A kinetic study of essential oil components distillation for the recovery of carvacrol rich fractions

Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx

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A kinetic study of essential oil components distillation for the recovery of carvacrol rich fractions ⁎

Dimitrios Tsimogiannis , Vassiliki Oreopoulou Laboratory of Food Chemistry and Technology, School of Chemical Engineering, National Technical University of Athens (NTUA), 5 Iroon Polytechniou str., Athens GR 15780, Greece

A R T I C LE I N FO

A B S T R A C T

Keywords: Essential oils Origanum vulgare L. ssp. hirtum Origanum onites L. Origanum dictamnus L. Satureja thymbra L. Carvacrol Distillation kinetics

The distillation kinetics of the different essential oil components from four Lamiaceae herbs was investigated. Origanum vulgare L. ssp. hirtum, Origanum onites L., Origanum dictamnus L., and Satureja thymbra L. (harvests of 2013–2015), that present similar composition of essential oil were distilled in a laboratory-scale steam distiller. The recovery of the essential oil (EO) and its components was adequately described by non-steady state diffusion kinetics. The results clearly evidenced that the components present different distillation rates, thus resulting in variation of the EO composition during the process. The distillation rate constants decreased with the increase of the individual components boiling points, therefore, the heavy volatiles, such as carvacrol, could be recovered separately after the distillation of the most volatile compounds. This hypothesis was experimentally confirmed for O. vulgare and S. thymbra; the separate fractions were obtained from 40 and 50 min respectively up to the end of distillations. GC–MS analyses of the fractions revealed very high carvacrol content that amounted up to 92.5% for O. vulgare. The procedure could be applicable in industrial practice in order to recover fractions with predesigned quality characteristics.

1. Introduction Medicinal and aromatic plants have been used for the treatment of various diseases all over the world since antiquity. The essential oil (EO) constitutes a valuable fraction of these plants, and extensive scientific studies concern its recovery and determination of biological activity (Gilling et al., 2014; Raut and Karuppayil, 2014; Langeveld et al., 2014; Lang and Buchbauer, 2012; Vimalanathan and Hudson, 2012; Pilau et al., 2011; Baser, 2008). Due to their pleasant odor, EOs from various medicinal and aromatic plants are used in cosmetics and food products, as fragrance ingredients and flavor enhancers, respectively. Moreover, EOs may be potent food preservatives (Prakash et al., 2015; Rasooli and Rasooli, 2013; Burt, 2004; Atarés and Chiralt, 2016; Otoni et al., 2016; Van Long et al., 2016; Palou et al., 2015). Among the aromatic plants, most of the largest Lamiaceae genera contain the monoterpenoid phenol carvacrol, which amounts up to 95% in the EO of specific species. Carvacrol has been recognized as an important compound with biological antimicrobial and antiviral activity (Gilling et al., 2014; Orhan et al., 2012; Lai et al., 2012; Guarda et al., 2011; Pilau et al., 2011; Baser, 2008). The activity of the compound against bacteria includes membrane disruption, inhibition of ATPase activity, leakage of cell ions, fluidization of membrane lipids and

⁎

reduction of proton motive force (Langeveld et al., 2014). Carvacrolbased additives for livestock feed are already commercially available for reducing or assisting conventional antibiotics in poultry, swine, ruminants and aquaculture production. Furthermore, the compound has been approved by the European Union as a flavoring substance for food products (Regulation No 872/2012 and 1334/2008), while the EOs from the Lamiaceae plants that contain carvacrol can be used by the food industry as natural flavoring preparations (defined by EU Regulation 1334/2008). The distillation rate of carvacrol and other EO components from a plant material depends, among others, on the tissue structure. The epidermal glandular trichomes in Lamiaceae are divided to peltate and capitate, with different morphology (Huang et al., 2008) and different composition of volatiles (Schmiderer et al., 2008). For the capitate trichomes, there is some evidence that their secretion consists mainly of a complex mixture of carbohydrates, lipids, and proteins (Turner et al., 2000). Baâtour (2012) observed by light microscopy, under the cuticule of the head cells of the peltate trichomes, three types of EO secretion: dark red droplets, clear lipid droplets and secretion of lucid appearance. The dark red color of the secretion might suggest that carvacrol is located in these specific oil glands. Furthermore the individual components of EO are primary located in the special secretory structures

Corresponding author. E-mail address: [email protected] (D. Tsimogiannis).

https://doi.org/10.1016/j.jarmap.2018.03.006 Received 20 October 2017; Received in revised form 25 March 2018; Accepted 29 March 2018 2214-7861/ © 2018 Elsevier GmbH. All rights reserved.

