Chemotaxonomy of Southeast Asian Peperomia (Piperaceae) Using High-Performance Thin-Layer Chromatography Colour Scale Fingerprint Imaging and Gas Chromatography-Mass Spectrometry

Abstract

The morphological characters of Southeast Asia's indigenous Peperomia species are very similar, especially in their flower structures. The flowers are simple, hermaphrodite and lack a perianth. Therefore, many species are hard to distinguish using morphological characters alone. Here, we apply chemometric data for species identification and classification, gathered using multiwavelength detection combined with the colour scale High-Performance Thin-Layer Chromatography (HPTLC) fingerprinting procedure and chemical compounds determined by Gas Chromatography-Mass Spectrometry (GC-MS). Fourteen taxa were investigated using hexane, ethyl acetate and ethanol solvent extractions. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used with the colour scale fingerprints to classify the Peperomia species. The PCA and HCA using the chromatogram profile from hexane divided the taxa into six groups compared to the profile from ethyl acetate and ethanol, which each detected seven groups. The chromatogram from the combined dataset of all three solvents can differentiate all the species. The GC-MS data detected a total of 40 compounds from the hexane extract, and these differed among Peperomia species. This approach based on HPTLC fingerprinting and GC-MS analysis can therefore be used as a tool for authentication and identification studies of Peperomia species.

Keywords: GC-MS; HPTLC; chemometric; chromatogram; image analysis; tropical plants.

Figure 1

HPTLC profile of Peperomia from hexane extracts; (a) scanned at UV 254 nm; (b) scanned at UV 366 nm; and (c) scanned after spraying with anisaldehyde–sulfuric reagent. The Y-axis shows the Rf values for each compound and the X-axis shows the species code: (1) P. cavaleriei; (2) P. dindygulensis; (3) P. heptaphylla; (4) P. heyneana; (5) P. kotana; (6) P. laevifolia; (7) P. masuthoniana; (8) P. moulmeiniana; (9) P. multisurcula; (10) P. nakaharai; (11) P. pellucida; (12) P. sirindhorniana; (13) P. tetraphylla; (14) P. ranongensis.

Figure 2

Image of chromatographic plates and corresponding colour scale fingerprints for Peperomia cavaleriei. Fingerprints were generated using TLC Analyzer software and different colour scale selections in (a) fluorescence quenching mode under 254 nm excitation wavelength and (b) fluorescence quenching mode under 366 nm excitation wavelength. Red line—red scale fingerprint (R); green line—green scale fingerprint (G); blue line—blue scale fingerprint (B); grey line—grey scale fingerprint (K).

Figure 3

Clustering dendrogram and PCA of the HPTLC results based on the colour scale fingerprint data using the hexane extracts for 14 Peperomia samples; (a) clustering dendrogram (numbers 1 to 6 is cluster group); (b) PCA profile (PC1 accounts for 56% of the variation; PC2 accounts for 10.5% of the variation).

Figure 3

Clustering dendrogram and PCA of the HPTLC results based on the colour scale fingerprint data using the hexane extracts for 14 Peperomia samples; (a) clustering dendrogram (numbers 1 to 6 is cluster group); (b) PCA profile (PC1 accounts for 56% of the variation; PC2 accounts for 10.5% of the variation).

Figure 4

Clustering dendrogram (a) and PCA profile (b) of the HPTLC based on colour scale fingerprint data using the combined data from the three different solvent extracts of 14 Peperomia samples.

Figure 4

Clustering dendrogram (a) and PCA profile (b) of the HPTLC based on colour scale fingerprint data using the combined data from the three different solvent extracts of 14 Peperomia samples.

Figure 5

Clustering dendrogram (a) and principal component analysis (b) ordination of the chemical composition data from the 14 n-hexane extract of Peperomia samples assessed by GC-MS. Axis PC1 accounts for 20.62% and axis PC2 for a further 15.81% of the total variance.