Packaging methods and storage duration affect essential oil content and composition of lemon verbena (Lippia citriodora Kunth.)

Abstract Changes in essential oils (EOs) content and composition of lemon verbena leave at different packaging methods (packaged with air, nitrogen, or under vacuum) and during storage period (0, 2, 4, 6 and 8 months) were determined. All the samples were hydrodistilled every 2 months during storage for EO content evaluation. EO composition was determined by gas chromatography and gas chromatography–mass spectrometry. The results showed that by extending the storage period in all packaging methods, EO content was significantly decreased. Parallel to the increase in the storage duration in all packaging methods, citral content was decreased, whereas the amounts of limonene and 1,8‐cineole were increased. Packaging of lemon verbena leaves with nitrogen preserved the highest EO content during 8 months of storage and achieved the desired amounts of citral, limonene, and 1,8‐cineole. This investigation also showed camphene may be a useful marker for the indication of storage duration of lemon verbena.

satisfaction (Potisate, Kerr, & Phoungchandang, 2015;Rosado, Pinto, Bertolucci, Jesus, & Alves, 2013). Therefore, determining a suitable packaging method to maintain higher concentrations of active substances during storage is very important and some researchers have paid attention to this topic, for example: Lin, Sung, and Chen (2011) reported that packing material affected the storability of coneflower (Echinacea purpurea L.) materials. They showed dried plant in polyethylene terephthalate/aluminum foil/polyethylene or nylon/polyethylene bags and stored under low temperature without light retained the highest content of bioactive compounds. Orav, Stulova, Kailas, and Müürisepp (2004) observed that after 1 year of storage of pepper (Piper nigrum L.) in a glass vessel at room temperature, the amount of the oil and terpenes decreased, but the amount of oxygenated terpenoids increased. Pääkkönen, Malmsten, and Hyvönen (1989) reported that the intensity of odor and taste of dill (Anethum graveolens L.) in 1 year storage were better preserved in vacuum packages than in glass jars or paper bags. Rosado et al. (2013) observed no significant differences in essential oils (EO) content of basil (Ocimum basilicum L.) stored in paper or plastic bags over a 12-month period; but, independent of packaging, the oil content was decreased by 0.1% per month. Pääkkönen et al. (1990) reported the intensity scores of odor and flavor of summer savory (Satureja hortensis L.) samples were packed into polyethylene-aluminum-polyethylene bags under vacuum and in nitrogen atmosphere were significantly higher than those of the samples stored in glass jars and paper bags after 9 months of storage. Díaz-Maroto, Pardo, Castillo-Muñoz, Díaz-Maroto, and Pérez-Coello (2009) observed that rosemary (Rosmarinus officinalis L.) samples packaged in polystyrene bottles retained a higher quantity of volatile compounds than those packaged in glass bottles after 21 months of storage.
Although the effects of packaging and storage on the quality of MAPs have been studied in research studies mentioned, little information regarding the effects of these processes on L. citriodora exists and this is a topic of great interest for both industry and consumer.
The aim of this research was to investigate the effect of packaging methods and storage duration on the EO content and composition of L. citriodora.

| Plant material
Fresh leaves of lemon verbena (Lippia citriodora Kunth.) used in this research were harvested from the greenhouse of the Department of Horticulture, Tarbiat Modares University (TMU), Iran. The samples were shade dried for 3 days at room temperature (20-25°C).

| Packaging and storage
In the laboratory, lemon verbena leaves were packed under vacuum, nitrogen, and air gas in polyamide/polyethylene laminate bags (size 35×25 cm, thickness 50 μm; Chap Iran Zamin Co. Ltd., Iran), by using a packaging machine (Henkelman 200A, The Netherlands). Also, identical samples were placed in boxes without cover as controls. The average weight of the leaves in each package was 15 g. The samples of all treatments were stored at 25±3°C and 35±5% RH for 8 months and every 2 months, sampling was done for EOs analysis. In addition, to determine the exact effects of storage conditions on EOs compositions during the experimental period, one sample of leaves was analyzed at the start of the experiment.

| EO isolation
The EOs of all samples were extracted by hydrodistillation using a Clevenger-type apparatus. Fifteen grams of leaves from each sample was placed in a round-bottomed flask containing 300 ml of distilled water. Distillation was continued for approximately 3 hr and the EO content was determined on the basis of dry matter (ml/100 g D.M.).
The EOs were dried over anhydrous sodium sulfate to eliminate traces of moisture and stored in tightly closed dark vials at 4°C until analysis.

