DNA barcoding and anti-tyrosinase activities of three species-representative populations of the genus Greyia Hook & Harv

• First report of anti-tyrosinase activity of leaf extracts from wild-growing Greyia spp. • Greyia sutherlandii leaf extracts shown to have anti-tyrosinase activity for the first time. • HPTLC method developed to quantify the active compound 2′,4′,6′-trihydroxydihydrochalcone. • DNA barcoding (ITS, matK, trnL-F, psbA-trnH ) conducted on core populations of three Greyia spp. • DNA barcode phyogeny discriminates Greyia spp. from other Geraniales genera. • G.sutherlandii and G.radlkoferi not resolved phylogenetically with DNA barcodes. • G. sutherlandii and G. radlkoferi hard to differentiate in the field morphologically. The tree genus Greyia is endemic to South Africa and Eswatini. The Eastern Cape species Greyia flanaganii Bolus is confined to a limited range west of the Kei River. Greyia radlkoferi Szyszyl. occurs in Limpopo Province, whereas Greyia sutherlandii Hook. & Harv. is associated with KwaZulu-Natal Province, but the ranges of these two species overlap in Mpumalanga Province. Greyia trees are of value to the bioeconomy as leaf extracts from G. flanaganii and G. radlkoferi possess anti-tyrosinase activity and low toxicity and are being developed in herbal formulations for the treatment of skin hyper-pigmentation. The main active compound is 2’,4’,6’ trihydroxydihydrochalcone. However, there are no reports of medicinal assays from Greyia trees growing in their natural habitat, it is not known whether the third species - G. sutherlandii - has activity, and DNA barcode data is limited. To address these knowledge gaps, we sampled five trees per Greyia species that matched morphological descriptions and were from sites close to type specimen collection records. Leaf ethanolic extracts from G. sutherlandii had similar average anti-tyrosinase activity (IC 50 = 84 µg/mL ± 18 [SD]) when compared to G. radlkoferi (58 µg/mL ± 21) and G. flanaganii (72 µg/mL ± 11). High Performance Thin Layer Chromatography showed the presence of the active compound in all three species, although it was below the detection limit of 4.4 µg/10 mg extract in two of the G. flanaganii samples. Considering the difficulty in differentiating G. sutherlandii from G. radlkoferi morphologically in the field and production orchards, we investigated DNA barcoding as a method of species-specific authentication. Phylogenetic analysis using Bayesian Inference from combined ITS, trnL-F, matK and psbA-trnH barcodes from the 15 Greyia trees plus Genbank sequences indicated (i) clear differentiation from other lineages in the order Geraniales, but (ii) extremely short internal branches within Greyia and poor discrimination between Greyia species and individuals. Our study has shown that natural populations of all three species sampled in late summer exhibit consistent leaf anti-tyrosinase activity between biological replicate trees. We have demonstrated activity from G. sutherlandii for the first time, indicating that this species can also be deployed in production orchards. However, alternative phylogenetically informative SNP markers need to be developed to provide species-specific authentication of Greyia extracts in herbal products.