The roots of O. spinosa, which grows wild in Bulgaria were collected in Sevlievo region, Bulgaria. The voucher specimen SO 105292 has been deposited in the herbarium of faculty of Biology, Sofia University “St. Kliment Ohridski”.

The air-dried roots of the plant O. spinosa (100 g) were extracted twice with ethanol at 50˚-55˚C for 3 h. The ethanol solutions were evaporated under vacuum to give ethanol extract (yield: 6.68 g). In the same conditions was obtained methanol extract (yield: 15.96 g). The ethanol extract of O. spinosa (6.68 g) was subsequently partitioned between petroleum ether and water. Petroleum ether was separated and evaporated to dryness to get petroleum ether soluble fraction (0.41 g). The aqueous layer was further partitioned by chloroform and ethyl acetate. These were finally evaporated to dryness under reduced pressure to get chloroform (0.31 g) and ethyl acetate (1.16) soluble fractions.

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.

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.

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.

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.

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.

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.

Figure 1

Table 1. Antioxidant potential of , investigated by DPPH and ABTS methods.

The total phenolic content in the evaluated samples was determined from a regression equation for the calibration curve (y=8.18181x, R ² =0.998) and expressed in GAE. The obtained values are summarized in Table 2. The ethyl acetate fraction contained the highest amount, 108.70±0.68 mg GAE/g of sample, which was not in agreement with its lower antioxidant activity in relation to chloroform fraction. The methanol extract showed the lowest total phenolic content, which correspond to its weakest antioxidant potential.

Figure 2

Table 2. Total phenolic content of L.

GC and GC-MS analysis of the chloroform soluble fraction of ethanol extract from O. spinosa roots (Table 3) resulted in the identification of 31 constituents, which comprised mostly pterocarpanes (25.84%), saturated and unsaturated fatty acids (free and as esters, 21.6%), steroids (21.03%) and triterpenoids (19.16%).

Figure 3

Table 3. Compounds identified by GC and GC-MS analysis of chloroform fraction 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 O. spinosa the highest scavenging potential was found for chloroform fraction using two different in vitro assays DPPH (0.235 mmol g ^-1 ) and ABTS (0.264mmol g ^-1 ). An important point when assessing the antioxidant activity of plant extracts is to consider their interaction with other antioxidants, especially combinations of hydrophilic and lipophilic antioxidants may exert synergistic effects. Phytochemicals such as triterpenes, carotenoids, α-tocopherol and vitamin C also contributed to the total antioxidant activities in the plant extracts. Triterpenoids are natural, biologically active compounds extracted from many plants. They possess anti-inflammatory, anticancer and antioxidant properties ( ₂₁ ). The presence of triterpene 9, 19-cyclo-27-lanostan-25-on probably contributed to the highest activity of the chloroform fraction.