Effects of Extraction Techniques on Total Phenolic Content and Antioxidant Capacities of Two Oregano Leaves

The effects of dried oregano leaves (Mediterranean and Mexican oregano) extracted using different extraction techniques, solvent types, and six different ratios of each solvent to distilled water on total phenolic (TP) content and antioxidant properties were examined. The Folin-Ciocalteu and 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) assays were performed to assess the antioxidant capacity. The different species of oregano had a significant effect on TP content (107.6 vs. 34.5 mg GAEg in Mexican vs. Mediterranean oregano, respectively) (P<0.05). Comparing extraction techniques, the vortex procedure significantly increased the measured TP content compared to sonication or shaking (P<0.05); however, its effectiveness was sample species and solvent type dependent. Solvent type also had a significant impact on TP content of extracts in decreasing order of acetone, methanol, ethanol, and water (P<0.05). The solvent:water ratio on TP content of each extract was significant (P<0.05); higher TP content was measured for 40:60 and 60:40 acetone:water ratios for Mediterranean and 60:40 and 80:20 acetone:water ratios for Mexican oregano. The antioxidant capacity had a strong relationship with total phenolic contents. The current findings indicated that the species, extraction techniques, solvent type and the ratio of solvent:water had a significance influence on the TP content of two different species of dried oregano leaf, which may be a possible reason behind most variability reported on TP compounds of herbal and medicinal plants.

Determination of total phenolic content from plant extracts would be used as a suitable indirect index to estimate the antioxidant capacity and to investigate the effectiveness of different preparation methods (Shan et al., 2005).Especially compared to other testing methods (static headspace gas chromatography and β-carotene bleaching test), the DPPH assay is rapid, simple and independent of sample polarity, thus it can be applied for quick screening of extracts from samples for radical scavenging activity (Koleva, Van Beek, Linssen, Groot & Evstatieva, 2002;Matsuura, Chiji, Asakawa, Amano, Yoshihara, & Mizutani, 2003).
Since the polarity and less hazardous nature of the solvent is of utmost important in the extraction of bioactive compounds of herbal products (Tanko et al., 2005;Luthria, Mukhopadhyay, & Kwansa, 2006;Tsimogiannis, Stavrakaki, & Oreopoulou, 2006), the objectives of this study were to systematically investigate the effect of solvent type, solvent to water ratio, and extraction techniques (shaker, sonication, and vortex mixing) on total phenolic content of dried oregano leaves from two different species (Mediterranean vs. Mexican).

Plant Material
Two species of dried oregano leaf (known as Mediterranean, Origanum vulgare and Mexican, Lippia graveolens) were kindly provided by Newly Weds Foods Company (Springdale, AR, USA).Both samples were kept in air-tight plastic bags and stored at 4 ºC until analysis.The relative percentage of volatile essential oil contents of both dried leaf oregano samples (Mediterranean vs. Mexican) were previously determined by gas chromatography (GC)/ mass spectrometry (MS) with head space analysis (Karimi et al., 2010).
Extractions of TP contents of oregano samples were carried out according to Luthria et al. (2006).250 mg of each oregano sample were measured and added to 10 mL of the relevant solvent mixture.For the shaker technique, vials were placed on a MaxQ 2000 model shaker (Thermo Scientific, Asheville, N.C., U.S.A.) for 30 min at the maximum speed of 10.For the sonication technique, vials were placed in a sonication bath (Bransonic 35100R-DTH, Branson Ultrasonic Co., Danbury, Conn., U.S.A.) at 40 ºC for 30 min.For the vortex technique, vials were vortex-mixed for two minutes (three times) using a vortex (VM 3000 Minivortexer, VWR Scientific Products Inc. (Fayetteville, Ark., U.S.A.).Following each extraction technique, the vials were centrifuged (10,000×g) for 10 min (J2-21 Beckman Coulter, Brea Calif.U.S.A.).After transferring the supernatant into a 25 mL volumetric flask, the residues were resuspended with an additional 5 mL of relevant solvent mixture.After gently mixing, the vials were recentrifuged for additional 5 min.The supernatant of both centrifuge stages combined together and the final volume of each flask were filled to 25 mL using the relevant solvent mixture.Extraction and analysis of each sample was performed on the same day.Each extraction procedure was carried out in triplicate.

