Extraction of Hyssopus officinalis L. essential oil using instant controlled pressure drop process

Accepted Manuscript Title: Extraction of Hyssopus officinalis L. essential oil using instant controlled pressure drop process Authors: Sepideh Rashidi, Mohammad H. Eikani, Mehdi Ardjmand PII: DOI: Reference:

S0021-9673(18)31283-4 https://doi.org/10.1016/j.chroma.2018.10.020 CHROMA 359744

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

17-8-2018 9-10-2018 14-10-2018

Please cite this article as: Rashidi S, Eikani MH, Ardjmand M, Extraction of Hyssopus officinalis L. essential oil using instant controlled pressure drop process, Journal of Chromatography A (2018), https://doi.org/10.1016/j.chroma.2018.10.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Extraction of Hyssopus officinalis L. essential oil using instant controlled pressure drop process

Sepideh Rashidia, Mohammad H. Eikanib,*, Mehdi Ardjmanda a

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Department of Chemical Engineering, South Tehran Branch, Islamic Azad University, Tehran, Iran b Department of Chemical Technologies, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran

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Crresponding author: Mohammad H. Eikani, Department of Chemical Technologies, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran, P.O. Box: 33535111. Tel: (+9821) 56276637, Fax: (+9821) 56276265, [email protected]

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Highlights

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 First use of ethanol-water mixture to produce pressurized vapor in the DIC process.  Optimized DIC was applied to extract essential oil from Hyssopus officinalis L. The optimal DIC yield was higher than HD, UAE and Soxhlet methods.

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SEM revealed significant structure modifications caused by the DIC process

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Abstract To determine the optimum condition for the extraction of Hyssopus officinalis L. essential oil (EO) by instant controlled pressure drop (in French: Détente Instantanée Contrôlée or DIC), the influential parameters of pressure, heating time per cycle, number of cycles, mean particle diameter of the herb, and water concentration in the steam generator feed were evaluated using response surface methodology (RSM). The impact of using an ethanol-water mixture to generate the required vapor in the DIC process on EO extraction was investigated. The optimum condition was found to be at pressure=1 bar, heating time=100 s, cycles=12, particle diameter=1 mm, and water concentration=80 v%. The addition of ethanol turned out to be more efficient at higher heating times and cycle numbers. The optimum yield in DIC was highest compared with hydrodistillation (HD), ultrasound-assisted extraction (UAE), and Soxhlet (SOX) methods; their values, expressed as total area percentages per internal standard area percentage, were 39.04, 10.98, 1.58, and 7.71, respectively. Comparison of the four methods also indicated that the DICPage 1 of 40

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EO was of a superior quality. The analysis of the DIC-treated residual herb in the optimum experiment revealed that unlike the low amount of valuable oxygenated monoterpenes, the availability of sesquiterpenes increased, which proved that DIC performs selectively while extracting more valuable EO components. The DIC-EO and its extract were examined for total phenolic content (TPC) and antioxidant activity using Folin-Ciocalteu and DPPH assay, respectively. Comparison of these results with those obtained from HD demonstrated higher TPC and antioxidant activity of the DIC-EO. Moreover, scanning electron microscopy (SEM) of the raw material and residual herb in the optimum experiment showed significant structure modifications induced by the DIC process.

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Key words: Instant controlled pressure drop, DIC, RSM, Hyssopus officinalis L., Essential oil, Extraction

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1. Introduction

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a light green or light yellow liquid with a sweet camphoric scent which is used to preserve and

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Hyssopus officinalis L. (hyssop), a perennial plant from the Lamiaceae family, which grows in the temperate regions of Asia, Europe, and north-western Iran, is consumed as an important

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culinary, aromatic, and medicinal plant [1, 2]. Essential oil (EO) obtained from the aerial parts of

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hyssop is a light green or light yellow liquid with a sweet camphoric scent used to preserve and flavor foods. Hyssop EO possesses antiseptic, antifungal, antiviral (especially against HIV),

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antitumor, antispasmodic, and antioxidant properties [3, 4]. Its phytotherapeutic effect allows it

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to accelerate wound healing and treat pulmonary problems (e.g. colds, coughs, and asthma), inflammation of the mucous membrane of the gastrointestinal tract, nervous exhaustion, and

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certain skin diseases [5-7]. As a natural flavoring agent, it is used in several industries including hygiene, cosmetics, beverage, and foodstuffs (prepared food, meat and candied products) [2, 4]. Yield per hectare of hyssop ranges from 10-20 kg of EO [7]. Conventional techniques of EO extraction such as steam/hydro distillation (HD) and Soxhlet extraction (SOX) have disadvantages of their own, including low extract quality caused by Page 2 of 40

degradation or loss of volatile compounds, low extraction efficiency, long processing time, solid residue degradation, high energy consumption, lack of automation, and toxic solvent residue. To overcome these drawbacks several alternative techniques have been introduced to extract EOs

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[8], among which were ultrasound-assisted extraction (UAE), microwave-assisted extraction, subcritical fluid extraction, and supercritical fluid extraction. Despite their respective merits, these techniques suffer from disadvantages of being developed only at a laboratory or pilot scale, requiring costly facilities, and having a cumbersome installation [9].

Originally introduced by Allaf and Vidal [10], the green, efficient, and economically attractive

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technology of Détente Instantanée Contrôlée (DIC), a French term for instant controlled pressure

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drop, is one of the innovative methods for isolation of volatiles. Among its various applications

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have been drying [11], decontamination [12], texturing [13], and extraction of volatile

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compounds [9, 14] and non-volatile molecules [15] from certain plants. DIC is a thermomechanical process that involves subjecting a raw material to high-pressure saturated steam for a

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short period of time followed by an abrupt pressure drop towards vacuum. It is indeed a vacuum-

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driven steam distillation. DIC enjoys several advantages over the other methods, including

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higher extract quality, no solvent use that makes it environment friendly at industrial scale, high speed operation, selectivity, auto mode capability, and performance in mild conditions. DIC is

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generally considered the best way to obtain the most representative EO with the highest overall yield from a wide range of materials.

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The objective of this study was to improve the extraction yield of hyssop EO with DIC applying solvent modifiers in the steam generator feed. To the best of our knowledge, no studies have yet been reported concerning the extraction of EOs by DIC using solvent mixtures. Applying ethanol as a green and extremely efficient extraction solvent is a desired option. Using response surface

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methodology (RSM), the optimization was performed to maximize the extraction yield. The study also aimed to compare this EO with those obtained from HD, SOX, and UAE techniques,

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with respect to total yields and their compositions.