Please cite this article as: Tsimogiannis, D., Journal of Applied Research on Medicinal and Aromatic Plants (2018), https://doi.org/10.1016/j.jarmap.2018.03.006

Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx

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(30 m × 20 μm × 0.25 μm, Hewlett Packard, Palo Alto, CA, USA). The oven temperature was started at 50 °C, increased to 100 °C at 10 °C min−1 rate, then to 220 °C at 15 °C min−1 rate and hold at 220 °C for 7 min. Helium was used as a carrier gas at 1 mL min−1 column flow, inlet temperature 220 °C and split 20:1. The mass range was 40–400 and compounds were identified by comparison of their mass spectra with the data of NIST and Wiley mass spectral libraries, as well as the spectral database of Adams (2007). The retention times of compounds were converted to the respective Kovats Retention Indices (RI) by analyzing a standard C7–C30 alkanes mixture, with the abovementioned gas-chromatographic method, and using the equation:

either on the surface of the plant or within the plant tissues (Svoboda et al., 2000). Consequently, the diffusion of some components from the interior of the plant tissue to the surface may affect their distillation rate. Moreover, the vapor pressure of the different volatile components is another main factor affecting their distillation. The production of EOs with a high carvacrol content could enhance their quality and commercial value, as well as their applicability as feed and food additives. However, a substantial part of the global production of EOs is performed by small and medium enterprises that focus on the yield and the cost of production, and not on high quality in terms of carvacrol content. The co-distilling compounds might not present the same distillation kinetics and consequently the composition of the EO being produced, could markedly change during the process. The current work focused on the kinetic study of the distillation of individual EO components. Herbs of the Lamiaceae family, with considerable carvacrol content were examined, with the aim to investigate if the separation of carvacrol-rich fractions is attainable, even by using conventional distillation equipment. This would allow the application in the production line of small and medium enterprises.

tr (compound)−tr (n) ⎤ RI = 100 × ⎡n + (N −n) ⎢ tr (N )−tr (n) ⎥ ⎣ ⎦

(1)

where: RI: Kovats retention index n: the number of carbon atoms in the smaller n-alkane N: the number of carbon atoms in the larger n-alkane tr: the retention time The quantification of the compounds was performed by the area normalization method according to the formula:

2. Material and methods

Ci =

2.1. Plant samples

Ai × 100 ∑ Ai

(2)

whereCi : the % content of analyte in the EO Ai : the area of individual compound in the chromatogram ∑Ai : the sum of all the peak areas in the chromatogram

Air dried herbs (leaves and flowers) of O. onites L. (harvested in July) and O. dictamnus L. (harvested in June) were purchased from Alexopoulos Alexandros and Co (Athens, Greece), and the local market of Crete, respectively. O. vulgare L. ssp. hirtum (harvested in June), and S. thymbra L. (May 2013, May 2014 and July 2015 harvest) were provided by the Agricultural Research Centre of Northern Greece (member of the Hellenic Agricultural Organization-DEMETER). The plant species and varieties were defined by the providers.

2.4. Statistical analysis One way ANOVA analysis was performed to test the statistical differences between the results obtained, using STATISTICA 7.0 (StatSoft Inc., Tulsa, OK, USA). In case that values were significantly different, Duncan’s Test was further applied. Correlation coefficients were determined using Excel® 2013 (Microsoft Corporation).

2.2. Steam distillations The steam distillation was performed in a pilot scale distiller of 10 L useful capacity (Chalkos, Greece) with inner perforated grid to hold the plant material above the boiling water. Dry herbs (500 g), with moisture content varying from 7.9% (O. dictamnus) to 9.1% (S. thymbra 2014), wet basis, were subjected to distillation for a period up to 5 h with simultaneous determination of the essential oil (EO) volume. The process was terminated when the volume of the EO was practically constant (recovery of EO less than 0.2% v/w of dry herb for a period of 30 min). The steam supply rate varied in the range of 5.7 ± 1.4 mL min−1 kg−1. The EO was being received in a graduated glass collector and the volume was recorded versus distillation time. The yield was expressed as the EO volume (mL) obtained by 100 g dry herb per time unit (min). During the distillation process, 2 μL samples of EO were withdrawn with a Transferpette S digital automatic micropipette (0.2–10 μL) (Brand, Wertheim, Germany), equipped with nanocapTM tips, from the upper oil phase of the glass collector, periodically, diluted in 10 mL of hexane, and subjected to GC–MS analysis. Distillation experiments for each plant material were performed in duplicate. A series of additional duplicate distillation experiments was performed for O. vulgare (ssp. hirtum) and S. thymbra (2015 harvest) and in each case two fractions of EO were recovered. Fraction F1 was collected up to 40 min for O. vulgare and up to 50 min for S. thymbra, followed by F2 that was collected up to the end of distillations. The specific fractionation time for each herb was defined according to the results obtained from the data analysis of the previous experiments.

