Exploitation of apiaceae family plants as valuable renewable source of essential oils containing crops for the production of fine chemicals

Industrial Crops and Products 54 (2014) 70–77

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Exploitation of apiaceae family plants as valuable renewable source of essential oils containing crops for the production of fine chemicals Epameinondas Evergetis, Serkos A. Haroutounian ∗ Chemistry Laboratory, Agricultural University of Athens, Iera odos 75, Athens 11855, Greece

a r t i c l e

i n f o

Article history: Received 30 October 2013 Received in revised form 3 January 2014 Accepted 5 January 2014 Available online 30 January 2014 Keywords: Apiaceae Essential oil Fine chemicals Industrial crops ␣-Pinene ␤-phellandrene

a b s t r a c t Twenty two Apiaceae taxa retrieved from Greek herbal biodiversity were collected and their potency as essential oil (EO) containing crops and novel renewable sources for the production of fine chemicals (FCs) was evaluated. Their EOs and FCs production potentials were estimated on the basis of various experimental and field data, identifying the Bupleurum fruticosum plant as an outstanding source for its EO and the molecules of ␣-pinene and limonene in the form of racemic mixtures. Its production capacity per hectare is estimated to exceed the 500 L for EO, 200 L for ␣-pinene and 100 L for limonene. Furthermore, the following six taxa were identified for first time as potential EOs producing industrial crops: the Greek endemics Sclerochorton junceum, Laserpitium pseudomeum and Pimpinella rigidula and the indigenous Seseli montanum, Oenanthe pimpinelloides and Thapsia garganica. Finally, the Greek endemic plants Geocarym capillifolium, G. parnassicum and Seseli parnassicum as well as the indigenous Selinum silaifolium were determined as potent renewable sources for the isolation of twenty commercially important FCs such as aldehydes, aromatic compounds, saturated and unsaturated hydrocarbons, monoterpenes and sesquiterpenes. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Various products and byproducts of petroleum industry are inherently utilized as inexhaustible sources of easily accessible, cheap precursors for the industrial preparation of a broad variety of chemicals. However, during the last decades their extended use has raised several availability and environmental impact issues. The latter are worsening in the course of the 21st century, generating new trends among the consumers and industries as well (Salimon et al., 2012). Among a broad variety of alternative sources considered, the agricultural commodities–recognized for longtime by U.S. Congress as potential sources of industrial raw materials–constitute a sustainable source of industrial crops emerged as a new source for strategic and essential industrial raw materials (U.S. Congress, 1991). The introduction of green chemistry by Anastas and Farris (1994) set the corner stone of relative conceptions, which soon after their fundamental framework definition (Anastas and Warner, 1998) became applicable in the form of green extraction, leading to the production of various bioactive chemicals through environmentally sound methods (Chemat et al., 2012). The subsequent

∗ Corresponding author. Tel.: +30 210 529 4247/529 4246; fax: +30 210 529 4265. E-mail address: [email protected] (S.A. Haroutounian). 0926-6690/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2014.01.009

application of biotechnological procedures in the form of biorefineries greatly contributed towards the goal of decreasing the industry’s dependency on petroleum products and provided products which are conceived as environmentally safe and cost effective. Currently, several products of this category serve as renewable raw materials for numerous fine chemicals (FCs) producing industries. A prioritization of these compounds has outlined the isoprene derivatives as a near-term opportunity and terpenes as future valueadded bio-based chemicals (Erickson et al., 2012). Among the latter, the simple acyclic monoterpenes constitute molecules of classical chemistry that are similar to the unsaturated hydrocarbons already known from oil and gas industries (Behr and Johnen, 2009). The essential oils (EOs) account as an inherently used source of natural terpenes, utilized from the very early beginning of human civilization in the form of aromatic crops. Their first documented production is localized at ancient Egypt during 3rd millennium BC (Scott, 2005), while on early 2nd millennium BC their use was also reported in Greece, since two of the first words deciphered from Linear B concerned coriander (Coriandrum sativum L.) and cumin (Cuminum cyminum L.) (Constance, 1971). Nowadays, the EOs are established raw materials for food, beverages, cosmetics, agrochemicals and pharmaceuticals industries (Scott, 2005; Breitmaier, 2006; Newman and Crag, 2007; Lucera et al., 2012; Rajashekar et al., 2012). Despite their importance for numerous industrial purposes, the EOs production in quantities exceeding the one ton per year

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Table 1 Annual production Volume and Value of commercially established Apiaceae EOs producing crops (data obtained from Douglas et al., 2005). Species

EO source

Volume (t)

Value (US$ 103 )

