About 500 g of the investigated spices (Table 1) were purchased from a local market, in Irbid city in Jordan. All the spices were purchased twice with two months time interval. Their scientific and local names were obtained from different references. All the purchased spices were pulverized to a fine powder using a laboratory mill to pass a 0.5 mm sieve and kept at 4˚C in Ziploc plastic bags until time of analysis.

The extractants of the investigated spices were prepared by using three different solvents (methanol, ethanol and acetone) to investigate the effect of solvent on phenolic compounds levels at three different temperatures (20, 40 and 60˚C) for each extractant.

One gram of each spice was weighed out and extracted with 50 ml of methanol. Extraction was carried out

under stirring for 60 minutes at 60˚C. Each one was filtered using filter paper into a 50 ml volumetric flask, the volume was made up to mark using the same extractant and kept in the dark at refrigerated temperature until the time of analysis.

The Folin-Ciocalteu assay [11] was used to determine the total content of phenolic compounds. Folin-Ciocalteu reagent is not specific to detect all phenolic groups found in the extracts. The total phenolic compounds were assayed calorimetrically:, 0.2 ml of spice extract (two replicates) were transferred into a test tube, then mixed with 0.4 ml of 10% diluted Folin-Ciocalteu reagent, after three minutes 0.8 ml of a 10% solution of sodium carbonate as a buffer was added and mixed well, all the tubes were allowed to stand for one hour at room temperature. The absorbance was measured at 725 nm using the spectrophotometer (CE CELL , model 1020). A mixture of reagents without the sample was used as a blank. The phenolic compounds content was expressed as gallic acid equivalents (mg GAE/100g) on dry weight basis; the gallic acid was used to prepare a calibration curve. Gallic acid standard was used to prepare the standard curve as follow; 200 mg of gallic acid were dissolved in 20 ml of distilled water, 0, 10, 25, 50, 100, 200 and 400 ppm concentrations, were prepared, all reagents wereadded as above and the absorbance was read at 725 nm.

Organic solvents; methanol, ethanol and acetone were used to extract phenolic compounds from the spices. One gram of each spice was weighed out and subjected to extraction. Extraction was done under stirring while holding one extraction temperature: 20, 40 and 60˚C for one hour at constant level. Each one was filtered using filter paper into a 50 ml volumetric flask, kept in the dark at refrigerated temperature until the time of analysis.

Approximately, one gram of each spice was subjected to extraction at 20˚C, 40˚C and 60˚C. Extraction was done under stirring while using one extraction solvent; methanol, ethanol and acetone. The extraction time was 60 minutes. Each one was filtered using filter paper into a 50 ml volumetric flask, kept in the dark at refrigerated temperature until the time of analysis.

DPPH (2,2-diphenyl-1-picrylhydrazyl)radical scavenging was estimated according to the method of Mättaus [12] . The scavenging effect of DPPH was followed by monitoring the decrease in absorbance at 515 nm that occurs due to the reduction by the antioxidants or reaction with a radical species. Approximately, 2 g (two replicates) of each spice sample were extracted under stirring with 50 ml methanol for 60 minutes, at 60˚C. Different levels of methanol extracts (10, 25, 50, 100, and 200 μg/ml) of each spice were reacted with 0.2 ml of DPPH (50 mg of DPPH in 100 ml methanol). The mixture was brought to a total volume of 4.0 ml with the extracting solvent. The mixture was mixed thoroughly and allowed to stand in the dark for 30 minutes. Absorbance (A) was read at 515 nm, against the blank. IC₅₀ (the efficient concentration of spice extract in mg/ml required to decrease initial DPPH radical concentration by 50%) was obtained by interpolation from linear regression analysis.

Tables 2-4 show data on total phenolic compounds content extracted by different solvents; methanol, ethanol and acetone at 60, 40 and 20˚C. Data shows that total phenolic compounds concentrations had been affected significantly by the extractant used and temperature employed. Total phenolic concentrations ranged between 6.1 (coriander) and 305.5 mg GAE/100g (cloves) when the methanol was used as extractant at 60˚C. All other spices found to have intermediate values (Table 2).

When ethanol was used, phenolic concentrations showed higher values than the methanolic extractant. Cloves, sumac and cinnamon had the highest values (548.0, 317.4 and 214.5 mg GAE/100g, respectively), whereas; green cardamom, coriander, cumin and ginger had the lowest concentrations (8.4, 13.8, 20.8 and 23.2 mg GAE/ 100g, respectively).

The phenolic compound levels in cloves found to be the highest as compared with values for other spices when acetone was used as extractant (781.0 mg GAE/100g) followed by sumac, cinnamon, turmeric and ginger (725.1, 293.4, 164.4 and 33.5 mg GAE/100g, respectively). Whereas, green cardamom, coriander and sweet laurel had the lowest values (4.5, 5.6 and 9.1 mg GAE/100g respectively).

