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Article

Seed Priming with Cold Plasma and Vacuum Increases the Amounts of Phenolic Compounds and Antioxidant Activity in Lavender Herb

by
Viktoriia Hurina
1,
Zita Nauciene
2,
Rasa Zukiene
2,
Laima Degutyte-Fomins
2,
Simona Tuckute
3,
Liudas Ivanauskas
4,
Mindaugas Marksa
4,
Victoriya Georgiyants
1,
Olha Mykhailenko
1 and
Vida Mildaziene
2,*
1
Department of Pharmaceutical Chemistry, National University of Pharmacy, 61168 Kharkiv, Ukraine
2
Department of Biochemistry, Faculty of Natural Sciences, Vytautas Magnus University, LT-53361 Akademija, Lithuania
3
Center for Hydrogen Energy Technologies, Lithuanian Energy Institute, LT-44403 Kaunas, Lithuania
4
Department of Analytical and Toxicological Chemistry, Lithuanian University of Health Sciences, LT-50161 Kaunas, Lithuania
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(12), 1413; https://doi.org/10.3390/horticulturae11121413
Submission received: 27 October 2025 / Revised: 11 November 2025 / Accepted: 19 November 2025 / Published: 21 November 2025
(This article belongs to the Section Medicinals, Herbs, and Specialty Crops)
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Versions Notes

Abstract

Seed processing effects induced by two types of cold plasma, CP (low-pressure plasma, LCP; dielectric barrier discharge plasma, DBD), and vacuum (V) treatments were compared by estimating changes in the emergence and growth of lavender seedlings, the density of leaf trichomes, and the biochemical composition of leaf extracts, including the content of photosynthetic pigments, TPC, antioxidant activity, and the amounts of two hydroxycinnamic acids associated with antioxidant activity—chlorogenic and rosmarinic acid. DBD treatment for 3 min stimulated the emergence and growth of seedlings but induced negative or neutral effects on biochemical parameters. All treatments, except DBD3, increased the density of glandular trichomes in leaves. Short-term treatments with LCP (0.5 min), DBD (2 min), and V (2 min) increased the total phenolic compound (TPC) content by 15–25%, and the first two treatments enhanced antioxidant activity (21–32%). HPLC analysis revealed that V (2 min) treatment was the most effective, increasing the content of chlorogenic (49%) and rosmarinic (14%) acid. LCP (1 min) and DBD (2 min) treatments increased chlorogenic acid content by 9% and 26%, respectively. The obtained results support the potential of pre-sowing seed treatments with CP and vacuum to produce raw lavender material enriched by biologically active compounds for pharmaceutical and nutraceutical applications.

Graphical Abstract

1. Introduction

Lavender (Lavandula angustifolia Mill.) is a medicinal plant included in numerous pharmacopoeias worldwide, such as the British Pharmacopoeia, German Homeopathic Pharmacopoeia, the State Pharmacopoeia of Ukraine [1], the European Pharmacopoeia, the American Herbal Pharmacopoeia, etc. [2]. This plant, due to its rich chemical composition and established pharmacological activity, has direct applications in medicine and pharmacy [3]. Lavender is primarily used as an essential oil plant because it contains terpenes (linalool, linalyl acetate, camphor, and terpinen-4-ol), which provide anxiolytic and sedative effects when used in aromatherapy [4]. In addition to essential oil (1–3%), the aerial part of the plant accumulates flavonoids, hydroxycinnamic acids, tannins (5–10%), amino acids, phytosterols, triterpene saponins, and other compounds [5]. The high content of hydroxycinnamic acids, specifically rosmarinic and chlorogenic acids [5], in lavender leaves is of particular importance. These acids contribute to antioxidant activity [5,6] and exhibit significant anti-inflammatory, antidiabetic, antiviral, and neuroprotective effects [7,8], which underpin the potential of lavender leaves and herbs for the phytomedicine industry [9,10]. Due to the synergistic action of their components, lavender leaf extracts exhibit diverse pharmacological effects, including antibacterial activity (4.0–9.0 mg/mL) [11], carminative, antidepressant, and anti-inflammatory effects [3,12].
Since the cultivation of lavender provides valuable raw materials for pharmaceutical and cosmetic purposes, methods to increase its production and the yield of biologically active substances are under development. Mineral fertilisers (nitrogen, potassium, and phosphate) are often used in lavender cultivation to increase yield and plant size [13]. However, organic fertilisers can cause an inconsistent composition of marker compounds in plants [14], risks of contamination, microbial growth danger, soil imbalance, longer decomposition time, and higher labour costs [15,16]. Therefore, it is important to investigate the possible impact of alternative eco-friendly cultivation methods on morphological characteristics as well as on biochemical processes in plants. From a bioeconomic perspective, it is important to increase the potential of lavender for medicinal, cosmetic, agricultural, and biotechnological purposes, while considering the need to conserve resources and ensure the sustainable and efficient use of plants [12]. This approach goes beyond the traditional use of lavender solely for essential oil production. The ecological use of natural resources aims to supply the pharmaceutical market with plant raw materials and help to preserve nature at the same time.
Plasma agriculture is an emerging research field due to the promising results observed in various plants, related to the control of seed microbial contamination, enhancement of germination, stimulation of plant development, improved production yield, and resistance to plant diseases (see reviews [17,18,19,20]). Plasma is an ionised gas generated in any gas or gas mixture at different pressures. It is a complex physico-chemical stressor consisting of electrons, ions, neutral molecules, atoms, radicals, excited species, etc., and ultraviolet (UV) light photons. Variations in plasma parameters (temperature, composition, densities, energies of particles, etc.) can be controlled by external parameters, such as the construction type and geometry of the plasma generator, gas nature and pressure, and discharge characteristics. It has been shown that pre-sowing seed treatment with physical stressors like non-thermal or cold plasma (CP) not only stimulates plant germination and increases yield, but also positively affects the content of biologically active compounds in raw plant materials [18,20]. Considering the goal to enrich the chemical composition of lavender extracts and the essential need for complex processing of raw materials, novel pre-sowing treatment methods and eco-friendly cultivation of lavender seeds might result not only in improved morphological characteristics, but also in stimulating biochemical processes, which can result in an increased content of chemical compounds. The positive effect on the content of biologically active compounds in plant raw materials was demonstrated using pre-sowing CP treatment on different medicinal plants, such as Trifolium pratense L. [21], Echinacea purpurea (L.) Moench [22], Coriandum sativum [23], soybean [24], Stevia rebaudiana (Bertoni) Bertoni [25], and other plants.
Recently, plasma technology has been employed to improve the extraction yield of essential oils from the raw material of different plants (reviewed in [26]), including lavandin ‘Grosso’ flowers [27]. This study has proven the effectiveness of plasma treatment in hydrodistillation-based essential oil extraction, which slightly increased the oil yield, although the chemical composition of the essential oil remained largely unchanged. Another similar study, conducted on Lavandula angustifolia Mill., proved the efficiency of pulsed electric field treatment for essential oil extraction [28]. Although the observed effects of pre-sowing seed treatment with CP on secondary metabolite content in medicinal plants were stronger compared to the CP-induced increase in extraction efficiency [26], the impact of CP on agronomic performance and biochemical parameters has not been studied in lavender.
In this study, we aimed to test the research hypothesis that pre-sowing seed treatment with CP can improve germination, growth, and stimulate biochemical processes in lavender leaves, including the accumulation of pharmaceutically relevant secondary metabolites. The effects of lavender seed processing using two types of CP-generating devices, low-pressure plasma (LCP) and atmospheric dielectric barrier discharge (DBD) plasma, were compared, since it was established on various plants that the response strongly depends on the plasma parameters used for treatment [17,18,19,20]. We have explored the effects of seed treatments on seed surface structure, seedling emergence, growth in the substrate, and leaf biochemical parameters, such as antioxidant activity, the content of photosynthetic pigments, total phenolic compounds (TPC), and hydroxycinnamic acids, which are key markers of the pharmacological activity of lavender leaves.
The obtained results indicate that the pre-sowing irradiation of lavender seeds with CP or exposure to vacuum can result in significant changes in seedling agronomic performance and biochemical composition. The observed effects were dependent on the nature of the stressor (the type of CP generator or vacuum used) and treatment duration. However, certain treatment protocols effectively induced positive effects on the content of chlorophylls, the density of glandular trichomes, antioxidant activity, and the content of TPC, rosmarinic, and chlorogenic acids in the lavender leaf extracts. Further development of a pre-sowing seed treatment strategy may provide tools for improving the yield and quality of L. angustifolia raw materials relevant for the pharmaceutical market and contribute to maximising the medicinal, cosmetic, agricultural, and biotechnological potential of lavender plants.

