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Fibre hemp inflorescences: From crop-residues to essential oil production
Industrial Crops and Products 32 (2010) 329–337
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Fibre hemp inflorescences: From crop-residues to essential oil production Alessandra Bertoli a,∗ , Sabrina Tozzi b , Luisa Pistelli a , Luciana G. Angelini b a b
Dipartimento di Scienze Farmaceutiche-sede Chimica Bioorganica e Biofarmacia, University of Pisa, Bonanno 33, 56126 Pisa, Italy Dipartimento di Agronomia e Gestione dell’Agroecosistema, University of Pisa, S. Michele degli Scalzi 2, 56127 Pisa, Italy
a r t i c l e
i n f o
Article history: Received 14 December 2009 Received in revised form 14 May 2010 Accepted 21 May 2010
Keywords: Cannabis sativa L. Fibre hemp Monoecious Dioecious Essential oils SPME GC–MS
a b s t r a c t The volatile composition of ten fibre hemp (Cannabis sativa L.) varieties was investigated during two successive growing seasons under temperate climatic conditions in Central Italy. The freshly plant inflorescences were hydrodistilled and the essential oils (EOs) were characterized by GC–MS. In addition, the composition of the aroma emitted spontaneously from the freshly plant inflorescences were analysed by SPME-GC–MS. The EO yields of eight dioecious (Carmagnola, C.S., Red Petiole, Pop 1, Pop 2, Pop 3, Pop 4, Pop 5) and two monoecious (Codimono and Felina 34) cultivars ranged from 0.11 to 0.25% (w/w) and showed a significant production of ␣-pinene (3–20%), -pinene (1–8%), E-ocimene (1–10%), myrcene (8–45%) and terpinolene (0.12–22%). The monoterpene composition was useful to distinguish the monoecious cultivars from the dioecious ones. -Caryophyllene (7–28%), ␣-humulene (3–12%), and caryophyllene oxide (2–6%) were the main sesquiterpenes. Tetrahydrocannabinol (THC) was present in traces in the EOs of only two dioecious cultivars cultivated in 2005. Cannabinol (CBN) was not detected in the essential oils, while the no-hallucinogenous cannabidiol (CBD) was found as typical volatile constituent in several analysed cultivars. These findings were also confirmed by the headspace GC–MS analysis carried out on the same samples. The analysed EOs obtained from fibre hemp varieties cultivated in Central Italy were characterized by an interesting and specific terpene composition with a legal and safe cannabinoid content. They were obtained from freshly plant inflorescences, which usually represent a waste material from C. sativa L. fibre varieties. The present study strengths the hypothesis to grow hemp as a multi-use crop through a complete utilization of the plant material using inflorescences to produce essential oils as natural flavour and fragrance additives. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, there is an increasing tendency in the use of plant-derived raw materials as alternative renewable sources to petrochemical derived products. In this situation, the resurgence of interest in fibre hemp (Cannabis sativa L.) as a multipurpose crop has been arising in Europe due to the multitude of end products derived from the different organs (Ranalli and Venturi, 2004). Besides the interest for textile uses, interest is growing in cultivating industrial hemp for non-textile applications. It is important to point out that the fibre hemp varieties are eligible for cultivation only after the verification of their tetrahydrocannabinol (THC) content recommended by the Regulation EC no. 1124/2008 (12 November 2008). Several studies have been carried out on the cannabinoid content, resin, and seed oil of C. sativa and their dif-
∗ Corresponding author at: Dipartimento di Scienze Farmaceutiche, University of Pisa, Via Bonanno 33, 56126 Pisa, Italy. Tel.: +39 050 2219705; fax: +39 050 2219660. E-mail address: [email protected] (A. Bertoli). 0926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2010.05.012
ferent products (Brenneisen and Elsohly, 1988; ElSohly and Slade, 2005; Kriese et al., 2004; Leizer et al., 2000; Turner et al., 1980). The hemp essential oil has generally been considered a niche high value product with promising potential marketing (Mediavilla and Steinemann, 2005; de Meijer, 1998; Thomas et al., 2000). It is a complex mixture of many volatile compounds, mainly monoterpenes, sesquiterpenes, and other terpenoid-like substances (Fournier and Paris, 1978; Novak and Franz, 2003). Monoterpenoids are principally responsible for differences in fragrance among hemp cultivars. Sesquiterpenoids are characteristic components too, but they are generally in lower amounts in comparison with monoterpenes (Hendriks et al., 1975; Hillig, 2004; Lemberkovics et al., 1981; Ross and ElSohly, 1996). Hemp essential oil is biosynthesized in the epidermal glands or glandular hairs where the cannabinoids are produced too (Kim and Mahlberg, 1981; Malingrè et al., 1975). Furthermore, the cannabinoid compounds can be hydrodistilled together with terpene constituents in the essential oil. The highest density of glandular hairs is found on the bract surrounding each female flower and the subtending leaflets of the female inflorescence (Hemphill et al., 1980; Lanyon et al., 1981). When fresh
Mean values within each column followed by the same letter are not significantly different for P < 0.05 probability level according to LSD test. Bold letters for the interaction “Type of cultivar × Year”. a With “upper part” we refer to the 30 cm from the top of plants. * Significance at P 0.1, according to F-test by ANOVA analysis. ** Significance at P 0.05, according to F-test by ANOVA analysis. *** Significance at P 0.01, according to F-test by ANOVA analysis.
* * * * *
*
0.15b 0.12 *
0.20a 0.13b 3.18b 4.08a
2.78b 3.67a
*** ***
35.62a 11.18c 33.48a 22.97b 43.24a 15.85c
** *** ***
39.15a 30.15b 3.04a 2.23c 108.7b 123.0b 134.7ab 150.2a
** ** **
2.66b 1.66d
***
**
*
0.12bc (0.08) 0.11c (0.07) 0.13d (0.09) 0.13cd (0.09) 4.13 (0.14) 3.21abc (1.06) 4.40 (1.17) 3.75ab (0.96) 11.86 (2.1) 10.5c (0.9) 16.81 (3.8) 29.13bc (5.7) 16.58 (3.1) 15.12b (1.9) 23.04 (4.8) 37.26abc (7.0) 1.36 (0.08) 1.85c (0.14) 135.3 (19.0) 2.04 (0.26) 110.8ab (10.2) 2.42c (0.08) 178.7 (7.7) 121.7b (7.8)
Monoecious Felina 34 Codimono Significance cultivar Dioecious Monoecious Significance interaction T×Y
0.15abc (0.05) 0.14abc (0.06) 0.10c (0.08) 0.19a (0.09) 0.14abc (0.09) 0.17ab (0.07) 0.12bc (0.09) 0.19a (0.07) 0.25a (0.09) 0.11d (0.07) 0.22ab (0.05) 0.23ab (0.08) 0.20abc (0.08) 0.19abc (0.05) 0.22ab (0.08) 0.15bcd (0.08) 2.71bcd (0.57) 2.01d (0.32) 2.86bcd (0.60) 3.31abc (0.82) 2.39cd (0.39) 3.62ab (0.56) 3.37abc (1.50) 1.95d (1.07) 3.40bc (0.24) 3.79ab (1.24) 2.34d (0.66) 3.48ab (0.82) 2.49cd (0.57) 3.32bc (1.08) 2.87bcd (0.92) 3.71ab (0.31) 2.71bcd (0.57) 2.01d (0.32) 2.86bcd (0.60) 3.31abc (0.82) 2.39cd (0.39) 3.62ab (0.56) 3.37abc (1.50) 1.95d (1.07) 3.40bc (0.24) 3.79ab (1.24) 2.34d (0.66) 3.48ab (0.82) 2.49cd (0.57) 3.32bc (1.08) 2.87bcd (0.92) 3.71ab (0.31) 47.58a (9.3) 46.76a (8.1) 36.19a (5.8) 41.04a (6.5) 46.51a (19.3) 41.65a (6.3) 41.81a (13.8) 44.37a (9.9) 52.55a (10.5) 49.09a (8.6) 43.24ab (11.9) 35.09abc (9.1) 25.73bc (9.3) 27.25bc (4.8) 36.37abc (6.8) 43.87ab (7.8) 2.91a (0.15) 2.68ab (0.34) 2.71ab (0.12) 2.57ab (0.26) 2.64ab (0,24) 2.44b (0.20) 2.75b (0.24) 2.57ab (0.30) 3.30a (0.10) 3.11ab (0.22) 3.17ab (0.30) 3.22ab (0.37) 2.83ab (0.35) 2.74bc (0.15) 2.89ab (0.08) 3.06ab (0.38) 125.5b (21.1) 130.0b (11.1) 133.0b (5.7) 162.0ab (21.2) 151.3ab (16.8) 119.5b (8.7) 135.3b (15.4) 120.8b (16.3) C.S. Red petiole Carmagnola Pop 1 Pop 2 Pop 3 Pop 4 Pop 5
108.8b (13.2) 95.5b (10.8) 95.3b (5.3) 116.3ab (15.5) 118.8ab (11,9) 107.8b (17.5) 121.3ab (16.4) 105.5b (12.6)
2006 2005 2006 2005 2006 2005 2006 2005 2006 2005 2005 Dioecious
2006
Plant density (number/m2 )
Plant height (m)
Plant dry yield (g/plant)
Stem dry yield (g/plant)
Dry Upper parta (g/plant)
Essential oil (% w/w)
A. Bertoli et al. / Industrial Crops and Products 32 (2010) 329–337
Cultivar
Table 1 Mean values (±standard deviation) of the main biometric and productive parameters of the hemp cultivars in 2005 and 2006 experimental seasons. Mean values of the main parameters related to the interaction T × Y between the type of cultivar (monoecious and dioecious) and the year (2005 and 2006) are also reported.
330
female inflorescences are dried, a greater loss of monoterpenoids than sesquiterpenoids is observed, but none of the major components of the oil completely disappears (Ross and ElSohly, 1996). Most of the studies on the essential oil production have been carried out for the strain selection of drug type C. sativa (Mediavilla and Steinemann, 2005; Novak et al., 2001; Novak and Franz, 2003). On the contrary, there are fewer studies on the essential oil extracted from the inflorescences of fibre type C. sativa cultivars. Recently, Nissen et al. (2009) characterized the essential oils of three legal hemp varieties for their in vitro antimicrobial activity, evidencing interesting perspectives as alternative antibacterial agents. The objectives of this study were to monitor the variations within ten monoecious and dioecious fibre hemp varieties grown in Central Italy in a two-year field trial: (a) on the main productive and biometric characteristics (plant density, plant height, plant and stem dry yield); (b) on the yields and composition of the essential oils. Therefore, the aim of this study was to bring new knowledge on the fibre hemp multi-use crop and the potentiality of inflorescences, generally considered waste parts for fibre industry, were considered as standardized plant material to produce valuable essential oils.
