Chemical Constituents of Euphorbia sanctae-catharinae Fayed Essential Oil : a Comparative Study of Hydro-distillation and Microwave-Assisted Extraction

Objective: Microwave-assisted extraction (MAE), as an effective tool for the extraction of essential oil from medicinal and aromatic plants, has gained a great attention over the last years, thus in this study, MAE was performed to extract essential oils from aerial part of Euphorbia sanctae-catharinae. Methods: microwave-assisted extraction (MAE), as well as hydro-distillation (HD) techniques was used for oil preparation. Oil composition was studied by gas chromatographymass spectrometry analysis. Results: Essential oil constituents and percentages of the obtained oils by MAE were compared with those obtained using conventional extraction HD technique. MAE offered reduction in the extraction time with better oil yield (1.2 % w/v) than that obtained when compared to HD (0.7 % w/v). Using MAE and HD for the extraction of E. sanctae-catharinae essential oil showed difference in the composition and percentage composition of the oil obtained with valencene (16.01%) and butyl hydroxy toluene (25.58%) being the major contents of the extracted oil using HD and MAE, respectively. Conclusion: To the best of our knowledge, this is the first study for the essential oil from E. sanctae-catharinae. The tested essential oil samples did not show any significant antimicrobial activity at a concentration of 1 mg per disc.


INTRODUCTION
Euphorbiaceae family, one of the largest families, is very diverse in range, composed of a wide variety of plants ranging from large woody trees to simple weeds that grow prostrate to the ground.The family composed of over 315 genera and nearly 8,000 species 1 .Euphorbia species have been widely used in folk medicine for treatment of diarrhea, inflammation, and swellings and is known as a wart remover [2][3][4] .Some of these species are endemic or confined in Egypt to the Sinai Peninsula [5][6] .As no reports were found to be done for the oil contents of E. sanctae-catharinae, particularly this variety growing wild in the mountains of Sinai (Gebal Sait Katherine), it was motivating to the authors to undertake this study.
Microwave-assisted extraction (MAE) is a superior greener alternative to several thermal extraction techniques.Early reports, to extract essential oils using MAE, were recorded in the 80's 7 [8][9][10] .
Recent studies have witnessed the development of microwaves applications in the separation and extraction of MAPs; such as microwave hydro-diffusion and gravity, solvent free microwave-assisted distillation and compressed air microwave-assisted distillation [11][12][13]

Plant Material
Aerial parts of Euphorbia sanctae-catharinae Fayed, were collected in June 2013, from North Sinai, Egypt and the plant was kindly authenticated by Dr. Mona Marzouk, Associate Professor of Taxonomy, National Research Centre, Cairo, Egypt.A voucher specimen has been deposited in the Herbarium of National Research Centre, Egypt (voucher ID 212).Collection was taken place under the permission of Saint Katherine protectorate for scientific purposes.

Microwave-assisted extraction
The extraction of the essential oil was carried out using focused microwave apparatus (CEM Corporation, Matthews, NC, USA), model (MARS 240/50, No. 907511, frequency 2450 MHz) operating at 2450 MHz with maximum power 1600 W. 100 g of sample (dried aerial parts of E. sanctae-catharinae) were placed in a 5000 mL round-bottomed flask that connected to Clevenger-type apparatus outside of microwave oven.The extraction was operated using 800 W power for 60 min.Temperature was adjusted at 100 °C.The essential oil was recovered and its volume was determined using micropipette.The obtained yield was calculated as percentage (volume of recovered oil per weigh of sample).Obtained oil was dried on anhydrous sodium sulfate to remove excess water and saved in a refrigerator till analysis.

Hydro-distillation extraction
For comparison, HD extraction of the essential oil was carried out using 100 gm of dried aerial parts of E. sanctae-catharinae.Extraction was carried out for 3 hours using a Clevenger-type apparatus.After HD extraction, same post extraction procedures for MAE, were applied on the recovered oil.

Essential oils chemical composition by Gas chromatography-mass spectrometry analysis
The Gas chromatography-mass spectrometry (GC-MS) analysis of the essential oil samples was carried out using gas chromatography-mass spectrometry instrument stands at the Department of Medicinal and Aromatic Plants Research, National Research Center with the following specifications.Instrument: a TRACE GC Ultra Gas Chromatographs (THERMO Scientific Corp., USA), coupled with a THERMO mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer).The GC-MS system was equipped with a TR-5 MS column (30 m x 0.32 mm i.d., 0.25 μm film thickness).Analyses were carried out using helium as carrier gas at a flow rate of 1.3 mL/min and a split ratio of 1:10 using the following temperature program: 60 °C for 1 min; rising at 4.0 °C/min to 240 °C and held for 1min.The injector and detector were held at 200 °C.Diluted samples (1:10 hexane, v/v) of 1 μL of the mixtures were injected.Mass spectra were obtained by electron ionization (EI) at 70 eV, using a spectral range of m/z 40-450.