3. Results and discussion 3.1. Distillation kinetics of EOs The yields of EOs versus distillation time are presented in Fig. 1A, while the respective rates (ry, (mL EO/100 g dry herb) min−1) during the distillation are depicted in Fig. 1B. The yield of EO increased very fast at the beginning of distillation and the herbs presented high variation of rates, while over time the rates decreased and the variations were minimized. Milojević et al. (2008) proposed the kinetic model of Eq. (3), based on non-steady state diffusion for the hydrodistillations of comminuted ripe juniper berries. The linearized form (Eq. (4)) can be used to calculate the parameters of the kinetic model.

q −q qo−q = e−kt or o = (1−b) e−kt qo qo−bqo

(3)

q −q ln ⎛⎜ o ⎞⎟ = ln(1−b)−kt ⎝ qo ⎠

(4)

Where qo:EO content initially present in the herbs (equal to the total EO yield at the end of distillation, mL/100 g) q :EO yield at any moment of steam distillation b :fast distillation coefficient k :distillation rate constant (min−1) In this model, b (fast distillation coefficient) represents the portion of the EO, probably located on the external surface of the plant particles that is removed during an initial, short period of distillation (theoretically at t = 0). It can be characterized by a rapid increase in the oil yield at the very beginning of the process. On the other hand k

2.3. GC–MS analysis GC–MS analysis was performed by using an HP 6890 GC system (plus +) coupled to an HP 5973 mass selective detector (Hewlett Packard, Palo Alto, CA, USA), and equipped with an HP-5 MS column 2

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Fig. 1. (A) The yields of essential oil during distillation. (B) The rates of produced yield during distillation.

Fig. 2. The dependence of ln[(qo −q)/qo] with distillation time: ((A) for the three harvests of S. thymbra and (B) for O. vulgare (ssp. hirtum), O. dictamnus and O. onites.

3.2. EOs yields and composition

(distillation rate constant) concerns the diffusion of the EO from the intact reservoirs in the interior of the plant particles towards the surface, followed by the oil distillation, a phenomenon that progresses slowly. The plots of ln[(qo −q)/qo] versus time are presented in Fig. 2, while the kinetic parameters and yields are reported in Table 1. The coefficient b was calculated through the y-intercept of the curves that equals ln(1-b), according to Eq. (4). The batches of S. thymbra harvested in 2013 and 2014 presented the same behavior with almost identical k and b values. As far as the 2015 harvest is concerned k was determined 10% higher and b 42% lower than the respective average values of the former harvests. The lower b value might suggest that the quantity of the rapid distilling species is lower. As indicated in Table 1, the rest herbs presented lower k and b values than the samples of S. thymbra. Especially the b value of O. onites was determined negative, a fact that is controversial to the assumed model. A negative b value should imply an initial inhibition of distillation. This might suggest that the surface glandular trichomes contain high quantities of non-volatiles, which prevent the initial rapid distillation (reduced vapor pressure of the volatiles). In any case further investigation is required to explain such deviations from the predicted by the model phenomena.

