Authentication of plant material.

The fresh leaves of Mitragyna speciosa (Korth.) Havil. have been traditionally consumed for centuries in Southeast Asia for its healing properties. Although the alkaloids of M. speciosa have been studied since the 1920s, comparative and systematic studies of metabolite composition based on different leaf maturity levels are still lacking. This study assessed the secondary metabolite composition in two different leaf stages (young and mature) of M. speciosa, using an untargeted liquid chromatography-electrospray ionisation-time-of-flight-mass spectrometry (LC-ESI-TOF-MS) metabolite profiling. The results revealed 86 putatively annotated metabolite features (RT:m/z value) comprising 63 alkaloids, 10 flavonoids, 6 terpenoids, 3 phenylpropanoids, and 1 of each carboxylic acid, glucoside, phenol, and phenolic aldehyde. The alkaloid features were further categorised into 14 subclasses, i.e., the most abundant class of secondary metabolites identified. As per previous reports, indole alkaloids are the most abundant alkaloid subclass in M. speciosa. The result of multivariate analysis (MVA) using principal component analysis (PCA) showed a clear separation of 92.8% between the young and mature leaf samples, indicating a high variance in metabolite levels between them. Akuammidine, alstonine, tryptamine, and yohimbine were tentatively identified among the many new alkaloids reported in this study, depicting the diverse biological activities of M. speciosa. Besides delving into the knowledge of metabolite distribution in different leaf stages, these findings have extended the current alkaloid repository of M. speciosa for a better understanding of its pharmaceutical potential.</div

S1 Data -

The fresh leaves of Mitragyna speciosa (Korth.) Havil. have been traditionally consumed for centuries in Southeast Asia for its healing properties. Although the alkaloids of M. speciosa have been studied since the 1920s, comparative and systematic studies of metabolite composition based on different leaf maturity levels are still lacking. This study assessed the secondary metabolite composition in two different leaf stages (young and mature) of M. speciosa, using an untargeted liquid chromatography-electrospray ionisation-time-of-flight-mass spectrometry (LC-ESI-TOF-MS) metabolite profiling. The results revealed 86 putatively annotated metabolite features (RT:m/z value) comprising 63 alkaloids, 10 flavonoids, 6 terpenoids, 3 phenylpropanoids, and 1 of each carboxylic acid, glucoside, phenol, and phenolic aldehyde. The alkaloid features were further categorised into 14 subclasses, i.e., the most abundant class of secondary metabolites identified. As per previous reports, indole alkaloids are the most abundant alkaloid subclass in M. speciosa. The result of multivariate analysis (MVA) using principal component analysis (PCA) showed a clear separation of 92.8% between the young and mature leaf samples, indicating a high variance in metabolite levels between them. Akuammidine, alstonine, tryptamine, and yohimbine were tentatively identified among the many new alkaloids reported in this study, depicting the diverse biological activities of M. speciosa. Besides delving into the knowledge of metabolite distribution in different leaf stages, these findings have extended the current alkaloid repository of M. speciosa for a better understanding of its pharmaceutical potential.</div

Multivariate analysis of <i>M</i>. <i>speciosa</i>’s metabolite profile.

(A) PCA score scatter plot of young (lime green) and mature (dark green) leaves in five biological replicates. Circles are labelled relative to the leaf age groups. (B) PLS-DA score scatter plot of young and mature leaves in five biological replicates. Circles are labelled relative to the leaf age groups. (C) PLS-DA loading scatter plot projecting metabolite features that influenced the clustering and separation of young and mature leaf groups.</p

Putative identification of other secondary metabolites in the young (Y) and mature (M) leaves of <i>M</i>. <i>speciosa</i>.

Putative identification of other secondary metabolites in the young (Y) and mature (M) leaves of M. speciosa.</p

List of alkaloids identified in <i>M</i>. <i>speciosa</i> from previous studies based on first occurrences in this species.

List of alkaloids identified in M. speciosa from previous studies based on first occurrences in this species.</p

Venn diagram representing the number of overall identified metabolite features shared between or unique to young and mature leaves of <i>M</i>. <i>speciosa</i>.

Venn diagram representing the number of overall identified metabolite features shared between or unique to young and mature leaves of M. speciosa.</p

Identification of alkaloids in the young (Y) and mature (M) leaves of <i>M</i>. <i>speciosa</i>.

Identification of alkaloids in the young (Y) and mature (M) leaves of M. speciosa.</p

Principal component analysis (PCA) of young (Y) and mature (M) leaves of <i>M</i>. <i>speciosa</i> with technical replicates.

Principal component analysis (PCA) of young (Y) and mature (M) leaves of M. speciosa with technical replicates.</p

Relative abundance of 32 indole alkaloids putatively identified in young and mature leaves of <i>M</i>. <i>speciosa</i>.

Relative intensity values for all indole alkaloids are provided in the S3 Table. Asterisks (*) indicate highly differential metabolites (* FDR 2FC| > 2). The graphs are arranged in alphabetical order. RT:m/z values are provided instead of compound names for metabolite features annotated to more than one compound, which can be referred to in the S3 Table.</p

Identification of important features by statistical analysis of fold change upon comparing alkaloid expression between young and mature leaves.

Identification of important features by statistical analysis of fold change upon comparing alkaloid expression between young and mature leaves.</p