Anethum graveolens

Leaves Seeds Seeds Seeds Roots Aerial parts Seeds Aerial parts Seeds and roots Seeds Seeds and leaves Leaves Seeds Seeds Gum Resin Resin Seeds (sweet) Seeds (bitter) Roots Aerial parts Seeds Aerial parts and seeds Aerial parts Seed Seeds Seeds and leaves Seeds

114 23 25 0.8 4 N/A 30 0.8 N/A 29 710 N/A 15 9 0.2 3 N/A 255 28 2 2 0.9 N/A

800 200 100 880 3080 N/A 1500 60 N/A 1000 49700 N/A 900 1230 N/A 1035 N/A 7700 700 1600 712 N/A N/A

4 8 26 N/A 25

560 1162 700 N/A 300

A. sowa Angelica archangelica Anthriscus cerefolium Apium graveolens Bunium persicum Carum carvi Coriandrum sativum Crithmum maritimum Cuminum cyminum Daucuc carota Dorema ammoniacum Ferula assafoetida F. gummosa Foeniculum vulgare Levisticum officinale

Myrrhis odorata Petroselinum crispum Pimpinella anisum Smyrnium olusatrum Trachyspermum copticum

is limited to less than 60 cultivated taxa (Lubbe and Verpoorte, 2011), despite that the mere commercial importance of EOs isolated from several hundreds (among the 3000 taxa) has been studied and revealed todate (Sankarikutty and Narayanan, 1993). Recent studies were further defined as two hundred precuts the number of commercially important EOs which are produced and traded internationally in volumes raging from 20 to 30,000 tons for orange oil to less than 100 kg for the EOs of selected flowers (Douglas et al., 2005). Since the number of EOs containing plant families is limited to nine, which globally refer to approximately 17,500 taxa and account almost 5% of the herbal biodiversity (Lawrencet, 2000), it is safe to assume that numerous crops with potential industrial application may also be traced within the EOs of the remaining 14,500 taxa of the aromatic herbal biodiversity. Research towards this direction is also advocated by the extensive exploitation the EOs of wild plant populations. The present report aspires to unveil potential industrial crops from Greece flora through the detailed investigation of the indigenous aromatic plants biodiversity. For this purpose, among the EO producing plant families of Greece we have identified the Apiacae (Umbelliferae) plants as very intriguing species of rich biodiversity that includes numerous endemic species (Georghiou and Delipetrou, 2010). The Apiaceae family has almost 3600 species worldwide (Pimenov and Leonov, 1993) and stands as one of the least investigated plant families, since only the 4% (almost 150 species distributed in 69 genera) of family’s taxa have been studied in respect to their EOs isolation and composition. Douglas et al. (2005) was identified as EO producing crops twenty-one Apiaceae taxa, which are annotated in Table 1. In Greece, the Apiaceae plants are well represented accounting 75 genera with 254 included species distributed in all three subfamilies. The present study concerns the investigation – as EOs and FCs producing crops – of 22 species distributed among 16 genera of Greece, providing a firm background for the exploitation of Apiaceae wild biodiversity of the region.

Fig. 1. Collections locations (a: Smolikas, b: Oiti, c: Parnassos, d: Elikon, e: Athens, f: Epidaurus, g: Parnon, h: Leonidio, i: Molai, j: S. Crete).

2. Materials and methods 2.1. Plant materials Herbal material retrieved from 22 different species of the Apiaceae family, Apioideae subfamily belonging to five tribes and 16 different genera, was used in the framework of this study. Nine of them are endemic of Greece and were collected from various habitats – subalpic pastures & screes, coniferous & deciduous forests, coastal & urban areas and agricultural land – of Greece. All samples were collected in the vegetative stage of late flowering-early seed development and consisted of whole plants, measured both in weight and land coverage per plant, as a mean average of the sample distilled. Each sample was documented by a voucher specimen of the related taxon and is deposited in the herbarium of the Agricultural University of Athens, Greece. Full records of the investigated taxa are provided in Table 2 and their distribution in Greece in Fig. 1. The taxa identification was performed using as primary taxonomic key the Flora Europaea (Tutin et al., 1968) and as supplementary – when appropriate – the additional regional Floras (Sibthorp, 1804; Halacsy, 1900; Rechinger, 1943; Strid, 1981; Turland et al., 1993; Georghiou and Delipetrou, 2010). The nomenclature used is in accordance with the proposed by Pimenov and Leonov (1993). 2.2. Isolation of the essential oils The freshly collected plant material (stems, leaves and flowers) was washed thoroughly, chopped off finely and subjected to steam distillation in a Clevenger-type apparatus with 3 L of H2 O and using as heat source a Microwave Accelerated Reaction System (MARS 5) for 20 min at 1400 W. The EOs obtained were dried over anhydrous sodium sulphate and stored at 4 ◦ C until analysis. Their yields are included in Table 2. 2.3. Chromatographical analyses 2.3.1. Gas chromatography (GC) All GC analyses were carried out on an Agilent Technologies 7890A gas chromatograph, fitted with a HP 5MS 30 m × 0.25 mm × 0.25 ␮m film thickness capillary column and FID. The column temperature was programmed from 60 to 280 ◦ C at an