The yields of EOs ranged within 1.2–4.6% (v/w) depending on the plant and the harvest year, while they are in agreement with literature data. According to the reviews of Kirimer et al. (1995) and Baser (2008), the plants of the current study yielded EOs within 1–6.5%. The compositions of the produced EOs are presented in Table 2. As expected there was a linear correlation (R2 = 0.961) between the obtained R.I. values and the boiling points of the individual compounds, which were retrieved from the literature. Carvacrol was identified as the major EO component in most cases with concentrations ranging within 31–81%. In general, a-terpinene (1.2–3.6%), p-cymene (5.3–13.1%), γ-terpinene (4.8–41%), and (E)-β-caryophyllene (1–7.6%) were characterized as minor components, while the rest as traces (0–3%). Kirimer et al. (1995) and Baser (2008) reported carvacrol content in the range of 23–82% for the five herbs. γ-Terpinene was the component with the highest variation and was detected as a major component in S. thymbra. More specifically, in the EOs from the batches harvested in 2013 and 2014, γ-terpinene overcame carvacrol and amounted to 40.9% and 36.5%, respectively. Interestingly, γ-terpinene concentration in S. thymbra is correlated with the yield (r = 0.9907) and inversely correlated with carvacrol content (r = 0.9991). γ-terpinene consists a known precursor of the phenolic monoterpenes, thymol and carvacrol, in S. thymbra and S. parnassica (Chorianopoulos et al., 2006). Therefore the increase of carvacrol with the simultaneous diminishment of its monoterpene precursor appears reasonable. The above compounds present significant seasonal variation. According to Chorianopoulos et al. (2006) during the flowering period carvacrol content increases, presenting a peak in August, while γ-terpinene minimizes; the opposite phenomenon occurs in February. In fact the S. thymbra samples of 2013 and 2014 were collected in spring, consequently, they presented a very high content of γ-terpinene.

Table 1 The determined parameters of the kinetic model (Eq. (4)) and the total essential oil yields of the examined herbs.

S. thymbra (2013 harvest) S. thymbra (2014 harvest) S. thymbra (2015 harvest) O. vulgare ssp. hirtum O. onites O. dictamnus

K (min−1)

b

yield (mL/100 g plant)

0.016 0.016 0.018 0.006 0.016 0.015

0.29 0.25 0.16 0.01 −0.09 0.04

4.6 4.3 2.8 3.7 1.2 1.7

3

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Table 2 The compositions of the EOs recovered from the different species (O. vulgare, O. onites, O. dictamnus, S. thymbra), as well as different harvest of the same specie (S. thymbra). O. vulgare

O. onites

O. dictamnus

(ssp. hirtum) No.

compound

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

α-thujene α-pinene1 camphene1 sabinene1 β-pinene1 myrcene1 β-phellandrene1 δ-car-3-ene 1 α-terpinene1 p-cymene1 limonene1 γ-terpinene1 cis-sabinene hydrate2 α-terpinolene1 linalool2 borneol2 terpinen-4-ol2 α-terpineol2 carvacrol methyl ether3 thymoquinone thymol3 carvacrol3 carvacryl acetate3 α-copaene4 (E)-β-caryophyllene4 aromadendrene4 α-selinene4 β-bisabolene4 caryophyllene oxide5 1

boiling point (°C) 151 155 159 163 166 167 171 168 174 177 176 183 201 187 198 213 215.5 215.5 217 231 233 236 247 249 255.5 262 261.5 274 280

RI 928.0 935.9 951.7 975.9 980.2 990.5 1007.6 1014.0 1020.0 1028.2 1032.4 1062.5 1072.6 1092.0 1100.3 1175.1 1184.6 1197.3 1249.1 1258.0 1296.2 1306.3 1377.6 1388.8 1437.3 1456.6 1472.5 1518.4 1581.0

sd 0.2 0.2 0.2 0.1 0.3 0.2 0.2 0.2 0.3 0.2 0.0 0.2 0.1 0.1 0.2 0.5 0.4 0.1 0.1 0.2 0.2 0.3 0.1 0.1 0.7 0.2 0.6 0.7 0.2

Ci (%) a

0.97 0.63a 0.15a,c – 0.18a 1.29a 0.23a 0.10a 1.20a 5.25a – 4.76a 0.30a 0.16a – 0.52a 0.78a – 0.11a – 0.25a 80.66a – – 1.91a – 0.22a 0.24a –

S. thymbra May 2013

sd

Ci (%)

sd

b

0.04 0.03 0.01

0.14 0.31b 0.25b – 0.10b 0.60b 0.28b 0.01b 1.99b 5.63a – 5.44a 0.23b 0.59b 0.75a 1.95b 2.91b 1.31 – – 0.24a 72.36b 0.20 – 0.96b 0.27 0.03a 0.11b 0.25a

0.01 0.01 0.00 0.02 0.07 0.23 0.19 0.02 0.05 0.04 0.03 0.02 0.21 0.64

0.08 0.02 0.03

0.00 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.10 0.07 0.01 0.01 0.07 0.07 0.01 0.05

