Abstract
Study of plant metabolome is a growing field of science that catalogs vast biochemical and functional diversity of phytochemicals. However, collecting and storing samples of plant metabolome, sharing these samples across the scientific community and making them compatible with bioactivity assays presents significant challenges to the advancement of metabolome research. We have developed a RApid Metabolome Extraction and Storage (RAMES) technology that allows efficient, highly compact, field-deployable collection and storage of libraries of plant metabolome. RAMES technology combines rapid extraction with immobilization of extracts on glass microfiber filter discs. Two grams of plant tissue extracted in ethanol, using a specially adapted Dremel® rotary tool, produces 25–35 replicas of 10 mm glass fiber discs impregnated with phytochemicals. These discs can be either eluted with solvents (such as 70% ethanol) to study the metabolomic profiles or used directly in a variety of functional assays. We have developed simple, non-sterile, anti-fungal, anti-bacterial, and anti-oxidant assays formatted for 24-multiwell plates directly compatible with RAMES discs placed inside the wells. Using these methods we confirmed activity in 30 out of 32 randomly selected anti-microbial medicinal plants and spices. Seven species scored the highest activity (total kill) in the anti-bacterial (bacteria from human saliva) and two anti-fungal screens (Fusarium spp. and Saccharomyces cerevisiae), providing functional validation of RAMES technology. RAMES libraries showed limited degradation of compounds after 12 months of storage at -20°C, while others remained stable. Fifty-eight percent of structures characterized in the extracts loaded onto RAMES discs could be eluted from the discs without significant losses. Miniaturized RAMES technology, as described and validated in this manuscript offers a labor, cost, and time-effective alternative to conventional collection of phytochemicals. RAMES technology enables creation of comprehensive metabolomic libraries from various ecosystems and geographical regions in a format compatible with further biochemical and functional studies.
Highlights
Natural products, those derived from plants, have made invaluable contributions to human civilization
The RApid Metabolome Extraction and Storage (RAMES) method for collecting and storing metabolomic libraries of phytochemicals is designed for speed, efficiency, low-cost, simplicity, portability, long-term storage, and compatibility with bioactivity assays
RAMES allows compact storage of thousands of plant metabolome samples in a format compatible with screening strategies that utilize glass fiber discs impregnated with phytochemicals in the variety of functional assays
Summary
Those derived from plants, have made invaluable contributions to human civilization. They enabled the development of human medicines, crop protection chemicals, dietary supplements, cosmetics, preservatives, disinfectants, flavors, fragrances, and colorants [1,2]. The first commercially produced pharmaceutical, aspirin, synthesized by Bayer in 1897, was derived from the plant natural product salicylic acid, and, to this date, the majority of painkillers and chemotherapeutic agents originate from plants [2]. Substantial advances in analytical instrumentation has accelerated the rate of natural product discovery in recent years; natural product-based formulations, medicines, are facing increasing competition from chemically synthesized compounds and biologics [3, 5]. Plant-based natural product discovery and research are hindered by worldwide destruction of natural habitats, disappearance of many species, as well as political and logistical concerns associated with the 1992 Rio Convention on Biological Diversity [6, 7]
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