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Research Journal of Biological Sciences
Year: 2011 | Volume: 6 | Issue: 3 | Page No.: 114-117
DOI: 10.3923/rjbsci.2011.114.117  
Antioxidant Activity of Essential Oil of Lallemantia iberica in Flowering Stage and Post-Flowering Stage
Yaghoub Amanzadeh, Nafiseh Khosravi Dehaghi, Ahmad Reza Gohari, Hamid Reza Monsef-Esfehani and Seyed Esmail Sadat Ebrahimi
 
Abstract: In this study, the essential oils from the arial parts of Lallemantia iberica (Lamiaceae), collected in 2 stages (flowering and post-flowering) from plants that caltivated in Institute of Medicinal Plants (ACECR) in Hashtgerd of Iran were obtained by hydrodistillation in Clevenger type apparatus. The chemical components of the essential oils were examined by GC and GC-MS then 36 components were characterized in flowering stage with β-cubeben (19.55%), Linalool (18.71%), spathulenol (18.04%), β-caryophyllene (11.11%), geraniol (3.50%) and bicyclogermacrene (3.46%) as the major constituents. All constituents are representing 97.39% of the essential oil, contained monoterpenes (33.85%) and sesquiterpens (63.54%). About 39 components of essential oil of post-flowering stage were introduced which caryophylene oxide (38.77%), linalool (15.15%), Germacrene-D (7.03%), Trans-caryophylene (5.61%), β-bourbonene (4.96%) and Trans-geraniol (4.34%) as the major constituents of it. All components are representing 95.74% of the essential oil contained monoterpens (26.51%) and sesquiterpens (69.23%). The studied essential oils showed antioxidant activities as calculated by 2 in vitro assays; DPPH radical scavenging and Ferric Reducing Power Assay (FRAP).
 
 

INTRODUCTION

Lallemantia iberica belongs to the tribe Stachyoideae-Nepeteae, family Lamiaceae and this family has 46 genera and 410 species and subspecies in Iran (Naghibi et al., 2005). Lallemantia iberica originated from Caucasian region that has been found in Asia (Syria, Iran and Iraq) but it now appears in central and Southern Europe. The Lallemantia genus has 5 different species which are distributed in different places of Iran (North, East North, East South, Alborz and other areas). Lallemantia iberica is introduced with popular name Balangu and traditional name Balangue shahri and with other synonyms Lallemantia sulphurea, Dracocephalum ibericum (Bieb.) (Amin, 1991; Mozaffarian, 1996). People use leaves, oil, seed (Hedrick, 1972) and it has traditional uses as reconstiuent, stimulant, diuretic and expectorant (Aynechi, 1986; Naghibi et al., 2005). It is seeds contain mucilage that it used in the treatment of various disorders such as some nervous, hepatic and renal diseases and also used as general tonic, aphrodisiac and expectorant remedies in Iranian Folk medicine (Amin, 1991; Emad, 2000). Lallemantia iberica cultivated for its seeds from which and oil is extracted, the seed contains up to 30% of a drying oil (Usher, 1974).

MATERIALS AND METHODS

Plants: The aerial parts of cultivated L. iberica were collected in May, 2009 (flowering stage) and in July, 2009 (post-flowering stage) from the Karaj, Iran. Identification of the plant as L. iberica was confirmed by the Herbarium Department of the Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. A voucher specimen of the plant was deposited in the Herbarium of the Department of Pharmacognosy, School of Pharmacy and Pharmaceutical Sciences, Tehran University of Medical Sciences, Tehran, Iran.

Chemicals: All reagents and solvents were of analytical grade or of pure quality which were purchased from Merck, Sigma, Aldrich and Fluka.

Extraction of essential oil: The aerial parts of the plant (100 g) dried in the shade and then powdered after that the volatile oils were isolated by hydrodistillation for 3 h according to the method recommended by the European Pharmacopoeia. The oil was dried over anhydrous sodium sulphate and stored in a refrigerator (4°C).

