Harvesting time influences the yield and oil composition of Origanum vulgare L. ssp. vulgare and ssp. hirtum

Industrial Crops and Products 49 (2013) 43–51

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop

Harvesting time influences the yield and oil composition of Origanum vulgare L. ssp. vulgare and ssp. hirtum Renata Baranauskiene˙ a , Petras Rimantas Venskutonis a,∗ , Edita Dambrauskiene˙ b , Pranas Viˇskelis b a b

Department of Food Technology, Kaunas University of Technology, Radvilen ˙ u˛ pl. 19, Kaunas LT-50254, Lithuania Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Babtai LT-54333, Lithuania

a r t i c l e

i n f o

Article history: Received 13 February 2013 Received in revised form 19 April 2013 Accepted 19 April 2013 Keywords: Origanum vulgare ssp. vulgare Origanum vulgare ssp. hirtum Seasonal variation Crop yield Essential oil composition

a b s t r a c t The influence of harvesting time on the herb crop yield, essential oil (EO) accumulation, its composition and some other characteristics of Origanum vulgare ssp. vulgare (OVV) and ssp. hirtum (OVH) were studied. The crop yield depending on the growing phase varied from 8.0 to 18.1 t ha−1 of fresh herb. The content of EO in OVV was remarkably lower (0.04–0.21 in fresh and 0.20–0.65 cm3 hg−1 in dried herb) than in OVH (0.56–1.86 in fresh and 2.29–5.75 cm3 hg−1 in dried herb). The highest yields were obtained at full flowering stage and slightly reduced after flowering, just before fruiting. The total productivity of OVH oil during different growing phases was remarkably higher (up to 335.9 dm3 ha−1 ) than that of OVV (up to 37.9 dm3 ha−1 ). OVV grown in Lithuania belongs to sabinene/␤-ocimene/␤-caryophyllene/germacrene D chemotype, while OVH belongs to carvacrol chemotype. The fluctuations in the percentage composition of the major compounds in EO throughout harvesting time were rather complex; more consistent changes were observed in the variation of individual constituents expressed in mg per 1 kg of fresh herb. The amounts of pigments, carotenoids and chlorophylls a and b, ascorbic acid, nitrates and soluble solids were measured at different growing phases. Selection of optimal harvesting time is the most important factor for obtaining the highest crop and EO yields from OVV and OVH; flowering phase was proved as the most productive period providing approximately 38 and 336 dm3 ha−1 of the EO, respectively. Determination of optimal harvesting period would increase the grower’s ability to control crop yield and EO quality, which is the most important factor in further commercialization of production and application of such products in the preparation of functional ingredients for various industrial uses. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Essential oils (EO) and their components are mainly used as food flavourings; however, they may be successfully applied for various non-food applications as the substances exhibiting fungicidal activity, antimicrobial, cytotoxic/anticancer, antigenotoxic, antimutagenic, antiviral, antiparasitic, disinfectant, and insecticidal properties (Bakkali et al., 2008; Burt, 2004; Lang and Buchbauer, 2012; Amiri et al., 2008; Christian and Goggi, 2008; Vale-Silva et al., 2012; Höferl et al., 2009; Berrington and Lall, 2012; Al-Kalaldeh et al., 2010; Ipek et al., 2005; Mezzoug et al., 2007; Pilau et al., 2011). In order to increase technological and economical effectiveness in the uses of EOs their production should be optimized by a proper

Abbreviations: OVV, Origanum vulgare spp. vulgare; OVH, Origanum vulgare spp. hirtum; EO, essential oil; KI, Kováts retention indices. ∗ Corresponding author. Tel.: +370 37 300188; fax: +370 37 456647. E-mail address: [email protected] (P.R. Venskutonis). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.04.024

selection of species/varieties/cultivars and agrotechnological practices, for instance by selecting the most beneficial harvesting time. Oregano (Origanum vulgare L., Labiatae) is irreplaceable condiment for pizza dishes, which is also used in flavouring other foods (Olivier, 1997). Origani herba, its extracts and EOs have been intensively used as folk remedies as antimicrobial, antimutagenic and antioxidative agents (Bozin et al., 2006; De Martino et al., 2009; Bakkali et al., 2008; Dapkevicius et al., 1998; Kulisic et al., 2004). O. vulgare is the most variable species of the genus (Ietswaart, 1980), however in most European countries it is commonly known as ‘oregano’ (D’Antuono et al., 2000). On the basis of discriminative morphologic characters, the oregano growing in Lithuania was identified as ssp. vulgare and assigned to sabinene/␤ocimene/␤-caryophyllene/germacrene D chemotype (Raduˇsiene˙ et al., 2005a,b; Mockute et al., 2001). Other chemotypes of OVV were reported in different countries (Bozin et al., 2006; Sezik et al., 1993; Fguérédo et al., 2006; Chalchat and Pasquier, 1998; Pande and Mathela, 2000). For instance, OVV from Turkey was composed mainly of terpinen-4-ol and germacrene D (Sezik et al., 1993); the germacrene D, ␤-caryophyllene and spathulenol were major

44

R. Baranauskiene˙ et al. / Industrial Crops and Products 49 (2013) 43–51

components in OVV grown in northern France (Fguérédo et al., 2006). OVH is the main commercial oregano subspecies containing phenolic compounds thymol or carvacrol as the main EO constituents (Kokkini and Vokou, 1989; Bozin et al., 2006; De Martino et al., 2009; Novák et al., 2003; Baser et al., 1994; Russo et al., 1998; Jerkovic´ et al., 2001; Johnson et al., 2004). However, intermediate chemotypes containing high concentrations of thymol and carvacrol, as well as their precursors, p-cymene and ␥-terpinene, were also reported (Kokkini, 1997; Novák et al., 2003; Russo et al., 1998; Jerkovic´ et al., 2001; Johnson et al., 2004). For instance, three OVH chemotypes were characterized in the natural habitats of Campania (Italy), namely carvacrol/thymol, thymol/␣-terpineol and linalyl acetate and linalool (De Martino et al., 2009). In Lithuania oregano is mainly collected from natural habitats and used in herbal teas. Morphological and chemical variation (Raduˇsiene˙ et al., 2005a; Mockute et al., 2001) as well as antimicrobial (Raduˇsiene˙ et al., 2005b) and antioxidant (Dapkevicius et al., 1998) properties of locally grown OVV were reported, however comprehensive studies of oregano cultivated in controlled environment have not been performed until now. Moreover, recently experimental cultivation of Greek oregano has been introduced in the Institute of Horticulture of Lithuanian Research Centre for Agriculture and Forestry. Therefore, comprehensive assessment and comparison of two oregano subspecies grown in controlled environment is of great interest, both from the scientific and practical points of views. Several measurements were selected for this purpose, however, EO was in the focus of the present study, because in terms of flavouring and antimicrobial properties of herbs the concentration of EO and its composition are the most important quality characteristics, which depends on a number of factors, such as plant chemo- and biotype, environmental growing conditions, time of harvesting, drying and storage conditions. Consequently, successful introduction and industrial cultivation of aromatic plants should be optimized taking into account all these variables. Considering the above mentioned aspects, this study was aimed at evaluating and comparing chemical composition of two O. vulgare subspecies, namely spp. vulgare and spp. hirtum at different plant vegetation phases. It should be emphasized that although oregano composition and properties were previously reported, systematic studies and comparison of commercially important O. vulgare subspecies were not performed until now. Therefore, the results presented in this study expand our scientific knowledge on oregano; also they may be practically applied in the developments of commercial cultivation of this valuable herb and its further valorization as a valuable industrial crop for the production of natural substances in various North European regions.

climatic characteristics of the cultivation area are the following: 55◦ 05 31 N of latitude, 23◦ 47 38 E of longitude; average temperature in July +18 ◦ C; average precipitation 660 mm per year; period of vegetation 169–202 days. The plants were harvested at various growth phases (Table 1) and dried at 40 ± 1 ◦ C in the dryer UDS-150/1 Vasara (Utenos krosnys, Utena, Lithuania) during 12 h.