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Table 2 Apiaceae taxa studied herein (C.N. = code number, WD = weight distilled, YEO = essential oil yield, Wm = mean average weight, Am = mean average land coverage). Genus Caucalideae Pseudorlaya Apieae Athamanta Bupleurum Geocaryum Oenanthe Pimpinella

Sclerochorton* Selinum Seseli Angeliceae Angelica Tordylieae Heracleum Malabaila Tordylium Laserpitieae Elaeoselinum Laserpitium Thapsia

Species

C.N.

WD (kg)

YEO (ml/kg)

Wm (kg/plant)

Am (m2 /plant)

Location

pumila

284 – 522 504 21 644 614 464 421 402 384 392 252 122 221 172 61

0.345 – 0.450 0.130 0.570 0.100 0.130 0.600 0.770 0.235 0.055 0.500 0.650 0.600 0.430 0.200 0.660

0.14 – 1.11 1.54 14.91 1.5 1.54 0.83 0.65 2.98 0.91 0.8 2.31 3.33 1.63 2.5 2.88

0.19 – 0.24 0.26 3.5 0.01 0.01 0.24 0.26 0.06 0.05 0.07 0.07 0.28 0.07 0.08 0.18

0.18 – 0.34 0.38 0.5 0.01 0.01 0.08 0.08 0.07 0.12 0.09 0.15 0.34 0.11 0.13 0.19

Epidauros – Mt. Parnassos Leonidio Mt. Parnon Mt. Parnassos Mt. Parnassos Is. Crete Mt. Elikon Molai Athens Is. Crete Mt Oiti Mt. Parnnassos Mt Oiti Mt Oiti Mt. Smolikas

apulum

102 – 11 293 414 364

0.920 – 0.850 0.620 0.275 0.070

0.54 – 0.59 0.97 0.18 1.43

3.7 – 2.8 0.09 0.08 0.03

0.5 – 0.5 0.17 0.18 0.09

Mt. Parnon – Mt Oiti Mt. Parnon Mt. Parnassos Mt. Parnassos

asclepium pseudomeuma garganica

341 212 334

0.250 0.270 0.600

0.2 3.33 1.67

1.7 0.14 0.26

0.4 0.22 0.19

Mt. Parnon Mt Oiti Athens

densaa arachnoideaa fruticosum capillifoliuma parnassicuma pimpinelloides rigidulaa creticaa peregrina tragium junceuma silaifolium parnassicuma montanum sylvestris sphondyllium aurea

(a = endemic, 1 = Evergbcdetis et al., 2009, 2 = Evergetis et al., 2012, 3 = Evergetis et al., 2013, 4 = presented here for first time).

initial rate of 3 ◦ C/min. The injector and detector temperatures were programmed at 230 and 300 ◦ C, respectively. Helium was used as the carrier gas at a flow rate 1 mL/min.

The EOs productions estimates were also calculated per hectare, utilizing the previously calculated Ph values and the measurements of >Table 2, applied in the following formula: PEO = Ph × (YEO × 10−3 )

2.3.2. Gas chromatography–mass Spectrometry (GC–MS) The GC–MS analyses were performed on the same instrument connected with the Agilent 5957 C, VL MS Detector with TripleAxis Detector system operating in EI mode (equipped with a HP 5MS 30 m × 0.25 mm × 0.25 ␮m film thickness capillary column) and He as the carrier gas (1 mL/min). The column was heated gradually from its initial temperature (60–280 ◦ C with a 3 ◦ C/min rate. The compounds identification was based on comparison of their retention indices (RI) obtained (Van den Dool and Kratz, 1963) as compared to various n-alkanes (C9–C24). In addition, their EI–mass spectra were compared with the NIST/NBS and Wiley library spectra and the literature (Massada, 1976; Adams, 2001). Finally, the identity of the indicated phytochemicals was confirmed by comparison with available authentic samples.

