The Composition of Essential Oils and the Content of Saponins in Different Parts of Gilia capitata Sims

Abstract

Bluehead gilia or bluefield gilia (Gilia capitata Sims, Polemoniaceae) is an annual herbaceous plant widely distributed in the western regions of North America but cultivated as an ornamental flower in various regions to support pollinators. The comprehensive chemical composition of this plant has not been previously reported. Essential oils (EOs) were obtained by hydrodistillation from different parts of the gilia plants. The yield of EOs ranged from 0.42 to 1.97 mL/kg, with the largest yields being obtained from the seeds; smaller yields obtained from the flowers, fruits, and leaves; and the lowest quantity obtained from the stems, roots, and shells. Using the GC-MS method, we identified 116 compounds. Hexahydrofarnesyl acetone was dominant in most parts of the G. capitata. The EO of flowers was dominated by hexahydrofarnesyl acetone (19.1%), fruits by hexahydrofarnesyl acetone (18.2%), seeds by hexahydrofarnesyl acetone (15.2%), fruit by (+)-epi-bicyclosesquiphellandrene (15.4%), leaves by phytol (23.3%), stems by isomanool (8.3%), and roots by (-)-myrtenol (25.7%). Triterpenoid saponins were identified, and 21 compounds were quantified (by HPLC). Saponin levels were high in aerial parts (excluding stems) and the lowest in plant roots. Based on the contents of EO and saponins, the aerial parts of G. capitata may have pharmaceutical properties, but saponins might be the main value of G. capitata.

1. Introduction

Plant parts used for medicinal applications vary greatly regarding the qualitative and quantitative contents of biologically active substances [1]. The chemical composition of some plant species is still poorly studied. One of them is the genus Gilia and the family Polemoniaeae in general [2,3,4]. Gilia capitata Sims, with the common name bluehead gilia or blue field gilia or globe gilia, is a close relative to phloxes and polemonium [5,6,7], which suggests that the level and composition of essential oils could be analogous to Phloxes. However, there are differences in the genus Gilia as it has a high level of saponins [2,8].

Gilia capitata (Figure 1) is native to all western states of the USA [9,10]. 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 [11,12]. It is widely introduced throughout Western North America, Alaska, Northern Mexico, and Europe as an ornamental flower [13]. Bluehead gilia is declared one of the pollinator magnets by gardeners, e.g., the Blog of Oregon Native Plants for Pollinators [14]. It is included in many wildflower seed mixes as a pollinator attractor. G. capitata flowers are visited by a wide range of small-sized pollinator insects, including wild bees, bumblebees, flies, and butterflies [15,16].

In Estonia, G. capitata is cultivated as an ornamental plant, but recently, it was included in the list of bee-friendly intercrop species, and a farmer can obtain extra agri-environmental support for growing it in fields near beehives [17]. This makes the bluehead gilia a potential production species for additional agricultural goods, such as essential oils or saponins.

G. capitata is a polytypic species and phenotypically plastic in response to environmental conditions [11,18,19]. Eight defined subspecies form a morphological gradation throughout the Pacific coast of North America [11]. The flagship sub-species, G. capitata subsp. capitata, is a herbaceous annual plant with a slender 20–100 cm erect stem. The 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 the base and stem, cauline leaves are bipinnately dissected, usually glabrous, 4–10 cm long, and with 5–20 mm lobes; the upper leaves are more straightforward and 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. The inflorescence diameter varies between 14 and 40 mm. Individual flowers therein are 0.6–2.5 mm [18] with acute lobes 0.6–1 mm wide and 2 mm long, straight-tipped, with a 6–8 mm corolla. The 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 [6,20].

Bluehead gilia is mostly autogamous and self-compatible; only some of its native populations or races are facultative outcrossers [6,20] or even self-incompatible [18]. 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, with 1 or 2 in each cell. The capsule remains indehiscent in dry weather and splits only partly open with the rain. When dry, the capsules break off easily by touch. The seeds are hard, brown, ovoid, or ovoid-angled and 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 or avoid its forage by birds or animals [2,18].

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 self-seeded reasonably well in autumn, overwintered in the Estonian winter, and underwent intensive germination in early spring. The soil should be moist when seeds are planted. In nature, bluehead gilia profits from wildfires, removing mature vegetation and opening the ground for light [21]. Its germination probably reacts to the signals from fire residues [22]. During growth, minimal maintenance is required except for weeding. The flowering process can be long-term and continuous depending on soil moisture and temperature conditions. Cutting deadhead flowers will encourage reflowering. It grows in gardens alone or with other low-competition annuals in sunny open locations. It grows well in well-drained medium to coarse soil with a sub-neutral pH (6.0–7.0). Adult plants are drought tolerant.

The attractiveness of bluehead gilia to pollinators has been partly explained by its tight capitate inflorescence structure, composed of up to 80 flowers, which forms a convenient landing pad for all insects [15]. Some of its essential oils could also be one of the reasons why the flower’s scent attracts pollinators [23,24] or the target of foraging [25]. Therefore, the authors of this study were interested in the content of essential oil in different parts of G. capitata, as well as the composition of the oil. The presence and a sufficient amount of essential oil in the plant may provide an opportunity to use the species to benefit human health, such as through medicinal plants, the food industry, perfumery, etc. Alternatively, earlier studies in the literature hint that saponins can be the main value of the species [2,18].