2.2. Chemicals For Kováts retention indices determination, a mixture of C8 -C32 n-alkanes (Sigma Chemical Co., St. Louis, MO) was used and run at the experimental conditions reported below. The identification of oregano EO volatiles was performed using as reference compounds the following chemicals (95–99% purity), which were purchased from various companies (Fluka and Sigma–Aldrich): ␣-thujene, ␣-pinene, camphene, sabinene, myrcene, 1-octen-3-ol, 3-octanol, ␣-phellandrene, 3-carene, ␣-terpinene, p-cymene, d-limonene, 1,8-cineole, Z- and E-␤-ocimene, ␥-terpinene, terpinolene, linalool, camphor, borneol, terpinen-4-ol, l-(−)-menthol, ␣-terpineol, carvone, bornyl acetate, linalyl acetate, thymol, carvacrol, myrtenyl acetate, ␣-copaene, ␤-cubebene, ␤-caryophyllene, aromadendrene, ␣-humulene, ␦-cadinene, spathulenol, caryophyllene oxide. All other chemicals used were either of analytical grade or of the high purity commercially available.

2.3. Measurement of compositional characteristics The amounts of pigments, carotenoids and chlorophylls a and b, ascorbic acid, nitrates and soluble solids were measured at different growing phases. Soluble solids were determined by refractometer method using an Abbe refractometer model I (Carl Zeiss, Jena, Germany) (AOAC, 1990a). Ascorbic acid (vitamin C) was determined by titrimetric method using 2,6-dichloroindophenol (AOAC, 1990b). Sucrose was determined by reducing sugars before and after inversion; reducing sugars were determined by the inversion method (AOAC, 1990c). Nitrates were determined on a potentiometer pH-150 with an ion selective electrode EM-020604 (NPO Izmeritelnaja technika, Russia) (Lithuanian Ministry of Agriculture and Ministry of Health, 1990). The chlorophylls from one gram of green foliage mass were prepared according to Vernon method and determined in 80% acetone extract by measuring the extinction on Genesys spectrophotometer at 665 nm and 649 nm (Gavrilenko et al., 1975). Carotenes were determined by spectrophotometric method measuring the extinction at 450 nm in hexane (Scott, 2001).

2.4. Isolation of EO and gas chromatographic (GC) analysis 2. Materials and methods 2.1. Plant material Two different subspecies of O. vulgare differing by several morphological features were studied. Local cultivars of spp. vulgare have been cultivated in the experimental garden of Institute of Horticulture of Lithuanian Research Centre for Agriculture and Forestry since 1970, while Greek oregano (spp. hirtum) was introduced in 2004. Naturally grown or cultivated in Lithuania and in other North European regions OVV distinguishes itself by light or dark pink flowers and dark green leaves, and during flowering reach up to 60 cm height. OVH, which is widely spread in South Europe, in Lithuania grows up to 50 cm; the flowers are white, the leaves of characteristic light green colour. In this study oregano seedlings were planted in the fields at the end of May at the distance of 30 cm; the distance between rows was 70 cm. The main geographical and

The content of EO in fresh and dried oregano was determined by hydrodistillation of 100 g herb in a Clevenger-type apparatus during 3 h. It was diluted in pentane (1%, v/v) and analyzed on a Fisons 8000 series GC system (Fisons Instruments Inc., Rodano, MI, Italy) equipped with a flame ionization detector and a DB-5 (polydimethylsiloxane, 5% phenyl) fused silica capillary column, 50 m length, 0.32 mm i.d., 0.25 ␮m film thickness (J&W Scientific, Folsom, CA). The carrier gas was helium at a linear flow velocity of 32.7 cm s−1 at 50 ◦ C which was equivalent to a 2.35 mL min−1 volumetric flow; the detector temperature was 320 ◦ C, the oven temperature was programmed from 50 ◦ C (2 min) to 280 ◦ C (10 min) at the rate of 5 ◦ C min−1 . A split/splitless injector was used at 250 ◦ C in a split mode at a ratio of 1:10; the injection volume was 1 ␮L. The content of the eluted compounds was calculated on a DP800 integrator and expressed as a GC peak area percent and in mg kg−1 of fresh herb; in the latter case GC response

R. Baranauskiene˙ et al. / Industrial Crops and Products 49 (2013) 43–51

45

Table 1 Collection data of Origanum vulgare spp. vulgare (OVV) and Origanum vulgare spp. hirtum (OVH). Growing phases

Harvesting time

Vegetative stage

Part distilled

I II III

May 23 June 7 June 20

IV V

July 11 September 2

Regrowth Intensive vegetative growth Butonization (just before flowering, formation of inflorescences) Full flowering After flowering (in fruiting–seeds ripening)

Stems, leaves, calyx (fresh and dried) Stems, leaves, calyx (fresh and dried) Stems, leaves, calyx, buds (fresh and dried); only stems (fresh); leaves, calyx and buds (fresh) Leaves, calyx and flowers (fresh and dried) Leaves, seeds (fresh and dried)

factors for different compounds were taken being equal to 1. The mean values were calculated from quadruplicate injections.

3. Results and discussion 3.1. Oregano crop and EO content

2.5. Gas chromatography–mass spectrometry (GC–MS) The crop yield and the quality of plant material are the most important characteristics in commercial cultivation of spices and aromatic herbs. It is very important to select the most productive subspecies/cultivars and to determine the optimal harvesting period, which may differ depending on climatic conditions and plant properties. The yields of oregano crop and the content of EOs in freshly harvested and dried plants are listed in Table 2. In the period of May 23–September 2 the crop of fresh oregano herb varied from 8.0 to 18.1 t ha−1 ; it is interesting to note that the productivity of green mass was not statistically different (P < 0.05) for both oregano subspecies, vulgare and hirtum. Crop yield increased until its peak of more than 18 t ha−1 at full flowering stage in July and afterwards remarkably decreased after flowering during seed formation to 10 t ha−1 . It was also observed that OVH forms more stems, therefore their two-year-old and older shrubs were denser comparing to OVV. The effect of different growing times on the yield of fresh oregano herb can be described by the third-order polynomial regression equation y = −0.8231x3 + 5.6932x2 − 8.1304x + 11.447 with a determination coefficient R2 = 0.9643 (OVH), or y = −0.8297x3 + 5.7473x2 − 8.2497x + 11.496, R2 = 0.968 (OVV). Oregano herb may be used in its dried form or for further processing, e.g. isolation of EO, extraction. Therefore it is important to assess the yield of dried raw materials. After drying, the mass of oregano herb decreased 2–5 times and was 1.54–5.66 t ha−1 . The output of the dry oregano weight of the investigated subspecies may be described by the third-order polynomial regression equation: y = −0.1963x3 + 1.511x2 − 2.1305x + 2.4089, R2 = 0.9898 (OVH), or y = −0.1932x3 + 1.4876x2 − 2.0921x + 2.382, R2 = 0.9898 (OVV). Significant differences were obtained in the yields of EO between two investigated oregano subspecies. So far as the EO

GC–MS analyses were performed using a Perkin Elmer Clarus 500 GC coupled to a Perkin Elmer Clarus 500 series mass selective detector (Perkin Elmer Instruments, Shelton, USA) in the electron impact ionization mode at 70 eV, the mass range was m/z 29–550. Volatile compounds were separated using an Elite-5 MS capillary column (dimethylpolysiloxane, 5% diphenyl), 30 m length, 0.25 mm i.d., 0.25 ␮m film thickness (Perkin Elmer Instruments, Shelton, USA). The oven temperature was programmed as described above. Carrier gas helium was adjusted to a linear velocity of 36.2 cm s−1 at 50 ◦ C or 1.0 mL min−1 volumetric flow. Split mode was used at ratio of 1:20 and an injector temperature of 250 ◦ C. The identity of the components was assigned by comparing their Kováts retention indices (KI) relative to C8 –C32 n-alkanes (Sigma Chemical Co., St. Louis, MO), obtained on nonpolar DB-5 column with those provided in literature (Adams, 2009), and by comparing their mass spectra with the data provided by the NIST (vers.1.7), NBS 75K/WILEY 275 and EPA/NIH mass spectral libraries and mass spectra with corresponding data of components of reference oils. Additionally, the identity of some compounds was confirmed by co-injection of reference compounds. Positive identification was assumed when good match of mass spectrum and KI was achieved. 2.6. Statistical data analysis All analyses were replicated four times and all data were reported as means ± standard deviation. Data were statistically handled by one-way analysis of variance (ANOVA, vers. 2.2, 1999). Duncan’s multiple-range test was applied for the calculation of the significant differences among the crop and oil yields, biochemical characteristics, and the individual EO components during harvesting time treatment at probability level (P < 0.05). Table 2 Yield of crop and content of EO of two different O. vulgare subspecies. Growing phase

Yield of herb crop (t ha−1 )

Oil content (cm3 hg−1 )