PEO = production of EO per hectare in L, Ph = production of herbal matter per hectare in kg, YEO = EO content of plants measured in ml of EO per kg of plant tissue. Finally, the productions of pure compounds estimates –calculated per hectare–were performed by applying the PEO values in the following formula: Pc = PEO × (Cc × 10−2 ) Pc = production of pure EO compound in L per hectare, PEO = production of EO per hectare in L, Cc = percentage of pure compound concentration in each EO. 3. Results and discussion 3.1. Essential oils composition

2.4. Crop potentials estimation The crop potentials estimates in respect to herbal matter as well as their EO content and ability to produce pure compounds, were relied on the measurements depicted in Table 2. More specifically, the herbal matter estimates were calculated per hectare using the following formula along with the basic assumption of 50% land coverage per hectare: ∗ Ph = Wm [(103 × 2−1 ) × A−1 m ]

Ph = production of herbal matter per hectare in kg, Wm = mean average weight of plant in kg, Am = mean average land coverage of plant in m2 .

Herein is presented and discussed for the first time the chemical composition of the EOs isolated from the following five Greek endemic taxa: Athamanta arachnoidea Boiss. & Orph., Pimpinella cretica Poiret, G. parnassicum (Boiss. & Heldr.) Engstrad, G. capillifolium (Guss.) and P. seudorlaya pumila (L.) Grande, Cosson. In addition, the EOs compositions of four unexplored specimens from already investigated taxa are also studied for first time herein. The aforementioned nine EOs were found to contain 52 phytochemichals, which represent the 79.90–98.40% of their total volume. The detailed qualitative and quantitative analytical data of these constituents (and their respective retention indices) are summarized in Table 3. The EOs compositions of the remaining 13 taxa have been reported previously (Evergetis et al., 2009, 2012, 2013) and not discussed herein.

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Table 3 Qualitative and quantitative (%) composition of the EOs investigated (28: Pseudorlaya pumila, 50: Athamanta arachnoidea, 64: Geocaryum capillifolium, 61: G. parnassicum, 46: Oenanthe pimpinelloides-Is. Crete, 38: Pimpinella cretica, 41: Malabaila aurea-Mt Parnassos, 36: Tordylium apulum, 33: Thapsia garganica). Components

RI

28

50

64

61

46

38

41

36

33

I/D

2-Hexenal Heptanal ␣-Thujene ␣-Pinene Camphene Sabinene ␤-Pinene Myrcene Octanal ␣-Terpinene o-Cymene Limonene ␦-3-Carene cis-Ocimene trans-Ocimene ␥-Terpinene n-Octanol Terpinolene ␣-Terpineol 1-Bornyl acetate Thymol Carvacrol N-Octyl isobutyrate ␤-Elemene ␤-Caryophyllene ␣-Bergamontene ␥-Elemene ␣-Humulene ␤-Farnesene C14 H30 O (m/z: 189, 147, 105, 91, 204) Germacrene-D ␣-Zingiberene Pentadecane Bicyclogermacrene ␣-Muurolene ␣-Farnesene ␤-Bisabolene Myristicin ␤-Sesquiphellandrene Germacrene-B Caryophyllene oxide ␣-Sinensal C12 H25 O2 N (m/z: 91, 55, 115, 129, 77) C12 H25 O2 N (m/z: 91, 115, 55, 129, 77) Phytol C13 H27 O2 N (m/z: 91, 115, 55, 129, 159) C17 H36 O3 (m/z: 109, 69, 43, 93, 133) n-Heneicosane Tricosane Pentacosane Heptacosane Nonacosane

855 902 930 939 954 975 979 991 999 1017 1026 1029 1031 1037 1050 1060 1068 1089 1177 1289 1292 1299 1390 1391 1419 1435 1437 1455 1457 1483 1487 1499 1500 1500 1502 1506 1508 1519 1523 1561 1583 1758 1923 1943 1943 2030 2094 2100 2300 2500 2700 2900

– – – – – 7.1 – – 4.4 – – 8.1 – – 3.2 4.5 – 3.0 1.8 – – – – – – – – 1.8 – – – – – – – – – 57.6 – – – – – – – – – – – – – –

– – – – – 5.2 – 4.8 – – – 29.1 – – – – – – – – – – – – 11.3 – – – – 10.7 4.6 – – – – 8.8 – – – – 1.2 – 1.6 4.6 – 2.3 – – – – – –

– – – – – – – – – – – 0.7 – – – – – – – – – – – – 1.9 – – – – – 0.6 – – – – 41.3 – – – – 0.7 34.2 – – – – – – 0.6 – – –

– – – – – – – – – – – – – – – – – – – 2.1 – – – 3.3 2.8 – 24.3 – – – 5.2 – – 1.0 – – 1.0 – – 49.5 – – – – – – – – 0.5 – 1.1 1.5

0.1 0.2 0.5 0.6 – – 6.8 2.7 0.8 0.3 17.8 – – 0.6 2.8 43.4 – 0.1 0.4 – 0.1 0.1 – 0.4 1.3 0.8 – 0.1 0.1 – 2.1 – – 0.6 0.1 – 0.7 – 8.3 0.1 0.1 – – – 0.1 – – – 0.1 0.1 0.2 0.2