2. Materials and Methods

2.1. Plant Material

The seeds of lavender (Lavandula angustifolia Mill.) were collected in the “Spice-Aromatic Plants” department of M.M. Gryshko National Botanical Garden, Kyiv, Ukraine, in 2021. The seeds were stored in a well-ventilated room in hermetically sealed containers to prevent moisture accumulation and raw material spoilage.

2.2. Pre-Sowing Plasma Treatment

Lavender seeds were treated with LCP for 0.5 and 1 min (further denoted as LCP0.5, and LCP1) or with atmospheric DBD plasma for 2 and 3 min (further denoted as DBD2 and DBD3). These exposure durations were selected based on the results of pilot experiments. Vacuum treatment for 2 min (further—V2) was used as an additional control for LCP.
Seed treatment with vacuum and LCP was carried out in a hermetic stainless-steel reactor with capacitively coupled RF discharge, as described earlier. A total of 200 seeds were spread out in a single layer within a 5 cm diameter glass Petri dish, which was placed in the reactor at a distance from the electrode. The chamber was sealed, and a vacuum pump and an airflow controller were used to reach and maintain a constant 100 Pa pressure inside the chamber. RF voltage with a frequency of 430 MHz was applied to the powered electrode (discharge power: 50 W). Air was used as the gaseous phase, and the airflow was set at 89 ± 5 mL/min. For V2 treatment, seeds were exposed to a vacuum (100 Pa) for 1 min without discharge.
Seed treatment with DBD plasma was performed using a controlled DBD device, as previously described in detail [29], with a uniform plasma exposure area of 4 × 4.38 cm2. The discharge voltage was 7 kV (Logy Electric, LHV-09K), the current was 0.2 A, and the power density was 3.1 W/cm2. The treatment was conducted in air at atmospheric pressure, room temperature, and a humidity of 50%. A single layer of 200 seeds was placed on a glass plate, 5 mm below the electrode. During the process, the surface temperature of the seeds was monitored, aiming not to exceed 35 °C.

