The Composition of Essential Oils from Different Parts of Gilia capitata Sims

1. Introduction

Gilia capitata Sims, with the common name bluehead gilia or blue field gilia (bule gilia), belongs to the family Polemoniaceae, and is a close relative to floxes and polemonium [1]. G. capitata Sims belongs to the subgenus Gilia and the Eugilia section. The plants that make up this section are herbaceous annuals with blue and purple flowers gathered in loose false umbrellas, corymbs or heads. The section consists of 9 species, among which are G. achilleaefolia, G. capitata, G. millefoliata, G. clivorum, G. laciniata, G. tricolor, G. angelensis, G. nevinii, and G. valdiviensis [2,3].

G. capitata is native to all western states of the USA [4,5]. The native habitats of G. capitata are sunny rocky hillslopes and open fields with well-drained soils at an elevation range of up to 2000 m [6,7]. It is introduced widely throughout western North America, Alaska, northern Mexico, and Europe as an ornamental flower [8].

G. capitata is a polytypic species. It is phenotypically plastic in response to environmental conditions [9,10]. It has a wide morphological diversity because of the ecotypic and genotypic variability [6]. Eight defined subspecies form a morphological gradation throughout the Pacific coast of North America [6]. Chromosomes: 2n=18. The flagship sub-species, G. capitata subsp. capitata, is an herbaceous annual plant with a slender 20-100 cm erect stem. Stem can be simple or branched in the upper third, glabrous, glandular or slightly floccose. The root system consists of a relatively shallow main root. On base and stem, cauline leaves are bipinnately dissected, usually glabrous, 4–10 cm long, and with 5–20 mm lobes; upper leaves are more straightforward linear. Blue (or blue-violet) campanulate flowers are clustered into globose to conoidal inflorescences with (25) 50–100 flowers at the terminal position of each stem or branch. Inflorescence diameter varies between 14–40 mm. Individual flowers therein are 0.6-2.5 mm [9] with acute lobes 0.6–1 mm wide and 2 mm long, straight-tipped and with a 6-8 mm corolla. Pollen has a light blue colour. The flowers of the gilia are protandrous, i.e. the anthers mature first, and stigmas rise a few days later [2,11]. Gilia is mostly self-compatible; only some populations are facultatively outcrossing [2,11], though [9] reports that gilia is self-incompatible. The fruit is a three-celled globose or obovoid capsule, which splits at maturity only partly as three valves (i.e. it has an indehiscent capsule)). The capsule contains 1-10(25) seeds, one or two in each cell. The capsule remains indehiscent in dry weather and splits only partly open with the rain. When dry, capsules break off easily by touch. Seeds are hard, brown, ovoid or ovoid-angled, 1–2.3 mm long. When wet, the verrucose seed coat becomes a sticky, mucilaginous coat; this feature probably serves to stick seeds to animals for dispersal and later to substrate [9].

G. capitata is easily grown from seed. No stratification or scarification is required (though two weeks of cold stratification has been suggested to speed up germination). Seeds can be sown directly into the garden in spring. Still, we observed that they reseed reasonably well in autumn and overwinter in the Estonian winter to germinate in early spring. Minimal maintenance is required except for weeding. Soil should be moist when seeds are planted, but adult plants are drought-tolerant. Cutting deadhead flowers will encourage reflowering. In nature, gilia profits from wildfires, removing mature vegetation and opening the ground for light [12]. Its germination probably reacts to the signals from fire residues [13]. It grows in gardens alone or with other low-competition annuals in sunny open locations. Grows well in well-drained medium to coarse soil with a neutral pH (6.0-7.0). In Estonia, G. capitata is cultivated as an ornamental plant.

The gilia is declared one of the pollinator magnets (e.g., Blog of Oregon Native Plants for Pollinators [14] ), and it is included in many wildflower seed mixes for bees. The flowering can be long-term continuous depending on water and temperature conditions. G. capitata needs an insect vector for pollination; flowers are visited by a wide range of insects, including small-sized pollinators and wild bees, bumblebees, long-tongued flies, and butterflies [15,16]. The attractiveness has been partly explained by its tight capitate inflorescence structure, composed of up to 80 flowers and forms a convenient landing pad for all insects [15]. Some of its essential oils could also be one of the causes of flower visit attraction of pollinators [17].

The chemical composition of G. capitata has not been studied before. Therefore, this work aimed to study the component composition of essential oil (EO) of different morphological groups of raw materials of G. capitata.

