Conium divaricatum Boiss. & Orph.
Creators: name: Vlassi, Anthi
name: Koutsaviti, Aikaterini
name: Constantinidis, Theophanis
name: Ioannou, Efstathia
name: Tzakou, Olga
Publisher: name: Zenodo
Year: 2022
Subjects: Biodiversity
Taxonomy
Plantae
Tracheophyta
Magnoliopsida
Apiales
Apiaceae
Conium
Conium divaricatum

description: 2.1. Chemical composition of essential oils of C. divaricatum and C. maculatum
The chemical composition of twenty-five C. divaricatum and ten C. maculatum essential oils obtained after hydrodistillation of separate plant tissues (infructescences, inflorescences, leaves, stems, roots) collected from either natural populations or cultivated plants was investigated using gas chromatography with flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) (Table 1).
In total 68 components were identified in the C. divaricatum essential oils (Table 2). The chemical group that dominated the essential oils of C. divaricatum was that of oxygenated hydrocarbons, mainly represented by esters. Among them, one of the major constituents (1 with RRI 1980; subsequently identified as 4 ′ -oxodecyl hexanoate) of the C. divaricatum essential oils (2.8%–88.4%), consistently abundant in stems’ essential oils, regardless of the collection date or site, could not be readily identified on the basis of its mass spectral data. Nonetheless, the highest percentage of compound 1 was detected in the essential oil of the infructescences collected from Mt Pateras in 2010. Furthermore, two more metabolites (2 and 3 with RRI 2153 and 1767, respectively, subsequently identified as 4 ′ -oxododecyl hexanoate and 4 ′ -oxooctyl hexanoate) were also abundant in the majority of the investigated C. divaricatum samples. Moreover, sesquiterpenes and non-oxygenated hydrocarbons (alkanes and alkenes) were constantly present, but in lower ratios. The roots’ essential oils exhibited a different chemical profile compared to the aerial parts, as they were rich in sesquiterpenes, with cis -α -bergamotene and β -bisabolene being the major components. Additionally, elemicin and osthol were compounds detected exclusively in the essential oils of roots in significant percentages.
According to the chemical analyses of the essential oils of C. divaricatum collected from Mt Imittos, hydrocarbon esters dominated the aerial parts’ essential oils, followed by sesquiterpenes, alkanes and alkenes, exhibiting a chemical profile that characterizes C. divaricatum. Hydrocarbon esters were also abundant in the essential oils of C. divaricatum aerial parts collected from Mt Pateras and Mt Parnon. However, the percentage of fatty acids was strikingly higher in the samples collected from Mt Pateras. In contrast to the consistently high content in hydrocarbon esters detected in the samples of C. divaricatum from Mt Imittos, Mt Pateras and Mt Parnon, the analysis of the essential oils from Erythres revealed a different chemical profile, as hydrocarbons were also in this case the most abundant chemical group, represented however mainly by alkanes, alkenes and ketones, whereas esters were detected in lower amounts. This variation in the chemical profile within the same species could be attributed to the different collection period and different vegetative stage, namely to a seasonal variation, since the samples from Erythres were collected in a more advanced fruiting stage, however lacking ripe fruits. Therefore, it can be concluded that hydrocarbon esters are either important for the plant defense or play a key role in reproduction, as they are found in abundance during the most important vegetative stage, the flowering stage. To the best of our knowledge this is the first report on the essential oil composition of C. divaricatum.
In the essential oils of C. maculatum, 67 compounds were identified in total (Table 3). The two investigated taxa showed significant differences in their chemical profile, since in the case of C. maculatum hydrocarbons were detected in lower amounts, and most importantly hydrocarbon esters that characterized C. divaricatum samples were either absent or detected only in traces.
Many quantitative differences were also observed among the essential oils of C. maculatum, with the percentages of the main chemical groups showing a rather significant variation. (Z)-Falcarinol was the main constituent in the infructescences’ essential oils collected from the areas of Thiva and Avlona, while the inflorescences’ oil was characterized by the abundance of alkanes and alkenes, followed by sesquiterpenes. Sesquiterpene derivatives were the main chemical group in the leaves’ essential oil, with germacrene D being the most abundant representative. (Z)-Falcarinol, a natural pesticide, was also the most abundant metabolite in the essential oils of the stems and the roots. Furthermore, the chemical profile of cultivated samples showed some differences in comparison to wild grown plant specimens. In particular, the essential oil of the cultivated plant was characterized by an almost equal ratio of monoterpenes (21.9%) and sesquiterpenes (21.5%). The sample also presented a strikingly lower percentage of (Z)-falcarinol (3.4%), when compared to the essential oils obtained from wild grown plants.
Table 3
Chemical composition of the essential oils a of Conium maculatum.