2. Material and methods 2.1. Growing conditions and field experiment layout The experiments were carried out at the Experimental Centre of Dipartimento di Agronomia e Gestione dell’Agroecosistema (DAGA) of Pisa University (15 km SW of Pisa, Italy, latitude 43◦ N, longitude 10◦ E, altitude 10 m above sea level) during the seasons 2005 and 2006. The morphology of the region is flat and is characterized by a Mediterranean climate, with rainfall mainly concentrated in the autumn and spring (mean 941 mm year−1 ). Mean air temperatures generally increase from April to July with mean minimum temperature of 9.6 ◦ C, and mean maximum temperature of 20.0 ◦ C. During summer (July–half August) a dry period generally occurs with low rainfall and high air temperatures often above 25 ◦ C. The loam soil, representative of the low Arno river plain, has a good fertility and water retention capacity (sand 44.3%; silt 41.0%; clay 14.6%; organic matter 1.8%; pH 8.0; total nitrogen 0.12%; available P2 O5 17.25 mg kg−1 ; exchangeable K2 O 97.0 mg kg−1 ; field capacity 23.0% dw; wilting point 9.4% dw). The previous crop was castor in 2005 and rapeseed in 2006. Soil tillage was done in November 2004 and 2005 with 30 cm deep ploughing and two superficial disks harrowing at the end of March to prepare the sowing bed. Plants were maintained under identical fertilization conditions throughout the field experiments. Mineral fertilizer was applied at pre-planting at rates of 120/80/120 kg ha−1 of N/P/K. No irrigation supplies were needed after sowing either in summer period thanks to the natural soil water availability. Ten Italian and European fibre hemp cultivars were compared in a randomized block experimental design with four replications: Carmagnola, C.S., Red Petiole, Pop 1, Pop 2, Pop 3, Pop 4, Pop 5 as Italian dioecious, and Codimono, Felina 34 as Italian and French monoecious, respectively. Seed were kindly supplied by ISCI-CRA (Bologna, Italy) and showed a legal THC content (i.e. <0.2%, w/v). Hemp cultivars were sown the 27th April 2005 and the 4th April 2006 by a pneumatic drill with 0.18 m inter-row in order to achieve the planned crop density of 130–140 plants per m2 . Each plot size was 20 m2 (5 × 4 m) and 15 m2 (3 × 5 m), in 2005 and 2006, respectively. Crop was protected against weeds by frequent hand and mechanical weeding but no pesticides were supplied. During the growing cycle, emergence and flowering dates were recorded when at least 2/3 of plants showed the specific phenological stage. The
Table 2 GC–MS results of the essential oils extracted by hydrodistillation from the different fibre hemp inflorescences cultivated in Central Italy. L.R.I.a Compound 931 941 942 959 979 984 992 1010 1013 1022 1030 1035 1037 1039 1040 1051 1064 1089 1101 1107 1177 1184 1197 1420 1431 1434 1437 1440 1454 1457 1462 1476 1489 1496 1499 1503 1506 1511 1513 1519 1534 1539 1540 1560 1563 1584 1634 1658 1673 2431
POP2
2005
2006
2005
0.08b 0.09 3.05 – 0.09 1.45 12.73 0.44 0.43 0.39 tr 0.91 0.82 – 0.27 2.96 0.31 15.78 – – – – – 11.37 – 0.35 – 0.09 0.86 3.78 0.16 0.1 0.28 0.79 0.62 0.5 0.13 tr 0.07 0.42 1.12 1.63 2.28 1.33 1.72 4.18 0.6 1.56 0.92 0.26 13.85
– 0.1 9.06 0.15 0.12 4.33 19.01 0.59 0.47 0.53 0.06 1.96 1.17 – 0.25 3.36 0.25 17.29 0.33 – 0.04 – 0.18 20.54 – 0.12 – – 0.17 7.98 0.34 – 0.2 0.4 0.39 tr tr – 0.21 – 0.66 0.14 0.32 0.13 1.41 2.69 0.31 tr 0.1 0.13 1.52
0.08 – 6.25 0.17 – 3.21 23.12 0.15 0.11 0.15 0.15 1.6 0.5 – 0.14 1.83 0.1 3.67 0.11 Tr Tr – 0.17 15.3 0.16 – – 0.1 0.1 6.6 0.13 0.18 0.27 0.83 0.72 0.62 – 0.09 0.03 0.46 1.27 3.05 3.79 4.09 1.03 4.13 0.18 tr 0.83 0.19 3.16
POP3 2006 0.07 – 16.73 0.27 – 6.91 11.32 0.4 0.3 0.36 0.23 3.37 0.92 – 0.29 2.78 0.16 10.75 0.21 tr Tr – 0.2 22.73 tr – – – Tr 7.62 0.27 – 0.12 0.23 0.26 tr – 0.1 0.05 – 0.6 0.95 1.45 1.71 0.09 2.98 0.18 0.32 0.11 0.47 1.39
2005 – 0.12 5.12 0.15 0.12 3.29 9.47 0.64 0.56 0.62 tr 2.36 1.26 tr 0.36 4.97 0.38 22.16 0.5 – 0.11 0.14 0.58 14.17 Tr – – 0.09 – 4.71 0.58 – 0.18 0.48 0.34 0.17 – 0.08 tr 0.23 0.59 0.21 0.29 0.09 1.15 5.72 tr tr 1.29 0.55 3.88
2006 – 0.09 11.24 0.19 – 6.13 11.8 0.3 0.24 0.28 tr 0.86 0.68 tr 0.73 10.28 0.15 8.71 0.26 – 0.03 0.14 25.28 – – – – – 8.01 0.43 – 0.1 0.22 0.12 – – 0.11 0.08 tr 0.39 0.09 0.22 0.09 1.35 4.14 0.43 0.11 0.32 0.13 2.03
2005 – 0.15 3.89 0.15 tr 1.88 15.54 0.49 0.55 0.55 0.54 6.39 – 3.69 0.35 16.25 0.15 –
0.35 21.98 – 0.19 – 0.14 – 8.72 0.19 – 0.27 0.63 0.38 0.51 tr – tr 0.31 0.85 0.24 0.73 0.12 1.48 4.54 tr tr 0.62 0.77 –
POP5 2006 0.06 0.1 9.39 0.18 – 4.71 18.14 0.41 0.29 0.39 0.28 3.44 0.83 – 0.35 4.35 0.18 11.53 0.25 0.05 0.05 0.04 0.19 21.65 – 0.1 – 0.08 – 6.98 0.23 – 0.25 1.03 0.79 0.48 – 0.12 0.23 0.21 0.67 0.13 0.37 0.17 1.51 2.5 0.22 0.11 0.17 0.21 2.82
2005 – 0.2 5.31 0.12 0.09 1.90 12.15 0.28 0.16 0.65 0.09 0.62 0.73 – 0.15 3.89 0.11 12.07 0.18 tr Tr 0.07 0.01 18.50 Tr 0.10 – 0.03 0.18 7.14 0.72 – 0.17 0.33 0.2 tr 0.16 tr – 0.03 0.61 0.14 0.24 0.12 0.73 5.46 0.43 0.52 0.14 – 11.05
CARM 2006 – 0.1 12.56 0.18 0.07 4.05 8.23 0.48 0.36 0.42 0.13 0.75 0.97 – 0.51 5.15 0.23 13.87 0.24 tr Tr 0.05 – 26.33 – 0.16 – 0.07 0.29 9.46 0.72 – 0.17 0.33 0.2 tr 0.16 – tr 0.01 0.61 0.14 0.24 0.12 0.73 4.31 0.51 0.22 0.28 – 2.29
2005 0.09 0.07 6.05 0.18 – 2.67 30.47 – – 0.08 tr 4.04 0.44 – 0.71 8.21 0.13 1.5 1.18 Tr 0.17 0.09 0.38 17.43 – – – 0.15 – 6.34 0.48 0.07 – 0.93 0.6 1.13 0.22 tr 0.34 0.54 1.67 0.45 0.76 0.19 0.84 2.64 tr tr 0.31 0.42 –
RED 2006 0.09 0.05 7.51 0.22 – 3.41 27.25 – – 0.06 tr 5.32 1.33 tr 0.81 0.17 0.12 1.17 0.05 0.09 0.12 0.38 21.12 – 0.1 – 0.14 – 7.54 0.18 – 0.43 0.74 0.49 0.73 0.17 0.06 0.47 0.32 1.52 – 0.75 0.22 0.72 2.36 0.35 0.25 0.16 0.95 4.59
2005
CS 2006
0.17 – 5.9 0.18 tr 3.17 27.79 0.3 0.25 0.32 0.33 6.37 0.87 0.36 tr 1.06 0.23 8.9 0.31
0.09 0.12 4.06 0.14 0.08 2.45 15.12 0.51 0.48 0.45 0.28 6.07 0.8 0.49 – 0.91 0.23 13.31 0.43
0.11 0.09 0.46 15.07 – – – 0.13 – 6.94 – – 0.49 1.44 0.28 0.65 0.09 0.11 0.09 0.37 1.13 0.3 0.49 0.13 0.85 3.09 tr – 0.43 0.2 2.92
0.07 0.1 0.42 28.02 – 0.11 – 0.12 0.26 12.61 0.17 0.01 0.1 0.17 0.16 0.5 0.12 0.21 0.12 0.28 0.35 0.09 0.16 0.07 0.57 2.60 0.36 tr 0.27 0.25 2.5
2005 0.17 0.08 8.08 0.26 tr 3.92 45.35 0.34 – 0.1 0.08 – 6.65 0.99 0.78 4.14 0.14 1.62 2.66 0.09 0.21 0.09 0.62 7.26 – – – – – 3.25 0.13 – 0.53 1.48 1.02 0.67 tr – tr 0.34 1.00 0.27 0.39 0.12 0.44 1.92 tr tr 0.19 0.89 –
CODI 2006 0.09 0.16 8.82 0.18 0.09 4.01 17.14 0.67 0.48 0.61 0.06 2.14 1.32 1.24 0.89 3.28 0.4 17.67 0.44 0.04 0.06 0.17 0.38 17.65 0.09 – tr 0.13 7.87 0.2 – 0.14 0.25 0.17 0.33 0.27 tr tr 0.15 0.55 0.13 0.38 0.08 0.79 1.96 0.27 0.09 0.17 1.03 4.12
FEL34
2005
2006
2005
2006
– – 15.69 0.21 0.15 5.84 13.63 0.27 – 0.23 tr 0.34 0.87 0.87 0.34 4.29 0.67 14.46 0.98 0.09 tr 0.15 0.33 16.46 0.03 0.12 0.09 tr tr 7.02 0.44 tr tr 0.50 0.1 tr 0.15 0.08 0.27 0.09 0.87 0.08 0.78 0.89 0.85 2.67 tr tr tr
0.05 0.16 14.12 0.24 0.13 7.21 15.13 0.43 1.53 0.43 0.04 0.54 0.86 1.01 0.65 5.09 0.31 15.47 0.14 0.05 0.11 0.13 0.35 15.98
0.02 – 20.35 0.08 0.05 6.31 12.27 0.08 – 0.09 0.56 0.47 0.78 0.56 0.09 5.94 0.98 14.98 0.09 tr – 0.01 0.20 19.43 tr tr tr Tr – 4.96 0.20 0.01 0.05 0.12 0.02 tr 0.09 0.23 0.10 0.13 0.97 0.03 0.82 0.05 0.34 4.23 tr 0.02 0.22
0.02 – 20.44 0.15 0.08 7.95 13.56 0.14 – tr 0.78 0.89 0.99 0.77 0.21 6.49 0.78 19.14 0.24 0.02 – 0.02 0.33 19.50 tr 0.11 0.05 – – 5.96 0.32 tr 0.01 0.20 0.09 tr 0.10 0.12 0.17 0.11 0.80 0.06 0.61 0.09 0.54 4.99 tr 0.05 0.35 0.17 1.89
0.16
0.17 0.23 0.11 0.15 8.02 0.81 – 0.35 0.94 0.71 tr tr 0.13 0.39 0.15 0.95 0.5 0.97 0.92 1.1 2.97 tr 0.29 0.34 0.26 0.36
1.69
331
LRI = Linear retention indeces on DB5-column; tr = traces; <0.01%; (–) = not detected. Bold values are referred to the main constituents of the analysed samples. a Relative percentage composition.