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2. Materials and methods

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2.1. Chemicals

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Methanol 99.8% (HPLC grade) was purchased from LOBA Chemie (Mumbai, India). NaCl,

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anhydrous Na2SO4, n-nonane, sodium carbonate, gallic acid powder, 2,2-diphenyl-1picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, and n-hexane (for HPLC) were supplied by

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Merck KGaA (Darmstadt, Germany). Ethanol 96% was purchased from Bidestan Alcohol &

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Foodstuff Production (Qazvin, Iran). Deionized water purified through a Milli-Q unit from Millipore (Bedford, MA, USA) was used in the analyses. Double distilled water produced by an

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Automatic Water Still unit from Parmis Teb Azma (Tehran, Iran) was fed to the steam generator.

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2.2. Plant material

Collected in July 2016, the air-dried aerial parts (flowers, leaves, and stems) of hyssop during its

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flowering period were purchased from the Research Center for Medicinal Herbs at Ayatollah Amoly University (Mazandaran, Iran). The samples were stored in polyethylene bags at -18 °C until used. They were ground in a laboratory mill (Toosshekan Khorasan, Mashhad, Iran) and

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screened by ASTM standard sieves (Damavand Sieve, Tehran, Iran) immediately prior to extraction to prevent the loss of volatiles. 2.3. DIC apparatus and procedure

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A laboratory-scale DIC apparatus (Fig. 1) was designed and fabricated at the Iranian Research Organization for Science and Technology (IROST). It consisted of a 2-L insulated SS304 extraction vessel filled with 1/4-inch SS304 beads and equipped with a thermocouple and a pressure gauge, and a 10-L SS304 vacuum tank with a cooling water jacket connected to a water ring vacuum pump. The apparatus could be controlled with three manual valves, V1, V2, and V3.

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Saturated steam was supplied by a 3.5-L mini steam generator (SilTer, Istanbul, Turkey). Two

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traps were inserted between the vacuum tank and vacuum pump to impede the possible suction

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of volatiles into the vacuum pump throughout the process.

The sample ground was first weighed in a filter bag and then sealed and put into the extraction

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vessel. The use of the filter bag and beads hindered the flow of solid sample from the extraction

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vessel to the vacuum tank while opening the V2. The sample was initially subjected to a vacuum

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provided in the extraction vessel so as to remove the air and allow better diffusion of the heating fluid through the plant, which curtailed the time required to attain the desired processing

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pressure. The V2 was then closed and the cycle started with the injection of high-pressure saturated ethanol-water vapor into the extraction vessel to reach the selected pressure. The

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pressure was maintained for a short predetermined time, during which the temperature in the extraction vessel corresponded to that of the saturated vapor injected into the vessel. By opening the V2 the extraction vessel was connected to the vacuum tank and the cycle ended with an abrupt pressure drop towards vacuum. The above steps offer a number of advantages including adiabatic autovaporization, rapid cooling that stops thermal degradation, and expansion of the Page 5 of 40

herb texture. In order to have a multi-cycle process, the next cycles began by closing the V2 and injecting vapor after the pressure drop. The process ended by opening the V3 to establish the atmospheric pressure in the vacuum tank and collecting the condensed liquid from the tank. At

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the end of the process no volatiles were observed collected in the traps. DIC provides EO in a stable emulsion after completion of each run. For analytical purposes, a liquid-liquid extraction was performed to separate the EO from the obtained emulsion. Using a separator funnel, a volume of 100 ml extract was decanted with 50 ml n-hexane in three steps, each lasting 10 min. As a demulsifier, 1 g NaCl was added in each step to facilitate the emulsion

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breakdown. The upper phase was collected and centrifuged for 5 min at 3000 rpm to completely

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break the emulsion.

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In a rotary vacuum evaporator, the organic phase was concentrated under vacuum using a

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BÜCHI Rotvapor R-114 and BÜCHI Waterbath B-480 (BÜCHI Labortechnik AG, New Castle, USA) at 35 °C, and the aliquot was dried under an N2 stream. The dried sample was diluted with

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1 ml n-hexane (GC-grade) containing 12 µl n-nonane as an internal standard (IS). The samples

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were kept in the dark at -18 °C until GC-FID analysis. The whole treatment design is presented

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2.4. HD

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in Fig. 2.

The HD experiment was carried out using a Clevenger extractor (Ashk Shisheh, Tehran, Iran)

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with a 500-ml distillation flask for 3.5 h from the first drop of distillate. The herb powder (1 mm mean particle size) was immersed in 400 ml distilled water. The obtained EO was removed, dried over anhydrous sodium sulphate, and stored at low temperature in a tightly closed vial for further analyses. To allow comparison of the results with DIC, the HD sample was diluted, prior to GC analysis, by 1 ml n-hexane containing 12 µl n-nonane. Page 6 of 40

2.5. UAE Using a 4-L ultrasonic cleaning instrument (Alex Machine, Istanbul, Turkey), UAE was performed on 10 g of the herb mixed with 100 ml n-hexane (1:10 w/v) in a 250-ml conical flask.

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The ultrasound bath was operated at a frequency of 40 kHz for 30 min at room temperature. During the ultrasound irradiation the temperature rise was controlled by adding ice to the water bath. Following the process the extract was immediately filtered under vacuum through a filter paper (Whatman grade 1). Using a rotary vacuum evaporator, the solvent was evaporated at 35

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°C to reduce the solution volume.

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The aliquot was completely evaporated under an N2 stream. The sample was diluted with 1 ml n-

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hexane containing 12 µl n-nonane. In this method some unwanted materials such as waxes,

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cuticles, and chlorophylls may be co-extracted. These impurities must be removed prior to the GC-FID injection by a Millex-GV Syringe Filter Unit, 0.22 µm, with a hydrophilic

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Polyvinylidene Fluoride (PVDF) membrane from Millipore (Bedford, MA, USA). 2.6. SOX

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SOX was carried out on 5 g ground herb added to 250 ml n-hexane in a 500 ml volume apparatus

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(Ashk Shisheh, Tehran, Iran). The extraction was allowed to proceed until the extract became colorless (9 h). The post-processing procedure resembled that in UAE. An appropriate dilution

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ratio was applied to the GC-FID results. 2.7. Chemical analysis Separation and identification of the components were executed using GC-FID and GC-MS analyses. The GC-MS analysis was conducted in a TRACE MS system (Thermo Scientific, Waltham, MA, USA) equipped with a DB-5 fused-silica-capillary column (30 m, 0.25 mm, film Page 7 of 40

thickness of 0.25 µm). The GC conditions consisted of an oven temperature program 60-250 °C (10 min) at 5 °C/min; injector and transfer line temperatures 250 °C; carrier gas helium; flow rate 1.1 ml/min; split ratio 1:100; injection volume 0.2 µl; and ion source temperature 200 °C.