The detailed chemical composition of G. capitata has not been studied before. The foam produced during the distillation of the drug and the irritating effect of the dust produced during the grinding of the drug on mucous membranes indicate the possible presence of saponins in the plant. Therefore, this work aimed to study the contents and compositions of EOs and saponins of different morphological groups of raw materials of G. capitata.

2. Materials and Methods

2.1. Plant Material

This study aimed to obtain the first generalised estimates about the content and composition of essential oils (EOs) in G. capitata and to pinpoint the main source of EOs. This means that the variability and environmental effects were suppressed by mixing plant material grown in field conditions at three sites around Tartu, Estonia. The sites differed in soil conditions: one was heavily clay-rich, one was light clay, and one was sand-rich clay. More detailed studies can be conducted when EO sources within the plants are successfully explored.

Seeds of G. capitata were obtained from different commercial flower suppliers in Europe. Seeds were mixed to obtain more general estimates about the species’ chemical properties and to avoid strong bottleneck effects based on the genetic type grown by a single seed producer. Mixed seeds were sown in spring in mid-May (15–22 May) 2023. Species’ taxonomic identity was re-assessed at the flowering stage using keys available on the Internet. During the growth stage, plant growth was supported with commercial complex fertilisers aimed at flowers.

Plant material was collected twice during the summer. The bluehead gilia is an annual plant species, meaning it has the best EO content status at its flowering stage, except for fruits. Therefore, plant material was mainly collected during mass flowering. We observed two waves of mass flowering—the first wave of inflorescences formed at the top of the main stem, and the second wave of mass flowering occurred several weeks later, when inflorescences on lateral stems bloomed. The material was collected in both rounds and later mixed. Depending on the specific site phenology, the first forage was performed at the end of June and early July (30 June–15 July), and the second in the early weeks of July, with some continued collection being carried out until early August.

Plant parts (flowers, stems with leaves, roots, and fruits) were collected separately in the field. Later, after drying, the leaves and stems were separated. All collected material from different fields was mixed in relatively equal proportions before the analysis to ensure a more general estimate of EOs for the species. The fruits (including seeds and surrounding valves) matured continuously and were therefore collected over several weeks in late summer in August and September 2023. Fruits were defined as ripe for collection when they were yellow and started to dry or were already dry. 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 called 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 the distillation of essential oil and the extraction of saponins. The voucher sample specimens (No Polemon/Gil1-7) are available at the Institute of Pharmacy, University of Tartu, Estonia.

This study used plant material from only one year of growth, as the aim was to provide a preliminary assessment of the pharmaceutical potential of this plant, which had not been fully analysed phytochemically.

2.2. Hydrodistillation of Essential Oil

Before analysis, the plant material to be examined was ground in a coffee grinder and sieved (diameter 3 mm). The essential oils (EOs) were hydrodistilled from the different dried parts of G. capitata using the method described in the European Pharmacopoeia [1]. The plant materials (20 g) were hydrodistilled in a 1000 mL round-bottom flask for 2 h with 400 mL of distilled water (3–4 mL/min). Hexane (0.5 mL) was added to a graduated tube to remove the distilled oil [26,27]. Due to the content of saponins, intense foam was produced during the distillation of most plant parts, and 30 g of potassium chloride was added to the flask to reduce the foam (except for the roots and stems).

The yield of EOs was measured using the European Pharmacopoeia method with 0.5 mL of xylol [1]. The essential oil content (mL/kg) was calculated based on the volume of the distillate (mL), which was multiplied by 1000 g and divided by the mass of the plant material taken for distillation (20 g).

2.3. Gas Chromatography/Mass Spectrometry

The samples of EO were analysed by gas chromatography–mass spectrometry (GC-MS) using an Agilent 6890/5973 G-CMS system controlled by MSD Chemstation (Agilent Technologies, Inc., Santa Clara, CA, USA). An amount of 1 µL of the sample was automatically injected at an injector temperature of 280 °C in split mode (10:1), using He as the carrier gas onto Agilent HP-5MSI column (Agilent Technologies, Inc., Santa Clara, CA, USA) (30 m length, 0.25 mm inner diameter, and 0.25 µm film thickness). The carrier gas was held at a constant flow rate of 1 mL/min. The oven was held at 50 °C for 2 min, followed by a ramp of 4 °C/min to a final temperature of 280 °C and held at 280 °C for 5 min. 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 using Agilent Masshunter Data Analysis Software G1617DA using a deconvolution algorithm with different window size factors. Obtained compounds were identified by using NIST23 library with a Match Factor of ≥90 and using retention indexes (relative to n-alkanes C₈–C₃₀). The area percentages of each peak were calculated from the total areas in the chromatograms without using correction factors [26,27].

2.4. Extraction of Saponins

The saponins were extracted as follows: 0.5 g of crushed raw materials was placed into a 100.0 mL conical flask equipped with a reflux condenser, and then 25 mL of methyl alcohol was added and boiled for 30 min in a water bath. After cooling the sample to room temperature (20 °C), the extract was filtered into a 25 mL volumetric flask. The obtained extract was then topped up to 25 mL with methyl alcohol.

2.5. Identification of Saponins

The class identification of components was carried out by matching UV spectra to standard substances. The spectra of triterpene saponins have a characteristic absorption maximum at (200–210) nm, corresponding to the absorption spectra of ursolic and oleanolic acids.

