Bioactivity and chemical constituents of Rhoeo spathacea and Rhoeo discolor, with emphasis on Rhoeo spathacea

Rhoeo spathacea and Rhoeo discolor are two common ornamental plants from the Commelinaceae family, commonly grown in tropical countries including Malaysia and have been traditionally taken orally for the treatment of superficial mycoses, inflammations and gonorrhoea. Despite the ethnobotanical applications of both plants and their popularity as a beverage particularly in South America, relatively little work has been conducted on their bioactivity and chemical constituents, thus warranting further research into these aspects. The preliminary stages of bioactivity assessment involved the determination of the antioxidant, antibacterial, antiproliferative and anti-tyrosinase activity of the crude extracts of both species. It was found that crude R. spathacea leaf extracts exhibited superior antioxidant activity compared to crude R. discolor leaf extracts. In addition, R. spathacea exhibited antibacterial activity against S. aureus and MRSA, while the crude extracts of R. discolor were inactive against the bacteria strains tested. The antiproliferative activity of R. spathacea was observable, but considerably poor. Five solvents of increasing polarity were then used for solvent fractionation. For both species, the phenolic compounds were concentrated in the more polar methanol and water solvent fractions. The polar solvent fractions of R. spathacea exhibited antibacterial activity but R. discolor did not exhibit any antibacterial activity, cementing R. spathacea’s role as the primary plant for the project. The solvent-fractionated extracts of R. spathacea continued to show rather poor antiproliferative activity, but showed considerable tyrosinase inhibitory activity. Column chromatography was then conducted on these fractions using bioactivity-guided fractionation. However this method was found to be inefficient, requiring a lot of material and a long processing time for very little yield of bioactive compounds. A new and efficient method utilizing a single solvent system in lieu of sequential extraction was developed, reducing the number of pre-column chromatography steps from eighteen to three. The Diaion HP-20 stationary phase also proved to be effective at rapidly concentrating the more polar phytochemicals of interest. Its comparatively rapid flow rate, coupled with the vastly reduced number of steps in the earlier stages significantly sped up productiveness. The sub-fractions obtained from this new method showed vastly superior antioxidant and antibacterial activity compared to the previous method, indicating a higher concentration of bioactive phytochemicals extracted despite also taking less time to achieve. Using a combination of high pressure liquid chromatography (HPLC) analysis and mass spectrometry, 27 compounds were found in R. spathacea. NMR was conducted on a rhoeonin, which was successfully isolated. This is the first time the NMR for rhoeonin has ever been reported. NMR was also conducted on 3-(3,4-dihydroxyphenyl)lactic acid (danshensu), which was also found in R. spathacea. Danshensu is the primary active compound in Salvia miltiorrhiza, a traditional herb with cardiotonic properties. However, this compound had not been previously reported in journals to be present in R. spathacea. Lastly, two practical applications of R. spathacea leaves were tested. The first was the use of R. spathacea leaves as an herbal tea, in line with its ethnobotanical application. The antioxidant and antibacterial properties of two different forms of boiling water extracts, decoction and infusion, were compared to room temperature aqueous extracts and solvent extracts. It was found that both the decoction and infusion methods were equally viable extraction methods, comparable in bioactivity with the optimized solvent-extracted crude extract. The application of heat dramatically improved the phenolic content and resulting bioactivity. The second application was as a natural food colorant due to the presence of rhoeonin, an exceptionally stable triacylated anthocyanin. The colour of R. spathacea extracts was exceptionally stable even at relatively low acidity, and showed no significant changes in colour when tested in two food models, thus indicating great potential for future development as a natural food colorant.