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GC/MS Profiling, In-Vitro Cytotoxic and Antioxidant Potential of the Essential Oil of Pulicaria Crispa (Forssk) Growing in Egypt

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GC/MS Profiling, In-Vitro Cytotoxic and Antioxidant Potential of the Essential Oil of Pulicaria Crispa (Forssk) Growing in Egypt International Journal of Pharmacognosy and Chinese Medicine ISSN: 2576-4772 GC/MS Profiling, In-Vitro Cytotoxic and Antioxidant Potential of the Essential Oil of Pulicaria Crispa (Forssk) Growing in Egypt Dekinash MF1, Abou-Hashem MM2, Beltagy AM1 and El-Fiky FK3* Research Article 1Department of Pharmacognosy, Damanhour University, Egypt Volume 3 Issue 3 2Department of Pharmacognosy, Zagazig University, Egypt Received Date: May 02, 2019 3Department of Pharmacognosy, Delta University for Science and Technology, Egypt Published Date: August 22, 2019 DOI: 10.23880/ipcm-16000175 *Corresponding author: Fathy Kandeel El-Fiky, Department of Pharmacognosy, Faculty of Pharmacy, Delta University for Science and Technology, Gamasa, Egypt, Email: [email protected] Abstract The essential oil EO obtained by hydrodistillation from the aerial parts of Pulicaria crispa was profiled using GS/MS analysis. The oil was found to contain carvotanacetone as a major component (81.9%). Biological investigation revealed that the oil possessed EC100 value of12.8 µl/ml in the in-vitro cytotoxicity assay against peripheral blood mononuclear cells (PBMCS). The oil showed no immune-stimulant activity. The antioxidant potential of the oil was assayed using DPPH radical inhibition resulting in inhibition percentage of 66.19% and trolox equivalent antioxidant capacity (TEAC) of 9.22µg/ml. The results indicate powerful antioxidant capacity of the oil, which makes it a good candidate to be used as a natural preservative or as a source of natural antioxidants to be incorporated in pharmaceutical applications. The cytotoxicity assay of the oil against colorectal“Caco-2” and hepatocellular “HepG-2” carcinoma cell lines showed IC50 values of 4.73 and 20.11 µl/ml, respectively. The obtained results spot the light on the colorectal anticancer potential of the oil, which should be subjected to future animal experimental study in form of enema. Keywords: Pulicaria Crispa; GC/MS Analysis; Immune-Stimulant; Antioxidant; Anticancer Activity Introduction tribe species have been reported as traditional medicines [2]. Accordingly, various Pulicaria species have been Herbs containing essential oils (EOs) and their thoroughly investigated phytochemically and biologically. components impressively demonstrate a wide array of Pulicaria crispa (Forrssk), is often synonymous with potential activities in the fields of therapeutic products, Pulicaria undulata, Aster crispus, and Francoeuria crispa. It cosmetics, perfumery, food industry and aromatherapy [1]. is an annual herb locally known in Saudi Arabia as The genus Pulicaria , belongs to the Inuleae tribe "Gethgath". Leaves are sessile, acute to obtuse, undulate to (Asteraceae), encompasses around 100 species distributed rarely toothed, Heads are hemispherical, heterogamous, worldwide. Different Pulicaria species' have been reported radiate, and golden-yellow to orange, 0.5-1 cm across to possesses impressive biological activities and most of golden-yellow to orange in colour [3,4]. P. crispa is GC/MS Profiling, In-Vitro Cytotoxic and Antioxidant Potential of the Essential Oil of Int J Pharmacogn Chinese Med Pulicaria Crispa (Forssk) Growing in Egypt 2 International Journal of Pharmacognosy and Chinese Medicine traditionally used by people of southern Egypt and Saudi ionization detector and a 15 m HP5 column, 0.32 mm ID, Arabia to treat inflammation and as an insect repellent .It 0.25 µm film thicknesses. is also used as an herbal tea [5]. Also, it was reported to be used in Saudi Arabia for bruises, skin infections and GC/MS analysis was performed on Shimadzu GC-MS QP gastrointestinal disturbances [6]. Owing to its strong 2010. High purity helium was used as the carrier gas at a smelling EO, it has been used since ancient times for constant linear velocity of 43.8 cm/s. The constant flow treatment of sinusitis and respiratory tract infections in was controlled by 82.5 KP a pressure giving a total flow of the traditional medicine in the southern part of Iran [7]. 19.2 ml/min, a column flow of 1.47 ml/min and a purge flow of 3.0 ml/min. The ion source and interface Pulicaria crispa is distributed in Saudi Arabia, Kuwait, temperatures were 230°C and operated at ionization Iran, Iraq, Egypt, Afghanistan, Pakistan, India and many energy of 70 eV. The QP mass spectrometer was working parts of north and west tropical Africa [8]. Many studies with mass scan in a range of 40-700 m/z. The used column investigated the chemical composition of essential oil was Varian capillary column (TR5- CPSIL- 5CB, 5% poly- extracted from various Pulicaria species in the Middle East diphenyl 95% dimethylsiloxane), which is 50 m length, region [9-12]. However, to the best of our knowledge, the 0.32 mm diameter and 1.25 μm film thickness. Column current study is the first