Effect of cold plasma on essential oil content and composition of lemon verbena

Abstract Cold plasma is known as a novel nonthermal processing method for decontamination of medicinal and aromatic plants (MAPs); however, there are little research studies about its effects on active ingredients of these plants. The aim of this research was to investigate the influence of low‐pressure cold plasma (LPCP) treatments (1, 3, and 5 min) on the essential oil (EO) content and composition of lemon verbena leaves. The EO content was determined using hydro‐distillation, and the composition of the extracted EOs was quantified using gas chromatography and gas chromatography–mass spectrometry techniques. The results showed that by increasing the LPCP treatment duration, the EO content was reduced from 1.2 to 0.9 (% v/w). The highest content of monoterpene hydrocarbons (e.g., limonene) and oxygenated sesquiterpenes (e.g., spathulenol and globulol) was also observed in LPCP‐treated ones, whereas the oxygenated monoterpenes (e.g., citral) content of control was measurably higher than those treated with LCPC.

quiring methods of disinfection which minimize potential damage to active substances. It should be noted that the use of contaminated spices can reduce the longevity of their by-products and may even cause health risks to consumers (Hertwig, Reineke, Ehlbeck, Erdoğdu, et al., 2015;Little, Omotoye, & Mitchell, 2003;McKee, 1995).
Currently, various decontamination technologies such as gamma irradiation, heating (steam sterilization), and fumigation (ethylene oxide) have been used to overcome mentioned damages although they have some disadvantages too. For instance, irradiation with gamma rays is an efficient method, but is not easily accepted by consumers. Fumigation by ethylene oxide is also banned in many countries due to having carcinogenic effects in humans. As the same, steam sterilization also has some undesirable effects on active substances of spices, especially in terms of essential oil (EO) content and composition. Therefore, scientists have been looking forward to find new decontamination methods of spices which do not have the drawbacks and limitations listed (Hertwig, Reineke, Ehlbeck, Knorr, & Schlüter, 2015;Kim, Lee, & Min, 2014;Schweiggert, Carle, & Schieber, 2007).
Plasma is the fourth state of matter and resembles an ionized gas.
However, information about its impact on the quantity and quality of the active ingredients is limited. Lemon verbena is of agricultural origin and so is usually highly contaminated by microorganisms before any decontamination processes. Therefore, in this study, the influence of LPCP on the EO content of lemon verbena leaves, as well as its composition, was examined.

| Plant material
Lemon verbena shrubs were cultivated in greenhouse at Tarbiat Modares University, Tehran, Iran. Then, the leaves were carefully harvested in the flowering stage and dried for 72 hr under shade conditions at room temperature (20-25°C).

| Plasma supply and treatment
The LPCP system, designed by academic faculty group of TMU F I G U R E 1 Illustration of a low-pressure cold plasma (LPCP) generator designed at Tarbiat Modares University actual (left) and schematic (right) settings (Harouni & Abbasi, 2017) Ten grams of the dried lemon verbena leaves was placed in the reaction chamber (glass tube with a volume of 333.82 cm 3 ) and treated by LPCP in different time periods, namely 0 (control), 1, 3, and 5 min. Each treatment was repeated at least three times (Harouni & Abbasi, 2017).

| Essential oil extraction
Three dried samples each weighing 20 g were hydro-distilled for 3 h using the Clevenger apparatus. EO content was determined on the basis of dry matter (ml/100 D.M.). The removal of moisture from EOs was performed by adding 0.5 g sodium sulfate and then keeping in dark vials at 4°C until the time of analysis (Dianat, Saharkhiz, & Tavassolian, 2016).

| Gas chromatography
Gas chromatography (GC) analysis was carried out with a Thermo-UFM (Ultra-fast model, Italy) gas chromatograph equipped with flame ionization detector (FID) and a Ph-5-fused silica column (10 m × 0.1 mm; 0.4 μm film thickness). The oven temperature was held constant at 60°C for 5 min and then programmed a rate of 80°C/min to 285°C. The carrier gas was helium with a flow rate of 0.5 ml/min; injector and detector temperatures were 280°C.