2.3. Analysis of the Emergence Kinetics, Cultivation, and Morphometric Analysis of Seedlings

CP-treated and control lavender seeds were stored for 4 days at room temperature before sowing because a certain time interval between treatment and sowing is required for the development of phytohormonal changes that determine the seed response [30]. Four days after treatment, seeds were sown in 5.7 L (32 cm × 17 cm × 11 cm) plastic containers filled with the substrate prepared from peat, quartz sand, and perlite in a ratio of 3:1:0.05. The substrate was moistened with 1.5 L of water, maintaining a humidity level of 25–34%, and monitored throughout the experiment by using an HH2 moisture meter (Delta-T Devices Ltd., Cambridge, UK). The seeds were sown on the surface of the substrate, and the containers were covered with polyethylene film with perforations to allow for air circulation. A total of 60 seeds per container were sown, with three replicates for each experimental and control group. The seedling emergence dynamics were recorded approximately every 24 h until no further increase in the number of emerged seedlings was observed. Richards plots were constructed [22,31] to quantify the main indices of seedling emergence kinetics: Vi (%)—the maximal emergence percentage indicating seed viability; Me (days)—the median emergence time (t50%), indicating the half-time of seedling emergence or the time required for half of the seedlings to emerge; and Qu (days)—the quartile deviation indicating the dispersion of emergence time or emergence uniformity. The entire experiment on lavender emergence and cultivation was conducted in a greenhouse with alternating light regimes (16 h light, 8 h dark) at a temperature of 22–25 °C.
Morphometric analysis of plants was conducted 50 days after sowing. To assess seedling growth in the substrate, total plant and root height, as well as leaf number and weight, were measured. A total of 30 plants were randomly selected from the control and each experimental group.

2.4. SEM Analysis of Seed Surface and Leaf Trichomes

Before sowing, the seed surface was analysed using a scanning electron microscope (SEM) (Hitachi TM3000, Hitachi High-Technologies, Tokyo, Japan). Seven seeds from the control and all experimental groups were analysed using several magnifications and taking four ×100 and four ×500 pictures from different areas on the surface of each of the analysed seeds. The most typical images are selected for Figure 1.
The same equipment was applied for counting the density of trichomes in 50-day-old seedling leaves. The number of glandular and dendritic trichomes was counted on a 3 mm long segment of the leaf margin on SEM images (×25 magnification) and expressed in units/mm. Samples were taken from 3 young leaves (0.7–1.0 cm long) from one seedling, with three seedlings from the control and each experimental group (a total of 9 leaves were assessed for trichome density for each group).

2.5. Preparation of Plant Extracts

Lavender plant extraction was performed 50 days after sowing, using the leaves from the plant apex (21 seedlings were used for 3 pooled samples). Two types of extracts were prepared using the following ratios: 1 g of leaves to 5 mL of 85% methanol and 1 g of leaves to 5 mL of 96% ethanol. The leaves were immersed in the extraction solvent at 4 °C and ground in a mortar, then placed in an ultrasonic bath at 4 °C for 15 min. The obtained extracts were centrifuged at 13,400× g for 10 min. After centrifugation, the supernatants were collected and stored at −20 °C until analysis.

2.6. Analysis of Leaf Biochemical Parameters

The effects of seed treatments on the amount of photosynthetic pigments, total phenolic content (TPC), and antioxidant activity were estimated in the prepared leaf extracts spectrophotometrically using a Beckman Coulter DU-800 UV/Vis spectrophotometer (“PerkinElmer”, Rodgau, Germany).
The amounts of chlorophylls a and b and carotenoids were determined in the ethanolic extract of lavender leaves. The absorption of the extract was measured at wavelengths of 664 nm (chlorophyll a, Chl a), 648 nm (chlorophyll b, Chl b), and 471 nm (carotenoids). The amounts of Chl a, Chl b, and total carotenoids were calculated using the formulas described by Lichtenthaler and Buschmann [32].
The total content of phenolic compounds was determined in methanolic extracts by using the standard Folin–Ciocalteu reagent method. For the reaction, the following components were used: 0.8 mL of 7.5% Na2CO3 solution, 1 mL of Folin–Ciocalteu reagent solution diluted 10-fold with distilled water, and 0.2 mL of the test extract diluted 3-fold. The tubes with solutions were wrapped in foil and incubated in the dark at room temperature for 30 min. Absorbance was measured using a spectrophotometer at 765 nm. Gallic acid was used as a standard, and the results were expressed as mg of gallic acid equivalents (GAE) per g of dry weight (DW). All measurements were performed in triplicate.
Antioxidant activity was measured by the scavenging of the 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH). Methanolic leaf extract (50 µL) was added to 1950 µL of a DPPH solution (25 mg/L) in acetonitrile–methanol–sodium acetate buffer (100 mM, pH 5.5) (1:1:2). The tubes with the reaction mixture were wrapped in foil and incubated in the dark at room temperature for 15 min. Absorbance was measured at 515 nm. Rutin was used as a standard, and antioxidant activity was expressed as mg of rutin equivalents (RUE) per g of DW.

2.7. HPLC Analysis of Hydroxycinnamic Acids

The chromatographic analysis of chlorogenic and rosmarinic hydroxycinnamic acids in lavender extracts was performed using high-performance liquid chromatography (HPLC) on a Waters e2695 Alliance chromatograph combined with a 2998 PDA photodiode array detector (Waters, Milford, MA, USA) following established protocols from previous studies [33]. The separation was carried out using an ACE C18 column (250 mm × 4.6 mm, 3 μm) (ACT, Aberdeen, UK). The column temperature was maintained at 25 °C. Chromatographic separation was performed using a gradient elution with the following eluents: A—0.1% trifluoroacetic acid in water and B—acetonitrile. The gradient programme was as follows (eluent B): 0 min—5%; 8–30 min—20%; 30–48 min—40%; 48–58 min—50%; 58–65 min—50%; 65–66 min—95%; 66–70 min—95%; and 70–81 min—5%. The flow rate was 1 mL/min, and the injection volume was 10 μL. The chromatograms were recorded at 350 nm. The identification of chromatographic peaks and the quantification of compounds were performed according to the European Pharmacopoeia standards based on UV/MS spectral data, as well as the comparison with hydroxycinnamic acid standards. A typical HPLC chromatogram is shown in Figure 1.
The rosmarinic acid peak was detected at a retention time of 37 min (proved by comparison with the retention time and UV spectrum of the standard rosmarinic acid sample). The chlorogenic acid and its standard had a retention time of approximately 11 min.