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The MS conditions included ionization energy 70 eV; mass range 40–460 amu in 0.4 s; and the data acquisition frequency 2.7 scans/min. The components were identified by comparing their retention indices (RI) with those of pure reference components as well as their mass spectra with those stored in the MS database (National Institute of Standards and Technology (NIST), Wiley, and Adams libraries). The GC-FID analysis was conducted using a YL-6500 gas chromatograph

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(Young Lin, Gyeonggi-do, Korea) equipped with a 30-m TRB-G43 fused-silica-capillary column

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(0.53 mm i.d., 3µm film thickness). Nitrogen (99.999%, Roham Gas, Tehran, Iran) was used as a

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carrier gas (4 ml/min). Injector and detector temperatures were both set at 250 °C. The column

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temperature was programmed from 60-230 °C (10 min) at 5 °C/min. A volume of 1 µl with a split ratio of 1:50 was injected manually. The peaks were identified by comparing the GC-FID

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chromatograms with those of GC-MS.

2.8. Scanning electron microscopy (SEM)

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Microstructures were observed employing a Mira II LMU SEM (Tescan SEM, Kohoutovice,

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Czech Rep.). The samples were coated with a thin gold film using a Desk Sputter Coater-DSC (Nanostructured Coatings, Tehran, Iran). A high vacuum was achieved to improve the quality of

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SEM images scanned with an acceleration tension of 15 kV. 2.9. Vapor phase ethanol measurement To ensure the presence of ethanol in the generated vapor phase until the last cycle of the optimum experiment, an additional test was carried out to separately collect the condensate after each cycle. Vacuum was restored to resume the run. As the presence of EO in the condensate Page 8 of 40

could disturb the ethanol measurement procedure, the experiment was done in the absence of the sample. Ethanol percentage in the condensate was identified using GC-FID under the following conditions: column TRB-G43, carrier gas N2, and column temperature 80 °C.

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In addition, the theoretical ethanol compositions of the vapor injected into the extraction vessel in each cycle were calculated using Aspen-Plus V10. The NRTL equation of state was used for the ethanol-water binary mixture. Three flash drums were applied to simulate the DIC process (Fig. A.1). The first unit was a bubble point flash drum simulating the steam generator, the second was an adiabatic flash drum simulating the extraction vessel, and the third was a dew

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point flash drum corresponding to the vacuum tank. They were run separately, and the output

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vapor features of each drum determined those of the input feed to the subsequent one.

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Composition of the liquid stream flowing out of the last flash drum was empirically examined.

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Fig. 3 shows the experimental and theoretical ethanol concentrations of the condensate (v%) in each cycle of the optimum experiment. The condensate ethanol concentration was equal to that

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of the vapor in the extraction vessel. The results demonstrated that ethanol existed in the vapor

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phase until the last cycle. The difference observed between the theoretical and experimental

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percentages could arise from some assumptions and simplifications in the simulation procedure.

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2.10. Experimental design RSM was used to optimize the DIC parameters. The five factors considered were saturated vapor

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pressure (P) from 1-3.5 bar, heating time per cycle (t) from 20-100 seconds, number of cycles (C) from 1-12, particle diameters (dp) from 0.25-1 mm, and water concentration in the steam generator feed (W) from 80-100 v%. Initial moisture content and sample weight were kept constant at 11.47±0.05 wt% (dry basis) and 10 g, respectively. The experimental design was based on a face-centered central composite design using 16 cube points, 10 axial points, and 6 Page 9 of 40

center points in cube, and all the 32 runs were randomly performed (Table 1). The center points were used to estimate the experimental error. The response was total yield of isolated oil (Y) expressed as the ratio of sum of the area percentages of all components to the area percentage of

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IS (ARt/IS) obtained by GC chromatograms. The goal was to reach a condition that yielded the maximum EO.

Analysis of variance (ANOVA) was performed to determine significant differences between the

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independent variables. The responses were used to develop a second-order polynomial empirical

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model as a function of independent variables for predicting the factors effects (Eq. 1). As well as

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empirical model coefficients, the regression coefficient (R2) was determined and the response

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surfaces were introduced.

Y  β0   βi X i   βii X i2   βij X i X j  ε

(1)

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i 1

where Y, β0, βi, βii, and βij represent the response variable, equation constant, linear coefficients,

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quadratic coefficients, and cross product coefficients, respectively. Both Xi and Xj are

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independent variables, ε is random error, and i and j are indices of factors. The model was built based on the variables with a 95% confidence level. The coefficients of the response surface

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equation were estimated using Minitab V16. 2.11. Determination of total phenolic content (TPC) TPCs of the EO and extract, obtained from the DIC optimum experiment and HD, were determined using the Folin-Ciocalteu method. A stock solution was prepared in methanol (5

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mg/ml) for the EO, and subsequent dilutions were made when necessary. A Folin–Ciocalteu reagent-deionized water mixture (750 µl, 1:14) was added to 50 µl sample and the reaction was stopped exactly 3 min thereafter by adding 200 µl of Na2CO3 20%. After an incubation period of

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30 min in the dark, absorbance of the reaction mixture was read at 760 nm (each measurement in triplicate) using a Lambda 25 spectrophotometer (Perkin-Elmer, Waltham, MA, USA). Instead of the sample, 50 µl methanol was used as a blank. Methanolic solutions of gallic acid (50-500 µg/ml) were used as a standard for drawing the calibration curve [16]. Finally, gallic acid absorbance vs. concentration were plotted to assess the TPC of each sample. The results were

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expressed as mg gallic acid equivalent (GAE) in 100 mg EO.

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For the extract, 20 µl of each sample, blank (deionized water), and gallic acid aqueous solutions

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(for calibration) were pipetted into separate cuvettes. A volume of 1.58 ml deionized water, 100

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µl Folin-Ciocalteu reagent, and after 8 min, 300 µl Na2CO3 20% were added. The solutions were left at 20 °C for 2 h until the absorbance of each was determined at 765 nm [17]. The results

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were expressed as mg GAE per g of dry matter.

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2.12. Determination of antioxidant activity

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The EO antioxidant activity and that of the extract, obtained from the DIC optimum experiment and HD, were determined in terms of hydrogen donating potency using DPPH. The EO

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antioxidant activity was evaluated according to Baj et al. [18] with some modifications. Different concentrations of the EO (5–20 mg/ml) were prepared in methanol. A 100-µl portion of the solution was mixed with 3.9 ml of a freshly prepared 0.06 mM DPPH methanolic solution and the mixture was allowed to stand for 30 min at room temperature for any reaction to take place. The absorbance values of the solutions were measured at 517 nm with a UV–Vis spectrometer. Page 11 of 40

The blank contained the same volume of methanol instead of the sample. The antioxidant activity was expressed as inhibition percentage of DPPH radical (I%) calculated as follows:  Ablank  Asample    100 Ablank  

(2)

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I% = 

where Ablank and Asample are the absorbance values of the control reaction and EO, respectively. The EO concentration providing 50% inhibition (IC50) was calculated from the graph plotting inhibition percentage against the EO concentration.

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For the extract, 2 ml methanolic solution of DPPH (0.1 mM) was added to 2 ml of each sample.

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The absorbance values at 518 nm were measured after 30 min at room temperature in the dark

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[19, 20]. The antioxidant activity was calculated from Eq. 2, while deionized water was used as a

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3. Results and discussion

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blank rather than the extract.

