitutions occurring on their chromone moiety, which matched the above deduced structural characterization of compound 1, so compound 1 was tentative identified as one of the four reported compounds. The positions of the substituent groups of compound 3 were uncertain, so it was tentative identified as one of the six reported compounds while different from compound 1. By comparing the retention time and MS spectra with the reference compounds, compound 1 was identified as F3, and further proved that the above identification method was reasonable.
According to above method of identification, compounds 1–7 were deduced as THPECs. Except for compound 5 (m/z 317), the other six compounds, with [M + H]+ ions of m/z 319, 303, 367, 337, have been reported as THPECs. Compound 5 was identified as a new compound.
2.3.2. Structural Analysis of EPECs
Taking compounds 10 and 11 for example, according to the characteristic fragment ions of m/z 313 ([M + H–18]+) and 285 ([M + H–18–28]+), they were identified as EPECs. The protonated precursor ions at m/z 331 meant their molecular weight (MW) was 330. According to the Formula (1), a, b and c were calculated as 1, 2 and 0, respectively, which meant they had two hydroxyl and one methoxyl substitutions on the whole compound. The benzyl ion at m/z 121 indicated one methoxy substitution occurred on its benzyl moiety according to Formula (2), therefore, the two hydroxyl groups substitutions occurred on the chromone moiety. Subsequently, by literature searching, only one EPEC with MW = 330 was reported, which meant that one of them would be a new compound. By comparing the retention time and MS spectra with reference compound, compound 11 was identified as F6, which proved that the above identification method was feasible, and meanwhile compound 10 should be a new compound.
By the same method, compounds 8
were deduced as EPECs. Except for compound 9
(which [M + H]+
ion is m/z
285), all the other compounds, which [M + H]+
ions were at m/z
347 or 301, have been reported. By comparing retention time and MS spectra with reference compounds, compounds 8
were identified as reference compounds F5
, respectively. Compounds 9
were proposed to be new compounds as listed in Table 2
, because only four EPECs have been reported until now [7
2.3.4. Structural Analysis of FTPECs
Taking compounds 22~24, 27 and 30 for example, only benzyl ions (m/z 137 and 121) and chromone moiety ion (m/z 177) were detected, leading to identification of these compounds as FTPECs. All of them showed protonated precursor ions at m/z 313, which meant their molecular weight (MW) was 312. According to Formula (1), the a, b and c values were calculated as 1, 2 and 0, respectively, which meant they have two hydroxyl and one methoxyl substituent group on the whole compound. Then, according to Formula (2), the benzyl ion at m/z 137 was found in compounds 22 and 23, which meant one methoxyl and one hydroxyl substitution occurred on their benzyl moiety, while the benzyl ion at m/z 121 was detected in compounds 24, 27 and 30, which meant one methoxy substitution occurred on their benzyl moiety. Consequently, one hydroxyl substitution occurred on the chromone moiety of compounds 22 and 23, and two hydroxyl substitutions occurred on the chromone moiety of compounds 24, 27 and 30. In particular a chromone moiety ion (m/z 177) was detected in compound 22, which indicated the hydroxyl group should be substituted at the 4′/2′ position of the benzyl moiety. Five FTPECs with MW = 312 have been traced from the literature, so the five FTPECs were tentatively identified as reported compounds. By comparing the retention time and MS spectra with reference compounds, compounds 22, 24 and 27 were identified as F12, F13 and F16, respectively, and the results proved that the above identification method was reasonable.
According to the method of identification, compounds 20
were deduced as FTPECs (Table 3
), among which nineteen compounds were identified according to the reference compounds.
Finally, fifty-six 2-(2-phenylethyl)chromone derivatives were identified or characterized from the three samples according to the fragmentation behavior and the comparison of retention time and MS spectra with reference compounds (Table 1
). A total of 37 (seven DEPECs, four EPECs, three THPECs and 23 FTPECs), 43 (five EPECs, five THPECs and 33 FTPECs), and 29 (one DEPEC, two EPECs, three THPECs and 23 FTPECs) 2-(2-phenylethyl)chromones were identified or characterized in S1, S2 and S3, respectively. The respective relative content of 2-(2-phenylethyl)chromone derivatives was 66.42%, 81.39% and 79.20% in S1, S2 and S3 (Table 4
Based on the above data, it was found that the number and relative content of DEPECs (7/8.01%) from S1 was the highest among the three samples, while they were merely traces in S2 and S3 (Table 4
, Figure 4
). The relative content of EPECs showed a downtrend from S1 to S3 (12.58%, 8.07% and 2.96%, respectively), while the relative content of THPECs and FTPECs showed an uptrend from S1 to S3 (1.58%/1.73%/2.47%, 44.25%/71.59%/73.56%, respectively). It was noticeable that both the total number and relative content of DEPECs, and EPECs decreased obviously (20.59%, 8.07% and 3.17%, respectively) from S1 to S3, and this was consistent with that observed in the HPLC chromatograms (Figure 3
), in which the height of peaks for the DEPECs, EPECs (before 43 min) showed a remarkable decreasing trend. This finding was in agreement with the proposed biosynthetic pathways of the four types of chromones (Figure 4
], which showed transformations among them during different agarwood formation time. DEPECs have been recognized as precursors, and only accumulated at the early stage of agarwood formation. Then, the next group was EPECs, where the low occurrence rate also suggested they were early intermediates during the agarwood formation. The following group was the highly oxidized THPECs, and the last presented group were FTPECs, both of which were widely distributed in agarwood.
In total, twenty-one 2-(2-phenylethyl)chromones (two EPECs, two THPECs and 17 FTPECs) were detected in all three samples, while ten (six DEPECs, one THPEC and three FTPECs), twelve (one EPEC, three THPECs and eight FTPECs), and one (THPEC) 2-(2-phenylethyl)chromone were only found in S1, S2, and S3, respectively. Furthermore, we found that the relative content of six FTPECs, 6,8-dihydroxy-2-[2-(4-methoxy)phenylethyl]chromone, 6-methoxy-7-hydroxy-2-[2-(4-methoxy)-phenylethyl]chromone, 6-hydroxy-2-(2-phenylethyl)chromone, 6,7-dimethoxy-2-(2-phenylethyl) chromone, 2-[2-(4-methoxy)phenylethyl]chromone, and 2-(2-phenylethyl)chromone, which are reported as the main constituents of agarwood, showed a uptrend from S1 to S3. This finding suggested that the relative content of the six 2-(2-phenylethyl)chromones was the significant factor to evaluate the formation time of agarwood.