European Review for Medical and Pharmacological Sciences 2011; 15: 157-164 Antioxidant and antihaemolytic activities of Ferula foetida regel (Umbelliferae) S.M. NABAVI1, M.A. EBRAHIMZADEH1*, S.F. NABAVI1, B. ESLAMI2, A.A. DEHPOUR2 1Pharmaceutical Sciences Research Center, School of Pharmacy and Traditional and Complementary Medicine Research Center, Mazandaran University of Medical Sciences, Sari (Iran) 2Department of Biology, Islamic Azad University, Qaemshahr Branch, Qaemshahr (Iran) Abstract. – Objectives: The Ferula genus Introduction (Umbelliferae) is a rich source of gum-resin and is much utilized in folklore medicine. This study It is commonly accepted that under situations is designed to examine antioxidant and anti- of oxidative stress, reactive oxygen species haemolytic activities of Ferula foetida regel flower, stem and leaf extracts. (ROS) such as superoxide, hydroxyl and peroxyl Materials and Methods: 1,1-Diphenyl-2- radicals are generated. The ROS play an impor- picryl hydrazyl radical (DPPH), nitric oxide and tant role related to degenerative or pathological 2+ H2O2 scavenging activities, Fe chelating ability, processes such as aging, cancer, coronary heart reducing power and hemoglobin-induced linole- disease, Alzheimer’s disease, neurodegenerative ic acid peroxidation were used to evaluate an- disorders, atherosclerosis, cataracts, and inflam- tioxidant activities. Antihaemolytic activity was mation1,2. Minimizing oxidative damage may evaluated by H2O2 induced hemolysis in rat ery- throcyte. Total phenolic compounds were deter- well be one of the most important approaches to mined as gallic acid equivalents and total the primary prevention of these aging-associated flavonoid contents were calculated as quercetin diseases ad health problems, since antioxidants equivalents from a calibration curve. terminate direct ROS attacks and radical-mediat- Results: The leaf aqueous-ethanol extract ed oxidative reactions, and appear to be of prima- showed the highest activity in DPPH radical ry importance in the prevention of these diseases scavenging activity. All extracts showed weak 3 nitric oxide scavenging activity. The stem ex- and health problems . The human body has sev- tract had better activity in nitric oxide scaveng- eral mechanisms to counteract oxidative stress by ing model than the other extracts (IC50 = 896.9 ± producing antioxidants, which are either natural- 21.9 µg ml-1), but it was not comparable to ly produced in situ or externally supplied through quercetin (p<0.001). The leaf extract exhibited foods and/or supplements. Endogenous and ex- better H O scavenging and Fe2+ chelating activ- 2 2 ogenous antioxidants act as free radical scav- ity than the other parts. The extracts exhibited good antioxidant activity in linoleic acid peroxi- engers by preventing and repairing damage dation test but were not comparable to vitamin caused by ROS and, therefore, can enhance the C (p<0.001). Extracts showed weak reducing immune defense and lower the risk of cancer and power activity. The stem extract showed better degenerative diseases2,4. The use of traditional antihaemolytic activity than the flower and leaf. medicine is widespread and plants still present a The flower extract had higher phenolic con- large source of natural antioxidants that might tents. The extracts exhibited different levels of serve as leads for the development of novel antioxidant and antihaemolytic activities in all 1 tested models. drugs . Consequently, the need to identify alter- Conclusions: This study showed remarkable native natural and safe sources of food antioxi- antioxidant and antihemolytic activities in Ferula dants arose and the search for natural antioxi- foetida. Biological effects may be attributed to dants, especially of plant origin, has notably in- the presence of phenols and flavonoids in the creased in recent years5,6. The Ferula genus (Um- extract. It is very promising for further biochemi- belliferae) has been found to be a rich source of cal experiments. gum-resin7. This resin enjoys a reputation as a 8 Key Words: folklore medicine . Sedative, carminative, anti- spasmodic digestive, expectorant, laxative, anal- Antioxidant activity, Chelating activity, DPPH, Ferula foetida, Flavonoid, Nitric oxide scavenging. gesic, anthelminitic, antiseptic and diuretic prop- erties have been reported from the Ferula genus9. Corresponding Author: Mohammadali A. Ebrahimzadeh, Ph.D; e-mail: [email protected] 157 S.M. Nabavi, M.A. Ebrahimzadeh, S.F. Nabavi, B. Eslami, A.A. Dehpour It is also believed to have aphrodisiac and in- Determination of Total Phenolic creasing sexual appetite10. This genus presents Compounds and Flavonoid Content interesting phytochemical features, such as the Total phenolic compound contents were deter- occurrence of sesquiterpenes and sesquiterpene mined by the Folin-Ciocalteau method19. The ex- coumarins8,11. Ferula foetida regel is a perennial tract samples (0.5 ml) were mixed with 2.5 ml of plant, which blooms once in its several years of 0.2 N Folin-Ciocalteau reagents for 5 min and life12,13. It is native to central Asia, Afghanistan 