(continued on next page)
tr: detected in traces (<0.1%), -: not detected.
a Numbering of samples according to Table 1.
b Compounds listed in the order of elution on a HP-5MS column under the specified chromatographic conditions.
c Relative retention indices calculated from the retention times of the compounds in relation to those of a series of n -alkanes (C –C) analyzed under the same 8 30 chromatographic conditions.
d The base peak and molecular ion peak (when observed) in the mass spectrum of the unidentified compound are included in parentheses.
e Including only the identified compounds.
Our results are generally in accordance with the literature data reporting the chemical analysis of essential oils from Iranian and Serbian hemlock (Masoudi et al., 2006; Radulovic et al., 2008). According to Radulovic et al. (2008), the leaves’ and the inflorescences’ essential oils from Serbia are rich in sesquiterpenes (54.4% and 46.3%, respectively), with germacrene D being the most abundant metabolite. Sesquiterpenes are also the main chemical group in the essential oils of C. maculatum aerial parts, wild grown in Iran, with germacrene D being the most abundant representative (Masoudi et al., 2006). On the contrary, the essential oils of hemlock from Sicily showed different chemical profiles. In particular, the essential oil of the inflorescences was characterized by the presence of 1-butylpiperidine and myrcene in high abundance (26.4% and 24.0%, respectively), while (E)-caryophyllene dominated (54.8%) the essential oil of the leaves (Di Napoli et al., 2019).
Comparison of the chemical profiles of the essential oils obtained from the different plant parts of C. divaricatum and C. maculatum revealed significant qualitative differences between the two taxa. In particular, (Z)-falcarinol was present in high abundance (18.1–81.1%) in all samples of C. maculatum, with the exception of the cultivated population M1, whereas it was completely absent from specimens of C. divaricatum. In contrast, all samples of C. divaricatum aerial parts, with the exception of samples D6 and D16 which were collected from Erythres in a more advanced fruiting stage, were characterized by the presence of compounds 1–3 in high abundance (50.5–95.0%).
2.2. Isolation and structure elucidation of metabolites 1–3 from C.
divaricatum
Since the major constituents of the C. divaricatum essential oils could not be readily identified on the basis of their mass spectral data, chromatographic separations were undertaken in order to isolate and elucidate the chemical structures of compounds 1–3 (Fig. 1). Towards this, C. divaricatum stems collected from Mt Imittos were subjected to biphasic alkaline extraction and the organic phase was repeatedly fractionated to yield compounds 1–3 and 12-nonacosanone in pure form.
Compound 1 was isolated as colourless oil. The molecular formula C 16 H 30 O 3 was deduced by EIMS data ([M] + observed at m/z 270). The 13 C NMR and HSQC-DEPT spectra revealed 16 carbon signals, including an ester and a ketone carbonyl at δ 173.9 and 211.1, respectively, one oxygenated sp 3 carbon (δ 64.1), and thirteen non-functionalized sp 3 carbons (δ 13.7–44.6), among which eleven methylenes and two methyls (Table 4). The 1 H NMR spectrum revealed two triplets ascribed to two aliphatic methyls at δ 0.83 (t, J = 6.9 Hz, H 3 -6) and 0.85 (t, J = 7.1 Hz, H 3 -1 ′), as well as two multiplets at δ 1.48–1.60 and δ 1.22–1.34, each assigned to four aliphatic methylenes. Three triplets were also observed at δ 2.23 (t, J =7.6 Hz, H 2 -2), 2.31 (t, J =7.1 Hz, H 2 -3 ′), and 2.33 (t, J = 7.0 Hz, H 2 -5 ′) attributed to three relatively deshielded methylenes, as well as a triplet at 3.99 ppm (t, J = 6.7 Hz, H 2 -10 ′) assigned to an oxygenated methylene (Table 4). Analysis of the HSQC-DEPT, HMBC and COSY spectra revealed the planar structure of compound 1 (Fig. 2). Specifically, the COSY correlations of H 3 -1’/H 2 -2 ′, H 2 - 2’/H 2 -3 ′, H 2 -5’/H 2 -6 ′, H 2 -6’/H 2 -7 ′, H 2 -7’/H 2 -8 ′, H 2 -8’/H 2 -9 ′, H 2 -9’/H 2 - 10 ′, H 2 -2/H 2 -3, H 2 -3/H 2 -4, H 2 -4/H 2 -5, and H 2 -5/H 3 -6 revealed three separate spin systems. Additionally, HMBC correlations of C-4 ′ with H 2 - 2 ′, H 2 -3 ′, H 2 -5 ′ and H 2 -6 ′ were observed, connecting the two separate spin systems. Moreover, HMBC correlations of C-1 with H 2 -2, H 2 -3 and H 2 -10 ′ were observed, concluding the structure of compound 1, which was identified as 4 ′ -oxodecyl hexanoate, representing an undescribed natural product.
nd: not detected.
a Recorded at 400 MHz.
b Recorded at 50.3 MHz.
c Determined through HSQC-DEPT correlations.