Fresh herb

Fresh herb

Dried herb

Yield of oil (dm3 ha−1 ) Dried herb

OVH I II III IV V

8.01 12.09 14.99 18.06 10.06

± ± ± ± ±

0.17a 0.42c 0.13d 0.14f 0.15b

1.55 2.78 4.08 5.66 4.96

± ± ± ± ±

0.07a 0.10b 0.14c 0.13f 0.08d

0.56 0.76 1.50 1.86 1.78

± ± ± ± ±

0.04e 0.02f 0.02g 0.03h 0.02i

2.29 3.04 5.29 5.75 3.65

± ± ± ± ±

0.13d 0.14e 0.21g 0.12h 0.16f

OVV I II III IV V

7.99 12.03 15.05 18.03 10.04

± ± ± ± ±

0.05a 0.08c 0.13d 0.14f 0.09b

1.54 2.76 4.04 5.61 4.92

± ± ± ± ±

0.08a 0.11b 0.13c 0.16f 0.09d

0.04 0.09 0.14 0.21 0.16

± ± ± ± ±

0.01a 0.02c 0.02b 0.03d 0.01b

0.20 0.34 0.48 0.65 0.34

± ± ± ± ±

0.02a 0.04ab 0.03bc 0.05c 0.02ab

Fresh herb

Dried herb

44.86 91.88 224.85 335.92 179.07

± ± ± ± ±

3.53f/c 3.89g/e 2.09i/g 4.28j/i 3.23h/b

35.50 84.51 215.83 325.45 181.04

± ± ± ± ±

0.84c/a 1.60e/d 6.88g/f 2.98h/h 5.39f/b

3.20 10.82 21.07 37.86 16.07

± ± ± ± ±

0.66a/a 1.95b/b 1.21d/d 1.25e/f 0.76c/c

3.08 9.38 19.56 36.65 16.73

± ± ± ± ±

0.19a/a 0.65d/b 1.63b/d 1.88c/f 0.47b/c

Values within columns followed by the same letter (a–j) do not differ statistically at P < 0.05 (Duncan test); values after slash (the last two columns) within rows followed by the same letter (a–i) do not differ statistically at P < 0.05 (Duncan test).

46

R. Baranauskiene˙ et al. / Industrial Crops and Products 49 (2013) 43–51

may be isolated from freshly harvested plants and dried herb, while drying can result in the changes of EO yield, its content was measured in fresh plants and dried herb. EO content in OVH at different growing periods was in the range of 0.56–1.86 (fresh herb) and 2.29–5.75 cm3 hg−1 (dried herb). The yield of EO from fresh OVV was significantly lower compared to that of OVH and constituted 0.04–0.21 and 0.20–0.65 cm3 hg−1 in fresh and dried herbs, respectively. The highest EO yields were obtained at full flowering phase and slightly reduced after flowering, just before fruiting (Table 2). Previously published results showed a great variation in the EO content within OV subspecies, depending on chemotype, cultivation area and harvesting period. Kokkini et al. (1994) investigated OVH grown in Greece and found very high content of EO (up to 8.2 ml hg−1 dry weight), whereas in spp. vulgare and spp. viride it was only up to 0.8 ml hg−1 dry weight. D’Antuono et al. (2000) studied native populations of OV from the Liguria and Emilia regions of northern Italy and found that the EO content of inflorescences of OVV from Emilia was very low (∼0.2%); the EO content of inflorescences of the majority samples from Liguria ranged from 4.3 to 5.0%. Jerkovic´ et al. (2001) studied OVH collected from the same geographic area in the south of Croatia at different seasons; the yields of EO in fresh plant material fluctuated from 312.6 to 2331.0 mg hg−1 , depending on the collection month. De Martino et al. (2009) studied EO from inflorescences of three OVH samples growing wild in different locations in Campania (Southern Italy) and determined that dry material gave yellow-reddish oils in a yield between 2.35 and 3.15%. The total productivity of EO from fresh OVV varied from 3.2 to 37.9 dm3 ha−1 , the maximum yield being reached during full flowering phase. After drying EO content slightly reduced and was from 3.1 to 36.5 dm3 ha−1 indicating that the losses of volatiles during drying were not remarkable, e.g. were not statistically different at P < 0.05. The productivity of OVH oil during different growing phases was 9–14 times higher compared to that of OVV. The oil yield of OVH varied from 44.9 to 335.9 (fresh herb) and from 35.5 to 325.5 dm3 ha−1 (dried herb) (Table 2). After drying EO content from OVH reduced more significantly; it was not statistically different only after flowering stage (September 2). 3.2. Biochemical composition The amount of pigment substances, chlorophylls and carotenes, was also monitored in the investigated oregano subspecies. The total amount of chlorophylls varied from 0.69 to 1.30 mg g−1 in OVH and from 0.92 to 1.68 mg g−1 in OVV (fresh herb) (Table 3). The total amount of chlorophylls increased in the raw material until flowering phase, and then rapidly decreased because of the changes in the biochemical composition of the plants, more abundant carotene synthesis during flowering stage, and the changes of plant physiology during seed ripening. OVV preserves more stable ratio of chlorophylls a and b, from 1.45 up to 1.73, while the ratio of chlorophylls a and b in OVH varied from 1.15 up to 1.91. Bigger amounts of carotenes at the beginning of plants vegetation are characteristic to OVV, while the plants of OVH synthesize them more in the second half of vegetation (Table 3). In another study (Capecka et al., 2005) the amount of carotenes in fresh O. vulgare collected just before flowering (experimental station of the Agricultural University of Krakow, Poland) was higher more than six times (51.0 mg hg−1 ) compared to oregano subspecies just before flowering (7.7 mg hg−1 ) investigated in our study. Some other chemical characteristics of O. vulgare are provided in Table 3. The content of dry soluble substances during OVH plant vegetation varied between 4.9 and 17.8%, while the changes in the dry soluble solids content in OVV were from 12.7 to 19.7%. Harvesting time also had a clear effect on the ascorbic acid content. Low concentration of vitamin C at the beginning of plant

Fig. 1. Percentage concentration of major components in the EO from fresh OVV at different growth phases.

vegetation (3.8–4.6 mg hg−1 ) increased up to 11.4–14.0 mg hg−1 during intensive flowering stage. Also, it was observed that OVV accumulates bigger amounts of this substance (Table 3). Capecka et al. (2005) determined that ascorbic acid content in fresh OVV from Poland was more than 2 times higher compared to our investigated oregano subspecies and constituted 23.1 mg hg−1 just before flowering, however significantly reduced after drying to 4.2 mg hg−1 . It is worth mentioning that the biosynthetic pathway of vitamin C in plants was proposed (Conklin, 2001), however, investigations on the effect of various environmental factors on this pathway in aromatic plants have not been reported. It is possible that the processes of biosynthesis of vitamin C change with plant ageing. The amounts of nitrates were from 680 to 970 mg kg−1 ; it decreased in both O. vulgare subspecies during plant vegetation, however, OVH accumulated higher amount of nitrates than OVV (Table 3). 3.3. EO composition Seasonal variations in chemical composition of EO isolated from OVV and OVH were analyzed by GC and GC–MS. The content of EO was lowest in the end of May and highest EO yields were obtained at the full flowering stage (July) and slightly reduced after flowering just before fruiting (end of August begin of September). Generally, flowering of O. vulgare starts in the middle of July and for a period of almost 6 weeks the plants are in full bloom. Jerkovic´ et al. (2001) suggested that the decrease in oil content after flowering may be due to the ageing of plant tissue during August, resulting in decomposition of phenols, thus preventing stress damage by acting as natural antioxidants. It is known that OVH is rich in EO, while OVV is a poor source of volatiles; however it is resistant to the cold and could be used for breeding of winter hardy varieties of this species (Raduˇsiene˙ et al., 2005a). The percentage distribution of the major components in OVV EO obtained at different growth phases is presented in Fig. 1, while the changes in concentration of individual constituents expressed in mg kg−1 of herb are presented in Table 4. The oil of OVV is a complex mixture of 62 components representing 99.5–99.9% of the total EO compounds detected by GC and GC–MS. The major monoterpene hydrocarbons were sabinene (6.6–28.2%), Z-␤-ocimene (4.7–7.7%), E-␤-ocimene (4.4–15.1%) and allo-ocimene (7.7–12.1%), other compounds of this class, namely myrcene (2.0–3.6%), ␤-phellandrene (0.9–1.6%), and ␥-terpinene (0.3–2.4%) were in lower concentrations. Phenolic compounds carvacrol and thymol constituted 2.1–6.7% and 0.5–0.8%, respectively. The sesquiterpene fraction was represented mainly by ␤-caryophyllene (7.3–15.5%), germacrene

