Region of study and collection of samples

The samples were collected randomly, in a wild environment during the Spring-Summer of 2014 and 2015, in three municipalities of the state of Zacatecas: Larrea tridentata and A. ludoviciana in Villa de Cos (23º 17’ 42’’ N, 102º 20’ 24” W), O. vulgare in Valparaiso (22º 46’ 12” N, 103º 34’ 14” W) and R. graveolens in Calera de Victor Rosales (23º 27’ 00” N, 102º 55’ 00” W). These locations have adequate climatic conditions for the development of the plants. After collecting the plants, they were maintained for two weeks at room temperature (approximately 20 ºC), then at 45 ºC for 24 h to dehydrate them, and stored in plastic bags until they were used.

Obtainment of the plant extracts

The extraction was made with 20 g of the triturated sample and 300 mL of ethanol at 70 % (J.T. Baker^®) as solvent, in amber 1 L jars (^{Pesewu et al., 2008}). The sample was mixed vigorously for 10 min, and left to set one month at room temperature (20 ºC). The supernatant was filtered with Whatman No. 2 paper and the solvent was evaporated in a Soxhlet type extractor at 85 ºC. Three aliquots were obtained and maintained at < 20 ºC for later analysis.

Qualitative tests of chemical profile

The chemical profile of the extracts was obtained according to what was described by Dominguez (1973), in 10 mL test tubes and includes the following procedures.

Chemical composition of the extract by gas chromatography

The chemical composition was determined in a gas chromatograph (GC: Agilent Technologies series 6890N, USA), with polar column DB_WAXetr, at 250 ºC and 12.13 psi, flow of 36.5 mL of He min^-1. Conditions for the column were: initial temperature 50 ºC, from 0 to 2 min, increase of 10 in 10 ºC up to 250 ºC, constant for 5 min, reduction to 50 ºC for 2 min with flow of 1.6 mL of He min^-1 at 12.13 psi and average velocity of 25 cm s^-1. The ionizing flame detector (IFD) was used at 210 ºC with flow of 40 mL of H₂ min^-1 and a flow of 450 mL of air min^-1. The standards (Sigma-Aldrich) were used in various concentrations (Table 1).

Chemical structure of the extract through Infrared Spectroscopy with Fourier Transformation (FTIR)

The spectroscopy analyses were made in a Thermo scientific (Nicolet^TM Is50^TM, USA), with dispersive cell; the graphs were made with OriginPro 8. The spectra were measured in the interval from 600 to 4000 nm, with 32 replications each one and were averaged. Each sample was concentrated in a rotoevaporator (Eppendorf Vacufuge^TM, USA) at 30 ºC for 12 h.

Qualitative tests of chemical profile

The tests of chemical profile of the extracts did not show differences (Table 3).

The diluted extract 3 of L. tridentata was positive because it was not subjected to temperature changes and conserved its chemical structure. The diluted O. vulgare extract was negative in the test of H₂SO₄ because its compounds were found in a lower quantity than in extracts four and five of O. vulgare that were concentrated.

The extract of L. tridentata presented unsaturations, esters, carbohydrates and flavonoids. The in vitro cultivation conditions can favor the synthesis of metabolytes which under natural conditions would not occur (^{Garza et al., 2010}). The extracts of O. vulgare were positive for unsaturations, esters, carbohydrates, flavonoids and saponins, and specifically for flavonoids. In extracts of O. vulgare flavonoids were identified, such as chrysoeriol, diosmetin, eriodyctiol, cosmocide and vicenine-2 (^{Koukoulitsa et al., 2006}), which were not identified in our study.

The extract of A. ludoviciana exhibited unsaturations, esters, coumarins and saponins, and that of R. graveolens showed unsaturations, esters, flavonoids and saponins. ^{Lin et al. (2007)} identified the following flavonoids in R. graveolens: luteolin, taxifolin, quercetin and naringenin in total concentration of 59.8 mg g^-1 dry. In our investigation variation was not detected between the years of harvest.