POP4
A. Bertoli et al. / Industrial Crops and Products 32 (2010) 329–337
Tricyclene ␣-Thujene ␣-Pinene Camphene Sabinene -Pinene Myrcene ␣-Phellandrene 3-Carene ␣-Terpinene p-Cimene Limonene -Phellandrene 1,8-Cineol (Z)-ocimene (E)-ocimene ␥-Terpinene Terpinolene Linalool Nonanal Borneol 4-Terpineol ␣-Terpineol Caryophyllene ␥-Elemene Trans-␣-bergamotene ␣-Guaiene Aromadendrene (E)--farnesene ␣-Humulene Alloaromadendrene ␥-Muurolene -Selinene ␣-Selinene ␣-Muurolene (E.E)-␣-Farnesene -Bisabolene Cis ␥-cadinene Trans ␥-cadinene ␦-Cadinene Cadin ␣-1.4-diene ␣-Cadinene Selina 3.7 (11) diene Germacrene B Trans-nerolidol Caryophyllene oxide ␥-Eudesmol -Eudesmol -Bisabolol ␣-Bisabolol Cannabidiol
POP1
332
Table 3a Composition of static headspace of the different fibre hemp varieties. Fibre hemp cultivars
Pop1
Pop2
2005 Compound
854 855 933 942 959 984 992 1010 1013 1022 1030 1035 1037 1040 1051 1064 1089 1420 1430 1437 1457 1489 1493 1496 1503 1534 1539 1560 1584
pdms tr tr 0.21 12.13 tr 6.72 39.41 0.91 1.12 1.04 – 9.59 – – 4.88 0.35 20.46 1.64 0.54 tr 3.32 1.28 0.23 0.12 0.78 1.02 0.34 0.23 0.12
2006 carb – 0.22 0.12 5.47 tr 3.70 28.26 0.29 0.52 0.38 tr 4.64 – – 2.73 0.15 14.96 19.25 0.47 tr 4.84 1.68 0.81 1.07 0.76 2.86 0.62 0.3 0.09
pdms b
0.05 0.15 0.16 1.99 0.08 1.55 38.99 1.35 1.43 1.02 – 0.75 3.11 tr 10.43 0.69 33.66 0.74 0.56 – 2.01 1.02 0.34 0.24 0.14 0.13 0.12 0.08 0.02
2005
2006
Pop4
2005
2006
Pop5
2005
2006
2005
2006
carb
pdms
carb
pdms
carb
pdms
carb
pdms
carb
pdms
carb
pdms
carb
pdms
carb
pdms
carb
0.01 0.09 0.08 2.12 0.04 1.65 31.41 2.12 2.09 1.5 – 0.68 4.44 tr 12.18 1.41 35.53 1.35 0.76 – 1.10 0.08 0.12 0.13 0.09 0.08 0.03 tr 0.02
0.54 tr 0.25 23.68 0.12 10.62 14.42 1.05 1.77 1.34 0.23 1.58 3.94 0.18 1.56 0.42 32.65 3.95 0.23 – 0.82 – 0.01 0.03 0.07 0.04 tr tr 0.01
0.32 – 0.15 16.83 0.26 8.28 15.41 0.99 1.67 1.42 0.50 3.31 3.13 0.27 2.34 0.53 38.41 3.51 0.12 – 0.86 tr 0.01 – tr 0.03 tr tr 0.03
0.21 tr tr 9.51 0.82 4.56 44.11 2.34 1.53 0.87 0.34 2.39 2.03 0.23 2.56 0.34 13.56 3.63 00.9 – 0.66 – 0.03 – tr tr tr tr 0.04
0.13 tr – 10.34 0.48 5.19 38.4 1.72 1.46 1.15 0.21 3.54 3.01 0.14 4.06 0.5 16.5 4.38 tr – 0.11 tr 0.03 0.01 tr – – – 0.05
0.19 – 0.38 18.18 0.26 10.8 4.70 1.82 2.96 2.51 0.38 1.21 8.93 0.12 0.85 0.83 45.1 0.86 0.11 0.08 1.28 0.12 0.34 0.45 0.23 0.98 0.10 tr 0.10
0.21 – – 9.22 0.18 6.69 2.60 1.01 1.33 1.23 0.25 1.18 5.79 0.11 0.53 0.44 50.11 8.62 0.21 0.02 3.69 0.79 0.51 0.51 0.33 1.4 0.29 0.11 0.14
tr – 0.16 5.18 0.27 3.39 34.0 1.53 1.32 1.06 0.12 1.8 2.63 0.09 15.32 0.51 27.55 0.76 0.19 0.01 1.35 – tr tr tr – – 0.01 0.08
tr – 0.14 4.11 0.23 2.73 22.33 2.07 1.88 1.54 0.09 2.36 3.31 tr 14.16 1.00 23.94 1.04 0.10 – 1.75 tr tr tr 0.13 0.14 – tr 0.02
1.45 tr 0.11 11.56 0.16 4.62 46.2 0.42 0.56 0.52 0.12 4.26 0.12 1.41 11.52 0.19 13.13 1.55 0.67 1.12 2.27 0.98 0.23 0.34 0.12 1.09 0.34 tr 0.01
1.59 tr – 6.66 0.14 3.2 28.16 0.42 0.34 0.44 0.23 2.52 0.11 0.86 9.06 0.11 9.13 20.08 0.12 1.96 4.3 1.31 – 1.17 0.82 1.51 0.38 – tr
0.76 tr 0.06 1.87 0.06 1.36 27.15 0.53 1.14 0.85 – 3.23 6.1 – 9.72 0.59 46.15 2.84 0.10 – 0.57 – 0.01 0.97 0.76 0.45 0.12 tr 0.03
0.87 0.10 tr 0.92 0.05 0.65 23.58 0.5 0.71 0.65 – 4.35 5.02 – 9.44 0.54 52.59 3.62 0.09 – 0.65 tr – 0.78 0.34 0.08 0.05 0.02 0.01
– tr 0.14 5.38 0.04 2.29 35.02 0.84 1.03 0.97 0.13 0.51 2.08 1.12 14.63 0.33 31.65 2.12 0.04 tr 0.49 tr – 0.25 0.23 0.03 0.02 tr 0.04
0.18 tr 0.12 4.94 0.03 2.21 32.99 0.83 0.92 0.92 0.17 3.04 – 1.25 14.13 0.35 32.19 2.84 0.03 tr 0.64 0.02 tr – – 0.01 tr tr 0.04
– – tr 7.16 0.02 2.51 72.64 tr tr tr tr 0.45 1.67 0.66 10.94 tr 0.17 0.10 tr – 1.34 tr tr 0.03 – 0.02 tr 0.01 0.05
– – tr 2.83 tr 0.82 82.87 tr tr – tr 1.21 1.31 0.63 7.52 tr 0.15 0.09 – – 1.87 0.01 tr tr – – – 0.01 0.08
Bold values are referred to the main constituents of the analysed samples. a LRI = Linear retention indeces on DB5-column; pdms = polydymethylsyloxane fibre; carb= carboxen fibre. b Relative percentage composition; tr = traces; <0.01%; (–) = not detected.