– – – – – 19.8 – – – – – – – – – – – – – – – – – 11.0 – – – – 1.5 – 1.5 – – – – – 19.8 – – 30.8 – – – – – – – – – – – –

– – – – – – – – – – – – – – – – 9.5 – – – – – 61.4 – – – – 1.9 – – – 4.9 – – – – 2.8 3.5 – – – – – – – – – – – – – –

– – – 9.1 3.7 50.9 – 2.6 – 3.4 – – – – – – – – 4.7 – – – – – 4.1 – – – – – 14.8 3.7 – – – – – – – – – – – – 1.6 – – – – – – –

– – – 14.5 – 14.2 – – – – – – 5.8 – – – – – – – – – – – – – – – – – – – 12.5 – – – – – – – – – – – – – – 17.4 17.9 – – –

a, b a, b a, b a, b, c a, b, c a, b, c a, b, c a, b, c a, b a, b, c a, b, c a, b, c a, b a, b, c a, b, c a, b, c a, b a, b, c a, b, c a, b a, b a, b a, b a, b a, b, c a, b, c a, b a, b a, b, c b a, b, c a, b a, b a, b a, b a, b, c a, b, c a, b, c a, b, c a, b, c a, b, c a, b b b a, b b a, b a, b, c a, b, c a, b, c a, b a, b

–

91.5

84.3

79.9

92.3

92.6

84.2

84.1

98.4

82.2

–

Total

a, RI, Retention indices calculated against C8 to C24 n-alkanes on the HP 5MS column; b, Comparison of mass spectra with MS libraries and retention times; c, Comparison with authentic compounds.

In respect the phytochemicals contained, it is noticeable that the EO of Pseudorlaya pumila is characterized by the prevailance of myrsticin, a valuable pharmaceutical molecule which was found in proportion exceeding the half of the EO’s quantity. On the other hand, the EOs of both Geocaryum species are dominated by sequiterpenes, with the EO of G. capillifolium containing as major consitutents the aldehyde ␣-sinensal and the unsaturated hydrocarbon ␣-farnesene, in amounts exceeding additively the 75% of the EO’s composition. In a similar manner two cyclic sesquitrepenoids, namely ␥-elemene and germacrene-B, comprise almost 75% of G. parnassicum EO content. In regard the remaining EOs, the oil of A. arachnoidea Boiss. & Orph. is characterized by the prevalence of myristicin, apiole and an

unidentified group of alkaloids. The latter are identical with those determined in the EO of A. densa, which constitutes another Greek specimen of the same plant (Evergetis et al., 2012). The interesting structures and suggested bioactivity of those molecules (Evergetis et al., 2012) has driven a more detailed, yet incomplete, study including other related taxa along with the molecules distribution in plant tissues. The EO of P. cretica Poiret was also studied for the first time and found to contain six phytochemicals with most abundant the commercially important molecule of germacrene-B. This molecule has reported as major component only for the EO of P. tragium ssp. tragium Tutin (Evergetis et al., 2012). The studied herein specimen of Oenanthe pimpinelloides L. was collected from the island of Crete and produced EO which

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Fig. 2. Essential oil producing crops in significant yields (liters per hectare).

displays a very complex profile of phytochemicals – composed by twenty eight additional components – as compared to the respective specimen from mountain Elicon (Evergetis et al., 2012). The EO of Malabaila aurea obtained from mountain Parnassos exhibited a significantly simpler pattern containing only six constituents, with the molecules of N-octyl-isobutyrate and ␣-zingiberene accounting almost the 70% of the total volume ot the EO. These phytochemicals have not been detected in a previous study concerning the EO of the respective specimen of mountain Parnon (Evergetis et al., 2013). Finally, from the six compounds identified herein as constituents of T. garganica EO, only the molecules of ␣-pinene and sabinene were traced before in Thapsia L, although the EO of Thapsia garganica L. has been studied extensively (Avato, 1991; Avato and Rosito, 2002; Iadjel et al., 2011), along with the EOs of other Thapsia L. species, such as T. maxima Miller (Avato et al., 1992; Avato and Smitt, 2000) and T. villosa L. (Smitt, 1995; Avato et al., 1996; Avato and Smitt, 2000). Thus, the presence of the aliphatic saturated hydrocarbons pentadecane, n-heneicosane and tricosane in T. garganica EO is reported herein for the first time. 3.2. Essential oils production potentials The EO production capacity of the investigated 22 taxa was evaluated on the basis of a specific threshold set to define whether a taxa is capable to produce a significant amount of the respective EO. This threshold refers to plant’s capability to produce at least 10 L of EO per hectare, an amount approximately 30% lower from the respective yield of Anethum graveolens (13.35 L/ha) which constitutes a well established industrial crop. Additionally, the cultivation of 100 ha is considered as a minimum for the establishment of a global significance for an EO producing crop. These requirements were fulfilled for ten out of the 22 investigated specimens that belong to nine different taxa. These specimens are included in Fig. 2 and their >taxa are briefly discussed in respect to their habitat and form of growth, in accordance with Flora Europaea (2010), unless otherwise stated. The plants of Angelica L. species comprise well-established EO producing crops and their inclusion in the present accreditation is expected. The estimated EO yield for the investigated herein specimen reached 20 L/ha advocating their potential value. The B. fruticosum L. is an evergreen shrub of 250 cm height that is endemic to Mediterranean coasts, growing mainly in the surrounding forests, road margins and olive-yards. The investigated specimen is originated from Peloponnese and was collected from a ravine in 800 m altitude. Its estimated yield reached the 522 L/ha providing an annual production capacity exceeding the 50 tons for