0.05 0.11 0.00 0.03 0.00 0.02 0.00 0.03

Ci (%) c

1.49 0.67a 0.10c 0.11 0.22a 1.06c 0.22c 0.10a 1.91b 13.12b – 9.85b 1.03c – – – – – – 0.92 – 65.29c – 1.03 2.51a – – – –

sd 0.06 0.02 0.01 0.02 0.02 0.06 0.02 0.01 0.05 0.33 0.24 0.01

0.04 0.65 0.02 0.09

Ci (%) d

1.79 0.88c 0.23a,b – 0.35c 1.34a 0.32c – 3.62c 9.28c 0.64a 40.88c – – 0.28b 0.14a 0.16c – 1.21b – 0.05a 30.79d – – 7.56c – 0.34a – 0.14b

May 2014 sd 0.03 0.01 0.01 0.01 0.05 0.01 0.01 0.07 0.03 0.50

0.00 0.17 0.09 0.01 0.05 0.47

0.09 0.06 0.00

Ci (%) c,d

1.60 0.85c 0.21a,b – 0.36c 1.30a 0.30b,c – 3.4c 10.29d 0.83a 36.47d – – 0.26b 0.20a 0.21c – 1.11b – 0.01a 34.51e – – 7.41c – 0.19a – 0.01c

July 2015 sd 0.08 0.04 0.03 0.02 0.05 0.00 0.06 0.20 0.06 0.63

0.01 0.23 0.06 0.03 0.03 0.75

0.20 0.10 0.01

Ci (%) a

1.19 1.13d 0.34d – 0.47d 1.19a,c 0.22a – 2.60d 8.29e 0.64a 26.02e – – 0.62a 0.48a 0.24c – 0.66c – 0.38a 48.74f – – 6.41d – 0.23a – 0.16b

sd 0.13 0.08 0.05 0.03 0.06 0.00 0.10 0.34 0.09 1.11

0.01 0.28 0.03 0.05 0.00 1.28

0.54 0.15 0.01

Different superscript letters in the same row indicate significant differences among herbs. 1 Monoterpene hydrocarbons. 2 Monoterpene alcohols. 3 Phenolic monoterpenes. 4 Sesquiterpene hydrocarbons. 5 oxygenated sesquiterpenes.

indicates that the compound belongs to the components with diffusion controlled distillation. However the r value between carvacrol concentration and k revealed poor correlation. The concentration of the compounds in the final EO might not be an appropriate index to conclude which compounds present rapid or slow distillation kinetics. For this reason a systematic study of the individual components distillation was performed.

Table 3 Results of the correlation of b and k values with the concentration (C) of individual major components. Compound(s)

rC-b

rC-k

p-cymene γ-terpinene carvacrol (E)-β-caryophyllene

0.4362 0.9715 −0.9132 0.9756

0.4332 0.5686 −0.6675 0.5339

3.3. Distillation kinetics of the individual components The examined herbs presented high resemblance in common constituents, which amounted from 88.1% (O. onites) up to 97.3% (O. vulgare ssp. hirtum) of the total content. The fast distillation coefficient (b) was attempted to be correlated to the concentration of the main common components of the herbs i.e. p-cymene, γ-terpinene, carvacrol, (E)-β-caryophyllene. From the results (presented in Table 3), it can be seen that b was negatively correlated with carvacrol and positively with γ-terpinene and (E)-β-caryophyllene. Consequently, an increased concentration of the last mentioned compounds, resulted in the increase of b value, indicating that these compounds comprise part of the highly available surfacial components that distill rapidly. On the contrary k values did not correlate with the concentration of any compound. It is reasonable, the rapidly distilling compounds not to affect k, which refers to the diffusion controlled distillation of compounds with low availability. Carvacrol concentration was negatively correlated to b, which

The yield of each component during distillation was determined though GC analysis of the periodically collected samples. Taking into consideration the produced EO yield and the concentration of each analyte at the time of sampling, each component was quantified on the basis of the processed herbal mass, according to Eq. (5):

qi =

Ci ∙q 100

(5)

where qi :the yield of analyte i per 100 g of the herbal mass at time t of distillation Ci :the % content of analyte in the EO q :the EO yield at the time t The evolution of Ci and qi for carvacrol and γ-terpinene recovered during the distillation of S. thymbra (2015 harvest), is presented in Fig. 3. According to Fig. 3A, the EO exhibited high concentration of γterpinene at the beginning of distillation, which gradually descended 4

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Fig. 3. The evolution of Ci (A) and qi (B) concerning carvacrol and γ-terpinene of the S. thymbra 2015 harvest, during distillation.