2.8. Statistical Analysis

All measurements of various parameters between the control and pre-sowing treatment groups were expressed as mean ± SEM and compared using Student’s t-tests for unpaired samples. The statistical significance of CP effects was assumed to be statistically significant when p < 0.05.

3. Results

3.1. The Effect on Seed Surface Structure

Seed treatment-induced changes in surface structure were not visible with a light binocular microscope. However, certain changes were revealed by SEM analysis, particularly at higher (×500) magnification (Figure 2). Surface ornamentation details in vacuum-, LCP-, and DBD-treated seeds were more pronounced (Figure 2H–L), possibly due to the destruction of the protective outer waxy coating. Moreover, LCP induced long cracks on the seed surface (seen in Figure 2C,K). The number, size, and depth of cracks increased with the duration of LCP treatment. Seed surface cracks were not observed in vacuum- or DBD-treated seeds.
Longer LCP treatment (3, 5, and 7 min) resulted in numerous deep cracks and inhibited germination (Therefore, shorter LCP treatments (0.5 and 1 min) were used in further experiments.

3.2. The Effect on Seedling Emergence

The effects of pre-sowing seed treatments on the emergence of lavender seedlings in the substrate were quantified by the changes in the indices of seedling emergence kinetics (Table 1). The results obtained showed that neither vacuum nor LCP treatment had a significant effect on Vi and Me. None of the seed treatments changed the germination uniformity index Qu. The maximal seedling emergence in the substrate (Vi) was affected by DBD treatments only; however, the observed effects in the DBD2 and DBD3 groups were the opposite. In the DBD3 group, the maximal seedling emergence was 20% higher, while the seedlings in the DBD2 group showed a 31% lower maximal emergence percentage compared to the control. Although the effect of DBD3 on Vi was positive, DBD reduced the germination rate (increased Me) in both treatment groups (DBD2 reduced the rate by 12% and DBD3 by 8%).

3.3. The Effects on Seedling Growth

Morphometric analysis of lavender seedlings cultivated in the substrate under greenhouse conditions was performed 50 days after sowing. The seedlings were carefully removed from the substrate, their roots washed, and moisture was absorbed with paper tissue (Figure 3).
The results of morphometric analysis (Table 2) revealed a positive effect of seed treatments on morphometric plant parameters, except for the vacuum-treated group, whose parameters did not differ from the control group. The positive effect on root weight was particularly noticeable: it increased by 33%, 28%, 30%, and 21% in seedlings from the LCP0.5, LCP1, DBD2, and DBD3 groups, respectively. DBD plasma had the most significant impact on plant growth, resulting in a higher seedling weight (DBD3—21%), longer roots (DBD2—14%), and a greater leaf number (DBD2—17%). The LCP0.5 treatment also resulted in a 33% increased root weight and 5% longer seedlings, although the leaf weight remained unchanged. Due to the large scatter of results, differences in leaf weight among the control and experimental groups were not statistically significant.

3.4. The Effect on the Density of Trichomes in Lavender Leaves

Two main types of trichomes were found through SEM analysis on the leaf surface in 50-day-old lavender seedlings—glandular and dendritic trichomes (Figure 4). The density of trichomes on the margins of young leaves (0.7–1 cm long) was measured on SEM images.
The seed treatment with V2 enhanced the total number of trichomes by 25% by increasing the density of both glandular (2.35 times) and dendritic (21%) trichomes (Table 3). LCP0.5 had the same strong positive effect on the density of glandular trichomes, although the number of dendritic trichomes did not differ from the control. The DBD2 treatment reduced the density of dendritic trichomes by 16%.
The effects of LCP1, DBD1, and DBD2 on the number of glandular trichomes were not statistically significant. The relative proportion of glandular trichomes on the leaves of the V2, LCP0.5, LCP1, and DBD2 seedlings was higher compared to the control (1.9, 2.3, 1.6, and 1.6 times, respectively), but the DBD3 treatment reduced this parameter by a factor of 1.7 (Table 3).

3.5. The Effects on the Leaf Content of Photosynthetic Pigments

The amounts of chlorophylls a and b and carotenoids in the lavender leaves were determined spectrophotometrically using leaf extracts in 96% ethanol. The results of the analysis are presented in Figure 5.
The obtained results indicate that the amount of Chl a in lavender leaves was almost 3 times (from 2.6 in the DBD2 group to 3 in the LCP0.5 group) larger compared to the amount of Chl b. In the control samples, the amount of carotenoids was 6.2 times smaller compared to the amount of Chl a. The amount of chlorophylls was positively affected by the V2 and LCP1 treatments: the amount of Chl a increased by 13% and 18% and the amount of Chl b increased by 8% and 25%, respectively. However, LCP0.5 and DBD2 reduced chlorophyll levels—LCP0.5 reduced Chl a by 5% and Chl b by 10%, while DBD2 reduced Chl a by 12%. Seed treatment with DBD3 had no statistically significant effect on the amount of pigments. The content of carotenoids in the leaves of the plants was affected by the V2 and DBD2 treatments: V2 increased their amount by 14%, but DBD2 reduced it by 13%.

3.6. The Effects on the Total Phenolic Compound Content and Antioxidant Activity

The TPC content was determined in leaf methanolic extracts. The results of the analysis are presented in Figure 6a.
The seed treatment with V2, LCP0.5, and DBD2 cold plasma had a positive effect on the TPC in lavender leaves (the TPC increased by 17%, 25%, and 15%, respectively). Compared to the effects on the TPC, a similar pattern of seed treatment-induced changes in the leaf antioxidant activity was revealed by the DPPH radical scavenging method (Figure 6b), except for the absence of a positive V2 treatment effect. The seed treatment with LCP0.5 and DBD2 resulted in enhanced radical scavenging activity (by 32% and 21%, respectively) compared to the control. Neither LCP1 nor DBD2 had a statistically significant effect on the TPC and antioxidant activity.