The study using HPLC [28,29,30] was conducted on a Shimadzu LC20 Prominence liquid chromatograph in a modular system equipped with a four-channel LC20AD pump, a CTO20A column thermostat, an SIL20A automatic sampler, an SPDM20A diode-array detector, and ChemStation LC20 under the following conditions: column: X-Bridge C18, 150 mm × 4.6 mm, 5 µm particle size (Waters); column temperature: 30 °C; detection wavelength: 205 nm; mobile phase flow rate: 1.0 mL/min; injected sample volume: 20 µL; mobile phase composition: methanol for HPLC, 0.2% ammonium acetate solution (pH 6.75) in a ratio of 80:20; elution mode: isocratic.

Component classification was performed by matching the UV spectra of substances with those of standards. The spectra of triterpenoid saponins exhibit maximum absorption at 200–210 nm, so the detection of this group of compounds was conducted at 205 nm.

2.6. Quantification of Saponins

The quantitative determination of the saponin content in the methanol extracts of the studied raw materials was carried out using ursolic acid as a reference standard. Retention time for ursolic acid was 17.45 min; λ_max (nm) 200.

The content of the substance in raw materials during extraction with the specified extractant under the given conditions for the liquid extract or the content of the substance in the dry extract (X) as a percentage is determined as follows:

$$\mathrm{X}\left(\%\right)={\displaystyle \frac{{\mathrm{A}}_{\mathrm{p}\mathrm{r}}\times {\mathrm{m}}_{\mathrm{s}\mathrm{t}}\times {\mathrm{V}}_{\mathrm{p}\mathrm{r}}\times \mathrm{P}}{{\mathrm{A}}_{\mathrm{s}\mathrm{t}}\times {\mathrm{V}}_{\mathrm{s}\mathrm{t}}\times {\mathrm{m}}_{\mathrm{p}\mathrm{r}}}},$$

where

A_pr—the peak area of the substance on the chromatogram of the test solution;

A_st—the peak area of the substance on the chromatogram of the standard solution;

m_st—the mass of the standard substance sample in the standard solution, mg;

m_pr—the mass of the preparation (raw material), mg;

V_pr—the dilution of the test solution (volume of extractant used), mL;

V_st—the dilution of the standard solution, mL;

P—the purity of the standard, %.

2.7. Foam Index of Saponins

The foam index is determined by measuring the height of the foam produced by the equivalent of 1 g of herbal drug under the stated test conditions according to the European Pharmacopoeia method [1]. The foam index test indirectly indicates the amount of saponins in the material to be determined. During the analysis, an aqueous extract of the plant material is allowed to fall from a pipette, which is 45 cm in height, into a measuring cylinder, where the height of the foam column formed is measured. The higher (cm) the foam column, the higher the foam index value, and the more saponins the drug contains. The aqueous extract was prepared from 1 g of drug powder, adding 50 mL of distilled water and allowing it to stand for 30 min, and mixing 3–5 times with a spatula to disperse the powder without producing any foam. The spatula and the internal walls of the beaker were rinsed with 50 mL of distilled water to ensure that as much of the herbal drug or herbal drug preparation as possible was contained within the liquid. The mixture was allowed to stand undisturbed for 30 min and filtered through a filter paper 125 mm in diameter. Further analysis of the filtrate as the test solution was performed following the instructions of the European Pharmacopoeia using a filled pipette and measuring cylinder. The average of 3 determinations was recorded as a result.

2.8. Data Analysis

The multitude of EO compounds does not allow for an easy assessment of the uniformity of compounds from the table or lists. Therefore, the similarity of plant parts in their EO composition was analysed and illustrated using cluster analysis, which is implemented in PC-ord v7 [31]. The Sorensen (Bray–Curtis) distance measure of similarity was used to build the cluster tree by applying the flexible beta linkage method with a parameter of −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. The tree branch length illustrates the EO compositional dissimilarity between plant parts.

The foam index of saponins was compared between plant parts using a single-factor general linear model in Statistica ver 9 (StatSoft, Inc. Tulsa, OK, USA), and model-predicted least-square means were compared using Tukey’s multiple comparison test.

3. Results and Discussion

3.1. Analysis of Essential Oil

Different parts of G. capitata yielded concentrations of EO ranging from 0.42 mL/kg (shells) to 1.97 mL/kg (seeds) (Figure 2). The seeds contained the greatest proportion of essential oil instead of the flowers, which is the traditional expectation. The EO content was practically the same in the flowers, fruits, and leaves, but there was minimal EO in the stems, roots, and shells. Since the European Pharmacopoeia method (2.8.12. essential oils in herbal drugs [1]) does not require multiple parallel hydrodistillations, this work was limited to a single distillation. In our previous experience, the experimental error of this method is in the order of 10–15%, which we can also take into account when interpreting the results.

Gilia parts contain relatively modest levels of oils in comparison to some other well-known medicinal plants [32]. For instance, the aerial parts of Origanum vulgare from different countries contained 2–11 mL/kg of EO [33]. In the Thymus vulgaris herb, the yield of EO was 3–28 mL/kg [34]; in commercial samples of Valeriana officinalis roots from various countries of different origins, the yield was 2–10 mL/kg [35]; and in the herb of Artemisia absinthium from different origins, the yield was 2–4 mL/kg [36]. The branches of Juniperus communis shrubs were collected from 27 different habitats in Estonia and contained 0.3–6 mL/kg of EO [37].

