Macleaya cordata R. Br.

2.2. Screening and identifying isoquinoline alkaloids from M. cordata and

M. microcarpa samples

Screening and identifying as many compounds as possible are critical steps for establishing metabolic networks of isoquinoline alkaloids in the Macleaya genus. The metabolites were detected by the established screening method, and their structures were identified according to their characteristic MS/MS spectra. In this screening method, 1608 exact theoretical masses were first formed by combining 29 well-known skeletons with five common groups, after which 640 potential candidates were obtained via EIC of the exact theoretical mass on the TICs of 45 different samples. Finally, 392 potential candidates were generated, and 204 of them were further identified by their characteristic MS/MS data (Fig. S3) and the fragmentation pathways of isoquinoline alkaloids that have been systematically investigated in previous studies (Qing et al., 2013, 2020; Jeong et al., 2012). Identifying isoquinoline alkaloids from M. cordata and M. microcarpa fruits was performed in detail in our previous studies (Qing et al., 2014, 2015a, 2015b; Zuo et al., 2017). Therefore, in this study, only screening and identifying benzyltetrahydroisoquinoline alkaloids was taken as an example.

Previous studies indicated that benzyltetrahydroisoquinoline (whose theoretical m/z values is 220.1122 [M + H] +), N -methyl-benzyltetrahydroisoquinoline (234.1287) and N, N -methyl-benzyl- tetrahydroisoquinoline (248.1435) are the basic skeletons, and OCH 2 O (46.0055), OCH 3 (31.0184), OH (17.0027), H (1.0078), and glucose (179.0556) are the main substituent groups of this type of alkaloid. In total, 138 exact theoretical masses (Table S1) were generated by adding four substituent groups to the parent skeletons. Using EIC of the formed theoretical masses on the TICs of the 45 different Macleaya genus samples, 80 candidate compounds were generated, and target-MS/ MS analysis was performed for each candidate. Finally, 37 benzyltetrahydroisoquinoline alkaloids (1-37, Table S2) were tentatively identified based on their characteristic MS / MS spectra and the fragmentation pathways of reference alkaloids.

Take alkaloid 1 as an example. The exact theoretical mass at m/z 272.1281 (Table S1) was formed by combining the skeletons with substituent groups. The candidate (alkaloid 1, t R 3.39 min) was found from the TICs of 0- to 90-day tissue culture seedlings, M. cordata fruits (III), and M. cordata roots (IV) via EIC of the formed theoretical mass on the TICs of the 45 Macleaya genus samples. The MS/MS spectrum of alkaloid 1 was obtained using the relatively high-content 0-day-old sample (Fig. S4 A and B). In the MS/MS spectrum of alkaloid 1 (Fig.S4 C), the neutral loss of the NH 3 moiety from the protonated exact mass at m/z 272.1293 and formation of the fragment ion at m/z 255.1073 were observed. In addition, the relatively highly abundant fragment ions at m/z 107.0486, 143.0483, and 161.0604 were also formed. Both fragmentation behaviors indicated that alkaloid 1 was a benzyltetrahydroisoquinoline-type alkaloid according to previous studies (Qing et al., 2013, 2020). The structure of alkaloid 1 was combined by a benzyltetrahydroisoquinoline skeleton, three hydroxyls, and one hydrogen, as shown in Table S1. The base peak ion at m/z 107.0486 indicated that one hydroxyl was connected to the C-ring of the benzyltetrahydroisoquinoline skeleton. The fragmentation ions at m/z 143.0483, 161.0604, and 255.1073 demonstrated that the remaining two hydroxyls were connected to the A-ring. Therefore, alkaloid 1 was tentatively identified as higenamine (Fig.S4 C). Moreover, the structure of alkaloid 1 was unambiguously determined by comparing the retention time, exact MS and MS/MS data with the standard. The remaining 36 benzyltetrahydroisoquinoline alkaloids were also screened and identified using a similar method (Table S2).

In addition to benzyltetrahydroisoquinoline, 28 other types of isoquinoline alkaloids (Fig. S1) were screened from the Macleaya genus (Fig. 1). However, only 11 types of isoquinoline alkaloids, i.e., benzyltetrahydroisoquinoline (1-37), tetrahydroprotoberberine (38-53), N - methyltetrahydroproto -berberine (54-67), protopine (68-90),

berberine (91-107), 7,8-dihydroberberine (108-114), aporphine (115- 117), benzophenanthridine (118-167), dihydrobenzophenanthridine (168-201), benzoquinoline (202-203) and arnottianamide (204), were found and identified (Fig. S3; Table S2). Interestingly, among the 204 identified isoquinoline alkaloids, 40 glycosylated alkaloids (21-26, 34- 36, 42-44, 49, 52, 53, 63, 64, 67, 74, 93, 94, 99, 100, 103, 116, 117, 130-135, 158, 159, 161, 167, 170, 174, 180, and 181, Fig. S3; Table S2), which have rarely been reported in the Papaveraceae family and may have excellent bioactivities, were detected and characterized from the Macleaya genus. In addition, some addictive compounds, such as morphine and codeine, which have been found within the Papaveraceae family, were not detected in either plant species in the present study (Liu et al., 2017; Poeaknapo et al., 2004). Finally, a metabolic network of isoquinoline alkaloids in the Macleaya genus was established based on the identified metabolites.