Figure 1.
Sclareol contents of mature clary sage inflorescence organs.
(A) Fully opened flowers of clary sage with visible calyx, corolla and bract. (B) Sclareol was extracted, analysed and quantified as described under materials and methods on freshly harvested material. Dissected flowers were at a fully opened stage. Each value represents the average value of five independent samples ± CI (α = 0.05). Each sample was a mix of organs originating from three separate plants. Dissected organs were leaves, bracts, calyces (with attached carpels) and corollas (with attached stamens). Legends: Br, bract; Ca, calyx; Co, corolla; FB, flower bud; Le, leaf.
Figure 2.
Micrographies of calyx epidermis of clary sage.
(A) ESEM view of a calyx epidermis showing the diversity of trichomes. (B) ESEM view of a capitate gland. (C) Nadi staining of a capitate gland with a terpene droplet in the neck cell (hand-made cutting has destroyed the cuticle and led to the loss of essential oil in the subcuticular storage space). (D) ESEM view of epicuticular precipitate-like structures. (E) ESEM view of a cluster of epicuticular crystal-like structures. (F) Light micrography of crystals on the cuticle. Legends: Arrows, cuticle; Full arrowheads, epicuticular crystal and precipitate-like structures; Empty arrowhead, terpene droplet. Scale bars: 100 µm (A, D, E), 50 µm (B), 25 µm (C), 20 µm (F).
Figure 3.
Microscopic imaging of crystal-like structures of clary sage and sclareol crystals.
(A) Polarized light micrography of sclareol crystals. (B, C, D) Polarized light micrography of crystal-like structures on the cuticle of calyces showing a cluster on C and different shapes on D. (E) Differential interference contrast micrography of crystal-like structures on the cuticle of calyces. (F) TPF micrography of the same structures as in E. (G) Imaging of SHG signal (green canal) merge with photo F. (H) TPF micrography of sclareol crystals. (G) Imaging of SHG signal (green canal) merge with photo H. Legends: Full arrowheads, epicuticular crystal-like structures. Scale bars: 10 µm (D), 15 µm (B, C, E, F, G), 30 µm (A, H, I).
Figure 4.
Observation and GC-MS analysis of epicuticular crystals.
(A) Light micrography of cuticular crystals just before the industrial process of sclareol extraction. (B) Light micrography of cuticular crystals after steam distillation. (C) Light micrography of the cuticle after solid/liquid extraction with hexane. (D) Polarized micrography of crystals observed in the pellet after centrifugation (note the cuticle scraps). Trichomes were photographed because they allowed the best imaging after the treatments. (E) GC-MS analysis of essential oil obtained by steam distillation of straw and diluted 10-fold in hexane (% of total peak area: 16% peak 1, 62% peak 2, 4% peak 3, 6% peak 4, 2% peak 5). (F) GC-MS analysis of an hexane wash of calyces dissected after steam distillation of the straw (% of total peak area: 97% peak 5). (G) GC-MS analysis of pellets collected after centrifugation of calyces in water and phase extracted with hexane (% of total peak area: 11% peak 2, 84% peak 5). (H) GC-MS analysis of commercial sclareol (purity >2%, 2 g/L in hexane; total peak area, 96% peak 5). Peak identifications are based on retention time, mass spectrum and comparison with authentic standards. Legends: 1, linalool; 2, linalyl acetate; 3, β-caryophyllene; 4, germacrene D; 5, sclareol; Arrows, cuticle; Arrow head, crystals; TIC, total ion count. Scale bars: 30 µm.