3.1. EO composition by GC-MS

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The composition of hyssop EO obtained by HD was identified by GC-MS, the chromatogram of which is presented in Fig. 4. The yield of HD was 1.2±0.06 v/w% (ml/g). Fifty-four components

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were isolated representing 99.99% of the total chemical composition, of which 53 were identified. The cis-pinocamphone (21.59%), trans-pinocamphone (7.93%), elemol (7.12%),

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bicyclogermacrene (6.58%), germacrene D (6.52%), limonene (6.36%), β-pinene (5.20%), and trans-caryophyllene (4.65%) were the major components comprising over 65% of the chromatogram. In general, the GC-FID chromatograms of all runs identified cis-pinocamphone, trans-pinocamphone, and β-pinene as three major odorous components. β-pinene has been

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identified as one of the major components of hyssop EO in previous works [21]. The two bicyclic monoterpene ketones; pinocamphone and isopinocamphone; are generally considered the main characteristic components of the oil of genus Hyssopus, though their proportion may

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vary [22, 23]. Fig. 4.

3.2. Analysis of the model

As shown in Table 2, the effects of the variables as linear, quadratic, or interaction coefficients on the response were tested for significance by ANOVA. The significant effects of the variables

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on the yield consisted in the linear terms of t and C and the interaction terms of tP, tW, PC, Pdp,

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Cdp, and CW. The quadratic terms failed to have a significant effect. Extraction is achieved

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through evaporation and autovaporization phenomena resulting from thermomechanical effects.

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The t and C signify the thermal and mechanical aspects of the DIC process, respectively [24]. An

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increase in the C may enhance the yield as explained in the following steps. The vapor generated

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in the herb leads to an expanded porous and broken structure, the advantage of which is an increased rate of mass transfer processes thanks to enhancing the effective diffusivity of the

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product and promoting the availability of the liquid in the plant. The improved availability, in

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turn, allows the EO bearing cells to contact the solvent more easily.

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In a porous/broken structure, more vapor can penetrate into the alveolated bulk at the beginning of next cycle. Hence, the herb heats more rapidly and the bulk temperature gets closer to that of the vapor. It causes more volatile compounds to be vaporized in the plant during the succeeding cycle [9, 25, 26]. In the literature, either of the variables t or P, in conjunction with C, were introduced as key factors in the DIC process. The t was a prominent factor as it contributed to Page 13 of 40

deep diffusion of the vapor within the material. Using the regression model, the predicted values of the yield were calculated and compared with its experimental values (Table 1). The R2, as a measure of the degree of fit, was high enough to

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ensure that the model represented the experimental results properly. Moreover, lack of fit was not statistically significant. A reduced fitted quadratic equation in terms of actual factors, in the original units, is formulated in Eq. 3.

EO yield = 1.044 t + 3.787 C – 0.049 tP – 0.01 tW – 0.318 PC – 4.897 Pdp + 1.588 Cdp – 0.043 (3)

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CW

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3.3. Analysis of response surfaces

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In Fig. 5, the significant interactions between the variables and extraction yield are illustrated

Fig. 5.

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with 3D response surface plots, based on the predicted model equation.

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As shown in Fig. 5a, in low Ps enhancing the t had a positive effect on the yield, while raising the P could cause the trend to reverse. The oil decomposition is induced by the increase of

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pressure and heating time [26]. The figure demonstrates that the t and P cannot be set high simultaneously. A huge volume of high-temperature saturated vapor is required to generate high

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pressure, which will raise the risk of EO thermal degradation if it persists. The negative effect of the elevated P was reinforced by increasing the t. Decreasing the P was more beneficial at high dp, because loss of the most EO molecules occurred through grinding to reach the low dp (Fig. 5b). Many researchers have concluded that sample sizes of less than 1 mm either induce volatile compounds degradation or fail to have a Page 14 of 40

significant effect on the yield [27]. On the other hand, very tiny particles may stick together and form a mass that could inhibit vapor from penetrating inside the material [28]. Increasing the C produced a positive linear effect with a moderate to high dp (Fig. 5c). Low dp

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allows the glands to be damaged, and the EO that is prone to degradation remains in the herb. Therefore, increasing the C with a low dp caused a negative impact on the yield, which indicates that the process of grinding to very small particles is not recommended.

Increasing the P had a negative effect on the yield, especially in high C, probably due to thermal degradation (Fig. 5d). The more C, the higher the yield, especially at low Ps. Our results are in

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accordance with those obtained by Ranjbar et al. [29], who argued that higher cycle numbers

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produced more desirable results at low pressures.

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In a low to moderate C, an increase in the W enhanced the yield (Fig. 5e). Once the C was raised,

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the trend reversed. In fact, the ethanol presence boosted the effect of increasing the C. Solidliquid extraction takes place in two distinct stages governed by two different modes of mass

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transfer. The EOs located on the naturally broken glands (exogenous sites) are freely present on

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the material surface and easily extracted with rapid free diffusion mechanism on the plant

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surface. The oil extracted in the second step, subsequent to the solute removal from the material surface, is probably regulated by osmosis with slow internal diffusion from the endogenous

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storage sites of oil toward the plant surface [30, 31]. The figure supports the assumption that hydrophilic components are located in exogenous sites and easily extracted with water (polar

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solvent) in low C [32]. Not as easily extractable as hydrophilic ones, lipophilic components are probably located in endogenous storage sites of the EO deep inside the herb, and hence their extraction required a higher C using a less polar solvent. The interaction effect between t and W is reflected by the surface distortion in Fig. 5f. The yield

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was increased by enhancing the t in low W (high ethanol concentrations); however, this effect was reversed as the W was raised. Indeed, in low t steam was efficient, while in high t the existence of ethanol in the vapor was more useful. The EO molecules situated deep inside the

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herb are heated and evaporated for longer t. Given the assumption that lipophilic components mostly reside deep in the herb, using ethanol in the vapor would be more efficient. It should be noted that the boiling point of ethanol-water vapor is lower than that of each in isolation, which makes it possible to reduce the risk of thermal degradation in high t. Steam was found to be more appropriate in low t since its higher heat capacity allows more heat exchange with the herb

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within that short time.

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3.4. Optimization of DIC process

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Optimization of effective parameters was performed by RSM applying the regression model. The

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optimum extraction parameters were P=1 bar, t=100 s, C=12, dp=1 mm, and W=80 v%. The high

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value of C indicated that pressure drop was the main mechanism of DIC [33], explained by

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improvement of the autovaporization process and the alveolation effect [14]. The higher t and C values imply that the majority of EO is located in endogenous sites where its molecules have

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strong interactions with the solid matrix, so it can be extracted by internal diffusion and

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autovaporization. The total yield (in ARt/IS), obtained experimentally under this optimum condition (39.04), turned out to be adequately close to the model prediction (37.56).