3.7. The Effects on the Rosmarinic and Clorogenic Acid Contents

Since an increase in the TPC and antioxidant activity was observed in certain experimental groups, we further aimed to determine the levels of individual phenolic acids in the leaf extracts and focused on two hydroxycinnamic acids, rosmarinic and chlorogenic acid, which are used as antioxidative markers in lavender tissues. For this, a quantitative HPLC analysis was performed in ethanolic and methanolic extracts of lavender leaves. The HPLC method allowed us to obtain chromatograms with clear separation of the chlorogenic and rosmarinic acids (Figure 1). However, the comparison of the results obtained through the extract analysis from the control seedling samples revealed that the solvent had a strong impact on the extraction efficiency. The content of rosmarinic acid, using ethanol as a solvent for the control sample, was approximately twice as high compared to the content in the same sample using methanol as an extractant (Figure 7).
The opposite result was obtained for chlorogenic acid (Figure 7). Thus, in the control seedling samples, the content of chlorogenic acid was significantly higher in methanolic extracts, while ethanol led to better extraction of rosmarinic acid from raw materials.
In light of these findings, the changes induced by pre-sowing seed treatment in the content of the two hydroxycinnamic acids in different (but optimal for each acid) extracts are presented: the results obtained in methanol extracts are shown for chlorogenic acid (Figure 8a), while the corresponding changes in the content of rosmarinic acid in the ethanol extracts are provided (Figure 8b).
The obtained results revealed that all seed treatments, except LCP0.5, resulted in changed chlorogenic and romarinic acid contents in the leaves of 50-day-old lavender seedlings. Interestingly, a strong positive effect of the V2 treatment was observed on the content of both chlorogenic (49%) and rosmarinic (14%) acid. The LCP1 and DBD2 treatments increased the chlorogenic acid content by 9% and 26%, respectively. In contrast, the longer DBD treatment (DBD3) resulted in a negative effect (15% decrease) on chlorogenic acid content. Compared to the control, significant negative effects on the amount of rosmarinic acid were observed in the LCP1, DBD2, and DBD3 groups (25%, 19%, and 29%, respectively).