100 ha, while its woody growth is also increasing its value as a crop producing plant. Heracleum sphondylium L. that displayed a more modest yield – approximately 16 L/ha – is of herbaceous perennial growth that reaches the 3 m height when growed in mountain habitats. It was collected in Oiti mountain in a wet ravine (1800 m) which constitutes its preferred cultivation habitat. Sclerochorton junceum (Sibth. & Sm.) Boiss. constitutes an endemic to Greece genus with woody growth and low height averaging up to 0.5 m. Its EO production potential is estimated to 14 L/ha, displaying limited distribution in mountains of South Greece with preferred habitat the sub-alpic zone, which promote the utilization of the plant as a local-specific crop. Similar EO production estimate was also assessed for three additional species, namely Seseli montanum L., O. pimpinelloides L. and the endemic to Greece Pimpinella rigidula (Boiss. & Orph.) H. Wolf. All these species are of the same herbaceous perennial growth, averaging about 1 m of height. The P. rigidula distribution is limited only to cultivated fields of Greece, while S. montanum and O. pimpinelloides have wider distribution that includes respectively the South Europe’s forests and the wet places of West and South Europe. The two O. pimpinelloides specimens studied in this endeavor were originated from Crete (C.N. 46) and mountain Helicon (C.N. 42) displaying an annual EO production capacity about 10 L/ha. The remaining three species of the list include Thapsia garganica L. and the endemic to Greece Laserpitium pseudomeum Orph., Heldr. & Sart. ex Boiss., both exhibiting EO production capacities that averages around 11 L/ha. These species are of herbaceous perennial growth with heights averaging from 2 m (T. Garaganica) to 0.25 m (L. pseudomeum). Their geographical distribution expands into South Mediterranean region and the mountains of Central and South Greece respectively. The natural habitat of the former is located in Mediterranean forests, while the first is naturally grown in lowland shrub-land and disturbed habitats. 3.3. Fine chemicals production potentials Apart from the estimation of their EOs production potentials, this study also aspires to highlight on the role of the investigated taxa as source for the isolation of fine chemicals (FCs). In this respect, from the sum of 96 detected phytochemicals composing the investigated EOs, a panel of 36 molecules was selected for further studies. These molecules comprise the EOs major components exceeding the 10% of their composition. Appendix 1 (supplementary material) summarizes the major components concentration of the EOs isolated from 22 taxa investigated herein. These data were combined in conjunction with their average – per hectare – yield for the estimation of the production potentials for each FC. An EO was defined as a significant source of FC if the following two thresholds were fulfilled: (A) the amount of a specific FC isolated from each EO, considering as significant only yields exceeding the 1 L/ha, and (B) the total amount of the FC in each EO, which has to be at least 3 L/ha. Elaboration of both filters on the raw data of Appendix 2 (supplementary material) revealed the twenty Fine Chemicals listed in Table 4, which are discussed herein in respect to Terpenes (Breitmaier, 2006) and Flavor and Fragranses (Fahlbusch et al., 2002). Molecules of significant interest are also the three unidentified compounds, along with the thirteen other major compounds, that were not included in present discussion, as inneficiently produced. Structure elucidation of the unknown compounds, and compounds localization studies in plant organs are in progress, in order to evaluate further those potentials. Though interesting those 16 compounds do not surpass the above-set tresholds and therefore are not subjected to further discussion. Hydrocarbons (H/Cs) comprise the first group of FC considered herein. Various mixtures of volatile H/Cs are currently used in

Table 4 Fine chemicals production estimates (yields are liters per hectare). Bupleurum fruticosum

Angelica sylvestris

Sclerochorton junceum

Seseli montanum

Oeanthe pimpinelloides (42)

Oeanthe pimpinelloides (46)