We aimed to first estimate the possible pharmaceutical potential of the phytochemically completely unstudied species G. capitata. A more thorough study of the plant should include three parallel distillations over 2–3 years of growth. Based on the results obtained in this work, we see that the quantitative content of essential oil in the plant G. capitata does not deserve a more thorough study in the future before its other important groups of active ingredients, which may affect the plant’s pharmaceutical potential, have been investigated.

The composition of EOs was studied using GC-MS (

Table 1. The content (%) of volatile compounds (>0.1%) in different parts of G. capitata.

Compound	RI	Library NIST23	Content (%)
			Flowers	Fruits	Shells	Seeds	Leaves	Stems	Roots
Hexanal	799	798	3.35	nd	4.49	0.20	1.42	3.20	2.31
(E)-2-Hexenal	848	848	0.88	nd	nd	nd	2.09	0.43	nd
1-Hexanol	864	864	2.69	0.19	0.93	0.04	0.28	nd	0.08
1,3-Dimethylbenzene	866	866	nd	1.11	0.44	10.27	nd	nd	nd
(E,Z)-4-Ethylidenecyclohexene	876	877	0.36	nd	nd	nd	nd	0.07	nd
2-Heptanone	890	889	0.29	nd	0.74	nd	0.07	0.13	0.08
Nonane	900	900	0.28	nd	nd	nd	nd	0.19	nd
Heptanal	902	901	0.26	nd	0.50	nd	0.17	nd	0.28
(E)-2-Heptenal	954	954	0.17	nd	0.46	nd	0.23	3.23	0.15
Benzaldehyde	958	958	0.28	nd	0.41	nd	0.41	0.99	1.03
1-Heptanol	968	968	0.05	nd	0.14	nd	nd	nd	nd
1-Octen-3-ol	978	978	0.14	0.21	0.46	0.19	nd	1.65	0.22
2,3-Octanedione	983	983	0.26	nd	nd	nd	nd	0.34	0.14
6-Methyl-5-hepten-2-one	986	986	0.11	nd	nd	nd	nd	0.10	0.09
2-Pentylfuran	991	991	2.90	nd	1.20	nd	0.76	1.37	2.89
(Z)-2-(2-Pentenyl)furan	1001	1001	0.18	nd	nd	nd	nd	0.21	0.20
Octanal	1003	1002	0.37	nd	nd	nd	0.53	0.30	0.26
(E,E)-2,4-Heptadienal	1010	1010	0.17	nd	nd	nd	0.34	0.74	nd
α-Terpinene	1016	1016	0.40	nd	nd	nd	nd	nd	nd
p-Cymene	1023	1023	0.12	nd	nd	nd	nd	nd	nd
2-Ethyl-1-hexanol	1028	1027	nd	nd	nd	nd	nd	nd	0.49
β-Phellandrene	1028	1028	0.67	nd	nd	nd	nd	nd	nd
3-Octen-2-one	1038	1038	0.13	nd	nd	nd	nd	1.22	0.09
Phenylacetaldehyde	1042	1042	0.65	0.39	2.45	0.31	1.27	0.98	1.43
(E)-2-Octenal	1056	1056	0.80	nd	0.62	nd	nd	2.11	1.04
γ-Terpinene	1057	1057	0.47	nd	nd	nd	nd	nd	nd
Acetophenone	1065	1064	nd	nd	0.12	nd	nd	nd	0.10
(Z)-2-Octen-1-ol	1066	1067	nd	nd	nd	nd	nd	0.51	nd
1-Octanol	1069	1069	2.89	2.53	0.88	2.26	2.02	nd	0.37
(E)-β-Terpinolene	1087	1087	0.16	nd	nd	nd	nd	nd	nd
3,5-Octadien-2-one	1092	1093	0.25	nd	nd	nd	nd	0.49	0.12
Linalool	1098	1098	0.34	nd	0.58	0.29	0.50	0.28	nd
Nonanal	1103	1102	2.17	1.96	1.18	1.78	1.25	1.49	1.38
(Z)-2-p-Menthen-1-ol	1119	1120	3.62	3.28	1.98	2.70	1.43	3.82	nd
(E)-p-2-Menthen-1-ol	1138	1138	2.07	2.36	1.32	1.91	0.81	2.14	nd
(E)-Verbenol	1143	1143	0.20	nd	nd	nd	nd	nd	nd
(R,S)-5-Ethyl-6-methyl-3E-hepten-2-one	1145	1145	nd	nd	nd	nd	nd	0.41	0.19
(E,Z)-2,6-Nonadienal	1152	1152	0.27	nd	nd	nd	0.35	0.47	0.18
(E)-2-Nonenal	1158	1158	0.94	0.46	0.57	0.42	0.44	0.92	0.95
α-Phellandren-8-ol	1165	1165	0.08	0.03	0.09	nd	0.03	nd	0.05
1-Nonanol	1170	1170	nd	0.32	0.70	0.29	0.15	0.56	0.22
L-α-Terpineol	1189	1189	0.39	nd	nd	nd	nd	nd	nd
(-)-Myrtenol	1195	1195	1.94	1.73	0.97	1.44	0.67	2.12	25.72
Decanal	1204	1204	1.06	1.34	nd	1.03	1.16	2.21	1.89
(E)-Piperitol	1206	1206	1.21	1.63	1.39	1.31	nd	1.05	nd
(E,E)-2,4-Nonadienal	1212	1212	nd	0.25	nd	0.23	nd	0.74	0.39
7-methylene-6(or 8)-methyl-Bicyclo[3.3.0]octan-2-one	1220	1220	nd	nd	nd	nd	nd	nd	2.20
Carvone	1244	1243	nd	nd	nd	nd	nd	nd	1.47
p-Mentha-1(7),8(10)-dien-9-ol	1245	1246	nd	nd	nd	nd	nd	nd	1.19
Geraniol	1253	1254	nd	nd	nd	nd	nd	nd	1.01
(E)-Myrtanol	1258	1258	0.70	0.54	0.34	0.43	nd	0.47	16.44