4. Discussion

This study demonstrates the potential of pre-sowing seed processing with CP as an eco-friendly method for improving the production yield and quality of raw materials from the medicinal plant L. angustifolia. CP, including LCP and DBD, is considered a strong physico-chemical stressor for plant seeds, eliciting a complex dose-dependent response and finally resulting in stimulated growth, increased biomass accumulation, activated biosynthesis of secondary metabolites, and improved resistance to environmental stressors [17,18,19,20]. Positive effects of pre-sowing seed treatments on the biosynthesis of biologically active compounds have been documented for numerous medicinal plants, including red clover [21], purple coneflower [22], coriander [23], blue sage [34], stevia [25,29], and other plants. The transcriptomic analysis indicated that the upregulated expression of transcription factors (such as WRKY1) involved in the stress response and regulation of secondary metabolism pathways is responsible for the mechanism of the observed CP effects [34]. Studies performed on wheat [35], Capsicum annuum [36], and Melissa officinalis [37] reported that seed priming with CP increased the amounts of soluble phenols in plant tissues, alongside stimulating the activity of phenylalanine ammonia lyase, an enzyme involved in the synthesis of phenolic compounds. Besides the effects on secondary metabolism, pre-sowing CP treatments induce other systemic changes in growing seedlings—a shift in phytohormone balance, increased content of photosynthetic pigments and stimulated photosynthesis, altered enzyme and antioxidant activity, enhanced plant biomass gain, and increased seed production [17,18,19,20].
In this study, we aimed to find out how CP treatment affects the growth, biochemical composition, and pharmacological potential of this economically valuable plant, including its TPC content, antioxidant activity, and amounts of two hydroxycinnamic acids associated with antioxidant activity—chlorogenic and rosmarinic acids [5,6]. Hydroxycinnamic acids have also been selected as important precursors for the biosynthesis of phenolic compounds (stilbenes, chalcones, flavonoids, lignans, and anthocyanins) [38].
The results obtained through SEM analysis revealed that the V2 and DBD treatments had a slight etching effect on seed surface structure, most likely due to the removal of the waxy layer, while the LCP treatments induced much stronger changes on the seed surface. LCP0.5 and LCP1 caused accidental long cracks in the seed coat, but their number and depth increased progressively with the increasing duration of treatment from 1 to 7 min. The observed changes in the morphometric parameters (Table 2) were poorly associated with the effects on the amounts of chlorophylls (Figure 5). The amounts of photosynthetic pigments in the best-growing seedlings from the DBD3 group were the same as in the control seedlings. In contrast, the morphometric parameters for the V2 group seedlings did not differ from the control despite the positive effects observed in the content of all pigments in their leaves. The reduced pigment content in the seedlings from the LCP0.5 and DBD2 groups did not result in reduced growth. However, an increased root weight (28%) in the LCP1 group was associated with a positive effect on the amounts of photosynthetic pigments.
In this study, we focused our attention on treatment-induced changes in the levels of secondary metabolites and antioxidant activity because numerous beneficial effects on health depend on these variables. Since secondary metabolites accumulate in trichomes, we also performed an SEM analysis of trichomes in lavender leaves. Trichomes are epidermal structures on the surface of plants that protect the plant from biotic and abiotic stress [39]. Vesicle-shaped glandular trichomes accumulate primary and secondary metabolites, while non-glandular trichomes do not participate in the secretion and accumulation of these compounds. The density of glandular trichomes and their size vary depending on the specialised metabolites accumulated by different plants, and differences in the number or density of glandular trichomes may indicate changes in the synthesis of secondary metabolites [40].
The performed SEM analysis showed a large number of dendritic non-glandular trichomes and a much smaller density of glandular trichomes (Figure 4 and Table 3), in line with the reports of other authors [41]. All treatments, except DBD3, significantly increased the relative proportion of glandular trichomes, and the observed changes were in line with changes in TPC and antioxidant activity (Figure 6a,b). The efficiency of the V2 treatment in this respect was the most striking finding, particularly in the context of the neutral V2 effect on seedling emergence and growth (Table 1 and Table 2). Interestingly, the V2 treatment also induced the largest increase in the content of the two hydroxycinnamic acids (Figure 8a,b).
The largest proportion of glandular trichomes was observed in the LCP0.5 group, in line with the effects on the TPC and antioxidant activity (Figure 6a,b). The only negative effect on the proportion of glandular trichomes was induced by the DBD3 treatment, which did not have an impact on the TPC and antioxidant activity but reduced the amounts of both hydroxycinnamic acids (Figure 8a,b). Taking into consideration that the DBD3 treatment strongly enhanced the parameters related to seedling biomass gain (Table 2), we explain this by the so-called plant physiological trade-off [42]—allocating available resources between growth and defence processes. The content of secondary metabolites in the leaves per plant (calculated by multiplying the leaf DW and the content of metabolites per unit of DW) was most efficiently enhanced by the V2 and LCP0.5 treatments (compared to the control, V2 increased the amount of TPC by 20%, chlorogenic acid by 54%, and rosmarinic acid by 17%, while the corresponding numbers for LCP0.5 were 45%, 15% and 9%). DBD2 had a positive effect on the TPC and chlorogenic acid content (13% and 25%) but negatively affected the amount of rosmarinic acid (20%).
Pre-sowing treatment can affect the amount of secondary metabolites and change the solvent’s extraction capacity due to modified tissue texture. The hydrophobicity of the solvent is an another important factor. The analysis of treatment effects on rosmarinic and chlorogenic acid content revealed that the applied solvent had a strong influence on the extraction efficiency of hydroxycinnamic acids. The extracted amount of chlorogenic acid was significantly higher in the methanolic extracts, while ethanol caused greater extraction of rosmarinic acid from the lavender leaves (Figure 7). Methanol and ethanol are two short-chain alcohols with a single hydrophilic hydroxy group, but compared to the single methyl group in methanol, ethanol possesses a larger nonpolar hydrocarbon chain, which confers stronger hydrophobicity [43]. The hydrophobicity of rosmarinic and chlorogenic acid also differs. The logarithm of the octanol–water partition coefficient (logP) of rosmarinic acid is 1.626 [44], and this is related to the better solubility of hydrophobic solvents. Meanwhile, chlorogenic acid has a negative logP of approximately −1.01, indicating that it is highly hydrophilic. The different hydrophobicities of these two acids is a reason why methanol is a more optimal solvent for the extraction of chlorogenic acid, while ethanol is more optimal for the extraction of rosmarinic acid. Ethanol is a commonly used solvent for obtaining rosmarinic acid. Some studies indicate the greater effectiveness of using 30–70% ethanol rather than 100% ethanol, which allows for a higher yield of the marker compound [45].
Numerous authors have reported an increase in the content of phenolic compounds after exposing seeds to plasma in plant species such as wheat, maize, brown rice, buckwheat, spinach, purple coneflower, red clover, coriander, and Norway spruce [18]. For example, it has been documented [22] that seed treatment with vacuum and LCP2 significantly increased the content of chlorogenic acid in E. purpurea leaves (by 3 times and 2.3 times, respectively). Our study confirmed a similar tendency for lavender, as evidenced by the results on the changes in the content of TPC and hydroxycinnamic acids. Similar patterns of effects observed on the TPC and antioxidant activity (Figure 6) are also consistent with statements that phenolic compounds are a major class of plant compounds that act as antioxidants [46]. However, for some species, seed treatments with plasma enhance the amounts of certain specific metabolites but not TPC and antioxidant activity. For example, in Stevia rebaudiana, seed treatment enhanced germination and root development and increased the content of steviol glycosides in leaves. However, this treatment negatively impacted the TPC and flavonoid content as well as the antioxidant activity [25]. Such findings imply that the induced changes in TPC and antioxidant activity can be species-dependent and should be determined for each plant species.

5. Conclusions

The observed effects of the pre-sowing processing of the lavender seeds were dependent on the nature of the stressor and treatment dose. The V2 and DBD treatments caused slight etching of the seed surface, but the LCP treatments induced seed surface cracks. DBD plasma had a dose-dependent impact on the percentage of seedling emergence: DBD2 decreased this parameter, but DBD3 increased it, while both treatment durations decreased the rate of emergence. Only the DBD3 treatment promoted the growth of lavender seedlings. However, it induced negative or neutral effects on biochemical parameters. Although the V2 treatment did not affect seedling emergence and growth, it increased TPC and was the most effective in increasing the content of both hydroxycinnamic acids. Taken together, these two findings imply that the estimation of morphometric parameters is not sufficient to assess the effects of seed stressors on medicinal plants, which are valued as raw materials for biologically active compounds. Pre-sowing seed treatments resulted in numerous effects on the biochemical composition of lavender leaves. The amount of photosynthetic pigments in leaves was positively affected by V2 and LCP1; however, other treatments (LCP0.5 and DBD2) reduced their levels. SEM analysis of leaf trichomes indicated that all treatments, except DBD3, significantly increased the density of glandular trichomes. The most efficient in this respect was the LCP0.5 treatment, and such effect was associated with strong positive effects on TPC and antioxidant activity.
In summary, our study provides evidence that the short-term exposure of lavender seeds to physical stressors, such as vacuum and CP treatments, results in significant positive effects on the content of TPC, rosmarinic, and chlorogenic acids in lavender leaf extracts. This demonstrates the potential of the pre-sowing treatment to produce raw lavender material enriched in biologically active compounds. Since these compounds are responsible for antioxidant activity and many positive health effects, the results obtained are of importance for the pharmaceutical industry. The increased yields and improved phytochemical profile of raw materials for the pharmaceutical market could contribute to the development of novel sustainable technologies and the conservation of natural resources, as well as maximising the medicinal, cosmetic, agricultural, and biotechnological potential of lavender plants.

