Phytochemical analysis and antioxidant activity of some thymus species from Romania

Thymus species (Lamiaceae family) are aromatic medicinal plants widespread in the Romanian flora and are rich sources of bioactive principles [1-4]. Thyme is used both in gastronomy and in the pharmaceutical industry in the production of medicines. Natural products contain volatile oil (with thymol, carvacrol), flavonoids, phenolic acids, pentacyclic triterpenoids with several therapeutic properties: antispasmodic, carminative, antibacterial, expectorant, antiseptic, antiviral, antioxidant, antiinflammatory, anthelmintic, etc. [2-13]. The purpose Abstract


Plant material
The aerial parts of thymus sp. were collected during the flowering period (June, 2014) from the spontaneous flora of Cluj, Sibiu, Brasov: C1, C2, Cb2, Cb5, TC, TG. Five natural products purchased from Romanian commercial companies, in the form of medicinal tea, of which two were samples of T. vulgaris tea: C3, C4, and three were samples of T. serpyllum: Cb1, Cb3, Cb4 (Table 1).

Preparation of extracts
The aerial parts of thymus sp. were dried at room temperature and then were ground to a fine powder. The plant materials (5.0 g) were extracted at 60° C using 70% ethanol, on water bath, for 30 minutes. The ethanolic extracts obtained were filtered and made up to volume (50 ml) in volumetric flasks [14][15][16][17].

Identification of flavonoid and caffeic acid derivatives by TLC
Thin-layer chromatography (TLC) on silica gel was used for separation and identification of the flavonoids and phenolic acids from the thymus sp. extracts. Chromatography was performed on 10 cm × 20 cm glass TLC plates coated with silica gel Si 60 F254 (Merck, Darmstadt, Germany). The ethanolic extracts were applied in 5 μL volumes as 5-mm bands, and the TLC plates were developed with the mobile phase: ethyl acetate:acetic acid: formic acid: water (100:11:11:26). Standard solutions were used: 1% methanolic solutions of: rosmarinic acid, chlorogenic acid, caffeic acid, hyperoside, rutin, quercetrin and isoquercitrin. They were air dried and then sprayed with a fine spray of NEU/PEG reagents, left to dry and then visualized under UV light at 365 nm. The spots of separated compounds were marked and retention factors (Rf) were calculated and recorded [16]. The resultant chromatograms were captured on camera ( Fig. 1).

Determination of total polyphenolic content (TPC)
Total polyphenols content was determined using the Folin-Ciocalteu technique. The vegetal extracts were diluted with 70% ethanol in volumetric flasks to 25 ml. 2 ml of these solution were mixed with 1 ml of Folin-Ciocalteu reagent and 10 ml of water and were brought into a 25 ml volumetric flask. This volumetric flask was completed with 29% sodium carbonate. After 30 minutes, the absorbance was measured at 760 nm. Gallic acid was used as standard for the preparation of a calibration curve (R2 = 0.999) and the results were expressed percentage (gallic acid equivalent (GAE) g/100 g dry plant material [15,17].

Determination of flavonoidic content
Total flavonoids content was determined using a spectrophotometric method based on flavonoidaluminum chloride (AlCl3) color reaction. 10 ml of the 10% plant extracts are diluted with methanol in a volumetric flask to 25 ml. The mixture obtained is left to stand for 10 minutes and after that is filtered. 5 ml

FIGURE 1. TLC plates viewed after staining with NEU/PEG reagent, under 365 nm
of filtered solution is taken and placed in a 25 ml volumetric flask. 5 ml of 100 g/l sodium acetate and 3 ml aluminum chloride (25 g/l) are added. The flask is completed with methanol. The absorbance of the solution was measured at 430 nm. Rutin was used as a standard for the preparation of a calibration curve (R2 = 0.999). Data were expressed as g of rutin equivalents (RE)/100 g dry plant material [14][15][16][17][18].

Determination of caffeic acid derivatives content
The caffeic acid derivatives content was determined using a spectrophotometric method based on phenylpropane -Arnow reagent color reaction. The plant material was extracted by refluxing with 50 ml ethanol. After this, 5 ml of the filtrate solution was diluted with 50 ml ethanol 70° in a volumetric flask (solution A). To 1 ml of solution A was added 1 ml sodium hydroxide (1N), 1 ml Arnow regent and 10 ml water. Absorbance was measured at 500 nm with the values of phenolic acids content, expressed as caffeic acid equivalent (g CAE/100 g plant material), using an equation derived from the from the calibration curve of caffeic acid (R2 = 0.994) [14][15][16][17][18].