one, performed to investigate the oven temperature programmed from 40°C to 280°C in 10 antioxidant, immune-stimulant and cytotoxic activities of min. Essential oil samples (20μl) were diluted with the essential oil of P.crispa cultivated in Egypt. methanol (200μl). The injection volume was 1.0 μl. The split ratio was 10 and the injector temperature was 210°C. Materials and Methods Identification and Quantification of Essential Oil Plant Material Composition The aerial parts of Pulicaria crispa were collected Identification of components was performed by during the flowering stage, in March 2016 from their wild comparing their relative retention indices (RI) determined habitat in the western Egyptian desert, El- Sadat city, Al- with the reference of a homologous series of n-alkanes (C8 Menoufia Governorate. The exact location of collection is to C24) [14,15]. The fragmentation patterns of the mass 30.38182°N, 30.51159°E. The collected aerial parts were spectra were compared with the WILEY air-dried in shade for 7 days, and then pulverized to fine (http://eu.wiley.com/WileyCDA/WileyTitle /productCd- pieces. The plant was kindly verified by Dr. Mohamed El- 04-70047852.html) and NIST 05 Azazi; Assistant professor of Plant Ecology in the (http://www.nist.gov/srd/nist1a.htm) libraries. The linear Environmental Studies and Research Institute, El-Sadat temperature-programmed RIs of all the constituents were City University. Voucher specimen was kept in the calculated based on the GC through the interpolation herbarium of Faculty of Pharmacy, Damanhour University between bracketing n-alkanes as follow: as (106P). RI = 100 [(tR(i)+tR(z))×Z / (tR(z+1) – tR(z)] Extraction of the Essential Oil Where Z was the number of carbon atoms in the smaller n- According to the European Pharmacopoeia 2007, the alkane, and t ,t air-dried finely ground aerial parts of P.crispa (200 g) were R(i) R(Z) subjected to hydrodistillation for the extraction of its And t were the retention time of the desired essential oil using a Clevenger- type distillation apparatus R(Z + 1) compound [16]. Quantitative data of the oil components for 3h [13]. were obtained from the electronic integration of the FID peak areas The collected essential oil was then dried by passing over anhydrous sodium sulphate and kept away from light, at 4oC in a sealed glass vial, till subsequent analysis. Preparation of Oil Samples for the Bioactivity Study Compositional Analysis of Essential Oil by Different concentrations of the prepared essential oil GC/MS (12.5, 6.25, 3.125, 1.5625, 0.78125 and 0.3906 µl/ml) were prepared using DMSO by serial dilution in a 96-well plate; The essential oil of P. crispa was analyzed by HP-5890 these concentrations were screened for their immune- Series II gas chromatograph, equipped with a flame stimulant and anticancer potential against human El-Fiky FK, et al. GC/MS Profiling, In-Vitro Cytotoxic and Antioxidant Copyright© El-Fiky FK, et al. Potential of the Essential Oil of Pulicaria Crispa (Forssk) Growing in Egypt. Int J Pharmacogn Chinese Med 2019, 3(3): 000175. 3 International Journal of Pharmacognosy and Chinese Medicine epithelial colorectal adenocarcinoma “Caco-2” and C: The mean absorbance of control cells. hepatocellular carcinoma “HepG-2” cell lines. Antioxidant Activity of the Essential Oil In-vitro Cytotoxicity Assay The antioxidant capacity of P.Crispa EO was determined In-vitro Cytotoxicity Assay on Normal Lymphocytes using a test based on the redox potential of DDPH (Sigma– (Immune stimulant activity screening): In-vitro Aldrich, St. Louis, MO, USA). A 1 mM DPPH solution (0.394 cytotoxicity assay was performed to evaluate the viability mg/mL) in methanol was prepared and then diluted 1:10 of normal Human Peripheral Blood Mononuclear cells to obtain a 100 μM solution (Abs at 515nm = 0.5-0.6). 500 (PBMCs) after incubation for 72 with the different μl of the sample and 500μl of 100 μM DPPH solution were concentrations of the P. crispa essential oil. Viability of the mixed in a cuvette and a negative control with 500 μl of cells was measured using neutral red uptake assay to methanol and 500μl of DPPH solution was also prepared. determine the maximum safe concentration of the oil on Both solutions were incubated in the dark at room normal cells [17]. temperature for 15min. Absorbance was read at λ = 515 nm using methanol as blank. The results were interpreted to calculate the maximum safe concentration that keeps 100% cell viability (EC100) The results were expressed as the percentage for the essential oil using SPSS 15.0 for windows [18]. Data reduction in the radical absorbance [20]. output was presented as Stimulation Index, which is the (Abs max (negative control) – (Abs sample+ DPPH) x 100 ratio of the optical density of lymphocytes stimulated by Abs max the tested material to the optical density of control unstipulated lymphocytes after removal of the effect of the The oil concentrations used for the DPPH assay is blank. A value higher or equal to 1.5 indicates a significant chosen based on the cytotoxicity experiment on normal immune-stimulant activity [19].
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