Author Contributions

Conceptualisation, V.M., O.M. and V.G.; methodology, V.H., L.I. and S.T.; investigation, V.H., M.M., Z.N., R.Z., L.D.-F. and S.T.; resources, L.I. and V.M.; formal analysis, V.H. and V.M.; writing—original draft preparation, V.H., O.M., V.M. and Z.N.; writing—review and editing, O.M., V.M., L.I. and V.G.; supervision, V.M., O.M. and V.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data provided within the manuscript.

Acknowledgments

This research was conducted with the support of the Erasmus+ international programme at the Department of Biological Chemistry, Vytautas Magnus University, and the Department of Analytical Chemistry, Lithuanian University of Health Sciences, Kaunas, Lithuania.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

HPLCHigh-Performance Liquid Chromatography
CPCold Plasma
LPCLow-Pressure Cold Plasma
DBDDielectric Barrier Discharge
VVacuum
DWDry weight
TPCTotal Phenolic Compounds
Chl aChlorophyll a
Chl bChlorophyll b

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Figure 1. Typical HPLC chromatogram of a methanolic extract from lavender leaves (DBD2 MeOH) with detected peaks of chlorogenic acid (1) and rosmarinic acid (2).
Figure 1. Typical HPLC chromatogram of a methanolic extract from lavender leaves (DBD2 MeOH) with detected peaks of chlorogenic acid (1) and rosmarinic acid (2).
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Figure 2. Typical SEM images of the surface of control seeds and vacuum-, LCP- and DBD plasma-treated lavender seeds. Control (A,G), vacuum (B,H), LCP0.5 (C,I), LCP1 (D,K), DBD2 (E,L), and DBD3 (F,M) seed surface images taken at ×100 (A–F) and ×500 (G–M) magnification.
Figure 2. Typical SEM images of the surface of control seeds and vacuum-, LCP- and DBD plasma-treated lavender seeds. Control (A,G), vacuum (B,H), LCP0.5 (C,I), LCP1 (D,K), DBD2 (E,L), and DBD3 (F,M) seed surface images taken at ×100 (A–F) and ×500 (G–M) magnification.
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Figure 3. Lavender seedlings from control (A), LCP0.5 (B), DBD2 (C), V2 (D), LCP1 (E), and DBD3 (F) groups 50 days after sowing.
Figure 3. Lavender seedlings from control (A), LCP0.5 (B), DBD2 (C), V2 (D), LCP1 (E), and DBD3 (F) groups 50 days after sowing.
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Figure 4. SEM images of glandular trichomes, marked by the black letter G (A,C), and dendritic trichomes, marked by the black letter D (B–D) on the leaves of lavender seedlings. Magnification: ×650 (A), ×500 (B), ×300 (C), and ×200 (D).
Figure 4. SEM images of glandular trichomes, marked by the black letter G (A,C), and dendritic trichomes, marked by the black letter D (B–D) on the leaves of lavender seedlings. Magnification: ×650 (A), ×500 (B), ×300 (C), and ×200 (D).
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Figure 5. Photosynthetic pigment content in the leaves of lavender seedlings. The values are presented as the mean ± standard error (n = 3). *—statistically significant difference compared to the control group (p ≤ 0.05).
Figure 5. Photosynthetic pigment content in the leaves of lavender seedlings. The values are presented as the mean ± standard error (n = 3). *—statistically significant difference compared to the control group (p ≤ 0.05).
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Figure 6. Total phenolic compound (TPC) content (a) and antioxidant activity (b) in the leaves of lavender seedlings. The values are presented as the mean ± standard error (n = 3). *—statistically significant difference compared to the control group (p ≤ 0.05).
Figure 6. Total phenolic compound (TPC) content (a) and antioxidant activity (b) in the leaves of lavender seedlings. The values are presented as the mean ± standard error (n = 3). *—statistically significant difference compared to the control group (p ≤ 0.05).
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Figure 7. Chlorogenic acid and rosmarinic acid content (mg/g) in the samples of the control seedling leaves detected in methanolic (MeOH) and ethanolic (EtOH) extracts. The values are presented as the mean ± standard error (n = 3). *—statistically significant difference compared to the control group (p ≤ 0.05).
Figure 7. Chlorogenic acid and rosmarinic acid content (mg/g) in the samples of the control seedling leaves detected in methanolic (MeOH) and ethanolic (EtOH) extracts. The values are presented as the mean ± standard error (n = 3). *—statistically significant difference compared to the control group (p ≤ 0.05).
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Figure 8. Chlorogenic acid content (mg/g) in methanolic extracts (a) and rosmarinic acid content in ethanolic (b) extracts of lavender leaves. The values are presented as the mean ± standard error (n = 3). *—statistically significant difference compared to the control group (p ≤ 0.05).
Figure 8. Chlorogenic acid content (mg/g) in methanolic extracts (a) and rosmarinic acid content in ethanolic (b) extracts of lavender leaves. The values are presented as the mean ± standard error (n = 3). *—statistically significant difference compared to the control group (p ≤ 0.05).
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Table 1. Kinetic indices of seedling emergence in the substrate from control and vacuum-, LCP-, and DBD-treated lavender seeds.
Table 1. Kinetic indices of seedling emergence in the substrate from control and vacuum-, LCP-, and DBD-treated lavender seeds.
TreatmentVi, %Me, hQu, h
Control57.8 ± 2.0123.4 ± 3.827.0 ± 2.2
V256.7 ± 8.2127.5 ± 5.328.9 ± 1.6
LCP0.557.8 ± 2.0129.9 ± 6.027.7 ± 3.5
LCP160.6 ± 2.0125.9 ± 0.925.0 ± 0.9
DBD240.0 ± 4.4 *137.6 ± 4.6 *23.4 ± 3.9
DBD369.2 ± 0.8 *132.7 ± 1.7 *22.8 ± 0.9
Vi, the maximal percentage; Me, the half-time; Qu, the quartile deviation of seedling emergence in the substrate. Mean values ± standard errors are presented (60 seeds used in each of the three replicates, n = 3); *—statistically significant difference compared to the control group (p < 0.05).
Table 2. Morphometric analysis of lavender seedlings grown in substrate under greenhouse conditions from control and treated seeds 50 days after sowing (n = 30).
Table 2. Morphometric analysis of lavender seedlings grown in substrate under greenhouse conditions from control and treated seeds 50 days after sowing (n = 30).
Morphometric ParameterControlV2LCP0.5LCP1DBD2DBD3
Seedling length, cm20.7 ± 0.420.8 ± 0.421.7 ± 0.4 *20.5 ± 0.421.6 ± 0.420.9 ± 0.4
Seedling weight, mg834 ± 74884 ± 54972 ± 67828 ± 69848 ± 541013 ± 79 *
Root length, cm9.8 ± 0.39.6 ± 0.39.9 ± 0.210.2 ± 0.211.2 ± 0.2 *9.5 ± 0.2
Root weight, mg81.3 ± 6.188.7 ± 6.1108.0 ± 6.4 *103.7 ± 7.1 *106.0 ± 5.9 *98.3 ± 6.3 *
Number of leaves29.4 ± 1.628.9 ± 1.732.2 ± 2.131.6 ± 2.034.3 ± 2.5 *32.3 ± 1.7
Leaf weight, mg559 ± 53575 ± 38644 ± 48507 ± 50551 ± 41653 ± 56
Mean value ± standard error is presented. *—statistically significant difference compared to the control group (p ≤ 0.05).
Table 3. The number of trichomes per mm of the leaf margin length in lavender seedlings 50 days after sowing (n = 9).
Table 3. The number of trichomes per mm of the leaf margin length in lavender seedlings 50 days after sowing (n = 9).
Trichome TypeControlV2LCP0.5LCP1DBD2DBD3
Glandular2.0 ± 0.64.7 ± 0.6 *4.7 ± 0.6 *3.3 ± 0.82.8 ± 1.11.1 ± 0.4
Dendritic63.2 ± 4.376.7 ± 4.8 *60.8 ± 3.261.8 ± 3.452.7 ± 3.6 *61.8 ± 5.1
Total65.2 ± 4.681.4 ± 5.8 *65.5 ± 4.165.1 ± 4.455.5 ± 4.662.9 ± 5.4
Part of glandular trichomes, %3.1 ± 0.25.8 ± 0.5 *7.2 ± 1.3 *5.1 ± 0.8 *5.1 ± 0.4 *1.8 ± 0.4 *
The values are presented as the mean ± standard error. *—statistically significant difference compared to the control group (p ≤ 0.05).
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MDPI and ACS Style

