Cell-type specific localization and biological activity of the volatiles from the endemic species Chaerophyllum coloratum L

Abstract

New findings are presented for Chaerophyllum coloratum L. on the volatile composition of the essential oil, based on data of hydrosol and fresh plant material, light and electron microscopy of leaves, and cytotoxic and antiviral activity. The widespread Apiaceae family includes many well-known and economically important plants that are cultivated as food or spices. Many produce essential oils and are generally a source of secondary metabolites and compounds that have numerous applications in daily life. In this study, the chemical composition of volatile organic compounds (VOCs), ultrastructure and biological activity of the Mediterranean endemic species Cheaerophyllum coloratum L. are investigated, as literature data for this plant species are generally very scarce. The essential oil and hydrosol were extracted from the air-dried leaves by hydrodistillation and the chemical composition of both extracts was analysed by GC-MS in conjunction with headspace solid-phase microextraction (HS-SPME) of VOCs from the hydrosol and the fresh plant material. In the composition of the essential oil, the oxygenated sesquiterpenes spathulenol and caryophyllene oxide were the most abundant components. In the fresh plant material, non-oxygenated sesquiterpenes dominated, with β-caryophyllene and germacrene D being the main components. The hydrosol was dominated by monoterpenes, with the oxygenated monoterpene p-cymen-8-ol being the most abundant. Light and electron micrographs of the leaf of C. coloratum show secretory structures, and we hypothesize that glandular leaf trichomes, secretory epidermal cells and secretory canals are involved in the production of volatiles and their secretion on the leaf surface. Since the biological potential of C. coloratum is poorly investigated, we tested its cytotoxic activity on cancer and healthy cell lines and its antiviral activity on plants infected with tobacco mosiac virus (TMV). Our results dealing with the composition, ultrastructure and biological activity show that C. coloratum represent a hidden valuable plant species with a potential for future research.

Keywords: Antiviral activity; Cytotxic activity; Essential oil; GC–MS; Headspace solid-phase microextraction (HS-SPME); Hydrosol; Secretory structures; Tobacco mosiac virus (TMV); Trichomes; Volatile organic compounds (VOCs).

Fig. 1

VOCs of Chaerophyllum coloratum essential oil sorted by structural groups

Fig. 2

The distribution of non-oxygenated and oxygenated monoterpenes (a) and sesquiterpenes (b) of Chaerophyllum coloratum. The first value is the proportion of the total identified components, the second value is the proportion of monoterpenes/sesquiterpenes among terpenes

Fig. 3

VOCs of Chaerophyllum coloratum plant and hydrosol isolated by HS-SPME, sorted by structural groups

Fig. 4

Leaf sections of Chaerophyllum coloratum histochemically stained for terpenes with NADI reagent (a, b) and lipids stained with Sudan III (c, d). ue, upper epidermis; le, lower epidermis; sc, epithelial (secretory) cells around the duct; dt, non-glandular trichome; c, cavity of the duct; arrowheads (black and white), NADI staining; arrows, Sudan III staining

Fig. 5

a–f Semithin sections through the C. coloratum leaf, Toluidine blue. ue, upper epidermis, le lower epidermis, pc parenchymal cells, sec secretory epidermal cell, gt grandular trichome, ngt non-glandular trichome; arrowheads, secretory content within the glandular trichome, in subcuticular space, in secretory epidermal cells and on the leaf surface; xy xylem, ph phloem; arrow, secretory content inside the cavity, c cavity of the duct, sc epithelial (secretory) cells around the duct

Fig. 6

a–d Ultrathin sections through the C. coloratum leaf. a ec epidermal cell, sec secretory epidermal cell, cu cuticle, scs subcuticular space, er endoplasmic reticulum, p plastid, vocs volatile organic compounds; arrowhead, lighter secretory content; *, osmiophylic secretory content. b The enlargement of the part of the secretory epidermal cell shown on the a. ob oil body. c White arrowheads show grayish osmiophilic content, and asterisks show osmiophilic secretory content in the cuticular space. Inset: a higher magnification of the leaf surface area. d sc, epithelial (secretory) cells around the duct; c, cavity of the duct; yellow arrowhead, cell wall protuberance

Fig. 7

Cytotoxic activity of the methanolic extract of C. coloratum on HeLa, HCT116, U2OS and RPE1 cells using the MTS cell proliferation assay. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. IC₅₀ values are means of three independent experiments performed in groups of four ± SD (standard deviation), and statistical significances are labelled *P < 0.05 and ***P < 0.001

Fig. 8

Number of local lesions (LLN) on the plants treated with hydrosol of C. coloratum for two consecutive days before TMV inoculation. Control plants were treated with distilled water. The error bars show the standard deviation of the triplicate analyses. **Statistically significant difference between control and treatment data (P < 0.01)