2.0 ml of 75 g l-1 sodium carbonate was then and Iran12. Previously polysulfide derivatives14 added. The absorbance of reaction was measured and sesquiterpene coumarins15 were reported at 760 nm after 2 h of incubation at room temper- from Ferula foetida. In vitro fungi toxicity of the ature. Results were expressed as gallic acid resin extract of Ferula foetida has been studied16. equivalents. Total flavonoids were estimated as We have recently reported good antioxidant ac- previously described20. Briefly, 0.5 ml solution of tivity from Ferula assafoetida9 and Ferula gum- each extracts in methanol were separately mixed mosa17,18. The aim of this study was to determine with 1.5 ml of methanol, 0.1 ml of 10% alu- the antioxidant and antihemolytic activities of the minum chloride, 0.1 ml of 1 M potassium ac- hydroalcoholic extract of Ferula foetida regel etate, and 2.8 ml of distilled water and left at leaf, flower or stems in order to understand the room temperature for 30 min. The absorbance of usefulness of this plant as a medicinal plant. the reaction mixture was measured at 415 nm with a double beam spectrophotometer (UV–Visible EZ201, Perkin Elmer, USA). Total flavonoid contents were calculated as quercetin Materials and Methods from a calibration curve. Chemicals Trichloroacetic acid (TCA), 1,1-diphenyl-2- Antioxidant activity picryl hydroxyl (DPPH), potassium ferricyanide and hydrogen peroxide (H2O2) were purchased DPPH Radical-Scavenging Activity from Sigma Chemicals Co. (St Louis, MO, USA). The stable 1, 1-diphenyl-2-picryl hydroxyl Butylated hydroxyanisole (BHA), ascorbic acid, radical (DPPH) was used for determination of sulfanilamide, N-(1-naphthyl) ethylenediamine free radical scavenging activity of the extracts21 dihydrochloride, ethylenediaminetetraacetic acid Different concentrations of extracts were added, (EDTA) and ferric chloride were purchased from at an equal volume, to the methanol solution of Merck (Darmstadt, Germany). All other chemi- DPPH (100 µM). After 15 min at room tempera- cals were of analytical grade or purer. ture, the absorbance was recorded at 517 nm (UV–Visible EZ201, Perkin Elmer, USA). The Plant Materials and Preparation of experiment was repeated three times. Vitamin C, Freeze-Dried Extract BHA and quercetin were used as standard con- Ferula foetida regel was collected from trols. IC50 values denote the concentration of Gadouk area, central Elburz, Iran, in 2009. The sample, which is required to scavenge 50% of sample was identified by Dr. Bahman Eslami DPPH free radicals. (Assistant Professor of plant systematic, Islamic Azad University, Ghaemshahr Branch, Iran). Determination of Metal Chelating Activity Voucher specimens were deposited in the The ability of the Ferula foetida regel extracts Herbarium of Sari Faculty of Pharmacy (No GRF to chelate ferrous ions was estimated in our re- 32-34). A known amount of each sample (250 g) cently published paper22. Briefly, different con- was extracted at room temperature for 24 h by centrations of each extract were added to a solu- percolation with ethanol/water (600 ml, 70/30 tion of 2 mM FeCl2 (0.05 ml). The reaction was v/v). The extract was then separated from the initiated by the addition of 5 mM ferrozine (0.2 sample residue by filtration through Whatman ml) and the mixture was then shaken vigorously No. 1 filter paper. This procedure was repeated and left to stand at room temperature for 10 min. thrice. The resulting extract was concentrated The absorbance of the solutions was measured over a rotary vacuum until a crude extract was spectrophotometrically at 562 nm (UV–Visible obtained, which was then freeze-dried for com- EZ201, Perkin Elmer, USA). The percentage in- plete solvent removal. hibition of ferrozine-Fe2+ complex formation was 158 Antioxidant and antihaemolytic activities of Ferula foetida regel (Umbelliferae) calculated as [(A0 -A1)/A0] × 100, where A0 was Elmer, USA). Increased absorbance of the reac- the absorbance of the control, and A1 of the mix- tion mixture indicated increased reducing power. ture containing the extract or the absorbance of a Vitamin C was used as positive control. standard solution. EDTA was used as a standard. Antioxidant Activity in A Hemoglobin- Assay of Nitric Oxide-Scavenging Activity Induced Linoleic Acid Peroxidation Test Sodium nitroprusside (10 mM), in phosphate- The antioxidant activity of extracts was deter- buffered saline (PBS), was mixed with different mined by a modified photometry assay25. Reac- concentrations of extracts dissolved in water and tion mixtures (200 ml) containing 10 ml of each incubated at room temperature for 150 min. The extract (10-400 mg), 1 mmol/L of linoleic acid same reaction mixture, without the extracts but emulsion, 40 mmol/L of phosphate buffer (pH with an equivalent amount of water, served as 6.5), and 0.0016% hemoglobin, were incubated control. After the incubation period, 0.5 ml of at 37ºC for 45 min. After the incubation, 2.5 ml Griess reagent (1% sulfanilamide, 2% H3PO4 and of 0.6% HCl in ethanol was added to stop the 0.1% N-(1-naphthyl) ethylenediamine dihy- lipid peroxidation.