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2-Hexenal hexenol ␣-Thujene ␣-Pinene Camphene -Pinene Myrcene ␣-Phellandrene 3-Carene ␣-Terpinene p-Cymene Limonene -Phellandrene (Z)-ocimene (E)-ocimene ␥-Terpinene Terpinolene Caryophyllene ␥-Elemene ␣-Guaiene ␣-Humulene -Selinene Valencene ␣-Selinene ␣-Farnesene Cadin ␣-1,4-diene Selina 3,7 (11) diene Germacrene B Caryophyllene oxide
LRIa
Pop3
Table 3b Composition of static headspaces of the different fibre hemp varities. Red petiole 2005
Carmagnola 2006
LRIa
pdms
carb
2-Hexenal Hexenol ␣-Thujene ␣-Pinene Camphene -Pinene Myrcene ␣-Phellandrene 3-Carene ␣-Terpinene p-Cymene Limonene -Phellandrene (Z)-ocimene (E)-ocimene ␥-Terpinene Terpinolene Linalool ␣-Terpineol Caryophyllene ␥-Elemene (Z)--farnesene ␣-Humulene -Selinene Valencene ␣-Selinene ␣-Farnesene -Bisabolene Germacrene B Caryophyllene oxide
854 856 933 942 959 984 992 1010 1013 1022 1030 1035 1037 1040 1051 1064 1089 1101 1197 1420 1430 1454 1457 1489 1493 1496 1503 1506 1560 1584
0.49b 0.42 0.17 0.54 0.04 1.19 41.56 0.8 1.02 0.92 0.05 10.89 – – 0.58 0.42 34.86 tr 0.10 32.84 tr tr 11.03 0.97 tr 0.53 0.54 – 0.35 tr
0.48 0.12 – 0.13 tr 0.35 14.83 tr tr – tr 13.32 tr tr 0.23 0.15 14.94 – 0.13 40.67 0.52 – 13.35 1.13 0.24 0.73 0.87 0.15 tr 0.12
pdms – – tr 4.20 tr 4.60 46.23 0.9 0.87 0.75 – 8.66 – – 2.15 0.48 22.58 0.17 tr 15.30 0.34 1.74 4.22 – 0.10 – – – 0.09 0.23
2005
2006
CODI
2005
carb
pdms
carb
pdms
carb
– – – 2.10 tr 2.04 53.7 1.14 1.15 0.99 tr 8.55 tr tr 2.64 0.56 18.23 0.33 tr 20.01 0.23 1.82 5.12 tr 0.11 0.02 – – 0.05 0.02
0.3 0.24 0.14 17.35 tr 8.01 27.63 1.06 1.55 1.38 0.18 3.09 5.01 tr 2.99 0.66 27.27 0.16 tr 11.55 0.11 0.34 2.35 0.76 0.34 0.03 tr tr 0.37 0.09
0.39 – 0.02 9.01 0.17 4.92 21.54 0.44 0.67 0.57 0.09 1.47 3.22 tr 1.96 0.35 27.41 0.37 0.17 13.21 0.13 0.34 3.75 0.97 0.56 0.57 0.79 0.63 tr 0.17
– – – 7.43 0.23 3.47 76.56 – – – – 8.5 – – 0.86 tr 0.18 tr 0.09 1.56 0.09 0.25 0.31 0.87 0.34 0.76 0.45 0.23 tr 0.25
tr – – 3.81 0.1 1.87 82.36 0.02 – tr tr 8.41 tr tr 1.02 – 0.21 tr 0.05 1.36 0.05 0.03 0.26 0.34 0.36 0.78 0.13 0.09 tr –
pdms 0.28 tr 0.28 15.9 0.23 6.46 30.63 0.89 1.35 1.01 0.09 tr 3.63 1.84 8.71 0.44 23.31 0.21 0.07 2.59 0.04 0.02 4.72 0.09 0.12 0.99 tr 0.02 0.25 –
2006
Felina34
2005
2006
2005
2006
carb
pdms
carb
pdms
carb
pdms
carb
pdms
carb
0.23 tr – 6.98 tr 2.94 23.7 0.32 0.57 0.39 0.06 tr 1.55 0.76 5.84 0.18 18.07 0.3 0.05 22.54 0.16 0.13 7.59 0.71 0.46 0.46 1.25 0.15 tr 0.09
– tr 0.2 7.37 0.27 2.76 68.76 tr tr tr – 6.97 – – 8.46
– tr – 4.21 0.1 1.48 74.57 – tr – – 6.32 0.84 – 7.9 0.05 0.19 0.24 0.01 2.71 tr 0.74 0.73 0.09 0.07 0.04 tr – 0.2 0.23
0.5 – 0.51 17.53 0.25 5.38 2.12 tr 18.01 tr 0.1 1.54 0.37 4.45 36.93 0.27 0.63 0.28 tr 5.48 0.47 tr 1.13 0.16 – 0.13 tr tr 0.22 0.13
0.59 1.10 0.52 14.54 0.23 5.31 2.72 – 20.9 0.11 0.12 0.37 tr 4.85 36.57 0.3 0.62 0.25 tr 4.87 0.42 tr 1.01 tr – 0.12 tr tr tr tr
– – tr 12.69 0.15 5.84 10.13 0.27 – 0.23 tr 0.34 0.87 0.87 4.29 0.34 12.16 0.27 0.98 3.09 tr 0.15 0.33 0.46 0.03 0.12 0.09 tr 0.15 0.23
– – tr 10.12 0.07 7.21 9.13 0.23 1.03 0.13 – 0.62 0.77 0.67 6.09 0.25 13.47 0.11 0.14 2.05 0.11 0.13 0.35 0.98 – 0.17 0.23 0.11 0.15 0.12
tr tr tr 15.12 0.13 7.01 17.13 0.35 0.93 0.25 tr 0.61 0.56 0.58 5.35 0.15 16.41 0.13 0.14 15.16 0.11 0.13 8.12 0.50 – 0.17 0.23 0.11 Tr tr
tr tr tr 14.71 0.09 5.21 14.79 0.35 0.98 0.43 tr 0.57 0.24 0.87 4.98 tr 15.32 tr tr 17.18 tr tr 9.25 tr – – – – – 0.13
0.18 0.19 0.04 3.38 0.02 0.10 0.98 tr tr – – – tr 0.08
pdms 0.02 – 20.35 0.05 0.05 4.31 12.27 0.08 – 0.09 0.36 0.57 0.18 0.25 5.19 tr 13.18 0.24 0.09 21.13 – 0.01 4.16 0.03 tr tr tr tr – 0.10
carb 0.02 – 20.44 0.09 0.08 5.15 13.56 0.14 – tr 0.38 0.79 0.34 0.37 4.37 0.21 12.24 0.36 0.24 22.45 – 0.02 3.96 0.05 tr 0.11 0.05 – 0.96 0.09
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Compound
C.S.
Bold values are referred to the main constituents of the analysed samples. a LRI = Linear retention indeces on DB5-column; pdms = polydymethylsyloxane fibre; carb = carboxen fibre. b Relative percentage composition; tr = traces; <0.01%; (–) = not detected.
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harvest was carried out at flowering, corresponding to the phenological codes 2103 and 2302 (Mediavilla et al., 1998) for dioecious and monoecius varieties, respectively. The harvesting dates went from the beginning of July to the end of August. Harvesting were hand accomplished and plants were cut at 4–5 cm from the soil. Production measurements (plant and stem yield for fibre production) were performed on a 5 m2 area excluding the plants on the outer rows. Plant density, biomass, stems and leaves fresh weight were measured then samples were subsequently allowed to dry into a ventilated oven, for dry weight determination. The stem height and the basal diameter on 20 plants for each plot were also determined. Afterwards, the fresh inflorescences were manually sampled from the same plants, cutting the 30 cm upper part of the stem, and weighed for fresh and dry weight (after ventilated oven drying at 50 ◦ C). The fresh inflorescences were further sampled directly in the field (by cutting the 30 cm upper part of the stem) from 10 to 20 plants per plot randomly chosen. They were collected in plastic bags, put into cool thermos, delivered to the analytical laboratory and hydrodistilled immediately.
2.2. Phytochemical analysis 2.2.1. Chemicals The linear hydrocarbons (C9 –C40 ) and the volatile compounds used as standards were commercial substances purchased from Fluka (Sigma–Aldrich) with 98–99% grade purity. The stock and working solutions were prepared using n-hexane (Carlo Erba, HPLC-grade)
2.2.4. Identification and quantitation The identification of the essential oil constituents was performed by the comparison of the retention times of their constituents with those of pure reference material and by mean of their Linear Retention Indices (L.R.I.) relative to a series of nhydrocarbons (C9 –C40 ) on the two different columns. In addition, it was used a computer matching of mass spectra with two commercial data base (ADAMS 1995; NIST 2000) and an experimental home-made library mass spectra built up from pure substances or known oils. Moreover, the molecular weights of the all identified substances were confirmed by GC/CIMS, using MeOH as CI ionizing gas. The composition of the different essential oils and headspaces were showed as relative percentage composition by internal peak area normalization, not inclusive of solvent peak and all relative response factors being taken as one. Repeatability of the measuring system showed variation coefficients under 5% for the identified components in the headspaces and essential oils (Tables 2, 3a and 3b). 2.2.5. Statistical analysis All measured and derived data were analysed by analysis of variance (ANOVA) using the CoStat software, version 6.2. In all cases, means were separated on the basis of least significant difference (LSD) only when the F-test of the ANOVA treatment was significant at the 0.05 or 0.01 probability level (Gomez and Gomez, 1984) 3. Results and discussion 3.1. Agronomic evaluation
2.2.2. Extraction procedures and sample preaparation The fresh inflorescences (150–200 g) were hydrodistilled (2 h, 2 l water distilled, flow 2.0 ml/min) by a Clevenger apparatus described in the Italian Pharmacopoeia F.U.I. XI Ed. The EOs yields are reported in Table 1. The essential oils were dissolved in Et2 O, dried over anhydrous MgSO4 , filtered and the solvent removed by evaporation on a water bath. All the essentials oil were diluted in n-hexane (HPLC solvent grade, 10%) and injected both in GC-FID (injection volume 1 ml, HPWAX and HP-5 capillary columns) and GC–MS (injection volume 0.1 ml, DB-5 capillary column). SPME analyses were performed with Supelco SPME devices, coated with polydimethylsiloxane (PDMS, 100 m) and with Carboxen (100 m) in order to sample the headspace of a fixed portion (10 g) of the fresh hemp fibre inflorescences at room temperature for 20 min.
2.2.3. Gas chromatographic analysis The GC-FID analyses of the standard compounds and EOs samples were accomplished by HP-5890 Series II instrument equipped with HP-WAX and HP-5 capillary columns (30 m × 0.25 mm, 0.25 m film thickness), working with the following temperature program: 60 ◦ C for 10 min, ramp of 5 ◦ C/min up to 220 ◦ C; injector and detector temperatures 250 ◦ C; carrier gas nitrogen (2 ml/min); detector dual FID; split ratio 1:30; injection volume of 1 ml (10% v/v n-hexane solution). The GC/EIMS analyses were performed by a Varian CP-3800 gas chromatograph equipped with a HP-5 capillary column (30 m × 0.25 mm; coating thickness 0.25 m) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions: injector and transfer line temperatures 220 and 240 ◦ C, respectively; oven temperature programmed from 60 to 240 ◦ C at 3 ◦ C/min; carrier gas helium at 1 ml/min; injection volume 0.1 ml (10% n-hexane solution); split ratio 1:30. The GCMS analysis was repeated three times for each sample.