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3.5. Comparison with other techniques A comparison of the total yields, extraction times, and chemical compositions of the hyssop EO obtained from DIC, DIC residual, HD, UAE, and SOX is shown in Table 3. The categorized components were compared in terms of the ratio of the area percentage of each targeted component to that of IS (AR/IS). Only the easily identified components, accounting for over Page 16 of 40

80% of the total identified constituents of the EO, were included.

The DIC-EO and HD-EO were bright yellow and greenish yellow, respectively. The UAE-EO

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(bright green) and SOX-EO (dark green) were much more opaque than the others. DIC minimized the co-extraction of waxes and other undesired compounds. The odor of the DIC-EO was excellent in quality, probably thanks to the conditions provided by this method wherein the odorous and flavoring constituents (oxygenated components) were preserved in heat-sensitive herbs.

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EO composition may vary substantially within a single species depending on several factors

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particularly extraction technology [7, 21]. Taking the lower extraction time into account, the

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higher total amount of oxygenated components extracted by DIC was considerable. The total

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non-oxygenated components in DIC was greater due to the high amount of monoterpenes.

D

Monoterpenes are light volatile components, and oxygenated monoterpenes are the most

TE

valuable group in the EOs. Their exogenous state and higher volatility result in a greater amount of monoterpenes and oxygenated monoterpenes in condensate [26]. Moreover, in comparison

EP

with HD and SOX, the short-time contact between the plant and heated zones of the apparatus in

CC

DIC hinders loss and degradation of the volatile compounds [33]. The addition of ethanol, as a less polar modifier, enhances the extraction of non-oxygenated components, especially the light

A

ones (i.e. monoterpenes). The ratios of extracted sesquiterpenes to total extracted EO obtained from sum of the total oxygenated and non-oxygenated components in Table 3 were as follows: DIC=11.04%, HD=11.66%, UAE=22.73%, and SOX=19.93%. UAE had the highest ratio by virtue of its direct effect of ultrasonic waves’ radiation on the texture. As a result, the availability of molecules increases, and sesquiterpenes can be extracted as easily as light components. The Page 17 of 40

use of less polar solvent encourages the extraction of non-oxygenated components as well [17]. In SOX the long-term contact of materials with hot solvent leads to degradation of other components into sesquiterpenes. This method provides sufficient time (9 h) to perfectly extract

SC RI PT

such heavy, non-volatile compounds [26]. The lower content of sesquiterpenes in the DIC-EO is probably caused by short extraction time and reduced thermal degradation. The ratio of oxygenated sesquiterpenes to total EO was higher in SOX (8.64%) and UAE (6.92%) methods. The values for HD (3.46%) and DIC (2.30%) were not remarkably different.

Low contents of some components in the DIC-EO could possibly result from poor condensation

U

[34]. Some components including α-thujene, α-pinene, 3-octanone, and linalool were found

N

abundant in the DIC-EO, while they were negligible or low in the EO of other methods. This

A

could come about due to loss of volatile components as well as chemical changes. The changes

M

were probably caused by hydrolysis reactions as a consequence of water presence in HD [9], long extraction time in SOX [14], and materials structure breakdown induced by cavitation in

D

UAE [35].

TE

As a high-temperature, long-lasting process, HD may cause chemical alterations in oil

EP

components and loss of volatile molecules [26, 31]. High consumption of organic solvents and extraction of undesirable non-polar components are among the disadvantages of SOX [17].

CC

Having the lowest total extraction yield, UAE did not extract selectively and thus did extract cuticular waxes and lipids as well. DIC turned out to be most effective compared to the other

A

methods since it enjoyed the highest overall extraction yield, more valuable EO according to more oxygenated components and less non-oxygenated sesquiterpenes, and quicker kinetics due to the less time required for the extraction process. The remaining EO, which existed in a small quantity in the residual herb after the DIC optimum

Page 18 of 40

experiment, was obtained by HD to identify the DIC effectiveness. The yield of major components is shown in Table 3. Monoterpenes, not identified in the residual EO, are the most volatile components and are almost entirely eliminated by DIC. The amount of oxygenated

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monoterpenes was lower than that in the direct EO, indicating the infeasibility of releasing all components by autovaporization. Meanwhile, the amount of sesquiterpenes and oxygenated sesquiterpenes increased. The above facts confirm the assumption that the majority of monoterpenes and oxygenated monoterpenes are located in exogenous sites of the oil-containing glands, while sesquiterpenes and oxygenated sesquiterpenes are situated mainly in the

U

endogenous sites. The exogenous components and a portion of the endogenous ones are directly

M

A

N

isolated by autovaporization, while those enclosed in deeper sites cannot be easily carried away.

3.6. Impact of DIC on microstructure of plant

D

A plant’s natural tissue structure resists solvent diffusion, which slows down the extraction

TE

process. It is usually desirable to augment the mass transfer phenomena by improving diffusivity

EP

and permeability within the matrix. To highlight the impact of DIC, the morphological structures of the raw material and DIC residue at optimum condition were compared in Fig. 6. The raw

CC

material matrix had a compact and fairly homogeneous internal structure (Figs. 6a & 6b). SEM unveiled an expansion in the herbal structure with appearance of cavities indicating a partial

A

collapse of EO-containing glands after DIC (Figs. 6c & 6d). The vapor generated by the instantaneous autovaporization of volatiles and water induces the blowing and rupture of cell walls, making a more porous structure and increasing the availability and/or the initial accessibility of some high added-value compounds. Changing the plant’s cellular structure helps to achieve a more efficient extraction process. DIC resulted in profound alterations at Page 19 of 40

cytohistological levels, which explain the observed effectiveness. Studies have previously shown that DIC expanded and broke the cells structure thus making the tissue more porous [31, 34]. The same observation was cited by Spiro and Chen [36], who reported that the EO synthesized in

SC RI PT

secretory cells was not released unless an external factor damaged the microstructure. Fig. 6.

3.7. TPC and antioxidant activity

U

The TPC value was higher in the DIC-EO than in the HD-EO. A slightly higher antioxidant

N

activity was reached in the DIC-EO than in the HD-EO in a shorter period of time (20 min vs.

A

3.5 h). The superior conditions for preserving heat-sensitive constituents account for the better

M

results achieved in DIC. TPC and antioxidant activity of the HD extract were remarkably higher than those of the DIC extract. It shows that phenolic compounds generally remain in the HD

D

flask and do not distill. Unlike HD, the extraction time in DIC is inadequate to isolate phenolic

EP

Conclusions

TE

compounds. This fact proves that DIC is a method to selectively extract the EOs.

CC

Applying an ethanol-water mixture was an efficient method to improve the DIC performance. It was proved by successful optimization of the hyssop EO extraction using RSM. The optimum

A

DIC experiment was compared with three other techniques including HD, SOX, and UAE. The EO isolated by DIC was the best, both quantitatively and qualitatively. It indicates that DIC can be an appropriate alternative not only to classical techniques (i.e. HD or SOX), but also to the recently introduced method, UAE. SEM of the optimum DIC residue illustrated remarkable

Page 20 of 40

modifications in the texture and EO-bearing trichomes. TPC and antioxidant activity of the DICEO were greater than those of the HD-EO.