Determination of tannin content
To determine the tannins by spectrophotometry, the reactive Folin-Ciocalteu colorimetric method was used. First, the total polyphenolic compounds were determined using the Folin-Ciocalteu method and then, the same extracts were treated with 10 mg of skin powder, and the insoluble tannin-protein complexes were removed by filtration. The clear filtrate (solution of non-adsorbed polyphenols) was treated with Folin-Ciocalteu reagent as mentioned above. The tannin content was measured as a difference between the total phenolic and non-tannin content, and pyrogalol was used as the standard solution. The concentration of the tannins was calculated using the relation: C(%) = [62,5 x m1 x (A1-A2)]/A3 x m2. In which: A1 = absorbance of the total polyphenol solution; A2 = absorbance of the solution of unabsorbed polyphenols on the skin powder; A3 = absorbance of the pyrogalol solution (0.31); m1 = mass of pirogalol (0.05 g); m2 = mass of the sample used in work (= 0.5000 g) [16,18].

Determination of antioxidant activity
The antioxidant capacity of the thymus extracts was determined using the DPPH bleaching method and is based on the scavenging of DPPH radical in the presence of hydrogen donating antioxidants (eg, flavonoids, phenolic acids, tannins etc.) due to the formation of non-radical form DPPH-H. The radical DPPH• is used as a radical source in the evaluation of antioxidant activity, its monitoring being spectrophotometric. In the presence of antioxidants, DPPH• (purple color) is reduced to a pale yellow compound. In this assay, briefly, 30 μl of each 10% extract was mixed with 2 ml of methanolic DPPH solution (0.1 g/l). After 30 min. of incubation at 40°C in a thermostatic bath, the decrease in absorbance was measured at 517 nm. As control was used methanol. The percentage of inhibition is calculated using the relation: I(%) = [(A control -A extract)/(A control)] x 100 where: A control = Absorbance of DPPH radical and methanol (solution containing all reagents except sample to be analyzed); A extract = Absorbance of the mixture of DPPH radical and sample extract [14][15][16][17].

Thin-layer chromatography
Preliminary TLC separation and identification of polyphenolic compounds in the thymus ethanolic extracts were performed using the chromatographic system with silica gel as stationary phase and the corresponding mobile phase: ethyl acetate:acetic acid:  Table 2 and Figure 1.

Total polyphenolic content
The total polyphenolic content (TPC) was spectrophotometrically analyzed by a Folin-Ciocalteu colorimetric method, and the results are presented in Figure 2.

Total flavonoidic content
Total flavonoid content of the thymus ethanolic extracts was determined using the aluminum chloride colorimetric method, and the results were presented in Figure 3.
The highest amount of flavonoids was found in the T. comosus extract (1.65%). In the other samples, much smaller amounts of flavonoids were determined (0.13-0.76%), below 1%. T. vulgaris and T. serpyllum are medicinal plants with average amounts of flavonoids, T. serpyllum having a slightly larger amount than the garden thyme.

Total caffeic acid derivatives content
The contents of caffeic acid derivatives determined by spectrophotometry are shown in Figure 4.
The highest amount of phenylpropane compounds was found in T. serpyllum harvested from the spontaneous flora of Gura-Raului, Sibiu (Cb5 -4.48%). Also, higher amounts of caffeic acid derivatives were obtained in another T. serpyllum extract, namely Cb4 (3.86%). The lowest amount of phenylpropane compounds was determined in the extract of T. comosus (TC -0.05 %). Large amounts of caffeic acid derivatives have also been found in the T. vulgaris extract (2.23%).

Total tannins content
The tannins content in the thymus species extracts were determined by spectrophotometry, using the

Antioxidant activity
Evaluation of the antioxidant activity of thymus sp. extracts was performed in vitro by the DPPH method.
An I% greater than 50% shows an important antioxidant effect for which the extracts can be used in phytotherapy. The following extracts showed important antioxidant action: Cb5 (80.03% -T. serpyllum harvested from the spontaneous flora of Gura Riului), Cb4 (71.00% -T. serpyllum from a commercial tea). The other extracts showed a weaker antioxidant action. The T. comosus extract had lowest I% (2,81%), so without antioxidant action.

CONCLUSIONS
The genus Thymus, used since antiquity is very well represented in the spontaneous flora of Romania, such as: T. vulgaris, T. serpyllum, T. glabrescens, T. comosus etc.
In this paper we have comparatively studied the polyphenolic composition of four species of thymus from the spontaneous flora of our country (thymus species: T. vulgaris, T. serpyllum, T. glabrescens and T. comosus), as well as from thyme species from commercial teas.
We spectrophotometrically determined the contents of total polyphenols, flavonoids, tannins and caffeic acid derivatives, as well as the in vitro antioxidant activity. The results of the determinations showed that thymus species are medicinal plants rich in antioxidant polyphenolic compounds, especially those from spontaneous flora, and may be important ingredients for pharmaceutical preparations.