3.1.1. Growth and biomass production The Italian dioeciuos (Carmagnola, Fibranova, C.S., Red Petiole, Pop1, Pop2, Pop3, Pop4, Pop5) cultivars showed biometric and phenological traits greatly different from the monoecious ones (Codimono and Felina 34). The monoecious varieties, developed through breeding and selection, show the staminate and pistillate flowers on the same plant, but this character is usually not completely stable. In fact Codimono, the Italian monoecious variety, proved to be still genetically unstable, as elevated percentages of male plants (10%) were present in the experimental plots. On the other hand, the French monoecious cultivars Felina 34, was rather stable with only 1% of male plants on the total plants present in each plot. These two monoecious cultivars showed greater earliness, reaching flowering roughly three/four weeks before the dioecious ones. The analysis of variance has outlined significant effect of type of cultivar (dioecious and monoecious), year and their interaction on the main biometric and productive characters. The monoecious cultivars were characterized by higher plant density, early growth, early flowering and a lower stem height. On the contrary, dioecious cultivars were characterized by lower plant density, late flowering and higher stem development (Table 1). In particular the dioecious cultivars showed a significant lower plant density than monoecious ones (121.7 and 136.6 plants m−2 ) but a greater plant height (2.89 and 1.92 m) (Table 1). Mean plant densities at harvest were 137.5 and 111.3 plants m−2 in the two years. In the 2006 growing season the unfavourable climatic conditions after sowing, due to heavy rains and low air temperatures, negatively affected the seed germination and consequently plant density both in dioecious and monoecious cultivars with lower values (−19 and −18%, respectively) than 2005 (Table 1). In 2005 Felina 34 achieved greatest density, although differences compared to Pop 1, Pop 2 were not statistically significant. In the second year, greatest density was recorded always for Felina 34 (135.3 plants m−2 ), while
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335
Fig. 1. Production of the essential oil target-compounds (relative composition %) in the analysed cultivars in the 2005 and 2006 growing seasons. For each year the mean values followed by the same letter are not statistically different at P ≤ 0.05 according to LSD test (for 2005 italic letters and for 2006 normal letter).
Carmagnola and Red Petiole presented the lowest values (roughly 95 plants m−2 ) (Table 1). Plant height, similarly to plant density, was on average higher in the first year (2.89 versus 2.47 m). In the 2006 growing season the monoecious cultivar flowered about 2 weeks before than 2005. According to Amaducci et al. (2008) findings, once the flowering starts the dry matter accumulation drops rapidly and consequently
monoecious cultivars showed shorter stems (−28%) and lower above ground dry (−47%) and stem dry production (−51%) than the year before (Table 1). The earlier sowing time of 2006 (4th April instead 27th April) allowed the dioecious cultivars to prolong the growing season before flowering and to achieve higher plant and stem productions than 2005 (+9.4 and +4.6%). In these cultivars, selected with a longer critical photoperiod, the flowering began just
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one week later than 2005 and approx 4 weeks later than monoecious cultivars. Mean dry stem yields were 31.6 and 30.7 g plant−1 in the first and second year, respectively. Given the importance of this parameter, it is helpful to divide the trial cultivars into three groups for each of the years. Thus in 2005 C.S., Red Petiole, Carmagnola and Pop 5 were ranked in the top group, achieving the highest values with yields above 36.9 g plant−1 . The second group, composed of five cultivars, gave production ranging between 22.1 and 31.2 g plant−1 . The third group was composed of Felina 34 that showed a significantly lowest production compared to the other varieties. In 2006, the best performing cultivars were C.S., Red Petiole (as the year before), Pop 2, Pop 5, Pop 4, and Pop 1. In contrast to the previous year, lower dry stem yield was obtained with Carmagnola (26.65 g plant−1 ). Finally, the lower yields were recorded for the two monoecious cultivar Codimono and Felina 34, with values ranging from 11.86 to 10.5 g plant−1 . The development of the upper part of the plant, is greater in the monoecious cultivars, namely in Felina 34 than in the dioecious ones (Table 1). The inflorescence production of the female plants in the dioecious cultivars is greater in Red petiole, Pop 5 and Pop 1 in 2005 and in Pop 3, Pop 4 and Pop 1 in 2006. The lowest value was achieved in Carmagnola in 2005 (2.34 g plant−1 ) and in Pop 5 in 2006 (1.95 g plant−1 ). 3.2. Phytochemical investigation The essential oils (EOs) were obtained by hydrodistillation of inflorescences using the Clevenger apparatus described in the Italian Pharmacopoeia F.U.I. XI Ed. A preliminary screening of the headspaces (HSs) by SPME analysis were carried out on the fresh inflorescences of the different varieties to define the spontaneous aroma emitted from the plant material without artefacts due to the extraction process. Furthermore, only fresh upper parts were considered for the production of the essential oils in order to avoid any chemical variation during the drying process. The essential oil yields ranged from 0.10 to 0.25% (w/w) (Table 1) and they were similar or higher than literature data previous reported for C. sativa (Fournier and Paris, 1978; Hillig, 2004; Meijer and Mediavilla, 1998; Nissen et al., 2009; Novak et al., 2001; Novak and Franz, 2003). The EOs content in the monoecious upper inflorescences was lower in comparison with the dioecious ones, even if the Carmagnola cultivar (dioecious) showed the lowest yield (0.10%, w/w ±0.02). It is important to point out that the EO yield trend was opposite to the biomass inflorescence production as the upper part development was greater in the monoecious cultivars than in the dioecious ones. The development of the female inflorescences was significantly lower (2.98 and 3.87 g plant−1 dry weight), but the essential oil contained in them was higher (0.17 and 0.12%, w/w) (Table 1). Ten characteristic terpenes were chosen as marker compounds to monitor the EOs production in the plant material, collected in 2005–2006 (Fig. 1). ␣-Pinene (3.0–20.5%), -pinene (1.4–8.0%), myrcene (8.2–45.3%), limonene (0.3–6.4%), (E)-ocimene (0.7–10.3%), terpinolene (0.1–22.1%), -caryophyllene (7.3–28.0%), ␣-humulene (3.2–12.6%), caryophyllene oxide (1.9–5.7%) were the main constituents (Table 2 and Fig. 1). The gas chromatographic profiles of the corresponding headspaces showed significant qualitative and quantitative differences in comparison with the EOs, but these main constituents were confirmed apart from caryophyllene oxide (Tables 3a and 3b). However, the analysed monoecious and dioecious cultivars did not show menth-compounds such as menthone, menthol, and their acetate derivatives, which have been already reported for Cannabis cultivars (Tognolini et al., 2006). In the studied inflorescence EOs, ␣-pinene (7.9% ± 0.98, 2005; 11.4% ± 0.84, 2006), myrcene (20.2% ± 3.68, 2005; 15.6% ± 0.92, 2006), terpinolene (13.7% ± 0.61,
2005; 12.8% ± 0.97, 2006), and -caryophyllene (15.7% ± 0.61, 2005; 21.9% ± 0.67, 2006) were the main constituents in monoecious hemps, both in 2005 and 2006 (Fig. 1). The essential oils of dioecious cultivars produced significant percentages of myrcene (22.0% ± 1.4, 2005; 16.0% ± 1.1, 2006), terpinolene (10.3% ± 1.48, 2005; 11.7% ± 1.09, 2006) and -caryophyllene (15.2% ± 0.87, 2005; 22.9% ± 0.66, 2006) (Fig. 1). Particular attention was focused on ␣-pinene and -pinene, which showed much higher amounts in the monoecious cultivars (18.1% ± 1.0, year 2005; 17.3% ± 1.4, year 2006) than in dioecious ones (2.7% ± 0.1, year 2005; 4.5% ± 0.2, year 2006, Table 2 and Fig. 1). As ␣-pinene and -pinene were detected in such a huge amounts both in 2005 and 2006 collection, they might be useful markers to distinguish the monoecious Felina 34 and Codimono from the dioecious cultivars. A previous study on C. sativa cultivars, originated in different countries reported higher percentages of ␣-pinene (44–71%) in the essential oils in contrast to another monoterpene, terpinolene, which was found only around 1%. In the present work, the ten analysed cultivars, cultivated in Central Italy, showed much lower amounts of ␣-pinene than literature data (Hood and Barry, 1978), apart from the monoecious Felina 34 cultivar, which contained it in higher level (20.3% ± 1.4, 2005; 20.4% ± 0.9 2006) (Fig. 1). On the other hand, a significant presence of terpinolene was also observed in the EOs, especially in Felina 34 (19.0% ± 0.14, 2005; 19.0% ± 0.05) and in Codimono (20.1% ± 0.08, 2005; 15.5% ± 0.02, 2006) (Table 2 and Fig. 1). Therefore, significant production of ␣- and -pinenes as well as terpinolene generally characterized the EOs extracted from the monoecious cultivars (Fig. 1). Furthermore, terpinolene was another important constituent in many dioecious cultivars both in 2005 and 2006 (10.3% ± 1.5, 2005; 11.7% ± 1.1, 2006), except in the case of Carmagnola that showed the lowest yield (1.59% ± 0.6, 2005; 0.09% ± 0.05, 2006) (Fig. 1). Therefore, terpinolene was included in the pool of target monoterpenes to control the EOs production in the 2005–2006 hemp collections. Fewer hydrocarbon and oxygenated sesquiterpenes were generally present in high amounts in comparison with the monoterpenes (Fig. 1). -Caryophyllene (15.7% ± 0.61, 2005 average value; 21.9% ± 0.67, 2006), ␣-humulene (5.98% ± 0.61, 2005; 8.2% ± 0.31, 2006) and caryophyllene oxide (3.89% ± 0.61, 2005; 3.17% ± 0.17, 2006) were the main sesquiterpenes in all the analysed cultivars (Fig. 1). Their production was more constant than monoterpenes among the dioecious and monoecious cultivars in the two-year harvest (Fig. 1). Many other minor sesquiterpenes (around 1%) were identified such as -phellandrene, -selinene, germacrene B, trans-nerolidol, -bisabolol. These sequiterpenes have already reported in the literature as typical components of the Cannabis spp. essential oil (Hillig, 2004; Lemberkovics et al., 1981; Nissen et al., 2009), but in the present study, they were not considered as marker compounds because of their scarce and variable amounts in the period 2005–2006. In addition to terpenes, other secondary metabolites typical of C. sativa EO such as cannabinoids, are extremely important for the pharmacological activity (Segelman et al., 1974). The ratio (THC + CBN)/CBD has been proposed to classify Cannabis plant as drug- or fibre-type (Bocsa and Karus, 1998). The cannabinoid amount is generally very low in the EOs of C. sativa (Lemberkovics et al., 1981; Malingrè et al., 1975), but their content is strictly dependant on the state, age, number of secretory glands in the plant material (Lanyon et al., 1981; Meijer and Mediavilla, 1998) as well as the extraction condition (de Meijer, 1998). Furthermore, the differences in cannabinoids content may reflect procedural variation in sampling rather than external environmental effects (Hemphill et al., 1980; Lanyon et al., 1981; Turner et al., 1977). Therefore, particular attention was focused on the plant material sampling in the present study as