1-Decanol	1270	1271	0.46	0.64	0.88	0.53	0.19	nd	nd
Nonanoic acid	1271	1272	1.04	nd	nd	nd	nd	nd	nd
(E)-Bornyl acetate	1285	1285	nd	nd	0.73	nd	nd	nd	nd
Thymol	1289	1290	nd	nd	nd	nd	nd	nd	0.55
(E,Z)-2,4-Decadienal	1292	1292	0.77	0.87	nd	0.71	nd	1.61	0.32
(E)-Undec-4-enal	1298	1296	nd	nd	nd	nd	nd	0.33	nd
Undecanal	1306	1305	0.19	0.43	nd	0.39	0.25	0.33	0.32
2-Methoxy-4-vinylphenol	1312	1312	0.41	nd	0.53	nd	0.51	nd	nd
(E,E)-2,4-Decadienal	1315	1315	2.71	3.36	1.43	2.85	1.10	5.29	1.18
Eugenol	1357	1357	nd	nd	nd	nd	0.97	nd	nd
Dihydro-5-pentyl-2(3H)-furanone	1362	1362	nd	nd	nd	nd	nd	nd	0.25
2-Undecenal	1363	1363	nd	1.26	0.83	1.12	nd	3.89	nd
n-Decanoic acid	1373	1372	nd	nd	nd	nd	nd	nd	1.32
2-Butyl-2-octenal	1372	1373	0.34	0.32	nd	0.26	nd	nd	nd
(E)-β-Damascenone	1385	1385	0.27	0.58	0.72	0.45	nd	nd	nd
β-Elemene	1393	1393	nd	nd	8.46	nd	nd	nd	nd
6,10-Dimethyl-2-undecanone	1404	1404	nd	nd	nd	nd	nd	0.25	nd
Dodecanal	1408	1408	0.29	0.36	nd	0.30	0.26	0.28	0.50
7-epi-α-Cedrene	1416	1417	nd	nd	nd	nd	nd	nd	0.56
Copaene	1421	1420	1.28	nd	2.87	nd	nd	nd	nd
Caryophyllene	1421	1421	1.34	nd	2.77	nd	0.54	nd	nd
(E)-Geranylacetone	1453	1453	0.54	0.72	nd	0.63	0.85	0.52	0.86
Humulene	1455	1456	nd	nd	1.52	nd	nd	nd	nd
γ-Decalactone	1469	1469	7.09	4.62	1.66	4.23	1.50	1.04	nd
1-Dodecanol	1474	1474	nd	nd	0.43	nd	0.20	0.13	0.94
(+)-epi-Bicyclosesquiphellandrene	1484	1484	0.48	nd	15.41	nd	nd	nd	nd
(E)-β-Ionone	1488	1488	1.14	1.30	nd	0.97	4.54	3.24	nd
β-Selinene	1488	1489	nd	nd	0.41	nd	nd	nd	nd
α-(3-Methylbutylidene)- benzeneacetaldehyde	1492	1492	nd	nd	nd	nd	nd	0.21	nd
α-Farnesene	1510	1510	1.01	nd	nd	nd	nd	nd	nd
Tridecanal	1511	1511	nd	0.53	nd	0.44	0.53	0.34	0.29
(Z)-γ-Bisabolene	1517	1518	0.57	nd	nd	nd	nd	nd	nd
n-Tridecan-1-ol	1575	1575	nd	nd	nd	nd	nd	nd	0.25
Caryophyllene oxide	1587	1587	0.89	nd	nd	nd	nd	nd	nd
2,2,4-Trimethyl-1,3-pentanediol diisobutyrate	1600	1599	0.38	nd	nd	nd	nd	nd	0.92
Tetradecanal	1613	1613	0.24	1.03	1.55	0.88	0.76	0.54	0.50
Benzophenone	1629	1629	nd	nd	nd	nd	0.20	0.14	nd
τ-Muurolol	1644	1646	0.62	nd	nd	nd	nd	nd	nd
α-Cadinol	1658	1658	0.41	nd	nd	nd	nd	nd	nd
1-Tetradecanol	1677	1677	nd	1.01	1.81	0.97	nd	0.49	1.52
Pentadecanal	1715	1715	0.37	4.38	6.25	3.65	4.01	2.99	2.06
1-Tetradecene	1736	1736	nd	0.54	nd	0.50	nd	0.65	nd
Myristic acid	1765	1764	nd	nd	nd	nd	nd	nd	0.85
n-Pentadecanol	1779	1778	nd	0.43	1.63	0.44	nd	nd	0.38
Ambrial	1804	1804	nd	nd	nd	nd	nd	0.48	nd
Farnesyl acetaldehyde	1845	1845	nd	nd	nd	nd	nd	0.25	nd
Hexahydrofarnesyl acetone	1851	1851	19.09	18.16	3.24	15.21	3.10	4.94	1.27
1-Hexadecanol	1889	1889	nd	1.47	0.83	1.55	1.05	0.78	0.74
Manoyl oxide	1896	1897	nd	0.62	nd	0.51	2.28	2.96	nd
Roughanic acid	1904	1904	nd	nd	nd	nd	nd	0.70	nd
Farnesyl acetone	1928	1928	0.50	nd	nd	nd	nd	nd	nd
Cembrene A	1969	1969	0.82	nd	nd	nd	nd	nd	nd
Palmitic acid	1976	1974	12.24	7.95	nd	11.13	nd	nd	8.05
13-epi-Manoyl oxide	2018	2018	nd	nd	nd	nd	1.15	0.51	nd
Epi-13-Manool	2061	2061	nd	nd	nd	nd	1.01	0.77	nd
Kolavelool	2070	2070	nd	nd	nd	nd	1.67	0.97	nd
Isomanool	2093	2094	0.39	3.23	nd	2.48	12.07	8.29	nd
γ-Palmitolactone	2103	2104	nd	nd	nd	nd	nd	0.41	nd
Phytol	2116	2114	1.72	3.51	nd	2.97	23.26	2.63	nd
Sclareol	2225	2227	nd	nd	nd	nd	4.45	6.70	nd
Nonadecane	2300	2300	0.29	1.95	nd	1.71	0.53	0.17	nd
Pentacosane	2500	2500	nd	3.34	nd	3.10	0.99	0.64	nd
Bis(2-ethylhexyl) phthalate	2551	2550	nd	nd	nd	nd	nd	0.38	nd
Heptacosane	2700	2701	nd	5.34	2.07	4.95	1.74	2.84	nd
Octacosane	2800	2800	nd	1.47	1.51	1.47	nd	0.52	nd
TOTAL			92.16	87.75	80.50	89.50	86.09	92.15	88.57