during the distillation process. The obtained yield data of each individual component, qi, during the distillation were fitted to Eq. (4). Plotting the ln(qi,o-qi) vs time, for the major and minor constituents identified in all the examined herbs, the ki values of each constituent were determined and are presented in Table 4. The linearity of ln(qi,o-qi) vs time was observed up to 60 min for the monoterpene hydrocarbons and alcohols, a reasonable fact since this time corresponded to the recovery of 90% of the respective compounds. On the contrary a linear relation was observed for the rest compounds up to the end of distillation. 3.4. Comparison of the distillation kinetics of the individual components The general tendency was that an increase of R.I. up to 1100 was associated with a rapid decrease of the ki value of the respective compound (Tables 3 and 4). Above R.I. = 1150 (or b.p. = 200 °C) the ki values did not differ significantly (p > 0.05). The ki values of the common compounds of all herbs were statistically processed with one way ANOVA and were found statistically different (p < 0.05). According to the Duncan’s Test the components were divided to five groups with significant differences among the ki values and the average values of the components are presented in Fig. 5. Carvacrol and (E)-βcaryophyllene consisted the group with the lowest distillation rates, the average ki values were determined equal to 0.010 and 0.018 min−1, respectively. The second group included γ-terpinene, α-terpinene, pcymene, β-pinene, and myrcene with average ki values 0.072, 0.075, 0.076, 0.078, and 0.089 min−1, respectively. Groups C and D included myrcene, a-pinene, and a-thujene, with the last two possessing the highest distillation rate constants, which were determined equal to 0.112 and 0.123 min−1, respectively. The ratio between the ki, values of monoterpenes and of the slowly distilling compounds (Group A, Fig. 5) lied in the range 4–12, i.e. monoterpene hydrocarbons and the majority of monoterpene alcohols were recovered 4–12 times faster than the rest heavy volatiles such as carvacrol and (E)-β-caryophyllene. Thus compounds with low boiling point can be separated from compounds with higher boiling point. In other words, these results indicate that a simple and easy recovery of carvacrol-rich fractions from aromatic plants, by using common industrial distillers, is feasible.

Fig. 4. The normalized qi% values for γ-terpinene of all studied plants.

during the process due to dilution by the co-distillation of other species. The opposite was observed for carvacrol that enriched in a very slow rate the EO. In Fig. 3B the yield of the individual compounds can be observed. γ-Terpinene was recovered rapidly and the herbal mass was exhausted from the compound at approximately 60 min, when the yield reached a plateau. This time limit of γ-terpinene distillation was observed for all species, as presented in Fig. 4, where the normalized qi (%) values for γ-terpinene of all studied plants are plotted against distillation time. It is evident that at 60 min more than 90% of the total γterpinene had been recovered. Carvacrol presented a clearly slower rate of distillation (Fig. 3B) and the respective plateau was approached above 200 min. Monoterpene hydrocarbons and alcohols presented a γ-terpinene–like behavior, i.e. were totally recovered in 60 min. Phenols, phenol derivatives, and sesquiterpenes presented a carvacrol-like behavior. It can be assumed that from ∼60 min onwards the only components that practically are distilled are carvacrol and (E)-β-caryophyllene. The rest of phenols and sesquiterpenes are trace components. The same observations were made for the respective compounds of all the studied herbs. Zheljazkov et al. (2012) followed a similar approach, determining the concentration of individual compounds in the EO during the steam distillation of O. vulgare. They also concluded that, during the distillation, the composition of EO changes and the concentration of low boiling EO constituents such as a-thujene, a-pinene, a-terpinene, pcymene, γ-terpinene, was maximum at the beginning of distillation, reduced as distillation time increased up to 40 min, and then stayed constant. On the contrary, the compounds with higher boiling points were totally recovered in longer distillation times. The authors reported that the yields of borneol, 4-terpinenol, carvacrol, (E)-β-caryophyllene, and β-bisabolene were maxed at 240 min. Toker et al. (2017) presented similar results for carvacrol, by studying Origanum minutiflorum. Zheljazkov et al. (2016) presented 19 examples of studied species, which presented significant differences in the composition the EOs

3.5. Recovery of carvacrol-rich fractions O. vulgare (ssp. hirtum) and S. thymbra (2015 harvest) were arbitrary selected to test the potential fractionation to obtain carvacrol-rich fractions. The EOs of both herbs were separated in two fractions, as described in the experimental section. The composition of the fractions was determined by GC–MS and the results are presented in Table 5 together with the obtained yields. The sum of F1 and F2 yields in either distillation approximated the respective yield determined in the primary experiments. The 5