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described before. The production of the hallucinogenous tetrahydrocannabinol (TCH) and the non-hallucinogenous cannabidiol (CBD) were investigated together with the main target terpenes in the essential oils of the analysed fibre hemp cultivars (Table 2 and Fig. 1). Tetrahydrocannabinol (TCH) was detected in traces (less than 0.01%) only in the two monoecious collected in 2005 (Codimono and Felina 34), while the non-hallucinogenous cannabidiol (CBD) was detected in the all analysed EOs samples, exception given for the two dioecious cultivars Pop4 and C.S., collected in 2005 (Fig. 1). In general, CBD content was higher in dioecious cultivars (4.4% ± 0.3, 2005; 2.7% ± 0.3, 2006) than in monoecious (0.9% ± 0.08, 2005; 1.2% ± 0.3, 2006) in both harvesting years (Table 2 and Fig. 1). The 2005 dioecious collection showed the highest CBD yields, especially for Pop1 (13.9% ± 0.03) and Pop5 (11.0% ± 0.01), even if this compound was not present in the EO of Pop4, Carmagnola and C.S. harvested in 2005. The monoecious cultivars showed a much more stable CBD production in the two years. In particular, Felina 34 EO showed much higher amount of CBD (1.7% ± 0.11, 2005; 2.0% ± 0.03, 2006) than Codimono (0.2% ± 0.02, 2005; 0.4% ± 0.38, 2006) (Fig. 1). 4. Conclusions This study investigated the biomass and EO production of ten fibre-hemp cultivars, cultivated in Central Italy in two-year field trial. Their fresh inflorescences yielded significant amounts of essential oils which were characterized by the typical C. sativa terpenes and a legal and safe cannabinoid content. The EOs and HSs profiles of the different inflorescences were suitable to confirm the fibre-type classification of these cultivars which showed specific volatile constituents. In addition, significant quali–quantitative differences in the production of some target terpenes such as ␣-pinene, -pinene, and terpinolene allowed to distinguish monoecious from dioecious hemp cultivars. Interest in industrial hemp has gained momentum world wide, suggesting that demand for natural fibres will continue to increase. Market segmentation for ethically produced goods and growing demand for biodegradable and natural products has led to a wide range of new industrial hemp products being developed. A wide variety of hemp-based products are already available on the market, but the hemp essential oil is still considered a nicheproduct. The present study confirm the hypothesis to grow industrial fibre hemp as a multi-use crop through a complete utilization of the plant material using stems for fibre industry and inflorescences to produce essential oils as alternative flavour and fragrance additives. References Amaducci, S., Colauzzi, M., Zatta, A., Venturi, G., 2008. Flowering dynamics in monoecious and dioecious hemp genotypes. J. Ind. Hemp 13 (1), 5–19. Bocsa, J., Karus, M., 1998. In: Hemp Tech (Ed.), The Cultivation of Hemp, Sebastopol, California.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Fibre hemp inflorescences: From crop-residues to essential oil production Alessandra Bertoli a,∗ , Sabrina Tozzi b , Luisa Pistelli a , Luciana G. Angelini b a b
Dipartimento di Scienze Farmaceutiche-sede Chimica Bioorganica e Biofarmacia, University of Pisa, Bonanno 33, 56126 Pisa, Italy Dipartimento di Agronomia e Gestione dell’Agroecosistema, University of Pisa, S. Michele degli Scalzi 2, 56127 Pisa, Italy
a r t i c l e
i n f o
Article history: Received 14 December 2009 Received in revised form 14 May 2010 Accepted 21 May 2010
Keywords: Cannabis sativa L. Fibre hemp Monoecious Dioecious Essential oils SPME GC–MS
a b s t r a c t The volatile composition of ten fibre hemp (Cannabis sativa L.) varieties was investigated during two successive growing seasons under temperate climatic conditions in Central Italy. The freshly plant inflorescences were hydrodistilled and the essential oils (EOs) were characterized by GC–MS. In addition, the composition of the aroma emitted spontaneously from the freshly plant inflorescences were analysed by SPME-GC–MS. The EO yields of eight dioecious (Carmagnola, C.S., Red Petiole, Pop 1, Pop 2, Pop 3, Pop 4, Pop 5) and two monoecious (Codimono and Felina 34) cultivars ranged from 0.11 to 0.25% (w/w) and showed a significant production of ␣-pinene (3–20%), -pinene (1–8%), E-ocimene (1–10%), myrcene (8–45%) and terpinolene (0.12–22%). The monoterpene composition was useful to distinguish the monoecious cultivars from the dioecious ones. -Caryophyllene (7–28%), ␣-humulene (3–12%), and caryophyllene oxide (2–6%) were the main sesquiterpenes. Tetrahydrocannabinol (THC) was present in traces in the EOs of only two dioecious cultivars cultivated in 2005. Cannabinol (CBN) was not detected in the essential oils, while the no-hallucinogenous cannabidiol (CBD) was found as typical volatile constituent in several analysed cultivars. These findings were also confirmed by the headspace GC–MS analysis carried out on the same samples. The analysed EOs obtained from fibre hemp varieties cultivated in Central Italy were characterized by an interesting and specific terpene composition with a legal and safe cannabinoid content. They were obtained from freshly plant inflorescences, which usually represent a waste material from C. sativa L. fibre varieties. The present study strengths the hypothesis to grow hemp as a multi-use crop through a complete utilization of the plant material using inflorescences to produce essential oils as natural flavour and fragrance additives. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, there is an increasing tendency in the use of plant-derived raw materials as alternative renewable sources to petrochemical derived products. In this situation, the resurgence of interest in fibre hemp (Cannabis sativa L.) as a multipurpose crop has been arising in Europe due to the multitude of end products derived from the different organs (Ranalli and Venturi, 2004). Besides the interest for textile uses, interest is growing in cultivating industrial hemp for non-textile applications. It is important to point out that the fibre hemp varieties are eligible for cultivation only after the verification of their tetrahydrocannabinol (THC) content recommended by the Regulation EC no. 1124/2008 (12 November 2008). Several studies have been carried out on the cannabinoid content, resin, and seed oil of C. sativa and their dif-
∗ Corresponding author at: Dipartimento di Scienze Farmaceutiche, University of Pisa, Via Bonanno 33, 56126 Pisa, Italy. Tel.: +39 050 2219705; fax: +39 050 2219660. E-mail address: [email protected] (A. Bertoli). 0926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2010.05.012
ferent products (Brenneisen and Elsohly, 1988; ElSohly and Slade, 2005; Kriese et al., 2004; Leizer et al., 2000; Turner et al., 1980). The hemp essential oil has generally been considered a niche high value product with promising potential marketing (Mediavilla and Steinemann, 2005; de Meijer, 1998; Thomas et al., 2000). It is a complex mixture of many volatile compounds, mainly monoterpenes, sesquiterpenes, and other terpenoid-like substances (Fournier and Paris, 1978; Novak and Franz, 2003). Monoterpenoids are principally responsible for differences in fragrance among hemp cultivars. Sesquiterpenoids are characteristic components too, but they are generally in lower amounts in comparison with monoterpenes (Hendriks et al., 1975; Hillig, 2004; Lemberkovics et al., 1981; Ross and ElSohly, 1996). Hemp essential oil is biosynthesized in the epidermal glands or glandular hairs where the cannabinoids are produced too (Kim and Mahlberg, 1981; Malingrè et al., 1975). Furthermore, the cannabinoid compounds can be hydrodistilled together with terpene constituents in the essential oil. The highest density of glandular hairs is found on the bract surrounding each female flower and the subtending leaflets of the female inflorescence (Hemphill et al., 1980; Lanyon et al., 1981). When fresh
Mean values within each column followed by the same letter are not significantly different for P < 0.05 probability level according to LSD test. Bold letters for the interaction “Type of cultivar × Year”. a With “upper part” we refer to the 30 cm from the top of plants. * Significance at P 0.1, according to F-test by ANOVA analysis. ** Significance at P 0.05, according to F-test by ANOVA analysis. *** Significance at P 0.01, according to F-test by ANOVA analysis.
* * * * *
*
0.15b 0.12 *
0.20a 0.13b 3.18b 4.08a
2.78b 3.67a
*** ***
35.62a 11.18c 33.48a 22.97b 43.24a 15.85c
** *** ***
39.15a 30.15b 3.04a 2.23c 108.7b 123.0b 134.7ab 150.2a
** ** **
2.66b 1.66d
***
**
*
0.12bc (0.08) 0.11c (0.07) 0.13d (0.09) 0.13cd (0.09) 4.13 (0.14) 3.21abc (1.06) 4.40 (1.17) 3.75ab (0.96) 11.86 (2.1) 10.5c (0.9) 16.81 (3.8) 29.13bc (5.7) 16.58 (3.1) 15.12b (1.9) 23.04 (4.8) 37.26abc (7.0) 1.36 (0.08) 1.85c (0.14) 135.3 (19.0) 2.04 (0.26) 110.8ab (10.2) 2.42c (0.08) 178.7 (7.7) 121.7b (7.8)
Monoecious Felina 34 Codimono Significance cultivar Dioecious Monoecious Significance interaction T×Y
0.15abc (0.05) 0.14abc (0.06) 0.10c (0.08) 0.19a (0.09) 0.14abc (0.09) 0.17ab (0.07) 0.12bc (0.09) 0.19a (0.07) 0.25a (0.09) 0.11d (0.07) 0.22ab (0.05) 0.23ab (0.08) 0.20abc (0.08) 0.19abc (0.05) 0.22ab (0.08) 0.15bcd (0.08) 2.71bcd (0.57) 2.01d (0.32) 2.86bcd (0.60) 3.31abc (0.82) 2.39cd (0.39) 3.62ab (0.56) 3.37abc (1.50) 1.95d (1.07) 3.40bc (0.24) 3.79ab (1.24) 2.34d (0.66) 3.48ab (0.82) 2.49cd (0.57) 3.32bc (1.08) 2.87bcd (0.92) 3.71ab (0.31) 2.71bcd (0.57) 2.01d (0.32) 2.86bcd (0.60) 3.31abc (0.82) 2.39cd (0.39) 3.62ab (0.56) 3.37abc (1.50) 1.95d (1.07) 3.40bc (0.24) 3.79ab (1.24) 2.34d (0.66) 3.48ab (0.82) 2.49cd (0.57) 3.32bc (1.08) 2.87bcd (0.92) 3.71ab (0.31) 47.58a (9.3) 46.76a (8.1) 36.19a (5.8) 41.04a (6.5) 46.51a (19.3) 41.65a (6.3) 41.81a (13.8) 44.37a (9.9) 52.55a (10.5) 49.09a (8.6) 43.24ab (11.9) 35.09abc (9.1) 25.73bc (9.3) 27.25bc (4.8) 36.37abc (6.8) 43.87ab (7.8) 2.91a (0.15) 2.68ab (0.34) 2.71ab (0.12) 2.57ab (0.26) 2.64ab (0,24) 2.44b (0.20) 2.75b (0.24) 2.57ab (0.30) 3.30a (0.10) 3.11ab (0.22) 3.17ab (0.30) 3.22ab (0.37) 2.83ab (0.35) 2.74bc (0.15) 2.89ab (0.08) 3.06ab (0.38) 125.5b (21.1) 130.0b (11.1) 133.0b (5.7) 162.0ab (21.2) 151.3ab (16.8) 119.5b (8.7) 135.3b (15.4) 120.8b (16.3) C.S. Red petiole Carmagnola Pop 1 Pop 2 Pop 3 Pop 4 Pop 5
108.8b (13.2) 95.5b (10.8) 95.3b (5.3) 116.3ab (15.5) 118.8ab (11,9) 107.8b (17.5) 121.3ab (16.4) 105.5b (12.6)
2006 2005 2006 2005 2006 2005 2006 2005 2006 2005 2005 Dioecious
2006
Plant density (number/m2 )
Plant height (m)
Plant dry yield (g/plant)
Stem dry yield (g/plant)
Dry Upper parta (g/plant)
Essential oil (% w/w)
A. Bertoli et al. / Industrial Crops and Products 32 (2010) 329–337
Cultivar
Table 1 Mean values (±standard deviation) of the main biometric and productive parameters of the hemp cultivars in 2005 and 2006 experimental seasons. Mean values of the main parameters related to the interaction T × Y between the type of cultivar (monoecious and dioecious) and the year (2005 and 2006) are also reported.