Laserpitium pseudomeum

Pimpinella rigidula

Geocaryum capillifolium

Geocaryum parnassicum

Thapsia garganica

Selinum silaifolium

Seseli parnassicum

Pentadecane – – n-Heneicosane – Tricosane – ␣-Sinensal – cis-Ocimene 197.3 ␣-Pinene – Sabinene – ␤-Phellandrene – o-Cymene 113.1 Limonene – -Terpinene – Terpinolene – -Elemene – Germacrene-D – ␤-Selinene ␣-Farnesene – – ␤-Sesquiphellandrene – Germacrene-B – trans-Isomyristicin trans– Epoxypseudoisoeugenyl 2-methylbutyrate

– – – – – 5.0 – 8.6 – – – – – – – – – – – –

– – – – 2.6 – – – – 5.6 – 1.8 – – – – – – 1.4 –

– – – – – 4.4 2.3 2.6 – – – – – – – – – – – –

1.4 2.0 2.0 – – 1.7 1.6 – – – – – – – – – – – – –

– – – – – – – – 2.2 – 5.4 – – – – – – – – –

– – – – – – 1.2 – – 1.1 5.2 – – – – – – – – –

– – – – – – – – – – – – – – 3.0 – – – – 3.4

– – – 2.6 – – – – – – – – – – – 3.1 – – – –

– – – – – – – – – – – – 1.9 – – – – 3.8 – –

– – – – – 3.5 – 1.2 – – – – – – – – – – – –

– – – –– – 2.0 1.5 – – – – – – – – – – – – –

– – – – – – – – – – – – – 1.0 – – 2.3 – – –

310.4

13.6

11.3

9.3

8.7

7.6

7.6

6.5

5.7

5.7

4.7

3.5

3.3

Total

E. Evergetis, S.A. Haroutounian / Industrial Crops and Products 54 (2014) 70–77

Fine chemicals

75

76

E. Evergetis, S.A. Haroutounian / Industrial Crops and Products 54 (2014) 70–77

preference to chlorofluorocarbons as propellants in aerosol sprays, due to chlorofluorocarbon’s impact on the ozone layer. On the other hand, the unsaturated H/Cs constitutes a class of highly favorable FCs since they are widely used in sustainable chemistry applications for the production of diverse products (Behr and Johnen, 2009) which are extensively utilized as solvents, plasticizers, lubricants and industrial raw materials. Herein, the presence of three saturated H/Cs, pentadecane, n-heneicosane and tricosane all from S. montanum, were identified in quantities allowing their isolation as FC with an average cumulative production potential of 8.7 L/ha. In respect the unsaturated H/Cs, the farnesene and cisocimene were identified as potential isolation targets. Farnesene is a sesquiterpene widely used by perfume industry due to its beautiful smell, mainly in cosmetics preparation such as masks and powders. In addition, this molecule is utilized during beer brewing since contributes significantly to their aroma. Herein, the estimated quantity for ␣-farnesene isolation from the EO of G. capillifolium exceeds the amount of 3 L/ha. Terpene cis-ocimene was found in slightly lesser amount in the EO of S. junceum averaging 2.6 L/ha. Aldehydes comprise another group of FC with commercial applications either as precursors for the production of oxo-alcohols (used in detergents), or are produced in a small scale (less than 1000 tons/year) in order to be used as ingredients by perfumes and flavors industries. Herein the sesquiterpenic aldehyde ␣-sinensal was identified as potential isolation target. This molecule is originated from the EO of G. capillifolium Cosson >with a production potential reaching the 2.6 L/ha. Aromatic compounds are chemicals containing conjugated planar ring systems with delocalized ␲-electron clouds, such as benzene and toluene. The following three aromatic compounds were identified as potential isolation targets: (A) Eugenol, which is utilized by perfumery and flavor industries, as local antiseptic (Jadhav et al., 2004), anesthetic (Right and Payne, 1962), in dentistry for the production of zinc oxide eugenol (Ferracane, 2001) and during the manufacturing of plastics and rubbers as stabilizer and antioxidant. The molecule of trans-epoxypseudoisoeugenyl 2methylbutyrate, is a derivative of eugenol which was found in the EO of the endemic to Greece plant Pimpinella rigidula, with an estimated production capacity of almost 3.5 L/ha. (B) Myristicin, a phenylpropene detected in small amounts in the EO of nutmeg (Myristica fragrans) and to a lesser extent in other spices such as parsley and dill, plants belonging to the Apiaceae family. This molecule displays – known for longtime – various psychoactive effects and is utilized for the production of pharmaceuticals (Truitt et al., 1963). Its derivative trans-isomyristicin was detected in the EO of endemic to Greece monotypic genus Sclerochorton Boiss. EO in an estimated amount of 1.5 L/ha. (C) Monoterpene ␣-pinene, an oily colorless liquid with a turpentine-like odor used either in camphor and perfumes manufacturing or as ingredient for the preparation of insecticides, solvents, plasticizers and synthetic pine oil. The naturally occurring molecule of a-pinene is commercially retrieved from Pinus sp., predominantly as the (−) optical isomer in 94:6 ratio (Kotzias et al., 1992). Herein we have obtained the racemic mixture (50:50) of the optical isomers of the molecule, which constitutes either a novel source for the (+) isomer and/or an interesting miture for potential applications since the respective chemical properties and bioactivity vary greatly among the (+) and (−) isomers (Cataldo, 2006; Lis-Balcnin et al., 1999). Its most ambitious production yield was estimated for the EO of B. fruticosum in amounts exceeding the 190 L/ha, while the same molecule can also be isolated in lesser yields from five additional EOs. Phellandrenes are molecules used for the production of fragrances due to their pleasant aromas. The odor of ␤-phellandrene is described as peppery-minty and slightly citrusy. Though ␤phellandrene was detected in the EOs of three taxa, its production estimates promoted as most potent the EO of A. sylvestris L. with