Note. “nd”—not detected.

). In total, 116 compounds were identified, representing 80.50–92.16% of the extracted essential oils (Figure 3, Table 1). All parts of the plant contain phenylacetaldehyde, nonanal, (E)-2-nonenal, (-)-myrtenol, (E,E)-2,4-decadienal, tetradecanal, pentadecanal, and hexahydrofarnesyl acetone.

The composition of essential oils in different parts of G. capitata is illustrated with a cluster tree, with the branch lengths showing the similarity–dissimilarity between plant parts (Figure 4). The fruits and seeds were most similar in composition, being almost identical. The flowers were next, similar to the fruits and seeds. They all contain high levels of hexahydrofarnesyl acetone and palmitic acid, but there are many other common EOs with lower levels (Table 1, Figure 3). Leaves and stems formed another narrow cluster with flagship compounds phytol and isomanool, as well as (E)-beta-ionone, manoyl oxide, and sclareol. The most distant from them were the shells of the fruit, showing that seeds contain the most EO in a fruit. Shells can be characterised by β-elemene and (+)-epi-bicyclosesquiphellandrene. The composition of roots was the most distinct from all other plant parts (Figure 4), mostly determined by the prevalence of (-)-myrtenol and (E)-myrtanol (Figure 5).

We have not found detailed scientific articles on the content and composition of the essential oil (EO) of G. capitata, though there are a few available studies for other plants of the Gilia genus (Polemoniaceae) [2,3], and the essential oils in Polemonium caeruleum and/or other species of the Polemonium genus have not been studied [8,24]. The most studied in the family is the Phlox genus [24]. Thus, we cannot compare our results with previous studies conducted by other authors or with the EO compositions of other related plant species. Interestingly, we detected many different EOs, which have been reported as potential pollinator attractors or flower oils for forage [24,25,38], but all were found at very low concentrations. However, the diversity of EOs studied was rather high—116 compounds were identified in different parts of G. capitata. We detected lower EO diversity using the same method in these plant species, except in the roots of V. officinalis, in which we found 150 compounds [35].

All parts of the G. capitata plant contained eight common components: phenylacetaldehyde, nonanal, (E)-2-nonenal, (-)-myrtenol, (E,E)-2,4-decadienal, tetradecanal, pentadecanal, and hexahydrofarnesyl aceton. These are rather nontypical components in other plant species, at least in classical EO-bearing plants.

We can only state that in the case of G. capitata, the same components are not always detected in all plant parts. This shows the importance of addressing the plant in detail and avoiding analysing a single part of the plant or the overall herb (Table 1). The composition of more prominent EOs (at least 5%) was similar in flowers and fruits (specifically in seeds). The vegetative parts of the plant, namely the stems and leaves, had a slightly different composition. The least valuable parts in EO quantity and composition were the shells of the fruits and roots. The analysis results with the assessment ‘nd’ (not detected) do not necessarily mean the complete absence of these substances in EO but may indicate very low contents of the components (probably less than 0.03%, the reliability level of the GC-MS method).

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 5). In addition to the flowers, hexahydrofarnesyl acetone was also the dominant component in fruits (18.2%) and seeds (15.2%), and it was found in significant quantities in the stems (4.9%) of the G. capitata (Figure 5). Hexahydrofarnesyl acetone has been proven to exhibit potent antimicrobial, anti-inflammatory, and cytotoxic activity and is used in pain relief research [39,40,41,42]. Palmitic acid, also present in seeds (11.1%) and fruits (8.0%), has antioxidant and antibacterial activities [43,44].