330
female inflorescences are dried, a greater loss of monoterpenoids than sesquiterpenoids is observed, but none of the major components of the oil completely disappears (Ross and ElSohly, 1996). Most of the studies on the essential oil production have been carried out for the strain selection of drug type C. sativa (Mediavilla and Steinemann, 2005; Novak et al., 2001; Novak and Franz, 2003). On the contrary, there are fewer studies on the essential oil extracted from the inflorescences of fibre type C. sativa cultivars. Recently, Nissen et al. (2009) characterized the essential oils of three legal hemp varieties for their in vitro antimicrobial activity, evidencing interesting perspectives as alternative antibacterial agents. The objectives of this study were to monitor the variations within ten monoecious and dioecious fibre hemp varieties grown in Central Italy in a two-year field trial: (a) on the main productive and biometric characteristics (plant density, plant height, plant and stem dry yield); (b) on the yields and composition of the essential oils. Therefore, the aim of this study was to bring new knowledge on the fibre hemp multi-use crop and the potentiality of inflorescences, generally considered waste parts for fibre industry, were considered as standardized plant material to produce valuable essential oils.
2. Material and methods 2.1. Growing conditions and field experiment layout The experiments were carried out at the Experimental Centre of Dipartimento di Agronomia e Gestione dell’Agroecosistema (DAGA) of Pisa University (15 km SW of Pisa, Italy, latitude 43◦ N, longitude 10◦ E, altitude 10 m above sea level) during the seasons 2005 and 2006. The morphology of the region is flat and is characterized by a Mediterranean climate, with rainfall mainly concentrated in the autumn and spring (mean 941 mm year−1 ). Mean air temperatures generally increase from April to July with mean minimum temperature of 9.6 ◦ C, and mean maximum temperature of 20.0 ◦ C. During summer (July–half August) a dry period generally occurs with low rainfall and high air temperatures often above 25 ◦ C. The loam soil, representative of the low Arno river plain, has a good fertility and water retention capacity (sand 44.3%; silt 41.0%; clay 14.6%; organic matter 1.8%; pH 8.0; total nitrogen 0.12%; available P2 O5 17.25 mg kg−1 ; exchangeable K2 O 97.0 mg kg−1 ; field capacity 23.0% dw; wilting point 9.4% dw). The previous crop was castor in 2005 and rapeseed in 2006. Soil tillage was done in November 2004 and 2005 with 30 cm deep ploughing and two superficial disks harrowing at the end of March to prepare the sowing bed. Plants were maintained under identical fertilization conditions throughout the field experiments. Mineral fertilizer was applied at pre-planting at rates of 120/80/120 kg ha−1 of N/P/K. No irrigation supplies were needed after sowing either in summer period thanks to the natural soil water availability. Ten Italian and European fibre hemp cultivars were compared in a randomized block experimental design with four replications: Carmagnola, C.S., Red Petiole, Pop 1, Pop 2, Pop 3, Pop 4, Pop 5 as Italian dioecious, and Codimono, Felina 34 as Italian and French monoecious, respectively. Seed were kindly supplied by ISCI-CRA (Bologna, Italy) and showed a legal THC content (i.e. <0.2%, w/v). Hemp cultivars were sown the 27th April 2005 and the 4th April 2006 by a pneumatic drill with 0.18 m inter-row in order to achieve the planned crop density of 130–140 plants per m2 . Each plot size was 20 m2 (5 × 4 m) and 15 m2 (3 × 5 m), in 2005 and 2006, respectively. Crop was protected against weeds by frequent hand and mechanical weeding but no pesticides were supplied. During the growing cycle, emergence and flowering dates were recorded when at least 2/3 of plants showed the specific phenological stage. The
Table 2 GC–MS results of the essential oils extracted by hydrodistillation from the different fibre hemp inflorescences cultivated in Central Italy. L.R.I.a Compound 931 941 942 959 979 984 992 1010 1013 1022 1030 1035 1037 1039 1040 1051 1064 1089 1101 1107 1177 1184 1197 1420 1431 1434 1437 1440 1454 1457 1462 1476 1489 1496 1499 1503 1506 1511 1513 1519 1534 1539 1540 1560 1563 1584 1634 1658 1673 2431
POP2
2005
2006
2005
0.08b 0.09 3.05 – 0.09 1.45 12.73 0.44 0.43 0.39 tr 0.91 0.82 – 0.27 2.96 0.31 15.78 – – – – – 11.37 – 0.35 – 0.09 0.86 3.78 0.16 0.1 0.28 0.79 0.62 0.5 0.13 tr 0.07 0.42 1.12 1.63 2.28 1.33 1.72 4.18 0.6 1.56 0.92 0.26 13.85
– 0.1 9.06 0.15 0.12 4.33 19.01 0.59 0.47 0.53 0.06 1.96 1.17 – 0.25 3.36 0.25 17.29 0.33 – 0.04 – 0.18 20.54 – 0.12 – – 0.17 7.98 0.34 – 0.2 0.4 0.39 tr tr – 0.21 – 0.66 0.14 0.32 0.13 1.41 2.69 0.31 tr 0.1 0.13 1.52
0.08 – 6.25 0.17 – 3.21 23.12 0.15 0.11 0.15 0.15 1.6 0.5 – 0.14 1.83 0.1 3.67 0.11 Tr Tr – 0.17 15.3 0.16 – – 0.1 0.1 6.6 0.13 0.18 0.27 0.83 0.72 0.62 – 0.09 0.03 0.46 1.27 3.05 3.79 4.09 1.03 4.13 0.18 tr 0.83 0.19 3.16
POP3 2006 0.07 – 16.73 0.27 – 6.91 11.32 0.4 0.3 0.36 0.23 3.37 0.92 – 0.29 2.78 0.16 10.75 0.21 tr Tr – 0.2 22.73 tr – – – Tr 7.62 0.27 – 0.12 0.23 0.26 tr – 0.1 0.05 – 0.6 0.95 1.45 1.71 0.09 2.98 0.18 0.32 0.11 0.47 1.39
2005 – 0.12 5.12 0.15 0.12 3.29 9.47 0.64 0.56 0.62 tr 2.36 1.26 tr 0.36 4.97 0.38 22.16 0.5 – 0.11 0.14 0.58 14.17 Tr – – 0.09 – 4.71 0.58 – 0.18 0.48 0.34 0.17 – 0.08 tr 0.23 0.59 0.21 0.29 0.09 1.15 5.72 tr tr 1.29 0.55 3.88
2006 – 0.09 11.24 0.19 – 6.13 11.8 0.3 0.24 0.28 tr 0.86 0.68 tr 0.73 10.28 0.15 8.71 0.26 – 0.03 0.14 25.28 – – – – – 8.01 0.43 – 0.1 0.22 0.12 – – 0.11 0.08 tr 0.39 0.09 0.22 0.09 1.35 4.14 0.43 0.11 0.32 0.13 2.03
2005 – 0.15 3.89 0.15 tr 1.88 15.54 0.49 0.55 0.55 0.54 6.39 – 3.69 0.35 16.25 0.15 –
0.35 21.98 – 0.19 – 0.14 – 8.72 0.19 – 0.27 0.63 0.38 0.51 tr – tr 0.31 0.85 0.24 0.73 0.12 1.48 4.54 tr tr 0.62 0.77 –
POP5 2006 0.06 0.1 9.39 0.18 – 4.71 18.14 0.41 0.29 0.39 0.28 3.44 0.83 – 0.35 4.35 0.18 11.53 0.25 0.05 0.05 0.04 0.19 21.65 – 0.1 – 0.08 – 6.98 0.23 – 0.25 1.03 0.79 0.48 – 0.12 0.23 0.21 0.67 0.13 0.37 0.17 1.51 2.5 0.22 0.11 0.17 0.21 2.82
2005 – 0.2 5.31 0.12 0.09 1.90 12.15 0.28 0.16 0.65 0.09 0.62 0.73 – 0.15 3.89 0.11 12.07 0.18 tr Tr 0.07 0.01 18.50 Tr 0.10 – 0.03 0.18 7.14 0.72 – 0.17 0.33 0.2 tr 0.16 tr – 0.03 0.61 0.14 0.24 0.12 0.73 5.46 0.43 0.52 0.14 – 11.05