2. Materials and Methods

2.1. Plant Material

Seeds, obtained from different sources, were mixed and sown in spring. Plants were grown in field conditions at three sites with different soil conditions around Tartu, Estonia. Later, all the collected plant material was mixed in relatively equal proportions before analysis. During the growth, plant growth was supported with complex fertilizers. Plant material was collected twice. The main collection of plant material was done at the time of mass-flowering. We observed two waves of flowering – the first inflorescence formed at the top of the main stem and, several weeks later, on lateral stems – the material was collected in both rounds and later mixed. Depending on the site phenology, the first forage was done at the end of June 2023 and early weeks of July. Plant parts (flowers, leaves, roots, fruits) were collected separately in the field. Later, after drying, the leaves and stems were separated. The second collection was done in late summer in August 2023 (the date depended on the site and weather), at the time of fruit and seed ripening – the heads and clusters of fruit capsules were collected and dried. Later, fruit capsules were crushed, and seeds were separated. Capsule residues containing mainly fruit valves and walls were kept as a separate fraction as shells of the fruit wall (hereafter shells). The plant material was dried for 14 days at room temperature in a well-ventilated room. It was stored at room temperature in paper bags before distillation of essential oil. The voucher sample specimens (No Polemon/Gil1-7) are available at the Institute of Pharmacy, University of Tartu, Estonia.

2.2. Hydrodistillation of Essential Oil

The EO hydrodistilled from the different dried parts of G. capitata using the method described in the European Pharmacopoeia [18]. The plant materials (20 g) with 400 mL of purified water were hydrodistilled in a 1000 mL round-bottom flask for 2 hours (3–4 ml/min). Hexane (0.5 mL) was added to a graduated tube to remove the distilled oil [19,20]. The yield of EO was measured using European Pharmacopoeia with xylol [18]. Due to the content of saponins, intense foam was produced during the distillation of most plant parts, to reduce which 30 g of potassium chloride was added to the flask (except for the roots and stems).

2.3. Gas Chromatography/Mass Spectrometry

The samples of EO were analysed by gas chromatography-mass spectrometry (GC/MS), using an Agilent 6890/5973 GCMS system controlled by MSD Chemstation. 1 µL of the sample was injected at an injector temperature of 280 ^oC in split mode (10:1), using He as the carrier gas onto Agilent HP-5MSI column (30 m length, 0.25 mm inner diameter, 0.25 µm film thickness). The carrier gas was held at the constant flow rate of 1 mL/min. The oven was held at 50 ^oC for 2 min, followed by a ramp of 4 ^oC/min to a final temperature of 280 ^oC and held at 280 ^oC for 5 minutes. The MSD was operated in EI mode at 70 eV. After a delay time of 4 min mass spectra were recorded in the range of 29 – 400 m/z at a rate of 3.8 scans per second. The data were analysed by Agilent Masshunter Data Analysis Software using a deconvolution algorithm with different window size factors. Obtained compounds were identified by using NIST23 library with Match Factor ≥ 90 and by retention indexes (relative to n-alkanes C8 – C30) or obtained by the analysis of the reference compounds. The area percentages of each peak were calculated from the total areas in the chromatograms without using correction factors [19,20].

2.4. Data Analysis

The similarity of plant parts in their composition of EO was analysed using the cluster analysis, implemented in PC-ord v7 [21]. The Sorensen (Bray-Curtis) distance measure of similarity was used to build the cluster tree by applying the Flexible beta linkage method with parameter -0.25 (a default value). The log-transformed proportion of oils was used, and only components with at least 1% were included in the data set to reduce noise.

3. Results

As a result of the study, it was found that different parts of G. capitata accumulated from 0.42 ml/kg (shells) to 1.97 ml/kg (seeds) of EO (Figure 1). It should be noted that the flowers contain less essential oil than the seeds. According to the content of EO, raw materials are arranged in the following order: seeds > flowers > fruits > leaves > stems > roots > shells. Seeds contain a bit less than twice as many essential oils as flowers and leaves.

The composition of hydrodistillates was studied by GC-MS (Table 1). In total, 118 compounds were identified, representing 81.31-93.74 % in the oil (Table 1). All parts of the plant contain benzeneacetaldehyde, nonanal, (E)-2-nonenal, (-)-myrtenol, (E,E)-2,4-decadienal, tetradecanal, pentadecanal, hexahydrofarnesyl acetone, and di-2-methylpropylphtalate.

The composition of essential oils in different parts of G. capitata was illustrated with a cluster tree (Figure 2). Fruits and seeds were most similar in composition; flowers were next similar ones. Leaves and stems formed another narrow cluster. The most distant were shells of the fruit. The composition of roots was the most distinct from all other parts of a plant (Figure 2).