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Acknowledgments The South Tehran Branch, Islamic Azad University and the Iranian Research Organization for Science and Technology (IROST) are greatly appreciated for their financial support. Our special thanks also go to Dr. Z. Bashiri-Sadr for his valuable contribution to the experiments.

U

Declarations of interest: none

N

This research did not receive any specific grant from funding agencies in the public, commercial,

A

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EP

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D

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A

or not-for-profit sectors.

Page 21 of 40

References

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[1] H. Kazazi, K. Rezaei, Effect of various parameters on the selective extraction of main components from hyssop using supercritical fluid extraction (SFE), Food Sci. Technol. Res. 15 (2009) 645-652. [2] O. Jahantigh, F. Najafi, H. Naghdi Badi, R.A. Khavari-Nejad, F. Sanjarian, Essential oil composition of Hyssop (Hyssopus officinalis L.) under salt stress at flowering stage, J. Essent. Oil Res. 28 (2016) 458-464. [3] Y. Hristova, J. Wanner, L. Jirovetz, I. Stappen, I. Iliev, V. Gochev, Chemical composition and antifungal activity of essential oil of Hyssopus officinalis L. from Bulgaria against clinical isolates of Candida species, Biotechnol. Biotec. Eq. 29 (2015) 592-601. [4] S. Kizil, V. Guler, S. Kirici, M. Turk, Some agronomic characteristics and essential oil composition of Hyssop (Hyssopus officinalis L.) under cultivation conditions, Acta Sci. PolHortoru. 15 (2016) 193-207. [5] G. Zawiślak, The Chemical Composition Of Essential Hyssop Oil Depending On Plant Growth Stage, Acta Sci. Pol-Hortoru. 12 (2013) 161–170. [6] V.D. Zheljazkov, T. Astatkie, A.N. Hristov, Lavender and hyssop productivity, oil content, and bioactivity as a function of harvest time and drying, Ind. Corp. Prod. 36 (2012) 222-228. [7] N. Kara, H. Baydar, Morphogenetic, ontogenetic and diurnal variabilities of hyssop (Hyssopus officinalis L.), Res. Crop. 13 (2012) 661-668. [8] M. Khajenoori, A. Haghighi Asl , F. Hormozi, M.H. Eikani, H. Noori Bidgoli, Subcritical Water Extraction Of Essential Oils From Zataria Multiflora Boiss, J. Food Process Eng. 32 (2009) 804-816. [9] B. Berka-Zougali, A. Hassani, C. Besombes, K. Allaf, Extraction of essential oils from Algerian myrtle leaves using instant controlled pressure drop technology, J. Chromatogr. A 1217 (2010) 6134-6142. [10] K. Allaf, P. Vidal, Feasibility Study of a New Process of Swell-drying by Instant Decompression Toward Vacuum of in Pieces Vegetables in View of a Rapid Re-hydration, Gradient Activity Plotting University of Technology of Compiegne UTC N° CR/89/103, Industrial Partner SILVA-LAON, 1988. [11] J. Yi, P. Wang, J. Bi, X. Liu, X. Wu, Y. Zhong, Developing Novel Combination Drying Method for Jackfruit Bulb Chips: Instant Controlled Pressure Drop (DIC)-Assisted Freeze Drying, Food Bioprocess Tech. 9 (2016) 452-462. [12] S. Mounir, N. Albitar, K. Allaf, DIC Decontamination of Solid and Powder Foodstuffs, in: T. Allaf, K. Allaf (Eds.) Instant Controlled Pressure Drop (D.I.C.) in Food Processing: From Fundamental to Industrial Applications, Springer New York, New York, NY, 2014, pp. 83-94. [13] S. Mounir, D. Halle, K. Allaf, Characterization of pure cheese snacks and expanded granule powders textured by the instant controlled pressure drop (DIC) process, Dairy Sci. Technol. 91 (2011) 441. [14] G. Naji, H. Mellouk, S.A. Rezzouget, K. Allaf, Extraction of Essential Oils of Juniper berries by Instantaneous Controlled Pressure-Drop: Improvement of DIC Process and Comparison with the Steam Distillation, J. Essent. Oil Bear. Pl. 11 (2008) 356-364.

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[15] V. Sánchez-Valdepeñas, E. Barrajón, S. Vegara, L. Funes, N. Martí, M. Valero, D. Saura, Effect of instant controlled pressure drop (DIC) pre-treatment on conventional solvent extraction of phenolic compounds from grape stalk powder, Ind. Corp. Prod. 76 (2015) 545-549. [16] G. Hatipoğlu, M. Sökmen, E. Bektaş, D. Daferera, A. Sökmen, E. Demir, H. Şahin, Automated and standard extraction of antioxidant phenolic compounds of Hyssopus officinalis L. ssp. angustifolius, Ind. Corp. Prod. 43 (2013) 427-433. [17] E. Feyzi, M.H. Eikani, F. Golmohammad, B. Tafaghodinia, Extraction of essential oil from Bunium Persicum (Boiss.) by instant controlled pressure drop, J. Chromatogr. A 1530 (2017) 5967. [18] T. Baj, E. Sieniawska, R. Kowalski, L. Swiatek, M. Modzelewska, T. Wolski, Chemical composition and antioxidant activity of the essential oil of Hyssop (Hyssopus officinalis L. ssp. Officinalis). Part II. Free radical scavenging properties, Ann. Univ. Mariae Curie-Skłodowska 24 (2011) 103-109. [19] F. Pourmorad, S.J. Hosseinimehr, N. Shahabimajd, Antioxidant activity, phenol and flavonoid contents of some selected Iranian medicinal plants., Afr. J. Biotechnol. 5 (2006) 11421145. [20] L.L. Yu, K.K. Zhou, J. Parry, Antioxidant properties of cold-pressed black caraway, carrot, cranberry, and hemp seed oils, Food Chem. 91 (2005) 723-729. [21] F. Fathiazad, S. Hamedeyazdan, A review on Hyssopus officinalis L.: Composition and biological activities, Afr. J. Pharm. Pharmacol. 5 (2011) 1959-1966. [22] D. Fraternale, D. Ricci, F. Epifano, M. Curini, Composition and Antifungal Activity of Two Essential Oils of Hyssop (Hyssopus officinalis L.), J. Essent. Oil Res. 16 (2004) 617-622. [23] G. Mazzanti, L. Battinelli, G. Salvatore, Antimicrobial properties of the linalol‐rich essential oil of Hyssopus officinalis L. var decumbens (Lamiaceae), Flavour Fragr. J. 13 (1998) 289-294. [24] T. Allaf, V. Tomao, C. Besombes, F. Chemat, Thermal and mechanical intensification of essential oil extraction from orange peel via instant autovaporization, Chem. Eng. Process. 72 (2013) 24-30. [25] M. Kristiawan, V. Sobolik, M. Al-Haddad, K. Allaf, Effect of pressure-drop rate on the isolation of cananga oil using instantaneous controlled pressure-drop process, Chem. Eng. Process. 47 (2008) 66-75. [26] M. Kristiawan, V. Sobolik, K. Allaf, Isolation of Indonesian Cananga Oil by Instantaneous Controlled Pressure Drop, J. Essent. Oil Res. 20 (2008) 135-146. [27] M. Khajenoori, A. Asl Haghighi, M.H. Eikani, Subcritical Water Extraction of Essential Oils from Trachyspermum ammi Seeds, J. Essent. Oil Bear. Pl. 18 (2015) 1165-1173. [28] M.H. Eikani, F. Golmohammad, Z.B. Sadr, H.S. Amoli, M. Mirza, Optimization of Superheated Water Extraction of Essential Oils from Cinnamon Bark Using Response Surface Methodology, J. Essent. Oil Bear. Pl. 16 (2013) 740-748. [29] N. Ranjbar, M.H. Eikani, M. Javanmard, F. Golmohammad, Impact of instant controlled pressure drop on phenolic compounds extraction from pomegranate peel, Innov. Food Sci. Emerg. Technol. 37 (2016) 177-183. [30] B. Ben Amor, K. Allaf, Impact of texturing using instant pressure drop treatment prior to solvent extraction of anthocyanins from Malaysian Roselle (Hibiscus sabdariffa), Food Chem. 115 (2009) 820-825. [31] S.A. Rezzoug, Optimisation of steam extraction of oil from maritime pine needles, J. Wood Chem. Technol. 29 (2009) 87-100.

