Characterizing the biological properties of traditional medicinal plants from northern Vietnam using zebrafish larvae, cell culture and microorganisms

Evaluating the risks and benefits of using traditional medicinal plants is of utmost importance for a huge fraction of the human population, particularly in Northern Vietnam, which is home to one of the largest repertoires of tropical biota, both in terms of plants and of microorganisms. Ten to fifteen plants were selected based on their traditional use as well as socio-economic concerns for the initial simple screening tests. After that, crude extracts obtained from the most promising medicinal plants have been analyzed for beneficial or harmful properties using a defined test panel. The extracts have been evaluated in various serial dilutions to determine a dose-dependent effect. Zebrafish are increasingly utilized as a simple vertebrate model for assessing toxic and physiological effects of compounds, especially on development. Here, we tested twelve ethanolic extracts from well-known medicinal plants collected in Northern Vietnam for their effects on zebrafish survival and development during the first 4 days after fertilization. The results characterized in detail the effects of six ethanol extracts on epiboly, hatching, growth, necrosis, body curvature, angiogenesis, skeletal development and mostly increased movement behavior. We also confirm the effect on epiboly caused by the Mahonia bealei extract by staining the actin filaments and demonstrate that this extract also inhibits cell migration of mouse embryo fibroblasts.
Additionally, we employed the fungus Penicillium digitatum, a serious postharvest pathogen causing huge economic losses for citrus production as a model for screening antifungal activity of twelve traditional medicinal plant species collected in northern Vietnam. The results revealed that only three plants including Mahonia bealei, Ficus semicordata, and Gnetum montanum possess strong antifungal activity against P. digitatum. We also discovered that the ethanolic crude extract of M. bealei exhibited a broad-spectrum antifungal activity against different pathogenic fungi. Analysis of this extract by enrichment coupled with high-performance liquid chromatography indicated that berberine and palmatine are major active compounds. Further inspections indicated that berberine significantly contributes to the antifungal activity of the M. bealei ethanolic extract.

On the other hand, recently several important bioactive compounds from medicinal plants have been reported to be associated with microorganisms existing inside plants. In this study, we explored the culturable endophytic microorganisms associated with medicinal plants collected in northern Vietnam. Specifically, we isolated the endophytic microorganisms by applying surface sterilization methods and identified them based on morphological and rDNA sequence analyses. Agglomerative Hierarchical Clustering (AHC) and Principal Component Analysis (PCA) were used to analyze the correlations between the taxonomic affiliations of the culturable endophytes and the characteristics of their hosts. Many isolates are reported to be endophytes of medicinal plants for the first time. Several plants are also reported for the first time to contain microbial endophytes, while some plants with powerful pharmaceutical potential harbor unique endophytes. Furthermore, our results reveal a strikingly close relation between the compositions of bacterial and fungal isolates from plants having anti-bacterial activity and those from plants having anti-inflammatory activity, or between the compositions of the microbial endophytic isolates from plants having anti-cancer activity and those from plants having antioxidant activity.

In conclusion, we show that zebrafish early life stages reveal that traditional medicinal plant extracts are able to affect embryo development to varying degrees, prompting caution to apply these medications to pregnant women. We further demonstrate that an extract causing delay in epiboly also inhibits mammalian cell migration, suggesting that this effect may serve as a preliminary test for identifying extracts that inhibit cancer metastasis. In addition, our data provide a better understanding of the potential of Vietnamese medicinal plant resources for combating fungal pathogens. This work also highlights that P. digitatum can be employed as a model fungus for screening antifungal activity of plant extracts. The results of plant-endophytic microorganisms provide new findings which can be inspiring for further in-depth studies to explore and exploit the relationships between medicinal plants and their associated endophytes in northern Vietnam and world-wide.