Antagonistic effect of alkaloids and saponins on bioactivity in the quinine tree (Rauvolfia caffra sond.): further evidence to support biotechnology in traditional medicinal plants.

BACKGROUND: The Quinine tree (Rauvolfia caffra) is used as a medicinal plant among traditional communities in many countries to manage tumors and other diseases associated with oxidative stress. To validate indigenous knowledge and possibly position this herb for technology uptake and utilization, we established the level of antioxidant activity in R. caffra, and probed for the presence of associated phytochemicals. METHODS: Antioxidant activity was determined on 1,1-diphenyl-2-picrylhydrazyl (DPPH) while major phytochemicals were identified by multiple tests on methanol fractions. RESULTS: R. caffra showed promise as a cure, with antioxidant activity comparable to the commercially used drug quercetin (R. caffra = 79.7% ±1.9; quercetin = 82.6% ± 2.0). However, we found two phytochemicals with possible antagonistic effect: co-occurrence of alkaloids and saponins significantly reduced antioxidant activity (alkaloids only = 63%; alkaloids plus saponins = 15%; steroids, terpenoids and cardiac glycosides = 82%), thus alkaloids and saponins should be exclusive to each other in drug formulations. CONCLUSIONS: Antagonistic relationship among phytochemicals would affect the efficacy of crude extracts as used in traditional medicine. Unlike in herbal medicine, use of modern biotechnology in extraction, purification and design of optimal combinations will ensure efficient drug formulations with optimum bioactivity and minimum toxicity. Metabolic pathway engineering under a controlled environment may optimize availability of desired compounds.

Elevated gene expression in chalcone synthase enzyme suggests an increased production of flavonoids in skin and synchronized red cell cultures of North American native grape berries.

Anthocyanins are antioxidants and are among the natural products synthesized via the flavonoid biosynthesis pathway. Anthocyanins have been recommended for dietary intake in the prevention of cardiovascular diseases, cancer, and age-related conditions such as Alzheimer's disease or dementia. With an increasingly aging population in many parts of the world, strategies for the commercial production of in vitro synchronized red cell cultures as natural antioxidants will be a significant contribution to human medicine. Red pigmented fruits such as grapes (Vitis sp.) are a major source of bioavailable anthocyanins and other polyphenols. Since the level of antioxidants varies among cultivars, this study is the first one that phytochemically and genetically characterizes native grape cultivars of North America to determine the optimal cultivar and berry cells for the production of anthocyanins as antioxidants. Using real-time PCR and bioinformatics approaches, we tested for the transcript expression of the chalcone synthase (CHS) gene, an enzyme involved in the flavonoid and anthocyanin biosynthesis pathway, in different parts of physiologically mature grape berries and in vitro synchronized red cells. A low level of expression was recorded in berry flesh, compared with an elevated expression in berry skins and in vitro synchronized red cells, suggesting increased production of flavonoids in skin and cell cultures. This preliminary study demonstrates the potential of functional genomics in natural products research as well as in systematic studies of North American native grapes, specifically in muscadine (Vitis rotundifolia).

Nuclear ribosomal pseudogenes resolve a corroborated monophyly of the eucalypt genus Corymbia despite misleading hypotheses at functional ITS paralogs.

Divergent paralogs can create both obstacles and opportunities for phylogenetic reconstruction. Phylogenetic relationships among eucalypt genera have been incongruent among datasets in previous studies, where morphological characters supported monophyly of the genus Corymbia, while intergenic spacers of the nuclear ribosomes (ITS) and chloroplast loci (trnL, trnH, psbA) showed Corymbia as either equivocal or paraphyletic. Ribosomal DNA occurs in multiple copies in a genome. We cloned and sequenced the nrITS to investigate if gene duplication was the cause of incongruence among trees in the eucalypts. Three ITS riboforms, two of them widespread, were recovered within some genomes. One of the ITS riboforms recovered a robust phylogeny showing Corymbia as a monophyletic genus, corroborating the evidence from morphology, fossil data, a recent ITS/ETS dataset and microsatellites (SSRs). Compelling evidence suggested that this divergent riboform is a pseudogene, i.e., non-functional paralog: comparatively lower GC content suggesting lower structural stability, deamination-like mutations at potential methylation sites, lack of conserved helices and hairpins and conspicuously lower thermodynamic stability in secondary structures. Phylogenies from the apparently functional riboform retained Corymbia as paraphyletic. We show here that pseudogenes can recover a well-corroborated phylogeny whereas their functional paralogs show misleading hypotheses. We explain that phylogenetic signals may be obscured when functional constraints in ITS necessitate compensatory mutations in the secondary structure helices involved in RNA transcription, whereas pseudogenes mutate under neutrality.

Microsatellites retain phylogenetic signals across genera in eucalypts (Myrtaceae)

The utility of microsatellites (SSRs) in reconstructing phylogenies is largely confined to studies below the genus level, due to the potential of homoplasy resulting from allele size range constraints and poor SSR transferability among divergent taxa. The eucalypt genus Corymbia has been shown to be monophyletic using morphological characters, however, analyses of intergenic spacer sequences have resulted in contradictory hypotheses- showing the genus as either equivocal or paraphyletic. To assess SSR utility in higher order phylogeny in the family Myrtaceae, phylogenetic relationships of the bloodwood eucalypts Corymbia and related genera were investigated using eight polymorphic SSRs. Repeat size variation using the average square and Nei's distance were congruent and showed Corymbia to be a monophyletic group, supporting morphological characters and a recent combination of the internal and external transcribed spacers dataset. SSRs are selectively neutral and provide data at multiple genomic regions, thus may explain why SSRs retained informative phylogenetic signals despite deep divergences. We show that where the problems of size-range constraints, high mutation rates and size homoplasy are addressed, SSRs might resolve problematic phylogenies of taxa that have diverged for as long as three million generations or 30 million years.

African pastoralism: genetic imprints of origins and migrations.

The genetic history of African cattle pastoralism is controversial and poorly understood. We reveal the genetic signatures of its origins, secondary movements, and differentiation through the study of 15 microsatellite loci in 50 indigenous cattle breeds spanning the present cattle distribution in Africa. The earliest cattle originated within the African continent, but Near East and European genetic influences are also identified. The initial expansion of African Bos taurus was likely from a single region of origin. It reached the southern part of the continent by following an eastern route rather than a western one. The B. indicus genetic influence shows a major entry point through the Horn and the East Coast of Africa and two modes of introgression into the continent.