European Review for Medical and Pharmacological Sciences 2011; 15: 658-664 Antioxidant activity of hydroalcholic extract of Ferula gummosa Boiss roots M.A. EBRAHIMZADEH1*, S.M. NABAVI1,2, S.F. NABAVI1,3, A.A. DEHPOUR4 1Pharmaceutical Sciences Research Center, School of Pharmacy, Mazandaran University of Medical Sciences, Sari (Iran) 2Department of Biology, University of Mazandaran, Babolsar (Iran) 3Student Research Development Committee, Mazandaran University of Medical Sciences, Sari (Iran) 4Department of Biology, Islamic Azad University, Ghaemshahr Branch (Iran) Abstract. – Objectives: Ferula gummosa Introduction Boiss is native to central Asia. This plant has traditionally been used in the treatment of Free radicals are usually short-lived species many diseases. The antihypoxic and antioxi- but they possess a single unpaired electron, ren- dant activities of Ferula gummosa roots were investigated. dering them highly reactive against biologically Material and Methods: 1,1-diphenyl-2- important macromolecules including DNA, pro- picryl hydrazyl radical (DPPH), nitric oxide and teins and membrane lipids. To counteract this hydrogen peroxide scavenging activities, Fe2+ threat to their integrity, cells have evolved a vari- chelating ability, reducing power and hemoglo- ety of defense systems based on both water-solu- bin-induced linoleic acid peroxidation were ble and lipid-soluble antioxidant species, and on used to evaluate antioxidant activities. Antihe- antioxidant enzymes. A high proportion of the molytic activity was evaluated by H2O2 induced hemolysis in rat erythrocytes. The total amount antioxidant systems of the human body are de- of phenolic compounds was determined as gal- pendent on dietary constituents1. Synthetic an- lic acid equivalents and total flavonoid con- tioxidants such as butylhydroxyanisole (BHA) or tents were calculated as quercetin equivalents butylhydroxytoluene (BHT) are used to deceler- from a calibration curve. ate these processes. However, due to their unsta- Results: The extracts showed moderate an- ble and highly volatile nature, they have fre- tioxidant activity in some models. IC50 for DPPH radical-scavenging activity was 579.6±19.4 quently brought some questions about their safe- µg/ml. The extracts showed weak nitric oxide- ty and efficiency ever since their first introduc- scavenging activity between 0.1 and 1.6 mg tion to the food industry2. Consequently, the need ml-1 but showed good Fe2+ chelating ability. to identify alternative natural and safe sources of IC50 was 895.5±24.1 µg/ml. The extract also ex- antioxidant arose and the search for safe and nat- hibited low antioxidant activity in the linoleic ural antioxidants, especially of plant origin, has acid model but were capable of scavenging 3 hydrogen peroxide in a concentration depen- notably increased in recent years . Ferula (F.) dent manner. Tested extract show moderate gummosa Boiss. (Apiaceae) is a perennial plant activity in H2O2 induced hemolysis in rat ery- native to central Asia, growing in the northern throcytes which was not comparable with vita- and western parts of Iran and blooms once in its min C. several years’ life4. Nomads of southwest Iran Conclusions: F. gummosa Boiss root showed call this plant “Barijeh” and traditionally use its different level antioxidant and antihemolytic ac- resin for the treatment of diarrhea. They eat a tivities. Biological effects may be attributed, at least in part, to the presence of phenols and small piece of the resin and believe it to be a very 5 flavonoids in the extract. effective anti-diarrheal herbal medicin . In Iran- ian ancient medicine, the gum obtained from the Key Words: aerial parts of this plant has been used for stom- ach pain, chorea, epilepsy and as a wound-heal- Antihemolytic activity, Antioxidant activity, DPPH, 4,6 Ferula gummosa, Flavonoid. ing remedy . In recent years there are some re- ports regarding the central effects of this plant. Corresponding Author: M.A. Ebrahimzadeh, Ph.D; e-mail: [email protected] Antioxidant activity of hydroalcholic extract of Ferula gummosa Boiss roots An antinociceptive activity has been shown for crude extract was obtained, which was then the hydroalcoholic extract of aerial parts7 and freeze-dried for complete solvent removal. acetone extract of F. gummosa seed and root were reported previously8. Furthermore, a Determination of Total Phenolic methanol-chloroform (1:1) extract of F. gummosa Compounds and Flavonoid Contents and its fractions have alleviated the morphine Total phenolic compound contents were deter- withdrawal syndrome induced by naloxone9. An- mined by the Folin-Ciocalteau method14. The ex- ticonvulsant potential of an essential oil10 and an- tract sample (0.5 ml) was mixed with 2.5 ml of 0.2 tibacterial activity of the seed11 and anti-inflam- N Folin-Ciocalteau reagent for 5 min and 2.0 ml of matory activity of seed and root of F. gummosa8 75 g/l sodium carbonate were then added. The ab- reported previously .Composition of the essential sorbance of reaction was measured at 760 nm after oil of the fruit of the plant has been determined10. 