Growth Stage and Drying Methods Affect Essential Oil Content and Composition of Pickling Herb (Echinophora tenuifolia subsp. sibthorpiana Tutin)

The present research was conducted during 2012 in order to determine the essential oil content and composition of Echinophora tenuifolia subsp. sibthorpiana Tutin. Plants were collected during rosette, vegetative growth, full flowering and fruit-ripening stages. Oil was extracted using Clavenger hydro-distillation apparatus from either fresh, shade dried or sun dried samples. Oil composition was determined with a GC/MS. Oil content of samples showed significant variation during the vegetative stages of development. Oil contents of fresh samples were found to be 0.76% at seedling stage whereas oil content has risen to 1.06% at seed set. The shade-dried samples had higher oil contents than the fresh and sun dried samples. The oil composition of pickling herb changed with drying method and growth stage. Throughout the growth stage of the plant, the oil was composed of 21 components and the main components were found to be α- phellandrene (47.43 - 66.39%) and methyl eugenol (21.29 – 38.72%). While methyl eugenol content decreased during vegetation period for both fresh and dried samples, α-phellandrene level increased. Attention should be given to the collection time and drying method of pickling herb for different uses since vegetative stage and drying method influence oil content and composition.


Introduction
The genus Echinophora (Apiaceae) comprises about 10 species, distributed from the Mediterranean region to Afghanistan [1]. The Mediterranean and Middle East regions seem to be the only areas where this genus is established [2]. There are three taxa of the genus found in Europe (E. spinosa, E. tenuifolia ssp. sibthorpiana and E. tenuifolia ssp. tenuifolia) [3]. The flora of Turkey contains six species, three of which are endemic. Fresh or dried E. tenuifolia is used in the treatment of wounds, gastric ulcers and digestive activities in folk medicine due to its antifungal, carminative and digestive properties [4]. It is also added to foods, such as; soup, meat, pickles, dairy products, and meatballs, for enhancing their sensory properties [5,6].
Oil composition depends upon both biotic (genetic, ontogeny, morphogenesis) and abiotic (climate, soil, temperature etc.) factors affecting plant growth. Morphogenetic variability is the variation of oil compositions according to different parts of the plant, such as; flower, leaf, root etc. [7]. The post-harvest process of medicinal plants has great importance in the production chain, because of its direct influence on the quality and quantity of the active ingredient in the product sold. Aromatic plants are often dried before extraction to reduce the moisture content of the product to a level that prevents deterioration of the product and allows storage in a stable condition. Proper drying of medicinal plants is fundamental to the achievement of a high quality product [8]. In addition, the main purpose of drying is to extend product shelf life, minimize packaging requirements and reduce shipping weights [9]. It has been showed that drying method had a significant effect on oil content and composition of aromatic plants [10][11][12][13][14].
Some studies on the essential oil composition of Echinophora species have been carried out [2,6,[14][15][16][17][18][19]. Most of the studies dealt with components identified from different locations and growth stages, however; changes of essential oil content and composition at different plant growth stages with response to drying methods has not been investigated. The objective of the current study was to investigate the effect of growth stages and drying methods on essential oil content and chemical components of E. tenuifolia subsp. sibthorpiana Tutin from the Lakes Region of Turkey (Isparta).

Plant material
The experiment was carried out at the Field Crops Department of Suleyman Demirel University, Isparta, Turkey in the summer of 2012 (elevation 1030 m, average annual rainfall: 500 mm, and mean annual temperature: 18°C). Plant samples were collected at the different plant growth stages (rosette, vegetative growth (before flowering), full flowering and seed maturing stage) from the Research Farms of Suleyman Demirel University. The investigated drying methods were shade and sun drying. Five plants were used for each developmental period. The first group of samples was shade-dried at room temperature for 5 days till they reached to a constant weight. The second group was dried under the sun for 3 days until reaching a constant weight. Plant material was distributed as a thin layer to accelerate drying process. The maximum daily temperature during drying process ranged from 25 to 30°C.

Essential oil extraction
The fresh and dried plant materials (250 g) were powdered and used for extraction by using a hydrodistillation technique during 3 hours in an all-glass Clevenger type apparatus that separates water from oil. The essential oils were separated from the aqueous layer and essential oil yield was calculated. The extracted oil was stored in a dark glass tube and kept under refrigeration at 4°C until further analysis. Extraction was carried out in triplicates.

Identification of components
GC-MS (Gas Chromatography-Mass Spectrometry) analysis of the oil samples was performed on a QP5050 GC-MS equipped with a Quadrapole detector. GC/MS analysis was conducted under the following conditions: capillary column, CP-Wax 52 CB (50 m x 0.32 mm; film thickness = 0.25 μm); oven temperature program (60 0 C increased to 220 0 C at a rate of 2 0 C /min and then kept at 220 0 C for 10 min); total run time 60 min; injector temperature, 240 0 C; detector temperatures, 250 0 C; carrier gas, helium at a flow rate of 20 ml/min. Relative percentage amounts of the essential oil components were evaluated from the total peak area (TIC) by apparatus software. Identification of components in the volatile oil was based on the comparison of their mass spectra and retention time with literature data and by computer matching with NIST and WILEY libraries [20].
Data was subjected to the analysis of variance (ANOVA) procedure with SAS statistical program [21]. Means were separated using LSD test at the 0.05 significance level.