Chemical composition

The analyses in the gas chromatograph was 18 min per sample and the retention time of terpinene, limonene, linalol, thymol and carvacrol was 3.52, 3.62, 4.21, 8.87 and 9.45 min. In the extracts of L. tridentata, O. vulgare, A. ludoviciana and R. graveolens, carvacrol, thymol, terpinene, linalol and limonene were determined; and in the extracts of L. tridentate the compounds of thymol and carvacrol were found in variable concentrations (Table 4).

These results confirm that the medicinal plants studied are rich in terpenes (carvacrol, citral, linalol and geraniol) and phenolic compounds (^{Cai et al., 2004}). The phenolic compounds, such as flavonoids, quercetin, kaempferol and nordihydroguaiaretic acid, are bioactive compounds that can be found in L. tridentata (^{Nakamura et al., 2005}; ^{Tapas et al., 2008}; ^{Martins et al., 2012}) with a different concentration from those of our study.

Extracts four and six of O. vulgaris contain limonene, and extract five contains terpinene. These results coincide with those obtained in oregano oils which contain terpineols, phenols and compounds metabolically related with carvacrol (^{Albado et al., 2001}).

Extract seven of A. ludoviciana contains terpinene, linalol, thymol and carvacrol, extract eight contained only carvacrol, and in extract nine there was linalol, thymol and carvacrol. These results differ from those reported by ^{Kordali et al. (2005)}, who identified in A. ludoviciana anetol (81 %), beta-ocimene (6.5 %), limonene (3.0 %) and methyleugenol (1.8 %), and none of these compounds was found in our study. The contrasts may be due to genetic, agronomic and environmental factors (^{Sharapin et al., 2000}).

The compounds of limonene, thymol and carvacrol were identified in the extracts of R. graveolens. In essential oil of R. graveolens, ^{De Feo et al. (2002)} identified nonanone (18.8 %), undecane-2-one (46.8 %), nonan-2-one (18.8 %), decan-2-one (2.2 %) and tridecan-2-one (2.5 %). The terpenoids constituted 11.2 % of the oil with α-pinene (1.3 %), limonene (3 %) and 1,8-cineole (2.9 %) as the principal monoterpenes. The presence of limonene in our study coincided with those results.

The compounds identified in the extracts are important for their pharmacological activity. Limonene is antibacterial, antifungal, antiseptic and antiviral; thymol may be less caustic than other phenols and is antibacterial, antifungal, anti-inflammatory, antioxidant, antirheumatic and antiseptic; carvacrol is antibacterial, antifungal, anti-inflammatory, antiseptic, antispasmodic and expectorant (^{Sorentino and Landmesser, 2005}).

Chemical structure of the extract by FTIR

In the determination of the structure the standards for gas chromatography were used. The graphs of IR correlation express the frequencies of vibration in wave numbers and were obtained from the FTIR; the technique is considered of high resolution and provides a spectrum of reflection of the bands of the functional groups of inorganic and organic substances, which allows their identification (^{Skoog et al., 2001}). The frequencies of FTIR are attributed to the stretching and flexion of vibrations that characterize the functional groups (^{Baranska et al., 2005}).

The FTIR spectra showed eight regions, each one with three areas (Figure 1): 1 (1400-1500 cm^-1) corresponding to CO and CC, specific vibrations in phenyl groups; 2 (1500 to 1600 cm^-1) corresponding to the aromatic dominion and NH of flexion (^{Baranska et al., 2005}); 3 (1600-1760 cm^-1) corresponding to the NH flexion, stretching C=O (aldehydes, ketones, esters) and free fatty acids (1710 cm^-1) and glycerides (1740 cm^-1) (^{Socaciu et al., 2009 a}, ^b).