Identification of essential oil constituents
The identification of the chemical constituents of the essential oil were deconvoluted using AMDIS free software (www.amdis.net)and identified by its retention indices (relative to n-alkanes C 8 -C 22 ), mass spectrum matching to authentic standards (when available)., and Wiley Spectral Library Collection and NSIT library database).

Antimicrobial activity Microorganisms
The microorganisms used in this study were obtained from the culture collection of Microbial and Natural Products Chemistry Department, National Research Centre (NRC), Giza, Egypt.Gram positive bacterium: Staphylococcus aureus ATCC and Gram negative bacterium: Escherichia coli ATCC 25922 were used for testing the antibacterial activity.While, the antifungal activity was tested against Aspergillus niger NRRL-599 and Candida albicans.The bacterial strains were incubated in nutrient agar medium at 37°C for 24 hr, while those of fungi were incubated on potato dextrose agar at 28°C for 72 hr.

Chemicals
The nutrient agar and potato dextrose agar media were purchased from Lab M limited (Lancashire BL9 6AS, UK) and Becton, Dickinson and company Sparks (MD 21152, USA), respectively.The positive controls, thiamphenicol and nystatin, were obtained from Sanofi-Aventis and Pharaonia Pharmaceutical companies, Egypt, respectively and used in a concentration of 50 μg/disc.

Antimicrobial activity
The antimicrobial activity was carried out using agar disc diffusion method 15 .The bacterial and fungal suspensions were swabbed and spread on nutrient agar and potato dextrose agar, respectively and adjusted to McFarland No. 0.5 standard turbidity.The sterilized paper discs (6 mm D) with the required doses of the methanolic solution of the essential oil (250, 500 and 1000 μg/disc) were placed on the surface of inoculated plates.The positive controls, thiamphenicol and nystatin (50 μg/disc), were used for determination of the sensitivity of bacteria fungi, respectively.

Extraction time and yields
From the obtained results, MAE is clearly faster than HD extraction.The extraction using MAE is 3 times faster than HD extraction.In addition, only 10 min using MAE was enough to reach the extraction temperature, which is equal to boiling temperature of water (100°C); however, 30 min was required by HD extraction to reach the boiling temperature of water.Moreover, an extraction time of 60 min using MAE offered better yield than obtained using HD extraction for 3 hrs (1.2 and 0.7 % (w/v); respectively), confirming significant saving of extraction time and energy consumed.

Composition of essential oil
The composition and the percentage composition of E. sanctae-catharinae essential oil extracted using HD and MAE, presented in Table 1 in order of their elution from the column.Figures (1a) and (1b) show the chromatogarms of essential oils of E. sanctae-catharinae isolated by HD and MAE; respectively.
Forty-nine components were identified in oil extracted using MAE, representing 95.4% of the oil obtained, while 35 components were identified in oil extracted using HD, representing 95.57% of the oil obtained.

Antimicrobial activity
Results of evaluation of antimicrobial activity as presented in

CONCLUSION
This is the first report of essential oil composition of E. sanctae-catharinae, showing microwave-assisted extraction (MAE) method to be an efficient alternative method for the extraction of E. sanctae-catharinae essential oil than conventional hydro-distillation extraction (HD).However, MAE and HD extraction methods of E. sanctae-catharinae essential oil showed differences in both composition and percentage constituents of the obtained oils.Valencene (16.01%) and butyl hydroxy toluene (25.58%) are dominant compounds of the extracted oils using HD and MAE; respectively.MAE offered several advantages over conventional hydro-distillation (HD); shorter extraction time (60 min vs 3hrs) and better extraction yield (1.2 vs 0.7 w/v).Essential oil of E. sanctae-catharinae has neither antibacterial nor antifungal activity.

Figure 1 :
Figure 1: The GC/MS chromatogram of the essential oils obtained from dried aerial part of E. sanctae-catharinae; (a): Hydro-distillation extraction and (b) Microwaveassisted extraction

Table 1 . The composition and the percentage composition of E. sanctae-catharinae essential oil prepared by hydro- distillation and Microwave-assisted extraction
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