Lactones, including γ-decalactone, constitute an essential group of fatty acid-derived volatile organic compounds conferring a peach-like aroma to a few essential oils and fruits, including peach, plum, pineapple, and strawberry [45]. γ-Decalactone inhibits strawberry pathogen growth and achene germination [46].

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 synthesising desoxy cannabidiols and THC-related psychoactive compounds. It is formed from (+)-limonene using a photosynthesised O₂ transfer [47].

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 anti-tumor activity of γ-bisabolene in human neuroblastoma cells has been proven through the induction of p53-mediated mitochondrial apoptosis [48]. γ-Bisabolene has demonstrated antiproliferative activities against several human cancer cell lines. Another study [29] disclosed the antiproliferative and apoptosis-inducing activities of γ-bisabolene toward human neuroblastoma TE671 cells, and the CC50 value of γ-bisabolene was 8.2 μM toward TE671 cells [49].

Caryophyllene oxide was found in flowers (0.9%), while caryophyllene was distributed more widely in flowers, leaves, fruits, and shells (1.3, 0.5, and 2.8%, respectively). Its anti-cancer, antioxidant, and antimicrobial properties have been proven [50].

Linalool was 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 amelanotic melanoma and renal adenocarcinoma cells [51].

The predominant compounds in the EOs of the leaves were phytol (23.3%) and diterpene alcohols isomanool (12.1%), sclareol (4.5%), and (E)-β-ionone (4.5%). Phytol has antibacterial and antioxidant activities, inhibiting the growth of Staphylococcus aureus [52,53]. Phytol is used in fragrance and cosmetics to produce shampoos, toilet cleaners, household cleaners, and detergents [54].

Labdane diterpenoids are the most common types of diterpenoids isolated in minute amounts from higher plants [55]. Labdane diterpenoids are interesting for their cytotoxic, antifungal, anti-inflammatory, antiparasitic, and analgesic properties [56,57,58]. Labdane diterpenoid sclareol has a wide range of bioactivities, including anti-tumour, anti-inflammation, and anti-pathogenic microbes, and even anti-diabetes and anti-hypertension properties [59]. Sclareol can also kill human leukaemic cells and colon cancer cells in vitro by apoptosis [60,61]. 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 6). Labdan-type diterpenes, such as manoyl oxide, 13-epi-manoyl oxide, epi-13-manool, kolavelool, isomanool, and sclareol, make up a significant part of oils in leaves and stems—22.6% and 20.2%, respectively. Essential oils from fruits and seeds contain less—3.9% and 3.0%, respectively. The main component among labdan diterpenes is isomanool (labda-8,14-diene-13β-ol); it is found in flowers (0.4%), leaves (12.1%), stems (8.3%), fruits (3.2%), and seeds (2.5%). It is followed by manoyl oxide, which is found in leaves (2.3%), stems (3.0%), fruits (0.6%), and seeds (0.5%). Both 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 [62]. Data in the literature indicate that β-ionone and its derivatives have a lot of important pharmacological activities, including antibacterial, antifungal, antioxidant, anti-inflammatory, antiproliferative, and anti-cancer effects [63].

The most dominant compounds in EO in stems are isomanool (8.3%), sclareol (6.7%), and (E,E)-2,4-decadienal (2.9%). It is known that 2,4-decadienal has nematicidal [64] and insecticidal activities against the barn bug [65].

In addition to hexahydrofarnesyl acetone, the dominant compounds in the fruits were palmitic acid (8.0%), heptacosane (5.3%), and γ-decalactone (4.6%). The dominant compounds in the shell were (+)-epi-bicyclosesquiphellandrene (15.4%), β-elemene (8.5%), and pentadecanal (6.3%). β-Elemene exhibits anti-tumour properties, as well as anti-inflammatory and antioxidant effects [66,67]. β-Elemene and β-elemene piperazine derivatives have been shown to inhibit tumour cell growth in vitro and in vivo [68]. The antibacterial authorities of pentadecanal were established [69,70].

In addition to hexahydrofarnesyl acetone, the dominant compounds 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 [71].

The dominant compounds in the roots 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 [72,73]. (-)-(E)-Myrtanol is an antimicrobial and acaricide agent [74,75]. 2-Pentylfuran has been suggested as a repellent for spotted-wing drosophila as it can significantly reduce fruit infestations under field conditions [76].

Many volatile compounds are aliphatic (24.4–48.6%). They are the dominant compounds in flowers, stems, fruits, and seeds (Figure 6). At the same time, diterpenoids are the dominant group in the composition of volatile compounds in leaves (45.9%), sesquiterpenoids in shells (34.7%), and monoterpenoids in roots (46.4%). It should also be noted that the contents of mono- and sesquiterpenoids are quite high in flowers, stems, fruits, and seeds. Figure 6 clearly shows that the EO content of roots is much different from that of aerial parts.

Sesquiterpenoids are represented by derivatives of 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 [65]. Norsesquiterpenoids or apocarotenoids are represented by two compounds, namely (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 [66]. β-Damascenol effectively prevents skin sunburn [67].

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

3.2. Analysis of Saponins

3.2.1. Identification of Saponins

The class identification of components was carried out by matching UV spectra to standard substances. The spectra of triterpene saponins have a characteristic absorption maximum of 200–210 nm, corresponding to the absorption spectra of ursolic and oleanolic acids. The analysis report of saponins in different G. capitata parts is shown in the Supplementary File.