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[32] M. Kristiawan, V. Sobolik, K. Allaf, Isolation of Indonesian cananga oil using multi-cycle pressure drop process, J. Chromatogr. A 1192 (2008) 306-318. [33] H. Mellouk, A. Meullemiestre, Z. Maache-Rezzoug, K. Allaf, S.-A. Rezzoug, Isolation of Volatiles from Oak Wood (Quercus alba) by a Thermomechanical Process: Screening of some Processing Parameters, Sep. Sci. Technol. 48 (2013) 1851-1858. [34] C. Boutekedjiret, R. Belabbes, F. Bentahar, J.M. Bessière, S.A. Rezzoug, Isolation of Rosemary Oils by Different Processes, J. Essent. Oil Res. 16 (2004) 195-199. [35] P. Mašković, V. Veličković, M. Mitić, S. Đurović, Z. Zeković, M. Radojković, A. Cvetanović, J. Švarc-Gajić, J. Vujić, Summer savory extracts prepared by novel extraction methods resulted in enhanced biological activity, Ind. Corp. Prod. 109 (2017) 875-881. [36] M. Spiro, S.S. Chen, Kinetics of solvent extraction of essential oil from rosemary leaves, Flavour Fragr. J. 9 (1994) 187-200.

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T P

V1

Handy Button 2

3 3

Steam P Feed On/Off

V2

P

Steam

V4

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P

7

1

5

U

4

Water Out

V6

N

V5

6

M

V3

A

Water In

TE

D

Sample Outlet

Fig. 1

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Fig. 1. Schematic of DIC apparatus; 1) Steam generator, 2) Handy steam injector, 3) Extraction vessel, 4) Vacuum tank, 5 & 6) Trap, 7) Vacuum pump, V1) Steam generator valve, V2) Instant

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connection valve, V3) Vacuum tank drain valve, V4) Vacuum pump valve, V5 & V6) Traps drain

A

valves, P) Pressure gauge, T) Temperature gauge

Page 25 of 40

Raw Material: Hyssop

Grinding, Sieving

UAE

Residual DIC Treated Leaves

DIC

Reference Essential Oil & From Residual

Direct Extracted Essential Oil Emulsion

Scanning Electron Microscopy

Vacuum Filteration, Evaporation & N2 Stream

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Decantation, Vacuum Distillation & Centrifugation

A

N

GC-FID GC-MS

M

Direct DIC Essential Oil

Assessment & Analysis

Fig. 2

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D

Determination of TPC and Antioxidant Activity

DIC Extraction of Essential Oil

A

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Fig. 2. Protocol of experiments

Page 26 of 40

SOX

SC RI PT

HD

Evaporation, Centrifugation, Filteration & N2 Stream

Experimental Theoretical 80 70

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Ethanol v%

60 50 40 30 20 10

1

2

3

4

5

6

7

9

10

11

N

C

8

U

0

M

A

Fig. 3

A

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TE

D

Fig. 3. Ethanol concentration of condensate per cycle in the DIC optimum experiment

Page 27 of 40

12

SC RI PT U N A M D

Fig. 4

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Fig. 4. GC-MS chromatogram 1) α-Thujene 2) α-Pinene 3) Sabinene 4) β-Pinene 5) 3-Octanone 6) β-Myrcene 7) p-Cymene 8) Limonene 9) 1,8-Cineole 10) Sabinene hydrate 11) Linalool

EP

12) Pinocarveol 13) trans-Pinocamphone 14) Pinocarvone 15) cis-Pinocamphone 16) Myrtenol 17) β-Bourbonene 18) Methyl eugenol 19) trans-Caryophyllene 20) α-Humulene 21)

A

CC

Alloaromadendrene 22) Germacrene D 23) Bicyclogermacrene 24) Elemol

Page 28 of 40

Yield

5

SC RI PT

10

1

0

2

30

60

90

P

A

N

U

t

3

EP

TE

D

M

(a)

15

A

CC

10

Yield

5 0 1

2

P

0.4 3

(b) Page 29 of 40

0.6

0.8

1.0

dp

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15 10

Yield 5

1.0

0.8

0.6

0 0

0.4

4

8

12

A

N

U

C

dp

TE

D

M

(c)

EP

15

A

CC

10

Yield

5

12 8

0 1

4 2

P

3

(d) Page 30 of 40

0

C

8

Yield 4

0

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12

100

90

0

4

8

12

W

A

N

U

C

80

TE

D

M

(e)

A

CC

EP

10

Yield

5

100 90

0 30

60

t

90

(f)

Fig. 5 Page 31 of 40

80

W

Fig. 5. Surface plots of the yield (ARt/IS) as a function of a) t & P, b) P & dp, c) C & dp, d) P & C, e) C & W, f) t & W. For each plot the other variables were fixed at “0” level-all experiments at

A

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sample weight of 10 g & initial water content of 11.47±0.05 wt% (db)

Page 32 of 40

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EP

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(a)

(b)

Page 33 of 40

D

TE

EP

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A

(c)

Page 34 of 40

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SC RI PT U N A M D TE EP CC A

(d)

Fig. 6. Herb texture for raw material at a) 300 x & b) 2 kx, and for DIC residue at optimum Page 35 of 40

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condition at c) 300 x & d) 3 kx