| Gas chromatography
The EOs were analyzed by Gas chromatography (GC), using a Thermo-UFM ultra-fast gas chromatograph equipped with a Ph-5-fused silica column (10 m×0.1 mm i.d., film thickness 0.4 μm). The oven temperature was held at 60°C for 5 min and then programmed to 285°C at a rate of 80°C/min. Detector (FID) temperature was 280°C and the injector temperature was 280°C; helium was used as carrier gas with a linear velocity of 0.5 ml/min. The percentages of compounds were calculated by the area normalization method, without considering response factors.

| Gas chromatography-mass spectrometry
Gas chromatography-mass spectrometry (GC-MS) analyses were performed using a Varian 3400 GC-MS system equipped with a DB-5fused silica column (30 m×0.25 mm i.d., film thickness 0.25 μm); oven temperature was 50-240°C at a rate of 4°C/min, transfer line temperature 260°C, carrier gas helium with a linear velocity of 31.5 cm/s, split ratio 1:60, ionization energy 70 eV, scan time 1 s, and mass range 40-300 a.m.u. The EO components were identified by comparison of their mass spectra with those of a computer library or with authentic compounds and confirmed by comparison of their retention indices, either with those of authentic compounds or with data published in the literature (Adams, 2007;Davies, 1990). Mass spectra from the literature were also compared (Adams, 2007). The retention indices were calculated for all volatile constituents, using a homologous series of n-alkanes.

| Data analysis
This research was conducted using a factorial experiment based on completely randomized design with two factors (method of packaging and storage duration) and three replicates. Differences in means were tested by using a Duncan's Multiple Range Test (SAS) at 5% level of significance.

| Effect of packaging methods and storage duration on EO content
The results of the EO content analysis in different packaging methods and storage duration are presented in  (Phillips, 1996). In addition, other researchers have reported positive effects of packaging with nitrogen on the quality of MAPs (Pääkkönen et al., 1989). The

| Effect of packaging methods and storage duration on EO composition
In this experiment, 24 compounds were identified in the EOs of L. packages than in glass jars or paper bags. Sakamura (1987) observed an increase in nerol and geraniol concentrations and a reduction in geranial acetate during storage of ginger (Zingiber officinale Roscoe.). He considered the conversion of geranyl acetate into geraniol, geranial, and neral, successively. Rosado et al. (2013) reported that the relative concentrations of the major constituents of basil, linalool and geraniol were 76.1% and 16.7%, respectively, for leaves stored in paper bags for 12 months and 77.1% and 16.6%, respectively, for leaves stored in plastic bags. Chaliha et al. (2013) (1992) reported that the amount of methylchavicol and eugenol decreased during 7 months of storage of basil, but the content of linalol and 1,8-cineole increased. The reasons for these changes may be due to the water activity or available water in the product, availability of oxygen, and presence of temperature during storage. For MAPs, the most important factors in preserving quality are water and oxygen transmission rates (Chaliha et al., 2013). Also, the change in the EO composition during storage depends on the type of compound, the plant species, and the storage conditions (Mahmoodi Sourestani et al., 2014). Moreover, certain volatile compounds such as citral can migrate into the packaging material, which will produce changes in levels found (Chaliha et al., 2013). Misharina (2001) imagined terpene biotransformation as a reason for these changes. He explained: terpenes are able to bind or release a water molecule, to isomerize or rearrange, and EO components themselves, or trace contaminants, may catalyze or initiate these reactions. It has been observed that the composition of EOs readily changes upon processing and storage, whereby factors such as temperature, light, and oxygen availability have a crucial impact on alteration processes (Díaz-Maroto et al., 2009).
Additionally, our results showed that α-pinene was eliminated gradually during storage in all packaging methods, whereas camphene appeared from the fourth month and then increased. On the other hand, camphene, which could not be detected in the early stages of storage, was only detected at 4 months of storage in all packaging methods (Tables 2, 3, 4 and 5). Therefore, camphene may be a useful marker for the indication of storage duration of L. citriodora. Similar results were observed by Topuz and Ozdemir (2004). They reported isodihydrocapsaicin can be used to identify paprika (Capsicum annuum L.) which had been stored for longer than 6 months because this compound was only detected from 6 months of storage onward.

| CONCLUSION
The results showed that the packaging of L. citriodora leaves with nitrogen preserved the highest EO content at the end of 8 months of storage. Although vacuum packed leaves preserved the highest amount of citral during storage, the highest amount of limonene and desired contents of citral and 1,8-cineole were found in leaves packed with nitrogen. This study also showed camphene may be a useful marker for the indication of storage duration of L. citriodora.