Compound 2, isolated as colourless oil, displayed a molecular ion peak at m / z 298 (EIMS), corresponding to C 18 H 34 O 3. The spectroscopic characteristics of compound 2 (Table 4) revealed high similarity to those of 1. Specifically, in the 1 H NMR spectrum two triplets were observed at δ 0.86 (t, J = 7.0 Hz, H 3 -6) and 0.89 (t, J = 7.2 Hz, H 3 -1 ′), assigned to two aliphatic methyls, and two multiplets observed at δ 1.50–1.62 and 1.23–1.37 were ascribed to four and six aliphatic methylenes, respectively. Moreover, three triplets resonating at δ 2.26 (t, J =7.5 Hz, H 2 -2), 2.35 (t, J =7.2 Hz, H 2 -3 ′), and 2.37 (t, J =7.2 Hz, H 2 -5 ′) were assigned to three relatively deshielded methylenes, while a triplet at 4.02 ppm (t, J = 6.7 Hz, H 2 -12 ′) was attributed to an oxygenated methylene. In a similar manner to 1, analysis of the 2D NMR spectra led to the identification of compound 2 as 4 ′ -oxododecyl hexanoate, an undescribed natural product.
Compound 3, obtained as colourless oil, had the molecular formula C 14 H 26 O 3, as determined by EIMS data ([M] + observed at m/z 242). The spectroscopic data of compound 3 (Table 4) were rather similar to those of compounds 1 and 2. Direct comparison of the 1 H NMR spectra of 1–3, revealed that 3 contained only four methylenes in the middle spin system, in contrast to 1 and 2 containing six and eight methylenes, respectively. Thus, compound 3 was identified as an undescribed natural product, namely 4 ′ -oxooctyl hexanoate.
2.3. Detection of alkaloids and determination of the total alkaloids
content
The genus Conium is characterized by the abundance of alkaloids. In order to confirm their presence in the investigated taxa, two extracts of different polarity, namely an organic extract resulting from maceration of the air-dried aerial parts in CH 2 Cl 2 /MeOH (1:1) containing less polar constituents and a hydromethanolic extract resulting from maceration of the air-dried aerial parts in MeOH/H 2 O (4:1) containing more polar constituents, were prepared from samples of both taxa and the presence of alkaloids was investigated employing specific reagents (Dragendorff, Wagner and Mayer) for the detection of alkaloids. All extracts upon addition of the reagents resulted in the formation of precipitate, confirming the presence of alkaloids in both taxa (Table 5).
The total alkaloids content of both taxa was determined in triplicate in their hydroethanolic extracts, resulting from maceration of the air-dried samples in EtOH/H 2 O (3: 1), following a standard acid-base titrimetric procedure described in the literature (Mellon and Tigelaar, 1932). The total alkaloids content of C. maculatum was determined as 0.203 ± 0.096%, while for C. divaricatum it was measured at 0.060 ± 0.026%, expressed in both cases as coniine (Paech and Tracey, 1955). Regarding the total alkaloid content of C. maculatum, our results are in agreement with literature data (0.01–0.9% w/w for various extracts) (L´opez et al., 1999), while there are no previous reports for C. divaricatum.
2.4. Evaluation of toxicity
In this work, the brine shrimp lethality assay was used to assess the toxicity of the organic extracts and essential oils of both taxa, as well as of 4 ′ -oxodecyl hexanoate (1), the main component of the essential oil of C. divaricatum. The lethality exerted on the nauplii of the brine shrimp Artemia salina L. (Artemiidae) has been widely used in laboratory bioassays as a predictor of chemical toxicity on organisms in aquatic environments through the estimation of the medium lethal concentration (LC 50) (Meyer et al., 1982). The results of the evaluation of the toxicity on the brine shrimp after 24 h of incubation are presented in Table 6. The toxicity assessment against the crustacean A. salina did not reveal significant differences for the two investigated taxa, displaying moderate toxicity with LC 50 values between 0.1 and 0.5 mg /mL (Mitic´et al., 2021).
3. Concluding remarks
The distinctive differences observed between the chemical profiles of the essential oils obtained from various plant parts of C. divaricatum and of C. maculatum, in conjunction with the morphological differences observed for the two taxa, strongly support that C. divaricatum should be classified as a separate species within the genus, as it was originally proposed by Boissier and Orphanides in 1856, and not as a synonym of
+: moderate formation of precipitate; ++: intense formation of precipitate. C. maculatum, as it is found in several botanical sources. Since both species, forming rich populations in Attica and around Athens, exhibit similar levels of toxicity, Socrates may have been poisoned by either of the two.
description: Published as part of Vlassi, Anthi, Koutsaviti, Aikaterini, Constantinidis, Theophanis, Ioannou, Efstathia & Tzakou, Olga, 2022, What Socrates drank? Comparative chemical investigation of two Greek Conium taxa exhibiting diverse chemical profiles, pp. 1-10 in Phytochemistry (113060) (113060) 195 on pages 2-7, DOI: 10.1016/j.phytochem.2021.113060, http://zenodo.org/record/8234268