2 h of incubation at room temperature. Result was We recently have reported good antioxidant ac- expressed as gallic acid equivalents. Total tivity of Ferula assafoetida12 and F. gummosa flavonoids were estimated as previously de- leaves, flowers or stems13. To best of our knowl- scribed15. Briefly, 0.5 ml solution of extract in edge there is no scientific report on antioxidant methanol was separately mixed with 1.5 ml of activity of F. gummosa roots, so the aim of this methanol, 0.1 ml of 10% aluminum chloride, 0.1 study was to determine the antioxidant and anti- ml of 1 M potassium acetate and 2.8 ml of distilled hemolytic activities of hydroalcoholic extract of water and left at room temperature for 30 minutes. F. gummosa Boiss root in order to understand the The absorbance of the reaction mixture was mea- usefulness of this plant as a foodstuff as well as sured at 415 nm with a double beam spectropho- in medicine. tometer (UV-Visible EZ201, Perkin Elmer, Nor- walk, CA, USA). Total flavonoid contents were calculated as quercetin from a calibration curve. Materials and Methods Antioxidant Activity Chemicals Trichloroacetic acid (TCA), 1,1-diphenyl-2- DPPH Radical-Scavenging Activity picryl hydrazyl (DPPH), potassium ferricyanide The stable 1,1-diphenyl-2-picryl hydrazyl rad- and hydrogen peroxide H2O2 were purchased ical (DPPH) was used for determination of free from Sigma Chemicals Co. (St Louis, MO, radical scavenging activity of the extracts16. Dif- USA). Butylated hydroxyanisole (BHA), ascor- ferent concentrations of extract were added, at an bic acid, sulfanilamide, N-(1-naphthyl) ethylene- equal volume, to methanolic solution of DPPH diamine dihydrochloride, ethylenediaminete- (100 µM). After 15 min at room temperature, the traacetic acid (EDTA) and ferric chloride were absorbance was recorded at 517 nm. The experi- purchased from Merck (Darmstadt, Germany). ment was repeated for three times. Vitamin C, All other chemicals were of analytical grade or BHA and quercetin were used as standard con- purer. trols. IC50 values denote the concentration of sample, which is required to scavenge 50% of Plant Materials and Preparation of DPPH free radicals. Freeze-dried Extract F. gummosa Boiss root was collected from Determination of Metal Chelating Activity Gadouk area, south of Ghaemshahr, Iran, in 2009 The ability of the F. gummosa Boiss root ex- and identified by Dr. Bahman Eslami (Assistance tract to chelate ferrous ions was estimated by our Professor of Plant Systematic, Islamic Azad Uni- recently published papers17,18. Briefly, different versity, Ghaemshahr, Iran). Voucher specimens concentrations of extract were added to a solu- are deposited with the Faculty of Pharmacy tion of 2 mM FeCl2 (0.05 ml). The reaction was Herbarium (No. GRF 32). Sample was dried at initiated by the addition of 5 mM ferrozine (0.2 room temperature and coarsely ground before ex- ml) and the mixtures was then shaken vigorously traction. A known amount of sample was extract- and left to stand at room temperature for 10 min. ed at room temperature by percolation method The absorbance of the solutions was measured using ethanol/water (70:30). The resulting extract spectrophotometrically at 562 nm. The percent- was concentrated over a rotary vacuum until a age inhibition of ferrozine-Fe2+ complex forma- 659 M.A. Ebrahimzadeh, S.M. Nabavi, S.F. Nabavi, A.A. Dehpour tion was calculated as [(A0 -A1)/A0] ×100, where ter (2.5 ml) and FeCl3 (0.5 ml, 0.1%), and the ab- A0 was the absorbance of the control, and A1 of sorbance was measured at 700 nm. Increased ab- the mixture containing the extract or the ab- sorbance of the reaction mixture indicated in- sorbance of a standard solution. EDTA was used creased reducing power. Vitamin C was used as as a standard. positive control13. Assay of Nitric Oxide-Scavenging Activity Antioxidant activity in a hemoglobin- The procedure is based on the principle that induced linoleic acid peroxidation test sodium nitroprusside in aqueous solution at The antioxidant activity of extract was deter- physiological pH spontaneously generates nitric mined by a modified photometry assay19. Reac- oxide which interacts with oxygen to produce tion mixtures (200 ml) containing 10 ml of each nitrite ions that can be estimated using Griess extract (10-400 mg), 1 mmol/l of linoleic acid reagent. Scavengers of nitric oxide compete with emulsion, 40 mmol/l of phosphate buffer (pH 6.5) oxygen, leading to reduced production of nitrite and 0.0016% hemoglobin were incubated at 37ºC ions. For the experiment, sodium nitroprusside for 45 min. After the incubation, 2.5 ml of 0.6% (10 mM), in phosphate-buffered saline, was HCl in ethanol was added to stop the lipid peroxi- mixed with different concentrations of extract dation. The amount of peroxide value was mea- dissolved in water and incubated at room tem- sured in triplicate using the thiocyanate method perature for 150 min.