Figure 1 FTIR spectra of five standards used for the chemical determination of extracts of Larrea tridentata, Origanum vulgare, Artemisa ludoviciana and Ruta graveolens through infrared spectroscopy. 

The frequencies of 675 to 920 cm^-1 in the standards of carvacrol, thymol, linalol, limonene and terpinene are associated with aromatic groups C=C, w (C-H) of aromatic rings (^{Adinew, 2014}). The frequencies of 1050 cm^-1 in the five standards were assigned to vibrations of tension of C-O, whose values were reported by ^{Anicuta et al. (2010)}; the values of 1161 to 1233 cm^-1 corresponded to the ester of groups C-O, which coincided with what was pointed out by ^{Vlachos et al. (2006)}.

The frequencies of 1360 cm^-1 in the five standards pertained to the deformation of C-O (^{Martins et al., 2013}) and this group was identified in the five standards in frequency of 1440 (^{Anicuta et al., 2010}). The frequencies of 1580 cm^-1 corresponded to amino groups present in carvacrol, thymol and terpinene. The values 2830 and 2872 cm^-1 corresponded to symmetric and asymmetric vibrations of aliphatic CH₃ groups, present in carvacrol, thymol and terpinene. The frequencies of 3300 to 3400 cm^-1 corresponded to hydroxyl groups (O-H), strong bonds of hydrogen, or absorption and stretching in carvacrol, linalol and terpinene.

In thymol, the frequencies of 745 cm^-1 indicated superposition of oscillating CH₂ groups. The frequency of 809 cm^-1 belonged to w (C-H) of aromatic rings, and that of 860 cm^-1 to aromatic groups C-C. (^{Adinew, 2014}). The frequencies of 937, 995 and 1288 cm^-1 (flexion of bonds CH, CO, CN, CC) were in the five standards. The frequency of 1110 cm^-1 corresponded to the aromatic skeleton and stretching of C-O present in carvacrol, thymol, linalol and terpinene. The groups O-H absorbed and conjugated to C-O were associated to the frequency of 1620 cm^-1 in thymol.

The frequency of 2872 cm^-1 in thymol indicated symmetric and asymmetric vibrations of the group aliphatic CH₃. The frequencies of 2959 to 2962 cm^-1 pertain to groups of stretch CH₂ and to methyl groups in phenolic rings (^{Wu et al., 2012}) in the five standards. The stretch O-H was found in the frequency 3259 cm^-1 in the thymol.

The frequencies of 995 cm^-1 in linalol and limonene corresponded to the zone of the fingerprint of the flexion of bonds CH, CO, CN and CC. The frequency of 1650 cm^-1 in linalol, limonene and terpinene indicated the presence of C=O rings of aldehydes. Linalol, limonene and terpinene presented symmetric and asymmetric vibrations of the aliphatic group CH₂ with 2921 cm^-1. ^{López et al. (2014)} indicated that between the frequencies of 3000 and 3100 cm^-1, aromatic rings were identified, which were observed in linalol and limonene.

The outstanding frequencies of the extracts were 881 cm^-1, 1047 cm^-1, 1360 cm^-1, 1440 cm^-1, 1650 cm^-1, 2921 cm^-1, 2959 cm^-1 and 3367 cm^-1 pertaining to C=C of aromatic groups (^{Adinew, 2014}), vibration of tension of C-O (^{Anicuta et al., 2010}), deformation of C-H (^{Martins et al., 2013}), groups C-O (^{Anicuta et al., 2010}), ring C=O of aldehydes (Adinew, 2014), symmetric and asymmetric vibration of the group aliphatic CH₂ (^{Vlachos et al., 2006}) and hydroxyl groups (^{Martins et al., 2013}) (Figure 2).

Figure 2 FTIR spectra of extracts of Larrea tridentata (1, 2, 3), Origanum vulgare (4, 5, 6), Artemisa ludoviciana (7, 8, 9) and Ruta graveolens (10, 11).