Phenolic compounds concentrations extracted by methanol, ethanol and acetone showed a significant variation among the investigated spices, except for cumin and sweet cumin, the phenolic compounds content showed

^aMeans are average of two replicates and expressed as mg of gallic acid/100g on dry weight basis. ^*Means with different letters in the same row are significantly different at p ≤ 0.05.

^aMeans are average of two replicates and expressed as mg of gallic acid/100g on dry weight basis. ^*Means with different letters in the same row are significantly different at p ≤ 0.05.

^aMeans are average of two replicates and expressed as mg of gallic acid/100g on dry weight basis. ^*Means with different letters in the same row are significantly different at p ≤ 0.05.

similar values with different solvents. At 60˚C, acetone proved to be anexcellent extractant for cloves, sumac, cinnamon and turmeric (781.0, 725.1, 193.4 and 164.4 mg GAE/100g respectively). While ethanol was excellent extractant for coriander and sweet laurel (13.8 and 47.3 mg GAE/100g respectively). But methanol was suitable extractant for cumin, ginger, green cardamom and sweet cumin (20.8, 38.2, 23.1 and 18.3 mg GAE/100g respectively).

As shown in Table 3 the phenolic compounds content extracted at 40˚C using methanol, ethanol and acetone, the total phenolic compounds content varied significantly among the extractants. Acetone found to be a superior extractant for phenolics from sumac, cloves, cinnamon and turmeric (583.2, 445.2, 315.1 and 123.6 mg GAE/100g respectively) at 40˚C. Followed by methanol as extractant for coriander, cumin, ginger, and green cardamom; (13.7, 35.8, 41.7 and 8.8 mg GAE/100g respectively). Ethanol was suitable extractant for sweet laurel at 40˚C (42.4 mg GAE/100g).

Table 4 shows the effect of different extractants (methanol, ethanol and acetone) on the total phenolic concentrations at the ambient temperature (20˚C). The data depict that the three solvents significantly influenced the levels of phenolic compounds. When methanol was used as extractant at 20˚C, the highest value of phenolic contentwas for cloves (394.7 mg GAE/100g), and the lowest value was for sweet laurel (0.9 mg GAE/100g). Cloves had the highest value of phenolic compounds when ethanol was used as extractant (350.8 mg GAE/100g), and the lowest value for phenolics was found in green cardamom (7.5 mg GAE/100g). On the other hand, sumac gave the highest concentration of phenolic compounds (754.5 mg GAE/100g) when acetone was the extractant, while green cardamom had the lowest concentration (9.4 mg GAE/100g) among the spices at 20˚C.

Data proved that total phenolic compounds concentrations varied significantly according to extractant type and the temperature of extraction. This is in agreement with Hayouni et al. [14] and Hsu et al. [15] who reported that different extracting solvents influenced the total phenolic concentrations, which can be explained as a result of polarity of the solvent and thus different extractability among the phenolic compounds.

The level of total phenolic compounds for 36 vegetables and spices using 80% aqueous ethanol at room temperature, were studied [16] , among them, turmeric, ginger, coriander, their values were higher than those obtained in this study, probably that was due to water ethanol mixture. Phenolic compound in spices like any other plant material, occur in groups or subgroups having different polarity which will influence the effectiveness of the solvent at a given temperature, and that explains why extraction of the same spice using the same solvent yields different level of phenolic compounds using different temperatures.

Shan et al. [17] studied the total phenolic compounds concentrations in cinnamon, coriander, cumin, sweet laurel and cloves, despite variation in extraction circumstances and the extraction temperature, our values are slightly higher than their values.The microbiological status and the chemical composition of the investigated spices were studied [18] .

Table 4 shows data on the concentrations of phenolic compounds extracted at 20˚C, 40˚C and 60˚C using methanol as extractant. Cloves, ginger, turmeric, sumac, cinnamon and sweet laurel did not vary significantly by using different temperatures.

The highest concentrations of phenolic compounds were obtained by methanol at ambient temperature from cloves, sumac and cinnamon (394.7, 271.4 and 187.1 mg GAE/100g respectively). When the temperature was 40˚C; the maximum extraction of phenolic compounds from sumac was 343.9 mg GAE/100g). However, 60˚C was better for obtaining the extraction of phenolic compounds from green cardamom, turmeric and sweet laurel (23.1, 45.1 and 29.0 mg GAE/100g respectively). The highest phenolic compounds content in cloves, coriander, cumin, ginger, cinnamon and sweet cumin were detected at ambient temperature as compared with 40 and 60˚C.

Table 2 shows the effect of temperature on the total phenolic compounds extracted by ethanol. The data show that there were no significant differences by different temperature especially for cumin, green cardamom, sumac, sweet cumin and sweet laurel. The rest of the spices varied significantly in total phenolic compounds using different temperatures. On the other hand, cloves, sumac and cinnamon showed significantly the highest content of phenolic compounds at 60˚C (548.0, 317.4 and 214.5 mg GAE/100g respectively), followed by 40˚C and at the ambient temperature, respectively.

