Macleaya, R. Br.

Qing, Zhixing, Yan, Fangqin, Huang, Peng & Zeng, Jianguo, 2021, Establishing the metabolic network of isoquinoline alkaloids from the Macleaya genus, Phytochemistry (112696) 185, pp. 1-8 : 3-4

2.4. Establishing the metabolic network of isoquinoline alkaloids in the Macleaya genus

Metabolic networks can provide new insight into the biosynthesis pathways of some important compounds present in plant-derived medicines. Sanguinarine and chelerythrine, which are the main components of the Macleaya genus, are used as animal food additives. We previously characterized the biosynthesis pathways of these two compounds in M. cordata thoroughly ( Liu et al., 2017). However, are there any other biosynthesis pathways involving either of these two alkaloids in the Macleaya genus plants? Establishment of the metabolic networks is an effective method to answer this question. In this study, a metabolic framework was established primarily based on the reported biosynthesis pathways. A specific metabolic network of isoquinoline alkaloids was then constructed based on the structural relations and differences for identified compounds’ metabolic levels at various growth stages and isotopically labeled [ring - 13 C 6]-tyrosine feeding experiment.

In the Macleaya plants, most isoquinoline alkaloids are synthesized from the benzyltetrahydroisoquinoline alkaloids, which can then be converted to tetrahydroprotoberberine and aporphine alkaloids. N - methyltetrahydroprotoberberine, protoberberine, and 7,8-dihydroprotoberberine are derived from tetrahydroprotoberberine-type alkaloids. Protopine is generated from an N -methyltetrahydroprotoberberine-type alkaloid, and it is continually metabolized to dihydrobenzophenanthridine and benzophenanthridine alkaloids ( Liu et al., 2017; Takemura et al., 2013) (Fig. S6(A)). A metabolic framework of isoquinoline alkaloids in the Macleaya genus was established based on the above metabolism of alkaloids. Take alkaloids 17 (3 ′ -hydroxy- N, N -dimethylcoclaurine) and 115 (10-demethylmagnoflorine) as an example. Compounds 17 and 115 were identified as benzyltetrahydroisoquinoline- and aporphine-type alkaloids, respectively ( Fig. S3 ). Both alkaloids have the same substituent groups, and the benzyltetrahydroisoquinoline can be converted to aporphine in the Papaveraceae family ( Sato et al., 2007; Ziegler et al., 2006). Therefore, alkaloid 17 was regarded as the precursor of 115. The relations of substituent groups (Fig. S6(B)) that have been reported in the Macleaya genus were employed for establishing the specific metabolic network of the same skeleton compounds ( Liu et al., 2017). Take alkaloids 2 (4 ′ -demethylcolletine) and 28 (colletine) as an example. Both of those compounds were identified as benzyltetrahydroisoquinoline-type alkaloids ( Fig. S3 ). Their structures differed in that alkaloid 28 has an –OCH 3 group at the site of C-4’; however, an –OH group was present in the same position for alkaloid 2. In the genus Macleaya , the –OH can be transferred to an –OCH 3 group by the methyl transferase (MT), which is an enzyme encoded by various functional genes, such as 6-OMT, Mc2833, and McSMT (Han et al., 2010; Liu et al., 2017). The structural relation of both compounds indicated that alkaloid 2 was the precursor of 28.

The changes in metabolite levels at different growth stages provided further obvious evidence for establishing the metabolic network. The peak area of alkaloid 2 in the TICs of tissue culture seedlings continually decreased from 0 to 36 days; however, the peak area of alkaloid 28 successively increased ( Fig. 3 ). Interestingly, the level of change was nearly the same, which further demonstrated that the alkaloid 2 could be converted to 28 during plant growth. When we used a similar method, the specific metabolic network of other isoquinoline alkaloids in the Macleaya genus was also established ( Fig. 2 ). Finally, the [ring - 13 C 6]-tyrosine feeding experiment provided visual evidence for establishing the metabolic network of isoquinoline alkaloids in the Macleaya genus. The [ring - 13 C 6]-labeled alkaloids 2 and 28 were detected simultaneously in the TICs of M. cordata seedlings, which further indicated that alkaloid 2 was the precursor of 28. All [ring - 13 C 6]- labeled isoquinoline alkaloids were synthesized from [ring - 13 C 6]-tyrosine, and their metabolic relationships could be relatively easily determined ( Fig. 2 ). In addition, four enzymes, i.e., MT, glycosyltransferase (GTA), cyclooxygenase (COA), and oxidase, were proposed for each specific biosynthesis pathway in this metabolic network.

Although the isoquinoline alkaloids were screened from 45 TICs of samples, which were collected from various parts of Macleaya plants at different growth stages, some intermediate alkaloids were still no detected or were absent in this metabolic network. Take alkaloids 144 (sanguinarine) and 157 (10-methoxy-sanguinarine) as an example ( Fig. S3 ). Sanguinarine can be converted to 10-hydroxy-sanguinarine by an oxidase, and the intermediate alkaloid can be methylated to form alkaloid 157 in the Papaveraceae family (Fig. S7) ( Tanahashi et al., 1990). However, no detectable amount of 10-hydroxy-sanguinarine was found from the TICs of 45 Macleaya genus samples. Therefore, 10-hydroxy-sanguinarine was absent from the metabolic network of isoquinoline alkaloids in the Macleaya genus.

Figures

Fig. 3. TICs of 0-day (A) and 36-day-old (B) tissue culture seedlings.

Fig. 2. Proposed metabolic network of isoquinoline alkaloids in the Macleaya genus. a the solid arrow represents verified biosynthesis pathways; b the dotted arrow represents unverified pathways, c the double arrow represents two or more steps needed for biosynthesis; d MT, GOA, and COA are abbreviation for methyl transferase, glycosyltransferase, and cyclooxygenase, respectively; e the red moieties represent the changed substituent groups, and f the blue moieties represent the 13C -labeled benzene ring. (For 6 interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)