| Data analysis
The experiments were conducted based on completely randomized design (CRD) with three replications. The analysis of variance and mean comparisons of data were performed using Duncan's multiple range test at 5% significance level, and the statistical analysis of the results was done by SAS 9.1 software.

| Effect of cold plasma treatment on EO content
The results of the EO content analysis in different LPCP treatments are presented in Figure 2. As it can be seen, the LPCP had a sig-

| Effect of cold plasma treatment on EO composition
Based on our observations, 24 different compounds were recognized in the EOs of L. citriodora which were affected by LPCP treatments. The chemical compounds of the EOs are shown in  Figure 3). The greatest concentration (59.2%) of citral (geranial + neral) was observed in the control samples, and the lowest amount (41.9%) was measured in the samples treated for 3 min of LPCP. Furthermore, no significant difference was observed on other LPCP treatments (43.7% and 44.2% for 1-and 5-min LPCP treatments, respectively). Apart from citral, LPCP treatments also showed some effects on other compounds. For example, the limonene, 1,8-cineole, and γ-elemene contents were slightly increased (7.3%, 2.6%, and 2.9% compared to the control) after 3 min of LPCP treatment. The maximum amounts of spathulenol and globulol were observed after 5 min of LPCP treatment (8.1% and 7.3%, respectively).
The constituents of lemon verbena EOs in all treatments could be classified into these following chemical groups (Figure 4): monoterpene hydrocarbons (9.7%-15.1%), oxygenated monoterpenes (53.6%-69.4%), sesquiterpene hydrocarbons (7.3%-9.4%), and oxygenated sesquiterpenes (11%-16.4%). The highest amount of oxygenated monoterpenes (69.4%) was observed in the control samples, but the maximum amount of other chemical groups was obtained for LPCP-treated samples. In other words, the effectiveness of LPCP on EO was demonstrated in terms of its quality and quantity. Kodama et al. (2014) reported that plasma treatments had a significant effect on citrus EO composition, and limonene, γ-terpinene, and β-pinene in the EO were reduced. It was observed that the highest amount of main compounds was obtained in 1-min plasma treatment. The oxidation and damaged oil glands were identified as the main reasons for this phenomenon. Hertwig, Reineke, Ehlbeck, Knorr, et al. (2015) reported that the amount of piperine in cold plasma-treated black pepper was only slightly lower than the amount determined in the untreated one. The results revealed that pepper quality was relatively unaffected by cold plasma treatments (after 15 and 30 min). It seems our findings are in line with the existing studies about the impact of cold plasma (either positive or negative) on the quality of agricultural products (Bußler et al., 2015;Garofulić et al., 2015;Grabowski et al., 2014;Herceg et al., 2016;Hertwig, Reineke, Ehlbeck, Erdoğdu, et al., 2015;Kovačević et al., 2016;Lacombe et al., 2015). These considerable outcomes could be attributed to the differences in plant species, type, and duration of cold plasma treatment, the feed gas used, the applied voltages, plant secretory structures, and chemical composition of active substances (Mir et al., 2016).

| CON CLUS ION
Plasma technology is a novel and effective nonthermal decontamination method that can decrease the negative effects of thermal methods on quality characteristics of spices. Upon literature reviews, very few investigations have been focused on the effect of cold plasma on spices active substances. The results of this research revealed that brief LPCP treatment had a positive effect on EO content of lemon verbena leaves, but it reduced the amount of citral while the changes in other compounds were not significant.

ACK N OWLED G M ENTS
The authors would like to thank Tarbiat Modares University for providing the laboratory facilities and financial support for this project and Mrs. P. Sanders for her helpful recommendations on the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L R E V I E W
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.