Radical scavenger and Antioxidant activities of extracts and fractions from Bulgarian Ononis spinosa L. and GC-MS analysis of Ethanol extract
M Valyova, V Hadjimitova, S Stoyanov, Y Ganeva, T Traykov, I Petkov
gc-ms analysis, ononis spinosa l., phenolic content, radical scavenging activity
M Valyova, V Hadjimitova, S Stoyanov, Y Ganeva, T Traykov, I Petkov. Radical scavenger and Antioxidant activities of extracts and fractions from Bulgarian Ononis spinosa L. and GC-MS analysis of Ethanol extract. The Internet Journal of Alternative Medicine. 2008 Volume 7 Number 2.
Extracts and fractions from
In recent years there has been increasing interest in the presence and availability of compounds in plant materials that may possess bioactive properties, in particular, antioxidant activity. Plant antioxidants are composed of a broad variety of different substances like polyphenolic compounds, tocopherols or terpenoids. Most antioxidants isolated from higher plants are phenolic compounds (e.g. phenolic acids, tannins, coumarins, anthraquinones, flavonoids) ( 1 ).
The antioxidant activity of phenolic compounds is reported to be mainly due to their redox properties, which can play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides ( 2 , 3 ).
One of the best approaches for discovering new antioxidants is the screening of plant extracts. The goal is to use phytochemicals in foods and pharmaceutical preparations to replace synthetic antioxidants, which are being restricted due to their carcinogenicity ( 4 , 5 , 6 ).
Materials and Methods
The roots of
Preparation of plant extracts
The air-dried roots of the plant
DPPH radical scavenging assay
The antioxidant activity using the DPPH assay was assessed by modifying the method of Blois ( 16 ). One ml of 0.1 mM DPPH • methanol solution was added to 3 ml solution of the extracts and fractions or 3 ml pure methanol for the blank sample. The absorbance was read at 517 nm after 30 min incubation. Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) was used as a reference compound. The Trolox equivalent antioxidant capacity (TEAC) was expressed as mmol Trolox g -1 of sample. Unicam UV 500 Spectrophotometer (Thermo Spectronic, UK) and 1 cm disposable cuvettes (Brandt, Germany) were used for all the absorption measurements reported. All the analyses were done in 3 replicates.
ABTS radical cation scavenging assay
The ABTS •+ scavenging test was used to determine the antioxidant activity. ABTS •+ radical was obtained by reaction between ABTS and potassium persulfate ( 17 , 18 ). Blank sample was prepared from the daily solution by adding 1 ml ethanol, which gives an absorbance of 0.7 ± 0.01. The radical scavenging activity was assessed by mixing 2 ml of ABTS •+ solution with 1 ml ethanol solutions of the investigated plants with different concentrations. The reactive mixture was allowed to stand at room temperature for 10 min and the absorbance was recorded at 734 nm. The Trolox was used as a standard. The TEAC values were calculated like DPPH assay.
Determination of total phenolic compounds
Total phenolic content (TPC) in the investigated extracts and fractions was determined by the Folin-Ciocalteu colorimetric method, based on the procedure of Singleton and Rossi ( 19 , 20 ), using gallic acid as a standard phenolic compound. Briefly, 0.5 ml (three replicates) of the samples was mixed with 3 ml of distilled water and 0.25 ml Folin–Ciocalteu reagent. After 2 min, 0.75 ml of 20% sodium carbonate were added and the volume made up to 5 ml with distilled water. The absorbance of the resulting blue-colored solution was measured at 765 nm after 2 h with intermittent shaking. Quantitative measurements were performed, based on a standard calibration curve of seven points from 0.01 to 0.2 mg/ml of gallic acid in methanol. The total phenolic content was expressed as gallic acid equivalents (GAE) in mg g -1 of sample.
Gas chromatography and GC-MS analysis
The chloroform soluble fraction of ethanol extract was analyzed by GC and GC-MS. GC analysis was carried out on a PERKIN-ELMER Auto System GC, equipped with FID and split/splitless injector and a glass capillary column – HP-5 MS (5 % phenyl dimethylsiloxane) with a length of 30 m, an inside diameter of 0.25 mm and a film thickness of 0.25 m was used; carrier gas He with linear velocity 42 cm.min -1 , temperature programmed – from 60°C to 310°C, with 10°C/min. GC/MS analysis was performed on Hewlett-Packard GCD System G 1800A. The optimum conditions of analysis were employed: ionization type: EI; ionization energy: 70 eV; temperature of ion source: 200˚C. The column and temperature program were the same as in GC analyses. The GC–MS peaks were identified by comparison with data from the literature and the profiles from the Wiley 275 and NIST 05 libraries.
Results and Discussion
Effect of DPPH radical scavenging activity
Free radical scavenging activity of methanol extract, ethanol extract and its sub-fractions (petroleum ether, chloroform and ethyl acetate), measured by DPPH assay was reported for the first time (Table 1). The effect of the investigated samples on DPPH radicals has been checked at various concentrations: from 15 to 120 μg/ml for chloroform fraction and from 30 to 240 μg/ml for the total extracts and ethyl acetate fraction. The DPPH scavenging activities expressed as TEAC values were presented in Table 1. According to these data chloroform fraction was the most efficient free radical scavenger by the highest TEAC value of 0.235 mmol g -1 among all the samples. Since TEAC is a quantification of the effective antioxidant activity of the samples, a higher TEAC would imply greater protective action. The methanol extract was the least active of all samples.
Effect of ABTS radical cation scavenging activity
The results of ABTS experiment expressed as TEAC values are presented in Table 1. The TEAC values ranged from 0.095 to 0.264 mmol g -1 sample. Various concentrations were used: from 10 to 250 μg/ml. The highest scavenging potential (0.264 mmol g -1 ) was found for chloroform fraction like DPPH method. As can be seen from the results ethanol extract showed the weakest antioxidant activity, which is in contrast to DPPH method. Petroleum ether fraction was inactive in both antioxidant assays.
Total phenolic content
The total phenolic content in the evaluated samples was determined from a regression equation for the calibration curve (y=8.18181
GC and GC-MS analysis
GC and GC-MS analysis of the chloroform soluble fraction of ethanol extract from
The triterpene 9, 19-cyclo-27-lanostan-25-on was the major constituent (13.17%), followed by ß-sitosterol (9.61%), medicarpin (9.4%), maackiain (8.01%) and linolic acid (7.98%). It should be noted that in the case of
The present study elucidated for the first time antioxidant activity of the methanol extract, ethanol extract and fractions from