Total Phenolic Content Assay
The total phenolic content of oregano extracts was measured using the Folin-Ciocalteu (FC) assay as described by Luthria et al. (2006) with minor modification and using gallic acid as the standard.Before total phenolic assay, each aliquot was filtered by filter paper.Then, 60 µL of extract transferred into a test tube, followed by addition of 8.74 mL of distilled water.The tubes were vortex mixed for 20 s and 300 µL of FC reagent was added.After an additional 20 s of vortex mixing, 900 µL of filtered 200 gL -1 of sodium carbonate solution was added.After another 20 s of vortex mixing and 2 h sitting in ambient temperature, the absorbance of reaction mixtures was measured at 760 nm using a spectrophotometer (DU 520, Beckman Coulter, Brea, Calif., U.S.A.).The TP content of each extracted mixture was calculated using gallic acid standard curve and results expressed in mg of gallic acid equivalent per gram (mg GAE g -1 ) of dried oregano leaf samples.

HPLC/ESI-MS Analysis of Flavonoids
Oregano samples (250 mg) were vortexed for 2 min with 10 mL of 60% acetone, repeated 3 times.The samples were centrifuged at 10,000xg for 10 min, and supernatant was collected.The residue was washed with 5 ml of 60% acetone, and centrifuged at 10,000xg for 5 min.The supernatants were pooled.A portion of the extract (3 mL) was flushed with nitrogen until dry and reconstituted with 1 mL 50% aqueous methanol.The concentrated extracts were passed through a 0.45 m PTFE syringe filter prior to HPLC analysis.
The flavonoids were separated using a Phenomenex (Torrance, CA) Aqua 5 µm C18 (250 × 4.6 mm) column and a binary gradient of 2% acetic acid for mobile phase A and 0.5% acetic acid in water/acetonitrile (1:1 v/v) for mobile phase B at a flow rate of 1.0 mL/min.The linear gradient was from 10 to 50% B from 0 to 40 min, held at 50% B for 5 min, from 50 to 100% B from 45 to 65 min, held at 100% B for 5 min, and from 100 to 10% B from 70 to 71, held and 10% for 5 min.
An analytical Hewlett Packard 1100 series HPLC instrument equipped with an autosampler, a binary HPLC pump and a UV/VIS detector was used.Reverse phase separations of flavonoids were performed using the same HPLC conditions as described above, with absorption at 280 nm recorded for flavonols.For HPLC/MS analysis the HPLC apparatus was interfaced to a Bruker Esquire LC/MS ion trap mass spectrometer.Mass spectral data were collected with the Bruker software, which also controlled the instrument and collected the signal at 200 to 400 nm.Conditions for mass spectral analysis in negative ion electrospray mode for flavonols included a capillary voltage of 4000 V, a nebulising pressure of 30.0 psi, a drying gas flow of 9.0 mL min −1 and a temperature of 300 °C.Data were collected in full scan mode over a mass range of m/z 200-1000 at 1.0 s per cycle.Characteristic ions were used for peak assignment.For compounds where chemical standards were commercially available, retention times were also used to confirm the identification of components.

DPPH Radical-Scavenging Assay
The DPPH radical scavenging assay was conducted with minor modifications, according to previous study (Loizzo et al., 2010).The extracts (0.5 mL) were added to 0.5 mL of a 0.004% (w/v) freshly made ethanol solution of DPPH.The mixture was mixed vigorously using a vortex and left to stand in ambient temperature in darkness for 20 min, then the absorbance was measured at 517 nm using a spectrophotometer.Inhibition of free radical DPPH in percent was calculated by the formula: Scavenging effect (%) = (A blank -A sample /A blank ) × 100 Where A blank is absorbance of control (containing all reagents, except the sample) and A sample is the absorbance of the each sample.All experiments were run in triplicate.

Statistical Analysis
Experimental data were analyzed according to General Linear Model (GLM) procedure of SAS 9.4 (SAS institute, Cary, N.C., U.S.A.).Significant differences among treatments were determined by analysis of variance (ANOVA).An α of 0.05 was used to determine statistically significant differences.