8.6 L/ha yield. The monoterpene cymene is widely used either as precursor for the synthesis of p-cresol or as important intermediate by the pharmaceutical, food and agrochemical industries for the production of various fungicides, pesticides and flavoring agents (Selvaraj et al., 2002). The exploited herein isomer o-cymene was determined in adequate quantities in the EO of O. pimpinelloides from Crete (CN 46), with an estimated yield of 2.2 L/ha. Limonene is a monoterpene isolated primarily from Citrus limon (L.) Burm. f. This molecule is extensively used as a flavoring agent for the production of a broad variety of industrial products, including pharmaceuticals, foodstuff and beverages, as a fragrance, solvent or ingredient in water-free hand cleansers. The isolated herein molecule of limonene is also in the form of racemic mixture of both optical isomers. Since todate the molecule of limonene is being produced as the D-isomer from the leftovers of orange processing industries (Pourbafrani et al., 2010), our product may be considered as a good source of the L-isomer which naturally occurs in lemons. The presence of limonene in adequate quantities was confirmed in three EO’s with estimated yield from the EO of Bupleurum fruticosom exceeding the 110 L/ha. Elemenes comprise a group related closely to sesquiterpenes, contributing to the floral aromas of plants and used by some insects as pheromones (Peng et al., 2006). The molecule of ␥-elemene was detected in the EO of the endemic to Greece plant G. parnassicum with estimated yeld reaching the 1.9 L/ha. Germacrenes are sesquiterpenes produced by numerous plant species, displaying potent antimicrobial and insecticidal properties along with insect pheromone activities. In this endeavor the presence of germacrene-B and the more abundant germacrene-D was encountered. The molecule of germacrene-B was detected in the EO of G. parnassicum in 3.8 L/ha yield. Selinenes are isomeric sesquiterpenes, originaly isolated from celery (Apium graveolens) seeds. Herein the presence of ␤-selinene was assessed in significant amounts in the EO of the Greek endemic plant P. rigidula with estimated yield 3 L/ha. Sesquiphellandrene is the sesquiterpene analog of the previously annotated phellandrenes. Herein only the presence of ␤-sesquiphellandrene was determined in significant amounts in the EO of the Greek endemic S. parnassicum with estimated yield 2.3 L/ha. Finally, terpinenes that comprise a group of monoterpene isomers varying in their carbon-carbon double bonds position. They are considered as perfume and flavoring chemicals and used by the cosmetics and food industries. Nowadays these molecules are also gaining interest by electronics semi-conductor manufacturing industries (Marzec et al., 2010). Herein we determined the presence of two terpinene isomers in significant amounts. The molecule of ␥-terpinene, detected in the EOs of O. pimpinellodes from Crete and L. pseudomeum in almost identical yields of 5.4 and 5.2 L/ha respectively and terpinolene (also known as ␦-terpinene) identified as a significant component of the EO of S. junceum in 1.8 L/ha yield.

4. Conclusions Among the taxa investigated, B. fruticosum was identified to possess an outstanding potential either as EO producing crop or as ␣-pinene and limonene source, molecules with a mature international market value and numerous well established industrial applications. In a similar manner the A. sylvestris was identified as a significant source for the isolation of ␤-phellandrene and ␣-pinene. Nine taxa were identified for first time as potent EO producing crops, including three endemic to Greece species (S. junceum, P. rigidula and L. pseudomeum). Additionally, three Greek endemic taxa namely the G. capillifolium, G. parnassicum and S. parnassicum were defined as potent sources of FCs, since they were found to

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