The highest level of phenolic compounds were found in cloves, coriander, cumin, ginger, green cardamom, cinnamon and sweet laurel at 60˚C as compared with 40 and 20˚C as shown in Table 4.

Table 4 also shows the effect of temperatures (20, 40 and 60˚C) on the total phenolic compounds content extracted by acetone. The data showed that there were significant differences in total phenolic compounds. At 60˚C, cloves and turmeric had the highest content of phenolic compounds (781.0 and 164.4 mg GAE/100g respectively). At 40˚C, cinnamon, cumin and sweet cumin had the highest levels of phenolics, values were 315.1, 27.2 and 18.4 mg GAE/100g respectively. Also, at 20˚C; the total phenolics were for coriander, ginger, green cardamom, sumac and sweet laurel, values were 23.0, 41.5, 9.4, 754.5 and 18.3 mg GAE/100g respectively as shown in Table 4. Increasing the temperature from 20, 40 and up to 60˚C will increase the total phenolic compounds level from cloves and turmeric and that is in agreement with data reported previously [18] .

Wojdylo et al. [19] studied the levels of phenolics for cinnamon, cloves and turmeric; he reported values of 0.13 for the cinnamon, 8.96 for cloves and 1.72 mg GAE/100g for turmeric. These values are lower than values reported in the current investigation.

The type of extractant for phenolic had been regarded as a major issue for researchers. Suhaj [20] found that different spices reacted in different manner with respect to the extractant type, for instance; methanol was the most suitable extractant for cloves while the ethanol (96%) was best suitable for ginger. The cinnamon phenolics were extracted efficiently when 80% methanol with Soxhlet extraction were employed. The current investigations showed that acetone was a suitable extractant for cloves. On the other hand, ethanol was suitable to extract phenolics from ginger and that is in agreement with data reported previously [20] .

Phenolic compounds reported for cloves, sumac and cinnamon found in agreement with similar values reported by Lin and Tang [21] on strawberry, green pepper and white onion.

The scavenging of DPPH radicals was followed by monitoring the decrease in absorbance. IC₅₀ (the efficient concentration of spice extract in mg/ml required to decrease initial DPPH radical concentration by 50%) was obtained by interpolation from linear regression analysis. The higher DPPH radical scavenging activity is associated with a lower IC₅₀ values (good antioxidant activity). Table 5 shows the antioxidant activity expressed as IC₅₀ for the investigated spices. Antioxidant activity varied significantly among the spices, BHT was tested as reference synthetic antioxidant and showed the 50 % inhibition at 0.0193 mg/ml. For the investigated spices the lowest IC₅₀ values were detected for cloves and cinnamon (0.077 and 0.09 mg/ml, respectively). On the other hand, coriander and green cardamom had the highest values of IC₅₀ (1.051 and 1.26 mg/ml, respectively). The antioxidant activity expressed as IC₅₀ for sumac, turmeric and sweet laurel did not vary significantly among the investigated spices (0.150, 0.160 and 0.170 mg/ml respectively), from the antiradical activity (1/IC₅₀) in Table 5, the top five spices according to their antioxidant activity properties are: cloves, cinnamon, sumac, turmeric and cumin, all spices had lower antioxidant activity than the reference antioxidant BHT, these values are in agreement with values reported by Ereifej et al. [18] .

Data on total phenolic compounds presented in Table 4 suggest that the type of phenolic compounds rather than the amount is responsible for antioxidant activities [5] [22] .

Al-Ismail et al. [23] studied the antioxidant activities for a set of plant extracts such as cumin, sumac, sweet cumin, sweet laurel and others; these extracts were obtained using the water and the ethanol extractants. Although the amount of phenolic compounds was high in sumac and cumin, these spices had low levels of antioxidant activities, which were attributed to the differences in type of phenolic compounds in each spice. Generally, it is suggested that it is difficult to compare between the antioxidant activities of spices without considering

^aMeans are average of two replicates and expressed as mg/ml. ^*Means followed by different letters in the same column are significantly at P ≤ 0.05; ^bIC₅₀ is the efficient concentration of the test spices that scavenge (decrease) 50% of stable DPPH radical.

the extractant and extraction method as well as the types of phenolic compounds in spices.

Kaur and Kapoor [16] studied the antioxidant activity for ginger, coriander and turmeric. He found that the antioxidant activity ranged from 60 to 80%. The current study reports that there was a relationship between the antioxidant activity and the total phenolic compounds content (Figure 1).

The antioxidant activity for cloves, sweet laurel, coriander, cinnamon and sweet cumin using percent of inhibition for DPPH approach was studied [24] . Our observations found to be in agreement with that reported by Politeo et al. [24] .

Figure 1 shows the correlation between the antiradical activity and total phenolic compounds of the investigated spices, antiradical activity is 1/IC₅₀ and directly proportional to the antioxidant activity. Positive correlation (r = 0.67) was found between antiradical activity and total phenolic compound levels.