R. Baranauskiene˙ et al. / Industrial Crops and Products 49 (2013) 43–51

47

Table 3 Effect of different growing phases on some biochemical characteristics of fresh O. vulgare raw material. Growing phases

Dry soluble solids (%)

Chlorophylls total (mg g−1 )

Chlorophylls a/b (ratio)

Vitamin C (mg %)

Carotenes (mg %)

Nitrates (mg kg−1 )

Total sugars (%)

OVH I II III IV V

4.9 5.1 10.0 14.9 17.8

± ± ± ± ±

0.03a 0.05b 0.05c 0.07f 0.04i

0.69 1.31 1.30 0.64 0.67

± ± ± ± ±

0.01b 0.01c 0.01c 0.01a 0.01b

1.78 1.91 1.70 1.42 1.15

± ± ± ± ±

0.01h 0.01i 0.01g 0.01c 0.01a

3.8 5.6 10.0 11.4 6.8

± ± ± ± ±

0.04a 0.04c 0.12g 0.09i 0.04e

5.1 6.9 7.7 12.5 12.0

± ± ± ± ±

0.04a 0.11e 0.09c 0.09i 0.07h

960 970 850 810 790

± ± ± ± ±

2.0i 1.3j 2.2f 2.7d 2.1c

1.35 1.43 2.04 2.81 3.22

± ± ± ± ±

0.01a 0.01b 0.01d 0.01i 0.01j

OVV I II III IV V

12.7 14.2 16.5 17.7 19.7

± ± ± ± ±

0.02d 0.04e 0.04g 0.08h 0.09j

0.92 1.39 1.68 1.18 1.53

± ± ± ± ±

0.01d 0.01f 0.01h 0.01e 0.01g

1.72 1.73 1.61 1.45 1.64

± ± ± ± ±

0.01b 0.01b 0.01e 0.01d 0.02f

4.6 6.0 10.3 14.0 8.4

± ± ± ± ±

0.03b 0.04d 0.04h 0.11j 0.03f

10.7 8.3 7.7 7.6 5.2

± ± ± ± ±

0.09g 0.04f 0.04bc 0.04b 0.04d

940 870 820 730 680

± ± ± ± ±

2.5 h 1.8 g 2.5e 1.1b 1.1a

1.93 2.10 2.23 2.45 2.68

± ± ± ± ±

0.01c 0.01e 0.01f 0.01g 0.01h

Values within columns followed by the same letter (a–j) do not differ statistically at P < 0.05 (Duncan test).

D (5.2–12.0%) and bicyclogermacrene (3.5–7.1%). The main oxygenated monoterpenes were linalool (0.5–1.1%) and terpinen-4-ol (0.3–0.5%), while ␣-cadinol (1.1–4.9%), spathulenol (1.3–3.2%) and caryophyllene oxide (0.6–2.6%) were the major oxygenated sesquiterpenes. The fluctuations in the percentage composition of the major compounds during all vegetation period were rather complex, however, it is evident that OVV grown in Lithuania depends to sabinene/␤-ocimene/␤-caryophyllene/germacrene D chemotype, and this is in agreement with previously published reports on OVV (Raduˇsiene˙ et al., 2005a,b; Mockute et al., 2001). More significant changes were observed in the variation of individual constituents expressed in mg kg−1 (Table 4). It is evident, that the EO concentration and the amounts of individual EO chemical compounds varied greatly during the period examined, and it was established that the Origanum oils obtained during the full flowering period were the most potent flavourings. EOs produced from herbs harvested during or immediately after flowering possessed the strongest antimicrobial activity (Burt, 2004). For instance, sabinene was intensively biosynthesized from May (26.2 mg kg−1 ) until full flowering stage in July (592.8 mg kg−1 ); while after flowering in September its content decreased almost twice, to 267.4 mg kg−1 . The concentration of ␤-ocimenes constantly increased from 36.4 mg kg−1 (end of May) to 365.9 mg kg−1 (fruiting period) (Table 4), although its percentage in the period of May 23–July 11 varied from 9.1 to 11.2%, and increased twice (22.9%) in fruiting (September 2). The concentration of alloocimene varied from 32.1 to 192.9 mg kg−1 during all vegetative period. The highest percentage of sesquiterpene ␤-caryophyllene was at the beginning of vegetation in May 23 (15.5%), while during full flowering decreased to 12.5% and at the end of vegetation (September 2) constituted 7.3%; the highest amount of ␤-caryophyllene expressed in mg per 1 kg of plant material was during intensive flowering stage (261.9 mg kg−1 ) and decreased more than twice after flowering (fruiting phase). The amounts of another important sesquiterpenes, germacrene D and bicyclogermacrene were determined in the range of 48.1–149.8 and 28.4–105.5 mg kg−1 , respectively (Table 4). The absolute amounts of individual components in OVH EO from fresh herbs harvested at different growing phases are summarized in Table 5. Totally, 56 compounds were identified in OVH which accounted 99.8% of total EO volatiles. The percentage distribution of the major components in OVH EO obtained in different growth phases is presented in Fig. 2. The fluctuations in the percentage composition of the major compounds from May 23 till September 2 were rather complex. It is evident that this oregano subspecies belongs to carvacrol chemotype, which accounted 72.4–88.2% of total EO volatiles; its isomer thymol was in trace concentrations (0.1–0.2%). The percentage content of the second major component,

Fig. 2. Percentage concentration of major components in the EO from fresh OVH at different growth phases.

␥-terpinene decreased from 8.7 to 4.1%. On the contrary, the percentage of p-cymene increased during vegetation, and varied from 2.0% at the end of May to 3.2% during intensive flowering phase in July. Another quantified monoterpene hydrocarbons were myrcene (1.4–1.8%), ␣-thujene (0.5–1.0%), ␣-pinene (0.4–0.8%) and ␣-terpinene (0.3–0.8%). The changes in the content of the major sesquiterpene ␤-caryophyllene were rather complex; the highest percentage was determined in the end of May during regrowing (3.0%), then it decreased to 0.9% in the mid of June just before flowering and then again increased to 1.5% on September 2 (fruiting). Similar tendency was observed for the percentage content of ␤-bisabolene (1.7–0.3%). In general, the absolute amounts of major components were increasing with the increase of the total EO content. The amounts of carvacrol varied from 4054.2 (May 23) to 15692.7 mg kg−1 (September 2); it was not statistically different (P < 0.05) in the EO obtained on June 20 from the whole plant, from stems and whole plant without stems (Table 5). The amounts of p-cymene increased from 113.7 (May 23) to 597.4 mg kg−1 (July 11); it was different (P < 0.05) from EO obtained from the whole herb compared to that from the separated stems. The lowest amount of myrcene was obtained on May 23 (83.1 mg kg−1 ) and the highest (333.8 mg kg−1 ) at full flowering on July 11. The meaningful decrease in the amounts of sabinene was determined during plant vegetation, from 95.7 mg kg−1 (May 23) to 11.1 mg kg−1 (September 2). The amount of ␥-terpinene at the beginning of plant vegetation was 487.3 mg kg−1 , the maximum content (949.9 mg kg−1 ) was reached at full flowering stage, which decreased to 729.6 mg kg−1 in fruiting after flowering. The amounts of ␤-caryophyllene and ␤-bisabolene

48

R. Baranauskiene˙ et al. / Industrial Crops and Products 49 (2013) 43–51

Table 4 Chemical composition of EO of fresh OVV (mg kg−1 ) at different growth phases. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

Compound

KI on DB-5a

KI on DB-5b

I

III

IV

V

␣-Thujene ␣-Pinenec , d Sabinenec , d Myrcenec , d ␣-Terpinenec , d p-Cymenec , d Limonenee , c , d Z-␤-Ocimenec , d E-␤-Ocimenec , d  8+9 ␥-Terpinenec , d Z-Sabinene hydratec Linalool oxide Bc Terpinolenec , d E-Sabinene hydratec Linaloolc , d Z-p-Menth-2-en-1-olc allo-Ocimenec E-p-Menth-2-en-1-olc Camphorc , d Borneolc , d Terpinen-4-olc , d Mentholc , d ␣-Terpineolc , d E-Piperitolc Thymol methyl etherc Carvonec , d Carvacrol methyl etherc Bornyl acetatec , d Thymolc , d Carvacrolc , d Myrtenyl acetatec , d ␦-Elemenec ␣-Terpinyl acetatec , d ␣-Copaenec , d ␤-Bourbonenec ␤-Elemenec ␤-Cubebenec , d ␤-Caryophyllenec , d ␤-Gurjunenec Aromadendrenec , d ␣-Humulenec , d allo-Aromadendrenec ␥-Muurolenec Germacrene Dc Bicyclogermacrenec ␣-Muurolenec (E,E)-␣-Farnesenec ␦-Cadinenec , d ␣-Cadinenec Germacrene D 4-olc Spathulenolc , d Caryophyllene oxidec , d Globulolc Viridiflorolc Humulene epoxide IIc 1,10-di-epi-Cubenolc T-Cadinolc T-Muurololc ␣-Cadinolc Z-Calamenen-10-olc E-Calamenen-10-olc Phytolc