Triterpene saponins are quite common in medicinal plants [32]. In previous studies, the presence of a variety of triterpene saponins, as well as oleanane derivatives such as polemoniumsaponines [77], polemonioside C, polemoniumgenin A, and polemoniogenin, were found in underground parts of Polemonium caeruleum [8]. It contains pentacyclic saponins of the β-amyrin type, the aglycones of which are mostly represented by esters of strongly hydroxylatedtriterpene alcohols [78]. Hypothetically, the structures of saponins in G. capitata may be similar, but further analysis is needed.

3.2.2. Quantification of Saponins

The results of the quantification of saponins are shown in

Table 2. Saponin contents in different parts of Gilia capitata.

Saponin No.	Retention Time (min)	Content (%)
		Flowers	Seeds	Leaves	Roots	Stems
1	2.600	-	-	0.019	0.115	0.063
2	2.824	-	0.296	-	-	-
3	3.034	-	0.476	0.104	0.054	0.032
4	3.662	0.170	0.143	0.366	0.035	0.064
5	3.921	0.168	0.083	-	-	-
6	4.187	-	0.021	-	-	-
7	5.037	0.015	-	0.036	-	-
8	5.116	-	0.071	-	-	-
9	5.586	0.032	-	-	-	-
10	5.760	0.019	0.009	0.034	-	-
11	6.971	0.064	-	-	-	-
12	7.457	-	-	0.013	-	-
13	8.295	0.014	-	0.019	-	-
14	8.380	-	0.016	-	-	-
15	10.007	0.353	0.098	0.244	0.016	0.027
16	14.295	-	0.067	-	0.046	0.021
17	14.358	0.176	-	0.051	-	-
18	15.687	-	-	0.013	-	-
19	18.189	0.031	-	-	-	-
20	27.159	-	-	0.0150	-	-
21	27.159	0.068	-	-	-	-
The sum of saponins in terms of ursolic acid		1.919	1.230	0.914	0.266	0.208

. The analysis detected 21 different saponins. The highest levels of different saponins were found in the flowers and seeds, and minimal concentrations were found in the stems and roots. A similar tendency in the quantitative content of saponins in different parts of G. capitata is also confirmed by the results of foam index determination (Figure 7). The maximum number of individual saponins was typical of aerial parts (excluding stems) of G. capitata (Table 2). The foam index differed between plant parts (ANOVA F_6,10-test = 29.9, p ≤ 0.0001). But it was not statistically different among flowers, leaves, fruits, and shells (index mean values of 2.9–3.4); it was somewhat lower in the seeds (2.6) and stems (1.9) (Figure 7). Surprisingly, the foam index was significantly lower in the plant’s roots (1.0). However, it is known that the roots of many plants rich in saponins are used as herbal drugs in medicinal practice [31]. These results are similar to the content of individual saponins found in different parts of G. capitata.

Since HPLC and GC are analytical methods with very low standard deviation (SD) values, Table 1 does not include SD parameters. The data obtained are sufficient to provide a preliminary assessment of the pharmaceutical potential of G. capitata.

No studies are available on the qualitative and quantitative contents of saponins in G. capitata and the entire plant genus Gilia. We can draw parallels with the relatively well-studied medicinal plant Polemonium caeruleum, which has been named Polemoniaceae. Because the hemolytic index of P. caeruleum is 1100 for roots and rhizomes, 1000 for herbs, and 3000 for seeds [78], we can expect a high content of saponins in the aboveground parts of the plant, especially the seeds.

Saponin distribution has been found to vary in individual plant parts [79]. The richest parts of Medicago truncatula [80] and Allium nigrum are the roots [81]. The total saponin concentration has been reported to be the highest in the leaves of Maesa lanceolata [82] and Panicum virgatum [83]. Thus, the aboveground parts of plants may also be a rich source of saponins, as appears to be the case in G. capitata. The localisation of EO also supports their preference for underground parts. According to the European Pharmacopoeia [1], the foam index of Senega root (Polygala radix) must be no less than 3.5. Moreover, senega root is a classic saponin drug containing 6–10% triterpene saponins [84]. Thus, the aerial parts of G. capitata contain a remarkable amount of saponins. These results suggest that if G. capitata has potential as a medicinal plant, the herb from which the thin stems have been isolated should be used as a source of the herbal drug.

3.2.3. The Limitations of the Study

This is the first explorative study about the chemical composition of G. capitata, and we were not able to cover the variability in the composition and quantities of substances, driven by yearly variation in weather conditions, soil types, or seasonality. G. capitata is known to have genetic and morphological variability within its native geographic range [11,12], and therefore, there might be differences between native populations and ecological conditions. It is also somewhat problematic to perform EO distillations only once from each plant part. It would be more correct to perform four distillations and find the SD value based on them. But on the other hand, even in its current form, it is convincingly clear in which part of the plant the EO yield is minimal and where it is maximal.

4. Conclusions

The composition of volatile compounds and saponins of various parts of G. capitata was studied for the first time. The analysis of bluehead gilia essential oil made it possible to detect and identify important potentially active compounds with various biological properties, including antioxidant, anti-cancer, and antidepressant properties, among others, which indicates the prospects for further phytochemical and pharmacological studies of the genus Gilia. The main sources of essential oils and saponins are the flowers, seeds, and potentially leaves; these parts should be given more attention in future studies. However, saponins in bluehead gilia are the most abundant compound for future interest.