Hurina, V.; Nauciene, Z.; Zukiene, R.; Degutyte-Fomins, L.; Tuckute, S.; Ivanauskas, L.; Marksa, M.; Georgiyants, V.; Mykhailenko, O.; Mildaziene, V. Seed Priming with Cold Plasma and Vacuum Increases the Amounts of Phenolic Compounds and Antioxidant Activity in Lavender Herb. Horticulturae 2025, 11, 1413. https://doi.org/10.3390/horticulturae11121413

AMA Style

Hurina V, Nauciene Z, Zukiene R, Degutyte-Fomins L, Tuckute S, Ivanauskas L, Marksa M, Georgiyants V, Mykhailenko O, Mildaziene V. Seed Priming with Cold Plasma and Vacuum Increases the Amounts of Phenolic Compounds and Antioxidant Activity in Lavender Herb. Horticulturae. 2025; 11(12):1413. https://doi.org/10.3390/horticulturae11121413

Chicago/Turabian Style

Hurina, Viktoriia, Zita Nauciene, Rasa Zukiene, Laima Degutyte-Fomins, Simona Tuckute, Liudas Ivanauskas, Mindaugas Marksa, Victoriya Georgiyants, Olha Mykhailenko, and Vida Mildaziene. 2025. "Seed Priming with Cold Plasma and Vacuum Increases the Amounts of Phenolic Compounds and Antioxidant Activity in Lavender Herb" Horticulturae 11, no. 12: 1413. https://doi.org/10.3390/horticulturae11121413

APA Style

Hurina, V., Nauciene, Z., Zukiene, R., Degutyte-Fomins, L., Tuckute, S., Ivanauskas, L., Marksa, M., Georgiyants, V., Mykhailenko, O., & Mildaziene, V. (2025). Seed Priming with Cold Plasma and Vacuum Increases the Amounts of Phenolic Compounds and Antioxidant Activity in Lavender Herb. Horticulturae, 11(12), 1413. https://doi.org/10.3390/horticulturae11121413

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