932 936 976 989 1015 1024 1029 1038 1049

930 939 975 990 1017 1024 1029 1037 1050

1060 1070 1079 1084 1085 1096 1125 1130 1143 1149 1173 1178 1188 1190 1205 1240 1248 1253 1275 1290 1301 1330 1341 1344 1380 1391 1398 1406 1430 1444 1452 1462 1476 1478 1491 1501 1520 1529 1543 1558 1571 1585 1589 1602 1615 1628 1638 1641 1651 1665 1685 1695 1952

1059 1070 1072 1088 1098 1096 1121 1132 1140 1146 1169 1177 1171 1188 1208 1235 1243 1244 1285 1290 1299 1326 1338 1349 1376 1388 1390 1388 1419 1433 1441 1454 1460 1479 1481 1500 1500 1505 1523 1538 1575 1578 1583 1590 1592 1608 1619 1640 1642 1654 1661 1669 1943

0.34 ± 0.07a – 26.22 ± 5.35a 8.46 ± 1.73a 0.81 ± 0.17a 0.79 ± 0.16a 3.78 ± 0.77a 18.69 ± 3.82a 17.72 ± 3.62a 36.41 3.04 ± 0.62a – – 0.51 ± 0.10a 0.36 ± 0.07a 1.95 ± 0.40a – 32.08 ± 6.55a – 0.19 ± 0.04a 0.23 ± 0.05a 1.12 ± 0.23a – 0.46 ± 0.09a 0.22 ± 0.04a 0.48 ± 0.09a – 0.17 ± 0.03a – 3.34 ± 0.68a 8.30 ± 1.69a 1.32 ± 0.27b 12.58 ± 2.57c 0.17 ± 0.03a 0.76 ± 0.16a 5.89 ± 1.20a – 0.20 ± 0.04a 62.13 ± 12.68a 11.23 ± 2.29a 5.70 ± 1.16a 14.39 ± 2.94a 4.64 ± 0.95a – 48.14 ± 9.83a 28.35 ± 5.79a 2.51 ± 0.51a 12.83 ± 2.62a 2.52 ± 0.51b 0.69 ± 0.14a 0.49 ± 0.09a 12.79 ± 2.61a 2.55 ± 0.52a 1.80 ± 0.37a 1.00 ± 0.18b 0.69 ± 0.14a 1.11 ± 0.23a – 11.80 ± 2.41a 19.69 ± 4.02a 0.53 ± 0.11a 2.11 ± 0.43a 0.29 ± 0.06a

1.18 ± 0.04b – 153.65 ± 5.39b 51.55 ± 1.81d 1.13 ± 0.04a 8.03 ± 0.28c 17.65 ± 0.62b 84.18 ± 2.95b 75.57 ± 2.65b 159.75 3.92 ± 0.14a – – 2.13 ± 0.07b – 9.73 ± 0.34b – 129.19 ± 4.53b 0.81 ± 0.03a 1.25 ± 0.04b 6.85 ± 0.24d 7.71 ± 0.27b 0.37 ± 0.01a 3.28 ± 0.12b 9.41 ± 0.33c 1.60 ± 0.06d – 1.16 ± 0.04d – 6.43 ± 0.23b 38.39 ± 1.35b 0.71 ± 0.02a 5.02 ± 0.18a – 1.88 ± 0.07c 10.68 ± 0.37b 9.78 ± 0.34a 1.02 ± 0.04c 195.25 ± 6.85c 35.55 ± 1.25c 15.68 ± 0.55c 33.08 ± 1.16c 15.58 ± 0.55b 1.18 ± 0.41b 146.24 ± 5.13c 49.12 ± 1.72b 6.36 ± 0.22c 29.41 ± 1.03c 1.74 ± 0.06a 3.83 ± 0.13b 2.48 ± 0.09c 36.14 ± 1.27d 32.25 ± 1.13b 5.42 ± 0.19c 1.22 ± 0.04b 7.06 ± 0.25c 5.25 ± 0.18b 0.96 ± 0.03b 44.98 ± 1.58c 50.43 ± 1.77c 2.04 ± 0.07c 12.62 ± 0.44d 1.31 ± 0.05b

3.17 ± 0.12d 9.23 ± 0.36b 592.75 ± 23.04d 42.41 ± 1.65c 11.46 ± 0.45c 3.27 ± 0.13b 31.17 ± 1.21d 100.56 ± 3.91c 119.29 ± 4.64c 219.85 50.04 ± 1.94c 1.80 ± 0.07a – 2.36 ± 0.09c 2.37 ± 0.09c 11.70 ± 0.45c 0.64 ± 0.02a 161.31 ± 6.27c 3.15 ± 0.12b – 0.96 ± 0.04b 9.97 ± 0.39c – 3.76 ± 0.15c – 0.67 ± 0.026b 0.62 ± 0.02b 0.46 ± 0.02b – 13.04 ± 0.51d 141.24 ± 5.49c 0.64 ± 0.02a 6.83 ± 0.26a – 1.54 ± 0.06b 6.70 ± 0.26a 9.46 ± 0.37a 0.34 ± 0.01b 261.85 ± 10.18d 41.07 ± 1.60d 20.73 ± 0.81d 46.76 ± 1.82d 16.12 ± 0.63b 0.77 ± 0.03a 149.76 ± 5.83c 105.54 ± 4.10d 5.73 ± 0.22c 19.79 ± 0.77b 2.51 ± 0.09b – – 26.92 ± 1.05c 3.72 ± 0.14a 1.99 ± 0.08a 0.66 ± 0.02a 3.42 ± 0.13b – 0.66 ± 0.03a 17.12 ± 0.66b 26.97 ± 1.05b 0.97 ± 0.04d 3.76 ± 0.15b –

2.86 ± 0.15c 6.16 ± 0.31a 267.36 ± 13.64c 37.15 ± 1.90b 7.39 ± 0.38b 24.00 ± 1.22d 25.49 ± 1.30c 123.65 ± 6.31d 242.24 ± 12.36d 365.89 30.58 ± 1.56b 2.04 ± 0.10b 1.18 ± 0.06a 3.07 ± 0.16d 1.73 ± 0.09b 17.88 ± 0.91d 0.68 ± 0.03a 192.94 ± 9.85d 4.58 ± 0.23c 1.76 ± 0.09c 1.38 ± 0.07c 8.20 ± 0.42b 0.75 ± 0.04b 3.15 ± 0.16b 1.44 ± 0.07b 0.95 ± 0.05c 0.52 ± 0.03a 0.57 ± 0.03c 0.79 ± 0.04a 10.58 ± 0.54c 34.80 ± 1.78b 1.45 ± 0.07b 9.18 ± 0.47b – 1.36 ± 0.07b 19.28 ± 0.98c – 1.38 ± 0.07d 116.16 ± 5.93b 23.13 ± 1.18b 10.11 ± 0.52b 24.10 ± 1.23b 8.63 ± 0.44c – 82.44 ± 4.21b 96.17 ± 4.91c 3.36 ± 0.17b 11.53 ± 0.59a 3.59 ± 0.18c 4.18 ± 0.21b 1.29 ± 0.07b 22.87 ± 1.17b 41.27 ± 2.11c 3.04 ± 0.16b

c,d

11.28 ± 0.58d – 1.68 ± 0.09c 12.85 ± 0.66a 17.40 ± 0.89a 2.16 ± 0.11c 9.16 ± 0.47c –

Values within rows followed by the same letter (a–d) do not differ statistically at P < 0.05 (Duncan test). a Kováts retention indices calculated against C8 –C32 n-alkanes on nonpolar DB-5 column. b Kováts retention indices on nonpolar DB-5 column reported in literature (Adams, 2009). c Identified on the basis of GC–MS spectra and calculated Kováts retention index of GC–FID response. d Identification confirmed by co-injection of the reference compound. e Limonene + ␤-phellandrene (∼1.1:1).