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Table 4 The determined ki values from the first order kinetic model concerning the individual volatiles of the six herbs. O. vulgare

O. onites

O. dictamnus

S. thymbra

(ssp. hirtum)

May 2013

May 2014

July 2015

No.

compound

ki (min−1) ± sd

ki (min−1) ± sd

ki (min−1) ± sd

ki (min−1) ± sd

ki (min−1) ± sd

ki (min−1) ± sd

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

α-thujene α-pinene camphene sabinene β-pinene myrcene β-phellandrene δ-car-3-ene α-terpinene p-cymene limonene γ-terpinene cis-sabinene hydrate α-terpinolene linalool borneol terpinen-4-ol α-terpineol carvacrol methyl ether thymoquinone thymol carvacrol carvacryl acetate α-copaene (E)-β-caryophyllene aromadendrene β-selinene β-bisabolene caryophyllene oxide

0.142a 0.134a n.a. – 0.069b,c 0.086b 0.074b n.a. 0.067b,c 0.073b – 0.069b,c 0.045d 0.050c,d – 0.008e 0.009e – 0.014e – n.a. 0.006e – – 0.010e – 0.009e 0.008e

0.078a 0.078a 0.079a – 0.052b,c,d 0.078a 0.075a,b 0.064 a,b,c 0.062a,b,c 0.066a,b,c – 0.061a,b,c 0.033d,e,f 0.050c,d,e 0.034f 0.025d,e,f 0.028e,f 0.017f – – n.a. 0.012f 0.015f – 0.028e,f 0.014f n.a. n.a. n.a.

0.088a 0.084a 0.076a,b n.a. 0.060a,b 0.093a n.a. n.a. 0.076a,b 0.068a,b – 0.068a,b 0.056a,b,c – – – – – – 0.044b,c,d – 0.010d – 0.056a,b,c 0.016d – – – –

0.093a 0.090a 0.092a – 0.081a,b 0.066c n.a. – 0.044d,e 0.069b,c 0.033e,f 0.065c – – 0.052d n.a. 0.021f,g,h – 0.029f,g – n.a. 0.006i – – 0.017g,h,i – 0.015h,i – n.a.

0.197a 0.164a,b 0.099d – 0.122b,c,d 0.126b,c,d 0.119a,b,c – 0.115c,d 0.100d 0.092d 0.088d – – 0.033e 0.021e 0.015e – 0.022e – n.a. 0.013e – – 0.016e –

0.143a 0.122a,b 0.085a,b,c – 0.086b,c,d 0.088 b,c,d 0.053 c,d,e,f – 0.084 b,c,d 0.080 b,c,d,e 0.052d,e,f 0.079c,d – – 0.034e,f 0.021 f 0.018 f – 0.029f – n.a. 0.013f – – 0.022f –

−

0.003 0.013

0.003 0.014 0.007 0.005 0.007 0.003 0.002 0.005 0.000 0.000 0.004

0.000

0.000 0.000 0.001

0.012 0.001 0.013 0.016 0.002 0.016 0.003 0.006 0.003 0.006 0.005 0.005 0.007 0.002 0.001 0.004

0.000 0.001 0.004 0.000

0.020 0.015 0.020 0.001 0.013

0.000 0.005 0.002 0.012

0.013 0.000 0.012 0.002

0.005 0.004 0.005 0.014 0.004

0.001 0.000 0.002 0.000

0.000 0.002 0.003

0.000

0.000

0.031 0.003 0.003 0.011 0.017 0.012 0.008 0.006 0.004 0.005

0.003 0.005 0.000 0.000

0.000

0.000

0.042 0.022 0.018 0.019 0.019 0.001 0.004 0.003 0.004 0.002

0.001 0.000 0.001 0.001

0.000

0.001

0.003 – n.a.