Results
The analysis of variance showed that the plant growth stages and drying methods had a significant effect (p < 0.01) on the essential oil content of E. tenuifolia subsp. sibthorpiana. Essential oil contents obtained from different organs with different drying methods are given in Table 1. Essential oil content increased progressively during different growth and developmental stages; from rosette stage to fruitripening stage. Essential oil contents of freshly analyzed plants at rosette, vegetative growth, full flowering and fruit-ripening stages were 0.76%, 0.87%, 0.97% and 1.06%, respectively. Essential oil contents of shade-dried (2.2 fold) and sun-dried (1.5 fold) samples were higher than the freshly analyzed samples ( Table 1). The highest amount of essential oil was obtained from shade-dried plants at fruitripening stage (2.09%) and full flowering stage (2.0%) while the lowest value (0.76%) was obtained from fresh samples at rosette stages ( Table 1).
The increase in essential oil content during the growth period is believed to be due to both the increased synthesis of the essential oils during the flowering period and decrease of the plant water content with maturation. Braga et al. [22] found that the essential oil content was increased by the removal of moisture from leaves. Observed increase of the essential oil is due to decrease of the moisture content of the dried samples. Aromatic and medicinal plants are often dried before extraction to reduce moisture content. During this process, many compounds which are dragged to the leaf surface by the evaporating water are lost [23]. The proposed mechanism explains the changes of essential oil content with drying procedure. The essential oil content of the samples dried under the light was 30% lower than the samples dried under shade conditions. The loss of volatile compounds may be due to the effect of exposure to direct sunlight and high temperature. The method of drying has a significant effect on the quality and quantity of the essential oils [24]. Generally, drying the plant material before distillation resulted in both increased and reduced essential oil yield depending on drying duration and temperature [9]. Shade-dried plants had higher essential oil than fresh and sun-dried samples were reported by Hamrouni-Sellami et al. [9]. Asekun et al. [10] stated that drying aromatic plants at air ambient temperature is the most efficient method in terms of essential oil yield. Similar to our results, the reduction of volatile oil content of plant by the impact of high temperature drying process has also been reported [13,[25][26][27].
The quantities of the major compounds of essential oil have changed during various growth and developmental stages and in response to different drying methods. Myrcene, 1,8 cineole and ɤterpinene percentages at the rosette stage and geraniol at the vegetative growth period were higher than the other growth stages in fresh samples. ɤterpinene and limonene percentages increased after the full flowering stage ( Table 2). One of the main components, methyl eugenol content decreased gradually from rosette to fruit-ripening stage, while the other main component, α-phellandrene increased gradually during growth and developmental stages ( Table 2). At rosette stage α-phellandrene epoxide and at fruit ripening stage sabinen, α-terpinene and borneol were not detected ( Table 2).
While ɤ-terpinene and p-cymene contents were higher at fresh samples of all growth stages, limonene, 1,8 cineole and α-phellandrene contents increased with drying, especially with the sun drying method. However, drying process caused decreases in the ɤ-terpinene, α-pinene and methyl eugenol contents. Caryophyllene oxide was detected only in fresh samples. On the other hand, α-thujene, which was detected in the oil at fresh and shade-dried samples, was not identified at the sun-dried samples. The results of essential oil analysis showed that the amounts of α-thujene and mentha-1,5-dien-8-ol components was relatively stable in all growth and developmental stages and drying methods. On the other hand, some components showed a fluctuation based on the different growth stages and drying methods ( Table 2).
Methyl eugenol and α-phellandrene were the major constituents at different growth and developmental stages ( Table 2). Methyl eugenol content of the essential oils decreased during the growth period depending on the drying methods. The decrease in methyl eugenol from rosette to fruit-ripening stages at shade (37.68-24.12%, respectively) and sun drying (36.87-22.14%, respectively) samples were higher than fresh samples (38.72-33.8%, respectively). The amount of α-phellandrene increased as plants developed and α-phellandrene levels were higher at dried samples as compared to fresh samples.

Discussion and Conclusion
The loss of total oil is parallel with the changes of the absolute amount of each compound, but there are differences in the change of the relative amounts of the compounds because of different sensitivity for temperature. At high temperatures, the biological structure of the oil glands of medicinal and aromatic plants can be affected and the epithelial cells in the dried samples of some sensible plants could collapse [9,31]. Drying may have a role in total or partial loss of essential oil [13]. Alteration on chemical composition of essential oils may be related to the connection between variations in temperature with plants metabolic activity. The effect of shade and sun drying resulted in the loss of methyl eugenol, αpinene, α-thujene and ɤ-terpinene. These components seem to have more affinity to the water fraction contained in bay leaves and thereby, they were lost with water during drying process. On the other hand, sun drying method had a stimulative effect on some other compounds biosynthesis and accumulation such as α-phellandrene, limonene and 1,8 cineole.
Detected components of the essential oil of E. tenuifolia subsp. sibthorpiana varied between 21 to 45. However; the major constituents were αphellandrene and methyl eugenol. Some other components such as; δ-3-carene, β-phellandrene reported on other papers were not detected in this study. These results suggest that E. tenuifolia subsp. sibthorpiana populations may have different chemo types. It can be concluded that the variation in the essential oil content and composition of essential oil bearing plants depends on different stages of plant growth, as well as plant parts and drying methods. Over all, research results indicated that there were considerable differences in the content and composition of the essential oil of E. tenuifolia subsp. sibthorpiana at different growth stages and drying methods. It is believed that these differences are associated with modifications in secondary metabolism along with the growth and development of the plant.