Page 36 of 40

Table 1. RSM experimental design with empirical and predicted yields C

dp (mm)

W (v%)

3.5 1 2.2 2.2 2.2 2.2 1 3.5 2.2 2.2 1 2.2 1 2.2 2.2 1 3.5 1 2.2 1 3.5 3.5 1 3.5 2.2 2.2 3.5 3.5 1 2.2 3.5 2.2

100 20 60 20 60 60 20 20 60 60 100 60 100 60 60 20 100 100 60 60 20 60 100 20 60 60 100 20 20 100 100 60

1 1 12 7 7 7 12 12 1 7 12 7 1 7 7 12 1 1 7 7 1 7 12 1 7 7 12 12 1 7 12 7

0.25 1 0.625 0.625 0.625 0.625 1 1 0.625 0.625 1 0.25 0.25 0.625 0.625 0.25 1 1 0.625 0.625 1 0.625 0.25 0.25 0.625 0.625 1 0.25 0.25 0.625 0.25 1

100 80 90 90 80 90 100 80 90 100 80 90 80 90 90 80 80 100 90 90 100 90 100 80 90 90 100 100 100 90 80 90

N

A

M

D

TE

Yield Experimental 2.06 3.14 6.06 3.65 4.67 6.61 20.92 8.99 1.31 8.12 39.04 4.41 7.65 2.26 4.90 2.39 1.52 8.24 5.10 5.74 11.98 5.48 2.68 1.56 3.35 5.44 3.83 4.05 6.86 2.05 3.89 5.79

CC A

Page 37 of 40

Predicted 2.869 2.307 6.675 2.536 7.128 4.248 20.452 8.769 1.239 6.205 37.562 1.253 7.302 4.248 4.248 2.273 1.109 7.597 4.248 9.019 12.345 2.744 2.789 2.195 4.248 4.248 3.820 5.304 7.359 3.707 4.206 9.490

SC RI PT

t (s)

U

P (bar)

EP

Trial 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

Table 2. Analysis of variance with regression coefficients for actual variables p-value

10.61

0.000

β0 (constant)

-

-

-

-

0.357

Linear

5

268.21

53.642

7.78

0.002

t P C dp W Quadratic t2 P2 C2 dp2 W2 Interaction tP tC tdp tW PC Pdp PW Cdp CW dpW Residual Error Lack of Fit Pure Error R2 R2 (adj)

1 1 1 1 1 5 1 1 1 1 1 10 1 1 1 1 1 1 1 1 1 1 11 6 5

188.79 9.31 54.82 1.67 10.01 122.98 3.12 7.62 0.00 3.11 14.40 814.53 97.38 17.88 2.39 248.22 76.33 84.31 23.30 171.75 88.92 3.88 75.87 63.75 12.12

188.790 9.311 54.819 1.667 10.013 24.597 3.119 7.617 0.005 3.109 14.400 81.453 97.381 17.884 2.387 248.220 76.330 84.314 23.297 171.746 88.924 3.881 6.898 10.625 2.425

M

D

A

CC

EP

TE

U

Model

SC RI PT

Adjusted Mean Square 73.188

F-Value

20

Adjusted Sum of Squares 1463.77

N

Degree of Freedom

A

Source

Page 38 of 40

27.37 1.35 7.95 0.24 1.45 3.57 0.45 1.10 0.00 0.45 2.09 11.81 14.12 2.59 0.35 35.99 11.07 12.22 3.38 24.90 12.89 0.56 4.38 -

0.000 0.270 0.017 0.633 0.254 0.037 0.515 0.316 0.980 0.516 0.176 0.000 0.003 0.136 0.568 0.000 0.007 0.005 0.093 0.000 0.004 0.469 0.063 0.9507 0.8611

Table 3. Comparative composition of hyssop EO extracted by different methods (in AR/IS) Components

DIC

DIC Residual

HD

UAE

SOX

927.2 934.9 975.1 981.1 992.3 1025.8 1032.1

A

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TE

D

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A

N

U

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Monoterpenes α-Thujene 0.73 nd* 0.03 nd 0.01 α-Pinene 0.57 nd 0.05 nd 0.01 Sabinene 0.61 nd 0.12 0.01 0.05 β-Pinene 11.54 nd 0.76 0.02 0.18 β-Myrcene 0.37 nd 0.2 0.01 0.06 p-Cymene 0.08 nd 0.09 0.01 0.03 Limonene 0.09 nd 0.55 0.03 0.17 Sum 13.99 0 1.8 0.08 0.51 Oxygenated Monoterpenes 3-Octanone 0.1 nd nd nd nd 1,8-Cineole 0.09 nd 0.02 0.002 0.01 Sabinene hydrate 0.44 0.13 0.1 0.01 0.07 Linalool 6.57 0.05 0.35 0.03 0.19 Pinocarveol 0.04 nd 0.02 nd 0.01 trans-Pinocamphone 0.72 0.53 0.64 0.08 0.39 Pinocarvone 0.24 0.18 0.22 0.03 0.13 cis-Pinocamphone 4.96 3.01 4.05 0.46 2.58 Myrtenol 0.49 0.13 0.1 0.01 0.08 Methyl eugenol 0.23 0.04 0.05 0.01 0.08 Sum 13.88 4.07 5.55 0.632 3.54 Sesquiterpenes β-Bourbonene 2.78 1.60 0.09 0.01 0.04 trans-Caryophyllene 0.18 1.11 0.19 0.03 0.2 α-Humulene 0.05 0.41 0.06 0.01 0.06 Alloaromadendrene 0.14 1.08 0.14 0.03 0.15 Germacrene D 0.21 2.37 0.29 0.08 0.37 Bicyclogermacrene 0.19 2.26 0.24 0.07 0.31 Sum 3.55 8.83 1.01 0.23 1.13 Oxygenated Sesquiterpenes Elemol 0.74 7.88 0.3 0.07 0.49 17.54 8.83 2.81 0.31 1.64 Total non-oxygenated components 14.62 11.95 5.85 0.702 4.03 Total oxygenated components 20 min 3.5 h 3.5 h 30 min 9h Extraction Time Total yield (ARt/IS) 39.04 1.74 10.98 1.58 7.71 a Retention indices relative to C9-C23 n-alkanes calculated on DB-5 capillary column * nd: not detected

RIa

Page 39 of 40

985.8 1036.7 1068.3 1101.4 1145.3 1167.1 1168.7 1186.4 1203.0 1405.3

1386.2 1422.4 1455.5 1463.3 1484.5 1500.0

1553.2

Table 4. IC50 and TPC values for EOs and extracts of DIC and HD DIC

HD Extract

EO

Extract

TPC

2.6 mg GAE/ 100 mg EO

56.76 mg GAE/ 100 g db

1.79 mg GAE/ 100 mg EO

IC50 mg/ml

36.73

851.07

39.24

6430.26 mg GAE/ 100 g db 0.6

A

CC

EP

TE

D

M

A

N

U

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EO

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