Rapid, field-deployable method for collecting and preserving plant metabolome for biochemical and functional characterization

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.

Fig 1. Illustration of RAMES method of preparing metabolome libraries.

(See text for details).

Fig 2. Illustration of RAMES method of grinding hard plant tissues prior to extraction.

A1, Specially manufactured bit for grinding and extracting plant tissues (also shown in Fig 1A, 1B and 1E); A2, Dremel 562 tile grinding bit; A3, Dremel 561 multipurpose bit. B, Rapid grinding of woody stems to produce small particles suitable for extraction; C, Grinding of bark; D, Grinding of branches.

Fig 3. Quantitative comparison of compounds eluted from Chromolaena odorata RAMES libraries following 4 and 12-month storage at -20°C.

Mean peak areas (n = 2). Kaempferol-3-O-rutinoside (1), Quercetin-disaccharide (2), Quercetin-trisaccharide (3), Rutin (4), Chlorogenic acid derivative (5), Kaempferol-3-O-glucoside (6), Methoxyhesperetin-1 (7), Methoxyhesperetin-2 (8), Methoxyhesperetin-3 (9), Sakuranetin (10), Chromomoric acid (11), Oxophyto-9,15-dienoic acid (12).

Fig 4. Quantitative comparison of compounds eluted from Datura stramonium RAMES libraries following 4 and 12-month storage at -20°C.

Mean peak areas (n = 2). 6-Hydroxy-hyoscyamin (1), 6-Hydroxy-hyoscyamin-2 (2), Scopolamin (3), Atropine (4), 3-Tigloyloxy-6,7-dihydroxytropane (5), Daturilin (6), Hydroxy-oxowithatrienolide (7).

Fig 5. Quantitative comparison of compounds eluted from Datura inoxia RAMES libraries following 4 and 12-month storage at -20°C.

Mean peak areas (n = 2). 6-Hydroxy-hyoscyamin (1), Scopolamin (2), Atropine (3), 3-Tigloyloxy-6,7-dihydroxytropane (4), Daturilin (5), Hydroxy-oxowithatrienolide (6).

Fig 6. Illustration of anti-fungal, anti-bacterial and anti-oxidant RAMES-STN assays in 24-multiwell plate format.

Activity is rated using a 0–3 scale with 0 representing no activity and 3 representing high activity. Activity ratings are included in parenthesis after well numbers. Rows 1–2. Fusarium spp. anti-fungal assay. Wells A1-B1 (3) positive control (glass fiber disc with econazole nitrate); C1-D1 (0) negative control (glass fiber disc with 70% ethanol); E1-F1 (0) blank (no glass fiber disc); A2-B2 (0) peeled lemon extract; C2-D2 (3) peeled garlic extract, E2-F2 (0) orange rind extract. Rows 3–4. Saccharomyces cerevisiae anti-fungal assay. Wells A3-B3 (3) positive control (econazole nitrate); C3-D3 (0) negative control; E3-F3 (0) blank; A4-B4 (0) peeled lemon extract; C4-D4 (2) peeled garlic extract; E4-F4 (0) orange rind extract. Rows 5–6. Saliva-inoculated anti-bacterial assay. Wells A5-B5 (3) positive control (penicillin); C5-D5 (0) negative control, E5-F5 (0) blank; A6-B6 (3) peeled lemon extract; C6-D6 (3) peeled garlic extract; E6-F6 (1) orange rind extract. Rows 7–8. Anti-oxidant assay. Wells A7-B7 (3) positive control (ascorbic acid); C7-D7 (0) negative control; E7-F7 (0) blank; A8-B8 (1) peeled lemon extract; C8-D8 (2) peeled garlic extract; E8-F8 (3) orange rind extract.