4. Discussion

As mentioned above, the yield of EO in different parts of G. capitata was 0.42-1.97 ml/kg (Figure 1). We compare that with results obtained in studies of some well-known medicinal plants studied. The aerial parts of Origanum vulgare from different countries contained 2-11 ml/kg of EO [22], in Thymus vulgaris herb the yield of EO was 3-28 ml/kg [23], in commercial samples of Valeriana officinalis roots from various countries different origin 2-10 ml/kg [24], and in the herb of Artemisia absinthium from different origin 2-4 ml/kg [25]. The branches of Juniperus communis shrubs were collected from 27 different habitats in Estonia and contained 0.3-6 ml/kg of EO [26]. Therefore, the oil content in G. capitata is modest compared to classic EO plants.

The essential oil of flowers was dominated by hexahydrofarnesyl acetone (19.1%), followed by palmitic acid (12.2%), γ-decalactone (7.1%) and (Z)-2-p-menthen-1-ol (3.6%) (Figure 3). In addition to the flowers, hexahydrofarnesyl acetone is also the dominant component in fruits (18.2%) and seeds (15.2%), and it is found in significant quantities in the stems (4.9%) of the G. capitata (Figure 3). Hexahydrofarnesyl acetone has been proven to exhibit a potent antimicrobial, anti-inflammatory, and cytotoxic activity and is used in pain relief research [27,28,29,30]. Palmitic acid, present also in seeds (11.1%) and fruits (8.0%), has antioxidant and antibacterial activity [31,32].

Lactones, including γ-decalactone, constitute an essential group of fatty acid-derived volatile organic compounds conferring peach-like aroma to a few essential oils and fruits including peach, plum, pineapple and strawberry [33]. γ-Decalactone inhibits strawberry pathogen growth and achene germination [34].

Menthane monoterpenoids (Z)-2-p-menthen-1-ol and (E)-p-2-menthen-1-ol are found in all organs of the aboveground part of the plant. They make up 5.7% of flowers, 2.2% of leaves, 6.0% of stems, 5.6% of fruits, 3.3% of shells and 4.6% of seeds. Menthenol derivatives have potentially different biological properties. 1-Methyl-4-(1-methylethenyl)-2-cyclohexen-1-ol is an acetal reagent used in the synthesis of desoxy cannabidiols and THC related psychoactive compounds. It is formed from (+)-Limonene using a photosynthesized O₂ transfer [35].

In addition to the dominant compounds in the flowers, (Z)-γ-bisabolene was also found (though, it is similarly abundant in stems and fruits). The antitumor activity of γ-bisabolene in human neuroblastoma cells has been proven through the induction of p53-mediated mitochondrial apoptosis [36]. γ-Bisabolene has demonstrated antiproliferative activities against several human cancer cell lines. Another study [29] disclosed the antiproliferative and apoptosis induction activities of γ-bisabolene to human neuroblastoma TE671 cells, and a CC50 value of γ-bisabolene was 8.2 μM to TE671 cells [37].

Caryophyllene oxide is found in flowers (0.9%), while caryophyllene was distributed more widely in flowers, leaves, fruits and shells (1.3, 0.5, 2.8%, respectively). Its anticancer, antioxidant and antimicrobial properties have been proven [38].

Linalool is found in flowers (0.3%), leaves (0.5%), stems (0.3%), shells (0.6%) and seeds (0.3%). Linalool and (E)-caryophyllene exhibited high cytotoxic activity against the amelanotic melanoma and renal adenocarcinoma cells [39].

The characteristic terpenoids of G. capitata are shown in Figure 4.

The predominant compounds in the EOs of the leaves were phytol (23.3%) and diterpene alcohols isomanool (12.1%) and sclareol (4.5%), (E)-β-ionone (4.5%).

Phytol has antibacterial and antioxidant activity, inhibiting the growth of Staphylococcus aureus [40,41]. Phytol is used in fragrance and cosmetics to produce shampoos, toilet cleaners, household cleaners and detergents [42].

Labdane diterpenoids are the most common types of diterpenoids isolated in minute amounts from higher plants [43]. Labdane diterpenoids are interesting for their cytotoxic, antifungal, anti-inflammatory, antiparasitic and analgesic properties [44,45,46]. Labdane diterpenoid sclareol has a wide range of bioactivity, including anti-tumor, anti-inflammation and anti-pathogenic microbes, and even anti-diabetes and hypertension [47]. Sclareol also can kill human leukemic cells and colon cancer cells in vitro by apoptosis [48,49]. Labdanum-type diterpenoids are usually found in the plant as a mixture of components. Therefore, we found it necessary to provide all the structural formulas of labdanum diterpenoids contained in G. capitata raw materials (Figure 4). Labdan-type diterpenes, such as manoyl oxide, 13-epi-manoyl oxide, epi-13-manool, kolavelool, isomanool, sclareol make up a significant part of leaves and stems oils – 22.63% and 20.2%, respectively. Fruits and seeds essential oils contain less– 3.85% and 2.99%, respectively. The main component among labdan diterpenes is isomanool (labda-8,14-diene-13β-ol), it is found in flowers 0.39%, leaves 12.07%, stems 8.29%, fruits 3.23% and seeds 2.48%. It is followed by manoyl oxide, found in leaves 2.28%, stems 2.96%, fruits 0.62%, seeds 0.51%. They are absent in shells and roots. Nor-labdane diterpenoids ambrial is found in trace amounts only in stems.