Results and Discussion
There have been increasing interests in oregano as natural oxidant for human health and thus lots of studies have been conducted to determine potential health effects of oregano (Yoshino et al., 2006;Avila-Sosa et al., 2010;Chun, Vattem, Lin, & Shetty, 2005).Moreover, extracts from oregano have not only natural antioxidants, but also antimicrobial activity and antifungal activity (Govaris, Solomakos, Pexara, & Chatzopoulou, 2010;Portillo-Ruiz, Viramontes-Ramos, Munoz-Castellanos, Gastelum-Franco, & Nevarez-Moorillon, 2005).The optimal extraction conditions for oregano samples are important because the health-promoting properties are determined by the biological evaluation of these extracts.
Figure 1 shows that the origin of dried oregano samples and extraction techniques had a significant influence on TP content of extract (P<0.05).Average TP content of Mexican origin samples (107.6±2.8 mg/GAE) and Mediterranean samples (34.5±1.24mg/GAE) was determined.This result indicates that the vortex is most efficient technique to extract phenolic compounds from both types of oregano.In order to measure the antioxidant capacity of the samples, usually two or more methods are used because extracts might have different chemical characteristics (such as polarity) that might affect results of antioxidant assays.However, some studies (Matsuura et al., 2003;Kulisic et al., 2004;Puertas-Mejía, Hillebrand, Stashenko, & Winterhalter, 2002) were already conducted using the DPPH assay and found no significant differences between methods such as β-carotene bleaching test, thiobarbituric acid reactive test, and the ABTS (radical monocation 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) method.Therefore this experiment used the DPPH assay was used to evaluate the antioxidant properties of oregano samples with different extraction methods.Figure 2   Solvent type also had significant influences on total phenolic content and antioxidant capacities of samples (Table 1 and 2) (P<0.05).Water was the least efficient solvent for extracting phenolic compounds from oregano samples.Extraction of oregano samples with organic solvents yield higher TP content and greater scavenging activity.Babili et al. (2011) reported that a decoction (water extraction with heat treatment) showed a lower amount of TP content than organic solvent extraction (ethanol, petroleum ether, ethyl acetate).In this study, extraction with acetone showed significantly higher phenolic compounds and antioxidant properties among treatments (P<0.05).Based on current results, using a vortex and acetone is the most efficient technique of extracting phenolic compounds from oreganos.In order to determine highest yielding solvent ratio, 6 different solvent ratios were analyzed (0%, 20%, 40%, 60%, 80%, and 100% of each solvent).Results are shown in Figure 3.For Mediterranean oregano, extraction with a 40:60 and 60:40 (acetone:water) ratio exhibited significantly higher TP content than other ratios (P<0.05).Also, extraction with Mexican oregano showed that a 60:40 and 80:20 (acetone:water) ratio showed significantly more TP content.Antioxidant properties of extracts were determined (Figure 4).For Mediterranean oregano, extraction with a 40:60 and 60:40 (acetone:water) ratio were displayed significantly higher scavenging activity than other ratios (P<0.05).Extractions of Mexican oregano show that a 80:20 and 60:40 (acetone:water) ratio exhibited significantly higher scavenging activity than other ratios (P<0.05).

Conclusion
The significant impact of solvent:water ratio, and its solvent and sample origin dependency on total phenol content emphasize that the selection of the appropriate solvent type or ratio is an important task.In conclusion, the finding of the present experiment clearly showed that the origin of oregano sample, solvent type, and solvent:water ratios are all important factors influencing the measured total phenol content.It is evident that the most efficient extraction method for both oregano samples is using a vortex with acetone, but showing different solvent ratio for maximum TP content: Mediterranean is 40:60 and 60:40, while Mexican is 60:80 and 80:20 (acetone:water).

Figure 1 .
Figure 1.Total phenolic contents (mg GAE g -1 ) of the oregano extracts prepared by different extraction techniques.Different capital letters indicate significant differences among Mediterranean oregano (P<0.05).Different lower-case letters indicate significant differences among Mexican oregano (P<0.05) shows the scavenging effect of samples with different origins and extraction techniques.When a sample has greater phenolic content (Figure1), it shows higher antioxidant properties.The vortex technique is the most efficient way to extract a fraction with the most potent antioxidant properties.Considering time and efficiency, vortexing is the most efficient technique of extraction.

Figure 2 .
Figure 2. Scavenging activity (%) of the oregano extracts prepared by different extraction techniques.Different capital letters indicate significant differences among Mediterranean oregano (P<0.05).Different lower-case letters indicate significant differences among Mexican oregano (P<0.05)

Figure 3 .
Figure 3.Total phenolic content (mg GAE g -1 ) of the oregano extracts prepared by different acetone:water ratios using the vortexing technique.Different capital letters indicate significant differences among Mediterranean oregano (P<0.05).Different lower-case letters indicate significant differences among Mexican oregano (P<0.05)

Table 1 .
Total phenolic extraction (mg GAE g -1 ) of the analyzed dried oregano samples

Table 2 .
Scavenging activity (%) of the analyzed dried oregano leaf samples Values are expressed as luteolin equivalents, b expressed as quercetin equivalents, c quantified based on linear regression of respective known compounds, d expressed as phloridzin equivalents, e expressed as galangin equivalents.ND=not detected. a