Gas chromatography-mass spectrometry: Analytical gas chromatography was carried out using a Termoquest 2000 GC with capillary column DB-5 (30 mx0.25 mm i.d., 0.25 μm film thikness); carrier gas, He; split ratio, 1:25 and using a flame ionization detector. The column temperature was programmed at 50°C for 1 min and then heated to 265°C at a rate of 2.5°C min-1 and then kept constant at 265°C for 20 min GC-MS was performed on a Thermoquest 2000 with a qudrupole detector, on capillary column DB-5 (GC); carrier gas, He; flow rate, 1.5 mL min-1 the column was held at 50°C for 1 min and programmed up to 265°C at rate of 2.5°C min-1 then kept constant at 256°C for 20 min. The MS operated at 70 eV ionization. Retention indices were calculated by using retention times of n-alkanes that were injected after the oil at the same chromatographic conditions. Quantitative data was obtained from the electronic integration of the FID peak areas. The components of the oils were identified by comparison of their mass spectra and retention indices with Wiley library and those published in the literature.

Antioxidant activity
DPPH assay:
This assay is based on the Spectrophotometric method. A test sample was added to a concentration of methanolic 2, 2-Diphenyl-1-Picrylhydrazyl (DPPH). Then, the mixture was incubated in the dark at room temperature for 30 min and the absorbance was measured at 517 nm. The difference between the initial DPPH radical adsorption and the adsorption of the sample after reaction was determined as antioxidant activity. The IC50 values (concentration of the test samples providing 50% scavenging) were calculated from the graph-plotted scavenging percentage against the oil concentration. A lower IC50 value means a higher antioxidant power of the examined compound. Scavenging percentage of the DPPH stable radical was calculated in following way (Ramamoorthy and Bono, 2007; Rohman et al., 2010):

where, A0 is the absorbance of the control (The DPPH solution without sample solution) and as is the absorbance in the presence of sample. The DPPH antioxidant activity was assessed by the method of Sanchez-Moreno et al. (1999). Butyl Hydroxyanisole (BHA) and α-tocopherol were used as positive controls. All values are shown as the mean of 3 measurements (Huang et al., 2005; Tofighi et al., 2009; Monsef-Esfahani et al., 2010).

Ferric Reducing Antioxidant Power (FRAP) assay: The FRAP assay was done according to the method of Benzie and Strain (1996). This assay is based on the ability of sample to reduce Fe3+ in the presence of a Tripyridyltriazine (TPTZ) solution. After forming the Fe2+ ion, the blue colored complex Fe2+-tripyridyltriazine was produced. An increase above a complex concentration signals the reducing power of the sample. Solution absorbance was determined at 593 nm (Benzie and Strain, 1996; Huang et al., 2005; Monsef-Esfahani et al., 2010).

Statistical analysis: All tests repeated 3 times and data was expressed as mean±SD. Statistical analysis, plots and fittings were carried out by using Excel 2007.

RESULTS AND DISCUSSION

The hydrodistillation of the flowering aerial parts of L. iberica gave yellow oil with a sharp odour in the yield of 0.2% (w/w) based on dry weights. About 36 components were identified in it representing 97.39% of total oil. The identified components and their percentages are shown in Table 1. The oil of L. iberica was characterized by a high content of β-cubeben (19.55%), linalool (18.71%), spathulenol (18.04%), β-caryophyllene (11.11%), geraniol (3.50%), bicyclogermacrene (3.46%). It was characterized by high amount of sesquiterpene hydrocarbons (43.91%) and monoterpene oxygenated (28.01%) (Table 1).

The aerial parts of L. iberica in post-flowering stage yielded 0.1% (w/w) of a yellowish oil with a strong aroma. About 39 components were characterized, representing 95.74% of the total oil components detected. They are shown in Table 2 with their percentage.

