Effect of Phytohormones on the Composition of Sambucus ebulus Leaf Essential Oil

Purpose: To evaluate the effect of growth hormones - naphthalene acetic acid (NAA) and indole-3-acetic acid (IAA) - on the essential oil of Sambucus ebulus leaf. Methods: The leaves of S. ebulus were sprayed three times in one week with distilled water (as control) or with a solution of either NAA or IAA (150 ppb). Following the treatment, the leaves were collected from each of the plant and dried in the dark in a dry environment. The essential oil content of the leaves was obtained by hydrodistillation and analyzed by gas chromatography (GC) and GC-mass spectrometry (GC-MS) Results: Sixty constituents were identified in the plant oil, some of which could have been responsible for the plant’s biological and/or toxicological activities. The results indicate that NAA and IAA exerted significant effect on the composition of the essential oil, increasing some components and decreasing some others significantly. In some cases, certain compounds were eliminated completely from the oil. Conclusion: The use of phytohormones seems a useful strategy for modifying the composition of the essential oil in plants.


INTRODUCTION
Four species of the genus Sambucus (Caprifoliaceous) grow in Iran. Of these species, S. ebulus extensively grows in the northern regions of Iran [1]. Iranian traditional medicine uses the leaves and rhizomes of S. ebulus in treating some inflammatory problems such as, bee and nettle bites, arthritis, and a sore-throat [2]. It has been reported to be an insect repellent, anti-hemorrhoid, anti bacterial toward Helicobacter pylori, useful in the treatment of burns and infectious wounds, edema, eczema, urticaria, the common cold and rheumatism [3]. Recently good antioxidant activities were reported [4].
Phytohormones play an important role in the regulation of germination, growth, reproduction, and protective responses of plants against stress. Mass spectrometry (MS) is the most powerful tool for the determination of phytohormones due to its high sensitivity and selectivity. The use of GC-MS for this purpose has also been reported [5].
Indole-3-acetic acid (IAA) is the major plant growth hormone and is involved in the regulation of almost every step of plant development [6]. Naphthalene acetic acid (NAA) is widely employed in agriculture as a plant growth regulator. NAA prevents premature flowering, fruit drop and controls re-growth of tree sprouts after trimming.
In this study, the effect of growth hormones (NAA and IAA) on the chemical composition of volatile oils were evaluated because it seems that some compounds in the leaf may play a role in some biological and/or toxicological activities that have been reported previously for S. ebulus.

EXPERIMENTAL Chemicals
IAA and NAA were purchased from Merck (Germany). The structures of the two phytohormones are shown in Figure 1.

Plant materials and treatments
S. ebulus plant was collected from the suburb of Sari, Mazandaran Province, north of Iran, in August 2009 and identified by Dr B. Eslami (Department of Biology, Islamic Azad University of Qhaemshahr, Iran). A voucher specimen (no. 382) was deposited at the Herbarium of the Sari School of Pharmacy.
The experimental work was divided into 3 parts. The leaves of S. ebulus were sprayed three times in one week with distilled water (control) or a solution of either NAA or IAA (150 ppb). Following the treatment, the leaves were collected from each of the plants and dried in the dark and a non-humid environment. The conditions under which the treatments were carried out were the same for the duration of the experiment. All experiments were repeated thrice.

Isolation of the Essential Oil
The air-dried leaves were subjected to hydrodistillation, using a Clevenger-type apparatus for 4 h. The oil obtained was dried over anhydrous sodium sulfate and stored in a sealed vial at a low temperature before analysis. The oils were analyzed using GC and GC/MS analysis.

Gas chromatography (GC)
Gas chromatographic analysis was carried out on a Hewlett Packard 6890N GC system with FID detector and a HP-5 MS (30 m × 0.320 mm) capillary column. The column temperature was kept at 60°C for 20 min and programmed to 220°C at a rate of 5°C/min, and kept constant at 220°C for 20 min. The injector and detector temperature was 270°C. The injection volume was 1 μL. Helium was used as a carrier gas at a flow rate of 1 ml/min.

Gas chromatography-mass spectrometry (GC-MS)
GC-MS analysis was performed using a Hewlett-Packard 5973N mass selective detector connected to an HP 6890N gas chromatograph. The same capillary GC conditions as described above were used. MS measurement was carried out at 70 eV.

Identification of constituents
The components of the oil were identified by their retention times, retention indices relative to C 9 -C 28 n-alkanes, computer matching with AUTOINT 1. E library and comparison of their mass spectra with those of authentic samples or with data already available in the literature [7]. The composition of the identified compounds was computed from the GC peak area without any correction factor and was calculated relatively.

RESULTS
Hydrodistillation of the dried leaves of S. ebulus resulted in a light yellowish oil with a yield of 0.1% v/w. As shown in Table 1(a), (b) and (c), sixty components were identified in the oil, representing 97.31% of its total content. Longifolen was eradicated from the plant leaves treated with IAA when compared with control. On the other hand, NAA increased its content about 5 times. Both NAA and IAA substantially increased γ-elemene in plant oil. δ-Elemene

DISCUSSION
Sixty components were identified in the oil. Longifolene is a naturally occurring sesquiterpene whose role in a number of oxidation and rearrangement reactions, because of its significance in the fragrance industry, has been intensively investigated [8]. It was eradicated in the plant treated IAA in comparison with blank. NAA increased it about 5 times. Elemene is a mixture of sesquiterpene compounds extracted from ginger plants curcuma, with outstanding advantages of a broad anti-tumor spectrum, curative effect, and less adverse reaction [9]. There are three major components of β, γ, δ isomers. Recently, elemene emulsion has been used widely in clinical treatment for many malignancies and tumors [10].
Both NAA and IAA significantly increased γ-Elemene in the plant oil. δ-Elemene has been eradicated in plant-treated NAA but decreased by IAA. Aromadendrene is the main constituent of the distillation tail of the essential oil of Eucalyptus globulus. Aromadendrene is a cheap and abundantly available chiral starting material for organic syntheses. It has been shown that many other useful intermediates and natural products can be obtained from this compound [11]. Aromadendrene was increased 7 times in content by treating with IAA. NAA eradicated it completely.
β-Selinene is the major sesquiterpene of calamondin fruits. It was increased 12 times by treating with IAA. NAA eradicated it completely. α-Humulene and transcaryophyllene are plant sesquiterpenes with pronounced anti-inflammatory properties [