C-13-isoprenoid, apocarotenoid β-ionone had fungicidal activities against Aspergillus fumigatus. It also ameliorated fungal keratitis in mice by reducing inflammation, which LOX-1, p-p38MAPK and p-JNK regulated [50]. Literature data indicate that β-ionone and its derivatives have a lot of important pharmacological activities, including antibacterial, antifungal, antioxidant, anti-inflammatory, antiproliferative, and anti-cancer [51].

The most dominant compounds in EO of stems are isomanool (8.3 %), sclareol (6.7%) and 2,4-decadienal, (E,E)- (2.9%). Known about nematicidal activity 2,4-decadienal [52] and insecticidal against the barn bug [53].

In addition to hexahydrofarnesyl acetone, the dominant in the fruits were palmitic acid (8.0%), heptacosane (5.3%) and γ-decalactone (4.6%). The dominant in the shell were (+)-epi-bicyclosesquiphellandrene (15.4%), β-elemene (8.5%) and pentadecanal- (6.3%). β-Elemene exhibits anti-tumour properties, and anti-inflammatory and antioxidant effects [54,55]. β-Elemene and β-elemene piperazine derivatives have been shown to inhibit tumour cell growth in vitro and in vivo [56]. The anti-bacterial authorities of the pentadekanal were established [57,58].

In addition to hexahydrofarnesyl acetone, the dominant in the seeds were palmitic acid (11.1%), 1,3-dimethylbenzene (10.3%) and heptacosane (5.0%). Heptacosan can improve P-glycoprotein-mediated drug transport, demonstrating the ability to retain the substrate doxorubicin inside the cell and enhancing its cytotoxic effects [59].

The dominant in the root were (-)-myrtenol (25.7%), (E)-myrtanol (16.4%), palmitic acid (8.1%) and 2-pentylfuran (2.9%). Several reports have demonstrated the pharmacological properties of myrtenol, including its antioxidant, antibacterial, antifungal, antidiabetic, anxiolytic, and gastroprotective activities [60,61]. (-)-(E)-Myrtanol is an antimicrobial and acaricide agent [62,63]. 2-Pentylfuran has been suggested as a repellent for spotted-wing drosophila, as it can significantly reduce fruit infestations under field conditions [64]. In the roots, cuparene (0.3%) was found. Cuparene derivatives show moderate to high activity levels on lung cancer cell lines NSCLC-N6 and A549 [65].

A significant number of volatile compounds are aliphatic (24.37 – 48.56%). They are the dominant compounds in flowers, stems, fruits, and seeds (Figure 5).

At the same time, diterpenoids are the dominant group in the composition of volatile compounds in leaves (45.89%), sesquiterpenoids in shells (34.68%), and monoterpenoids in roots (46.43%).

It should also be noted that the content of mono- and sesquiterpenoids is quite high in flowers (12.78% and 26.73%, respectively), stems (9.88% and 5.71%), fruits (9.57% and 18.88%), seeds (8.08% and 15.84%), monoterpenoids in shells (7.93%).

Sesquiterpenoids are represented by derivatives of the naphthalene, such as copaene, humulene, (+)-epi-bicyclosesquiphellandrene, β-selinene, τ-muurolol, α-cadinol and azulene, such as 7-epi-α-cedrene series. Antiviral activity against dengue virus (VDEN) was established for a few sesquiterpenoids, including isomers of copaene, β-caryophillene, caryophillene oxide and (+)-epi-bicyclosquiphellandrene [66]. Norsesquiterpenoids or apocarotenoids are represented by two compounds such as (E)-β-ionone and (E)-β-damascenone. They are absent in the roots. β-Damascenone inhibits the expression of pro-inflammatory cytokines and leukocyte adhesion molecules [67]. β-Damascenol has been shown to be effective in preventing skin sunburn [68].

Aromatic compounds represent an insignificant part of the composition of volatile compounds, are found in all parts of the plant and accumulate in fruits to the maximum extent (11.0%).

5. Conclusions

The composition of volatile compounds of various parts of G. capitata was studied for the first time. The results of the analysis of Gilia essential oil made it possible to detect and identify important potentially active compounds with various biological properties: antioxidant, anti-cancer, antidepressant and others, which indicates the prospects for further phytochemical and pharmacological studies of the genus Gilia. Future studies should focus on Gilia saponins and polyphenolic substances and their biological activity.

6. Patents

No patents.