The major constituents of the oil were caryophylene oxide (38.77%), linalool (15.15%), Germacrene-D (7.03%), Trans-caryophylene (5.61%), β-bourbonene (4.96%) and Trans-geraniol (4.34%). It was characterized by high amount of sesquiterpene oxygenated (49.3%) and monoterpene oxygenated (24.93%) (Table 2). Previous investigations on the oil of some species of Lallemantia showed various results.

In a study on L. iberica that collected from Larijan, Iran, p-Cymene (22.1%), isophytol (19.8%), T-cadinol (11.1%), 3-octanol (8.1%), caryophyllene oxide (7.4%) and terpinen-4-ol (5.7%) were mentioned as the main constituents (Morteza-Semnani, 2006).

Table 1: Chemical composition of essential oil of Lallemantia iberica (%) in flowering stage

According to another study, the essential oil of leaves and stems of Lallemantia peltata (L.) (Baser et al., 2000) (Labiatae, collected from Turkey) was analysed by GC/MS. About 13 compounds were identified representing all of the components detected.

Germacrene D (27.4%), (E)-β-ocimene (20.1%) and geijerene (12.0%) were the major constituents of the oil (Baser et al., 2000). In the other study, water-distilled essential oil from aerial parts of Lallemantia royleana (Benth. in Wall.) grown in Isfahan province, Iran was analysed by GC and GC-MS. About 46 compounds were identified that constituting 94.5% of the total detected components, among them verbenone (16.4%) and trans-carveol (9.8%) were the major components of the oil (Ghannadi and Zolfaghari, 2003).

Table 2: Chemical composition of essential oil of Lallemantia iberica (%) in post-flowering stage

Table 3: Antioxidant activities of essential oils of aerial parts of Lallemantia iberica
Data presented is mean±SD from 2 different experiments; radical scavenging assay (DPPH ); Ferric Reducing Power Assay (FRAP); in flowering stage and post-flowering stage

In this research, the chemical constituents of two stages are different with each other and with other results that we have about essential oil of genus Lallemantia. About Lallemantia iberica , we compared the results with the result of study from Larijan, they are different because cultivar variations, geographical differences, times of plant growing and preparation procedures may have influenced oil compounds either at the qualitative or quantitative level (Javidnia et al., 2007; Masoudi et al., 2009).

DPPH radical scavenging activity: In the DPPH assay, the ability of the examined essential oils to perform as a giver of the hydrogen atom or electron in transforming the purple-colored radical DPPH into the yellow-colored DPPH-H with a reduced shape was studied. All samples possessed inhibitory activity. Essential oil in post-flowering exhibited the highest radical scavenging potential (IC50 = 70 μg mL-1) followed by essential oil in flowering stage (IC50 = 100 μg mL-1). The greatest effect was obtained by essential oil in post flowering stag (IC50 = 70 μg mL-1) though it was more effective than BHA (IC50 = 100 μg mL-1) and less effective than α-tocopherol (IC50 = 40 μg mL-1). There are not any results for camparing with the results.

FRAP assay: The reducing capacities of essential oils of L. iberica were calculated according to the FRAP assay. An aqueous solution of ferrous sulphate (50-500 μmol mL-1), y = 0.005x - 0.0234, R2 = 0.997) was prepared as acalibration curve. The results were expressed as μmol Fe2+ equivalents 9 g-1 DW and are shown in Table 3. FRAP values point to a considerably higher reducing power of post-flowering (100±3.6 μmol Fe2+ g-1 DW) compared with flowering (70±3.3 μmol Fe2+ g-1 DW).

CONCLUSION

The present study showed that it the chemical composition of the essential oil of aerial parts of L. iberica for possible use in foods and cosmetics products and their antioxidant effects of them because there are few texts about Lallemantia genus. This study will open new way to research on special effect of this plant.

ACKNOWLEDGEMENTS

This research has been supported by Tehran University of Medical Sciences and Health Services grant. Researchers also thank Institute of Medicinal Plants (IMP), Iranian Academic Center for Education Cultur and Research (ACECR) for supplying the plant materials.