was 76.9–272.5 and 46.3–112.8 mg kg−1 , respectively, however no consistent dependencies were observed in their changes. The changes of individual EO volatiles were also analyzed after drying 100 g of fresh OVH herb (Fig. 3). The changes in the

percentage composition of OVH volatile compounds during drying were negligible, however some variations in the amounts of each individual component in dried herbs expressed in absolute amounts were observed. These variations did not reveal consistent

Table 5 Chemical composition of EOs of fresh OVH (mg kg−1 ) at different growth stages. Compound

KI on DB-5a

KI on DB-5b

I

II

III

III (stems)

III (no stems)

IV (no stems)

V (leaves)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

␣-Thujenec , d ␣-Pinenec , d Camphenec , d Sabinenec , d 1-Octen-3-olc , d 3-Octanolc , d Myrcenec , d ␣-Phellandrenec , d ␦-3-Carenec , d ␣-Terpinenec , d p-Cymenec , d 1,8-Cineolec , d Z-␤-Ocimenec , d E-␤-Ocimenec , d ␥-Terpinenec , d Z-Sabinene hydratec Terpinolenec , d Linaloolc , d Borneolc , d Terpinen-4-olc , d ␣-Terpineolc , d Z-Dihydro carvonec E-Piperitolc Thymol methyl etherc Carvonec , d Carvacrol methyl etherc Linalyl acetatec , d Thymolc , d Carvacrolc , d Cuminolc ␦-Elemenec Carvacryl acetatec ␣-Copaenec , d ␤-Bourbonenec ␤-Elemenec ␤-Caryophyllenec , d ␤-Gurjunenec Aromadendrenec , d Z-␤-Farnesenec ␣-Humulenec , d allo-Aromadendrenec ␥-Muurolenec Germacrene Dc Bicyclogermacrenec ␣-Muurolenec ␤-Bisabolenec ␦-Cadinenec , d ␣-Cadinenec Germacrene Bc Spathulenolc , d Caryophyllene oxidec , d ␥-Eudesmolc ␶-Cadinolc ␣-Cadinolc ␤-Bisabololc ␣-Bisabololc

932 936 953 976 980 989 989 1003 1010 1015 1024 1031 1038 1049 1060 1070 1084 1096 1173 1178 1190 1195 1205 1240 1248 1253 1259 1290 1301 1318 1341 1376 1380 1391 1398 1430 1444 1452 1453 1462 1476 1478 1491 1501 1520 1525 1543 1558 1581 1585 1589 1637 1641 1665 1685 1693

930 939 954 975 979 991 990 1002 1011 1017 1024 1031 1037 1050 1059 1070 1088 1096 1169 1177 1188 1192 1208 1235 1243 1244 1257 1290 1299 1290 1338 1372 1376 1388 1390 1419 1433 1441 1442 1454 1460 1479 1481 1500 1500 1505 1523 1538 1561 1578 1583 1632 1640 1664 1675 1685

24.98 ± 1.63a 23.84 ± 1.55a 3.12 ± 0.20a 95.68 ± 6.24d – – 83.06 ± 5.42a 6.65 ± 0.43a – 45.68 ± 2.98a 113.69 ± 7.41a 30.49 ± 1.99a 22.26 ± 1.26c 24.44 ± 1.59d 487.30 ± 31.77b 0.78 ± 0.05a 1.71 ± 0.11a 16.63 ± 0.94b 17.92 ± 1.01b 7.92 ± 0.52c 15.39 ± 1.00a 1.08 ± 0.07a 4.83 ± 0.31d 1.68 ± 0.11a – 33.25 ± 2.17d 2.56 ± 0.17a 10.71 ± 0.70a 4054.2 ± 264.4a 2.02 ± 0.13a 8.41 ± 0.48c 16.42 ± b 0.56 ± 0.04a 4.07 ± 0.27e 3.90 ± 0.03a 166.85 ± 10.88e 17.71 ± 1.15e 0.35 ± 0.02a 6.78 ± 0.44f 30.74 ± 2.00c 2.53 ± 0.17d 1.06 ± 0.07b 62.41 ± 4.07d 5.87 ± 0.38c 10.57 ± 0.69a 97.41 ± 6.35e 5.45 ± 0.36f 1.32 ± 0.09d 0.95 ± 0.06a 1.11 ± 0.07a 3.70 ± 0.24b 0.50 ± 0.03a 0.99 ± 0.06a 1.82 ± 0.10a 3.50 ± 0.23a 1.32 ± 0.09b

58.39 ± 1.25b 50.94 ± 1.09c 9.92 ± 0.21b 6.44 ± 0.14a 8.17 ± 0.18a 7.60 ± 0.14a 114.86 ± 2.47b 11.19 ± 0.24b 4.60 ± 0.09a 57.67 ± 1.24c 197.68 ± 4.25c – 0.29 ± 0.005a 4.81 ± 0.10a 457.14 ± 9.82a 8.99 ± 0.19e 2.30 ± 0.05b – – 17.71 ± 0.38d 20.79 ± 0.45b 1.35 ± 0.03b 1.67 ± 0.04a 1.73 ± 0.04a 1.63 ± 0.03a 9.52 ± 0.20a 4.18 ± 0.09b 12.92 ± 0.28c 6347.2 ± 136.4e 5.59 ± 0.12b 1.67 ± 0.03a 16.80 ± b – 0.55 ± 0.01a 0.53 ± 0.01b 76.87 ± 1.65a 2.05 ± 0.04a 0.53 ± 0.01a 0.68 ± 0.01a 12.08 ± 0.26a 0.38 ± 0.01a – 5.97 ± 0.13a 1.08 ± 0.02a – 51.68 ± 1.11b 1.84 ± 0.04a 0.76 ± 0.02a – – 1.06 ± 0.02a – – – – –

116.44 ± 1.27c 101.18 ± 1.10d 16.05 ± 0.17 g 11.25 ± 0.12b 11.18 ± 0.12b 23.06 ± 0.22b 219.79 ± 2.39c 18.15 ± 0.20c 7.95 ± 0.09c 92.14 ± 1.00d 395.44 ± 4.30b – 3.00 ± 0.03b 9.00 ± 0.10b 694.09 ± 7.56c 4.84 ± 0.05b 4.13 ± 0.05c 15.38 ± 0.14a 3.38 ± 0.03a 22.35 ± 0.24f 30.19 ± 0.33d 1.99 ± 0.02c 3.08 ± 0.03c 3.56 ± 0.04c 4.01 ± 0.04b 38.78 ± 0.42e 9.71 ± 0.11e 14.81 ± 0.16b 12,668.9 ± 137.9b 7.69 ± 0.08c 8.66 ± 0.08c 24.71 ± e – 1.95 ± 0.02c – 132.83 ± 1.45d 4.50 ± 0.05 cd 3.98 ± 0.04b 21.86 ± 0.24 g 21.49 ± 0.23b 8.40 ± 0.09 g – 10.54 ± 0.11bc – 35.85 ± 0.39b 70.54 ± 0.77d 2.90 ± 0.03b – – 1.80 ± 0.02c 4.13 ± 0.04c – – – – –

145.50 ± 1.58e 121.31 ± 1.32b 11.51 ± 0.13c 12.60 ± 0.14bc 21.19 ± 0.23e 27.90 ± 0.26d 267.38 ± 2.91f 27.90 ± 0.30f 11.36 ± 0.12b 126.08 ± 1.37f 450.11 ± 4.90d – – 9.68 ± 0.11b 771.86 ± 8.40e 8.25 ± 0.09d 5.14 ± 0.06f 33.11 ± 0.31e – 7.16 ± 0.08a 23.14 ± 0.25c 4.65 ± 0.05f 5.25 ± 0.06e 5.14 ± 0.06d 3.70 ± 0.04c 10.84 ± 0.12a 4.76 ± 0.05c 18.30 ± 0.20d 12,453.5 ± 135.6b 17.18 ± 0.19f 1.80 ± 0.02a 4.88 ± a – 1.01 ± 0.01b – 263.93 ± 2.88bc 2.93 ± 0.03ab – 3.79 ± 0.04d 38.81 ± 0.42e 3.90 ± 0.04e – 7.88 ± 0.09ab 1.09 ± 0.01a – 46.28 ± 0.50a 4.16 ± 0.04d 1.20 ± 0.01c – 1.28 ± 0.01b 6.30 ± 0.07e 2.89 ± 0.03d – 2.25 ± 0.02b – –