– n.a.

n.a. not applicable due to low concentrations. Different superscript letters in the same column indicate significant differences among the compounds of the respective herb.

in two stages: a primary fast stage that represents the fraction of the highly available EO components that distill rapidly, and the subsequent slow stage when the compounds with slow distillation rates are accumulated. Also, the same kinetic model is applicable for the individual EO components. The distillation rate constants decrease with the increase of the boiling points i.e. the decrease of the volatility of the compounds. Therefore the heavy volatiles, such as carvacrol, could be recovered separately in rich fractions after the distillation of the most volatile compounds. The hypothesis was experimentally confirmed for O. vulgare (ssp. hirtum) and S. thymbra, resulting in fractions with carvacrol content up to 92.5%, and 82.5%, respectively. A similar distillation procedure could be applicable in industrial practice in order to easily recover fractions with pre-designed quality characteristics.

monoterpene hydrocarbons and alcohols were recovered mainly in F1 and only residual quantities were identified in F2. The F2 fraction of each distillation was very rich in carvacrol, especially the one produced from oregano, were the concentration of carvacrol amounted to 92.5%. Also, the sesquiterpene content of F2 fractions were higher than the respective in F1 fractions, as expected by the kinetic analysis performed above. Therefore by using a laboratory scale distiller, fractionation of the EO was accomplished and a carvacrol-rich fraction was obtained. 4. Conclusions The recovery of EOs from the examined Lamiaceae herbs could be successfully described by a mathematic model that is based on the nonsteady state diffusion. According to this, the distillation is distinguished

Fig. 5. The ki values of the common compounds in the six herbs, categorized by the Duncan’s Test according to statistically significant differences, in regard to the respective A: R.I. values, B: boiling points. The numerical values correspond to the compound numbers of Table 2. 6

Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx

D. Tsimogiannis, V. Oreopoulou

Table 5 The composition of the two EO fractions (F1, F2) obtained from O. vulgare and S. thymbra (2015 harvest). O. vulgare (ssp. hirtum)

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

S. thymbra 2015

Yield (mL/100 g plant)

F1

0.8

F2

2.9

F1

1.7

F2

1.1

compound α-thujene α-pinene camphene sabinene β-pinene myrcene β-phellandrene δ-car-3-ene α-terpinene p-cymene limonene γ-terpinene cis-sabinene hydrate α-terpinolene linalool borneol terpinen-4-ol a-terpineol carvacrol methyl ether thymoquinone thymol carvacrol carvacryl acetate α-copaene (E)-β-caryophyllene aromadendrene β-selinene β-bisabolene caryophyllene oxide

Ci (%) 4.38 a 2.77 a 0.69 a – 0.83 a 5.63 a 0.91 a 0.40 a 4.77 a 21.60 a – 19.03 a 0.95 a 0.54 a – 0.51 a 0.75 a 0.23 a 0.23 a – 0.14 a 33.10 a – – 2.36 a – 0.21 a 0.15 a –

sd 0.01 0.03 0.01

Ci (%) 0.05 b 0.06 b 0.01 b – 0.01 b 0.13 b 0.06 b 0.01 b 0.29 b 0.96 b – 1.07 b 0.16 b 0.07 b – 0.58 a 0.91 a 0.11 b 0.11 b – 0.33 b 92.47 b

sd 0.03 0.00 0.00

Ci (%) 2.05 a 1.95 a 0.59 a – 0.79 a 2.02 a 0.38 a – 4.35 a 13.23 a 1.12 a 42.85 a – – 0.82 a 0.46 a 0.20 a – 0.85 a – 0.19 a 21.57 a – – 6.36 a – 0.20 a – 0.01 a

sd 0.13 0.08 0.05

Ci (%) 0.11 b 0.09 b 0.02 b – 0.06 b 0.13 b 0.01 b – 0.41 b 1.98 b 0.02 b 4.97 b – – 0.38 b 0.61 b 0.29 b – 0.43 b – 0.63 b 82.45 b – – 6.83 a – 0.30 b – 0.29 b

sd 0.01 0.01 0.01

0.00 0.01 0.00 0.01 0.07 0.22 0.18 0.02 0.05 0.02 0.02 0.00 0.00 0.00 0.57

0.00 0.00 0.00 0.02 0.01 0.05 0.03 0.00 0.01 0.03 0.02 0.02 0.02 0.02 0.03

−

0.04 0.00 0.00

– 2.12 – 0.26 0.29 –

a

0.06

a

0.01 0.03

b

0.02 0.00 0.00 0.10 0.33 0.09 1.10

0.01 0.00 0.02 0.03 0.00 1.26

0.51 0.01 0.00

0.02 0.01 0.00 0.02 0.06 0.02 0.07

0.01 0.02 0.00 0.01 0.00 0.12

0.11 0.00 0.01

Different superscript letters in the same row indicate significant differences between fractions F1 and F2 of the same herb.

Acknowledgements

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