128.66 ± 1.40d 111.53 ± 1.21e 14.63 ± 0.16e 11.44 ± 0.12b 12.26 ± 0.13c 24.60 ± 0.23c 233.85 ± 2.55d 20.81 ± 0.23d 8.74 ± 1.0d 115.88 ± 1.26e 388.43 ± 4.23b – 1.13 ± 0.01a 9.41 ± 0.10b 857.21 ± 9.33f 3.00 ± 0.03c 4.50 ± 0.05d 19.20 ± 0.18c 3.50 ± 0.03a 21.15 ± 0.23e 39.83 ± 0.43f 2.25 ± 0.02d 3.64 ± 0.04b 3.26 ± 0.03b 4.05 ± 0.04b 30.41 ± 0.33c 7.35 ± 0.08d 20.81 ± 0.23e 12,505.5 ± 136.1b 13.16 ± 0.14e 4.20 ± 0.04e 43.91 ± f – 1.88 ± 0.02c – 210.64 ± 2.29f 4.91 ± 0.05d – 2.40 ± 0.03c 32.59 ± 0.35d 1.73 ± 0.02b – 13.91 ± 0.15c 2.21 ± 0.02b – 61.95 ± 0.67c 3.68 ± 0.04c 1.05 ± 0.01b – 1.73 ± 0.02c 4.61 ± 0.05d 1.39 ± 0.02b – – – –

181.16 ± 1.59 g 155.68 ± 1.37f 15.02 ± 0.13f 15.53 ± 0.14c 18.23 ± 0.16d 39.11 ± 0.29e 333.78 ± 2.93 g 34.13 ± 0.30 g 14.42 ± 0.13e 154.43 ± 1.36 g 597.43 ± 5.24f – – 11.25 ± 0.09c 949.86 ± 8.34 g 5.02 ± 0.04b 6.79 ± 0.06 g 38.74 ± 0.29f – 8.79 ± 0.08b 33.90 ± 0.29e 4.56 ± 0.04e 6.51 ± 0.06f 6.60 ± 0.06e 4.04 ± 0.03b 16.69 ± 0.15b 4.93 ± 0.04c 14.88 ± 0.13b 15,496.8 ± 136.0d 18.00 ± 0.16 g 2.33 ± 0.02d 6.37 ± c – 1.49 ± 0.01d – 270.49 ± 2.37bc 3.49 ± 0.03b – 4.46 ± 0.04e 40.60 ± 0.36e 4.46 ± 0.04f – 9.02 ± 0.08ab 1.26 ± 0.01a – 49.15 ± 0.43ab 4.88 ± 0.04e 1.40 ± 0.01d – 2.23 ± 0.02d 8.56 ± 0.07f 3.95 ± 0.03e – 3.02 ± 0.02c – –

149.39 ± 1.37f 119.79 ± 1.10b 12.64 ± 0.12d 11.13 ± 0.10b 53.36 ± 0.49f – 243.46 ± 2.23e 24.34 ± 0.22e 11.35 ± 0.10b 50.69 ± 0.47b 464.09 ± 4.26e – – 5.25 ± 0.05a 729.55 ± 1.93d 30.79 ± 0.28f 4.72 ± 0.04e 23.45 ± 0.19d – 9.03 ± 0.08b 20.29 ± 0.19b 5.96 ± 0.05 g 3.87 ± 0.04b 6.94 ± 0.06f 5.87 ± 0.05d 76.01 ± 0.70f – 23.14 ± 0.21f 15,692.7 ± 143.9d 11.62 ± 0.11d – 10.28 ± d – 1.11 ± 0.01b – 272.52 ± 2.50c 3.74 ± 0.03bc 31.15 ± 0.29c 1.51 ± 0.01b 45.03 ± 0.41f 2.00 ± 0.02c 0.98 ± 0.01a 6.76 ± 0.06a – – 112.81 ± 1.03f 6.45 ± 0.06 g 1.60 ± 0.01e – – 11.30 ± 0.10 g 1.78 ± 0.02c – – – 1.11 ± 0.01a

R. Baranauskiene˙ et al. / Industrial Crops and Products 49 (2013) 43–51

No

Values within rows followed by the same letter (a–g) do not differ statistically at P < 0.05 (Duncan test). a Kováts retention indices calculated against C8 -C32 n-alkanes on nonpolar DB-5 column. b Kováts retention indices on nonpolar DB-5 column reported in literature (Adams, 2009). c Identified on the basis of GC–MS spectra and calculated Kováts retention index of GC–FID response. d Identification confirmed by co-injection of the reference compound. 49

50

R. Baranauskiene˙ et al. / Industrial Crops and Products 49 (2013) 43–51

p -Cymene

Myrcene 400

fresh

700

dried

fresh

600

Amount, mg kg-1

Amount, mg kg-1

350 300 250 200 150 100

dried

500 400 300 200 100

50

0

0 I

II

III

IV

II

I

V

IV

V

III IV Growth phases

V

Growth phases

Growth phases

Carvacrol

γ-Terpinene 1200

18000 fresh

dried

fresh

15000

Amount, mg kg-1

1000

Amount, mg kg-1

III

800 600 400 200

dried

12000 9000 6000 3000

0

0 I

II

III

IV

V

I

Growth phases

II

Fig. 3. The changes in absolute amount (mg kg−1 ) of the major EO volatiles after drying 100 g of fresh OVH herb at different growth phases [mg kg−1 = (essential oil yield (g hg−1 ) × GC area percentage of individual component/100 × 1000/100) × 1000].

dependencies between harvesting time and the amount of individual constituents. The amount of monoterpene hydrocarbon ␥-terpinene decreased from 1.1 to 1.8 times after drying at different growing phases during all vegetation compared to that in fresh OVH. The amount of myrcene was almost the same and not statistically different (P < 0.05) in the fresh and dried OVH during plant vegetation. The content of the major compound carvacrol only slightly increased (∼1.2 times) after drying (regrowth phase) and further it was almost the same in fresh and dried OVH (Fig. 3).

4. Conclusion O. vulgare ssp. vulgare and ssp. hirtum cultivated in Lithuania belong to completely different chemotypes and industrial applications of their essential oils should be selected taking into account the bioactivities of the main essential oil components. Flowering phase was proved as the most productive period for both O. vulgare ssp. vulgare and ssp. hirtum providing approximately 38 and 336 dm3 ha−1 of the essential oil, respectively. O. vulgare ssp. hirtum producing high amounts of essential oil containing strong antimicrobial agent carvacrol as the most abundant constituent may be considered as a highly promising industrial crop for the production of valuable natural substances.

Acknowledgement Financial support from the Research Council of Lithuania (Grant No.: MT-1131) is gratefully acknowledged.

References Adams, R.P., 2009. Identification of Essential Oils Components by Gas Chromatography/Mass Spectrometry, 4th ed. Allured Business Media, Carol Stream, IL. Al-Kalaldeh, J.Z., Abu-Dahab, R., Afifi, F.U., 2010. Volatile oil composition and antiproliferative activity of Laurus nobilis. Origanum syriacum, Origanum vulgare, and Salvia triloba against human breast adenocarcinoma cells. Nutr. Res. 30, 271–278. Amiri, A., Dugas, R., Pichot, A.L., Bompeix, G., 2008. In vitro and in vitro activity of eugenol oil (Eugenia caryophylata) against four important postharvest apple pathogens. Int. J. Food Microbiol. 126, 13–19. AOAC, 1990a. Solids (soluble) in fruits and fruit products. In: Helrich, K. (Ed.), Official Methods of Analysis. , 15th ed. AOAC, Inc., Arlington, p. 915. AOAC, 1990b. Vitamin C (ascorbic acid) in vitamin preparations and juices. In: Helrich, K. (Ed.), Official Methods of Analysis. , 15th ed. AOAC, Inc., Arlington, p. 1058. AOAC, 1990c. Sucrose in fruits and fruit products. In: Helrich, K. (Ed.), Official Methods of Analysis. , 15th ed. AOAC, Inc., Arlington, p. 922. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils—a review. Food Chem. Toxicol. 46, 446–475. Baser, K.H.C., Ozek, T., Kurkcuoglu, M., Tumen, G., 1994. The essential oil of Origanum vulgare subsp. hirtum of Turkish origin. J. Essent. Oil Res. 6, 31–36. Berrington, D., Lall, N., 2012. Anticancer activity of certain herbs and spices on the cervical epithelial carcinoma (HeLa) cell line. eCAM 2012, 1–11. Bozin, B., Mimica-Dukic, N., Simin, N., Anackov, G., 2006. Characterization of the volatile composition of essential oils of some lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils. J. Agric. Food Chem. 54, 1822–1828. Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in foods—a review. Int. J. Food Microbiol. 94, 223–253. Capecka, E., Mareczek, A., Leja, M., 2005. Antioxidant activity of fresh and dry herbs of some Lamiaceae species. Food Chem. 93, 223–226. Chalchat, J.C., Pasquier, B., 1998. Morphological and chemical studies of Origanum clones: Origanum vulgare L. ssp. vulgare. J. Essent. Oil Res. 10, 119–125. Christian, E.J., Goggi, A.S., 2008. Aromatic plant oils as fungicide for organic corn production. Crop Sci. 48, 1941–1951. Conklin, P.L., 2001. Recent advances in the role and biosynthesis of ascorbic acid in plants. Plant Cell Environ. 24, 383–394.

R. Baranauskiene˙ et al. / Industrial Crops and Products 49 (2013) 43–51 D’Antuono, F., Galletti, G.C., Bocchini, P., 2000. Variability of essential oil content and composition of Origanum vulgare L. populations from a North Mediterranean area (Liguria Region, Northern Italy). Ann. Bot. – Lond. 86, 471–478. Dapkevicius, A., Venskutonis, R., van Beek, T.A., Linssen, J.P.H., 1998. Antioxidant activity of extracts obtained by different isolation procedures from some aromatic herbs grown in Lithuania. J. Sci. Food Agric. 77, 140–146. De Martino, L., De Feo, V., Formisano, C., Mignola, E., Senatore, F., 2009. Chemical composition and antimicrobial activity of the essential oils from three chemotypes of Origanum vulgare L. ssp. hirtum (Link) ietswaart growing wild in Campania (Southern Italy). Molecules 14, 2735–2746. Figuérédo, G., Cabassu, P., Chalchat, J.-C., Pasquier, B., 2006. Studies of Mediterranean oregano populations. VIII—chemical composition of essential oils of oreganos of various origins. Flavour Frag. J. 21, 134–139. Gavrilenko, V.J., Ladigina, M.E., Chandobina, L.M., 1975. Big Practicum on Plant Physiology. Visshaja Shkola, Moscow, Russian. Höferl, M., Buchbauer, G., Jirovetz, L., Schmidt, E., Stoyanova, A., Denkova, Z., Slavchev, A., Geissler, M., 2009. Correlation of antimicrobial activities of various essential oils and their main aromatic volatile constituents. J. Essent. Oil Res. 21, 459–463. Ietswaart, J.H., 1980. A Taxonomic Revision of the Genus Origanum (Labiatae). Leiden Botanical Series 4, Leiden University Press, The Hague, The Netherlands. Ipek, E., Zeytinoglu, H., Okay, S., Tuylu, B.A., Kurkcuoglu, M., Husnu Can Baser, K., 2005. Genotoxicity and antigenotoxicity of Origanum oil and carvacrol evaluated by Ames Salmonella/microsomal test. Food Chem. 93, 551–556. ´ I., Mastelic, ´ J., Miloˇs, M., 2001. The impact of both the season of collection Jerkovic, and drying on the volatile constituents of Origanum vulgare L. ssp. hirtum grown wild in Croatia. Int. J. Food Sci. Technol. 36, 649–654. Johnson, C.B., Kazantzis, A., Skoula, M., Mitteregger, U., Novak, J., 2004. Seasonal, populational and ontogenic variation in the volatile oil content and composition of individuals of Origanum vulgare subsp. Hirtum, assessed by GC headspace analysis and by SPME sampling of individual oil glands. Phytochem. Anal. 15, 286–292. Kokkini, S., 1997. Taxonomy, diversity and distribution of Origanum species. In: Padulosi, S. (Ed.), Oregano. Proceedings of the IPGRI International Workshop on Oregano. Valenzano (Bari), Italy, 8–12 May 1996. IPGRI, Rome, Italy, pp. 2–12. Kokkini, S., Vokou, D., 1989. Carvacrol-rich plants in Greece. Flavour Frag. J. 4, 1–7. Kokkini, S., Karousou, R., Vokou, D., 1994. Pattern of geographic variation of Origanum vulgare trichomes and essential oil content in Greece. Biochem. Syst. Ecol. 22, 517–528. Kulisic, T., Radonic, A., Katalinic, V., Milosa, M., 2004. Use of different methods for testing antioxidative activity of oregano essential oil. Food Chem. 85, 633–640. Lang, G., Buchbauer, G., 2012. A review on recent research results (2008–2010) on essential oils as antimicrobials and antifungals. A review. Flavour Frag. J. 27, 13–39.

51

Methodology directions for determination of nitrates in the plant production. Confirmed by Lithuanian Ministry of Agriculture and Ministry of Health (1990), Order No. 160/3-2841. Ministry of Agriculture. Vilnius, Lithuania, p. 41. Mezzoug, N., Elhadri, A., Dallouh, A., Amkiss, S., Skali, N.S., Abrini, J., Zhiri, A., Baudouxb, D., Diallo, B., El Jaziri, M., Idaomar, M., 2007. Investigation of the mutagenic and antimutagenic effects of Origanum compactum essential oil and some of its constituents. Mutat. Res. 629, 100–110. Mockute, D., Bernotiene, G., Judzentiene, A., 2001. The essential oil of Origanum vulgare L. ssp. vulgare growing wild in Vilnius district (Lithuania). Phytochemistry 57, 65–69. Novák, I., Zámbori-Nementh, É., Horváth, H., Seregély, Zs., Kaffka, K., 2003. Study of essential oil component in different Origanum species by GC and sensory analysis. Acta Aliment. Hung. 32, 141–150. Olivier, G.W., 1997. The world market of oregano. In: Padulosi, S. (Ed.), Oregano. Proceedings of the IPGRI International Workshop on Oregano. Valenzano (Bari), Italy, 8–12 May 1996. IPGRI, Rome, Italy, pp. 142–146. Pande, C., Mathela, C.S., 2000. Essential oil composition of Origanum vulgare L. ssp. vulgare from the Kumaon Himalayas. J. Essent. Oil Res. 12, 441–442. Pilau, M.R., Alves, S.H., Weiblen, R., Arenhart, S., Cueto, A.P., Lovato, L.T., 2011. Antiviral activity of the lippie graveolens (Mexican oregano) essential oil and its main compound carvacrol against human and animal viruses. Braz. J. Microbiol 42, 1616–1624. ˙ J., Stankeviˇciene, ˙ D., Venskutonis, R., 2005a. Morphological and chemical Raduˇsiene, variation of Origanum vulgare L. from Lithuania. WOCMAP III: bioprospecting and ethnophrmacology. Acta Hortic. 675, 197–203. ˙ J., Judˇzentiene, ˙ A., Peˇciulyte, ˙ D., Janulis, V., 2005b. Chemical composiRaduˇsiene, tion of essential oil and antimicrobial activity of Origanum vulgare. Biologija 4, 53–58. Russo, M., Guido, C., Galletti, G.C., Bocchini, P., Carnacini, A., 1998. Essential oil chemical composition of wild populations of Italian oregano spice (Origanum vulgare ssp. hirtum (Link) Ietswaart): a preliminary evaluation of their use in chemotaxonomy by cluster analysis. 1. Inflorescences. J. Agric. Food Chem. 46, 3741–3746. Scott, K.J., 2001. Detection and measurement of carotenoids by UV/VIS spectrophotometry. In: Wrolstad, R.E., Acree, T.E., An, H., Decker, E.A., Penner, M.H., Reid, S.D., Schwartz, S.J., et al. (Eds.), Current Protocols in Food Analytical Chemistry. John Wiley & Sons Inc., New York, pp. F.2.2.1–F.2.2.10. Sezik, E., Tümen, G., Kirimer, N., Özek, T., Baser, K.H.C., 1993. Essential oil composition of four Origanum vulgare subspecies of Anatolian origin. J. Essent. Oil Res. 5, 425–431. Vale-Silva, L., Silva, M.-J., Oliveira, D., Gonc¸alves, M.-J., Cavaleiro, C., Salgueiro, L., Pinto, E., 2012. Correlation of the chemical composition of essential oils from Origanum vulgare subsp. virens with their in vitro activity against pathogenic yeasts and filamentous fungi. J. Med. Microbiol. 61, 252–260.