CARM 2006 – 0.1 12.56 0.18 0.07 4.05 8.23 0.48 0.36 0.42 0.13 0.75 0.97 – 0.51 5.15 0.23 13.87 0.24 tr Tr 0.05 – 26.33 – 0.16 – 0.07 0.29 9.46 0.72 – 0.17 0.33 0.2 tr 0.16 – tr 0.01 0.61 0.14 0.24 0.12 0.73 4.31 0.51 0.22 0.28 – 2.29
2005 0.09 0.07 6.05 0.18 – 2.67 30.47 – – 0.08 tr 4.04 0.44 – 0.71 8.21 0.13 1.5 1.18 Tr 0.17 0.09 0.38 17.43 – – – 0.15 – 6.34 0.48 0.07 – 0.93 0.6 1.13 0.22 tr 0.34 0.54 1.67 0.45 0.76 0.19 0.84 2.64 tr tr 0.31 0.42 –
RED 2006 0.09 0.05 7.51 0.22 – 3.41 27.25 – – 0.06 tr 5.32 1.33 tr 0.81 0.17 0.12 1.17 0.05 0.09 0.12 0.38 21.12 – 0.1 – 0.14 – 7.54 0.18 – 0.43 0.74 0.49 0.73 0.17 0.06 0.47 0.32 1.52 – 0.75 0.22 0.72 2.36 0.35 0.25 0.16 0.95 4.59
2005
CS 2006
0.17 – 5.9 0.18 tr 3.17 27.79 0.3 0.25 0.32 0.33 6.37 0.87 0.36 tr 1.06 0.23 8.9 0.31
0.09 0.12 4.06 0.14 0.08 2.45 15.12 0.51 0.48 0.45 0.28 6.07 0.8 0.49 – 0.91 0.23 13.31 0.43
0.11 0.09 0.46 15.07 – – – 0.13 – 6.94 – – 0.49 1.44 0.28 0.65 0.09 0.11 0.09 0.37 1.13 0.3 0.49 0.13 0.85 3.09 tr – 0.43 0.2 2.92
0.07 0.1 0.42 28.02 – 0.11 – 0.12 0.26 12.61 0.17 0.01 0.1 0.17 0.16 0.5 0.12 0.21 0.12 0.28 0.35 0.09 0.16 0.07 0.57 2.60 0.36 tr 0.27 0.25 2.5
2005 0.17 0.08 8.08 0.26 tr 3.92 45.35 0.34 – 0.1 0.08 – 6.65 0.99 0.78 4.14 0.14 1.62 2.66 0.09 0.21 0.09 0.62 7.26 – – – – – 3.25 0.13 – 0.53 1.48 1.02 0.67 tr – tr 0.34 1.00 0.27 0.39 0.12 0.44 1.92 tr tr 0.19 0.89 –
CODI 2006 0.09 0.16 8.82 0.18 0.09 4.01 17.14 0.67 0.48 0.61 0.06 2.14 1.32 1.24 0.89 3.28 0.4 17.67 0.44 0.04 0.06 0.17 0.38 17.65 0.09 – tr 0.13 7.87 0.2 – 0.14 0.25 0.17 0.33 0.27 tr tr 0.15 0.55 0.13 0.38 0.08 0.79 1.96 0.27 0.09 0.17 1.03 4.12
FEL34
2005
2006
2005
2006
– – 15.69 0.21 0.15 5.84 13.63 0.27 – 0.23 tr 0.34 0.87 0.87 0.34 4.29 0.67 14.46 0.98 0.09 tr 0.15 0.33 16.46 0.03 0.12 0.09 tr tr 7.02 0.44 tr tr 0.50 0.1 tr 0.15 0.08 0.27 0.09 0.87 0.08 0.78 0.89 0.85 2.67 tr tr tr
0.05 0.16 14.12 0.24 0.13 7.21 15.13 0.43 1.53 0.43 0.04 0.54 0.86 1.01 0.65 5.09 0.31 15.47 0.14 0.05 0.11 0.13 0.35 15.98
0.02 – 20.35 0.08 0.05 6.31 12.27 0.08 – 0.09 0.56 0.47 0.78 0.56 0.09 5.94 0.98 14.98 0.09 tr – 0.01 0.20 19.43 tr tr tr Tr – 4.96 0.20 0.01 0.05 0.12 0.02 tr 0.09 0.23 0.10 0.13 0.97 0.03 0.82 0.05 0.34 4.23 tr 0.02 0.22
0.02 – 20.44 0.15 0.08 7.95 13.56 0.14 – tr 0.78 0.89 0.99 0.77 0.21 6.49 0.78 19.14 0.24 0.02 – 0.02 0.33 19.50 tr 0.11 0.05 – – 5.96 0.32 tr 0.01 0.20 0.09 tr 0.10 0.12 0.17 0.11 0.80 0.06 0.61 0.09 0.54 4.99 tr 0.05 0.35 0.17 1.89
0.16
0.17 0.23 0.11 0.15 8.02 0.81 – 0.35 0.94 0.71 tr tr 0.13 0.39 0.15 0.95 0.5 0.97 0.92 1.1 2.97 tr 0.29 0.34 0.26 0.36
1.69
331
LRI = Linear retention indeces on DB5-column; tr = traces; <0.01%; (–) = not detected. Bold values are referred to the main constituents of the analysed samples. a Relative percentage composition.
POP4
A. Bertoli et al. / Industrial Crops and Products 32 (2010) 329–337
Tricyclene ␣-Thujene ␣-Pinene Camphene Sabinene -Pinene Myrcene ␣-Phellandrene 3-Carene ␣-Terpinene p-Cimene Limonene -Phellandrene 1,8-Cineol (Z)-ocimene (E)-ocimene ␥-Terpinene Terpinolene Linalool Nonanal Borneol 4-Terpineol ␣-Terpineol Caryophyllene ␥-Elemene Trans-␣-bergamotene ␣-Guaiene Aromadendrene (E)--farnesene ␣-Humulene Alloaromadendrene ␥-Muurolene -Selinene ␣-Selinene ␣-Muurolene (E.E)-␣-Farnesene -Bisabolene Cis ␥-cadinene Trans ␥-cadinene ␦-Cadinene Cadin ␣-1.4-diene ␣-Cadinene Selina 3.7 (11) diene Germacrene B Trans-nerolidol Caryophyllene oxide ␥-Eudesmol -Eudesmol -Bisabolol ␣-Bisabolol Cannabidiol
POP1
332
Table 3a Composition of static headspace of the different fibre hemp varieties. Fibre hemp cultivars
Pop1
Pop2
2005 Compound
854 855 933 942 959 984 992 1010 1013 1022 1030 1035 1037 1040 1051 1064 1089 1420 1430 1437 1457 1489 1493 1496 1503 1534 1539 1560 1584
pdms tr tr 0.21 12.13 tr 6.72 39.41 0.91 1.12 1.04 – 9.59 – – 4.88 0.35 20.46 1.64 0.54 tr 3.32 1.28 0.23 0.12 0.78 1.02 0.34 0.23 0.12
2006 carb – 0.22 0.12 5.47 tr 3.70 28.26 0.29 0.52 0.38 tr 4.64 – – 2.73 0.15 14.96 19.25 0.47 tr 4.84 1.68 0.81 1.07 0.76 2.86 0.62 0.3 0.09
pdms b
0.05 0.15 0.16 1.99 0.08 1.55 38.99 1.35 1.43 1.02 – 0.75 3.11 tr 10.43 0.69 33.66 0.74 0.56 – 2.01 1.02 0.34 0.24 0.14 0.13 0.12 0.08 0.02
2005
2006
Pop4
2005
2006
Pop5
2005
2006
2005
2006
carb
pdms
carb
pdms
carb
pdms
carb
pdms
carb
pdms
carb
pdms
carb
pdms
carb
pdms
carb
0.01 0.09 0.08 2.12 0.04 1.65 31.41 2.12 2.09 1.5 – 0.68 4.44 tr 12.18 1.41 35.53 1.35 0.76 – 1.10 0.08 0.12 0.13 0.09 0.08 0.03 tr 0.02
0.54 tr 0.25 23.68 0.12 10.62 14.42 1.05 1.77 1.34 0.23 1.58 3.94 0.18 1.56 0.42 32.65 3.95 0.23 – 0.82 – 0.01 0.03 0.07 0.04 tr tr 0.01
0.32 – 0.15 16.83 0.26 8.28 15.41 0.99 1.67 1.42 0.50 3.31 3.13 0.27 2.34 0.53 38.41 3.51 0.12 – 0.86 tr 0.01 – tr 0.03 tr tr 0.03
0.21 tr tr 9.51 0.82 4.56 44.11 2.34 1.53 0.87 0.34 2.39 2.03 0.23 2.56 0.34 13.56 3.63 00.9 – 0.66 – 0.03 – tr tr tr tr 0.04
0.13 tr – 10.34 0.48 5.19 38.4 1.72 1.46 1.15 0.21 3.54 3.01 0.14 4.06 0.5 16.5 4.38 tr – 0.11 tr 0.03 0.01 tr – – – 0.05
0.19 – 0.38 18.18 0.26 10.8 4.70 1.82 2.96 2.51 0.38 1.21 8.93 0.12 0.85 0.83 45.1 0.86 0.11 0.08 1.28 0.12 0.34 0.45 0.23 0.98 0.10 tr 0.10
0.21 – – 9.22 0.18 6.69 2.60 1.01 1.33 1.23 0.25 1.18 5.79 0.11 0.53 0.44 50.11 8.62 0.21 0.02 3.69 0.79 0.51 0.51 0.33 1.4 0.29 0.11 0.14
tr – 0.16 5.18 0.27 3.39 34.0 1.53 1.32 1.06 0.12 1.8 2.63 0.09 15.32 0.51 27.55 0.76 0.19 0.01 1.35 – tr tr tr – – 0.01 0.08
tr – 0.14 4.11 0.23 2.73 22.33 2.07 1.88 1.54 0.09 2.36 3.31 tr 14.16 1.00 23.94 1.04 0.10 – 1.75 tr tr tr 0.13 0.14 – tr 0.02
1.45 tr 0.11 11.56 0.16 4.62 46.2 0.42 0.56 0.52 0.12 4.26 0.12 1.41 11.52 0.19 13.13 1.55 0.67 1.12 2.27 0.98 0.23 0.34 0.12 1.09 0.34 tr 0.01
1.59 tr – 6.66 0.14 3.2 28.16 0.42 0.34 0.44 0.23 2.52 0.11 0.86 9.06 0.11 9.13 20.08 0.12 1.96 4.3 1.31 – 1.17 0.82 1.51 0.38 – tr
0.76 tr 0.06 1.87 0.06 1.36 27.15 0.53 1.14 0.85 – 3.23 6.1 – 9.72 0.59 46.15 2.84 0.10 – 0.57 – 0.01 0.97 0.76 0.45 0.12 tr 0.03
0.87 0.10 tr 0.92 0.05 0.65 23.58 0.5 0.71 0.65 – 4.35 5.02 – 9.44 0.54 52.59 3.62 0.09 – 0.65 tr – 0.78 0.34 0.08 0.05 0.02 0.01
– tr 0.14 5.38 0.04 2.29 35.02 0.84 1.03 0.97 0.13 0.51 2.08 1.12 14.63 0.33 31.65 2.12 0.04 tr 0.49 tr – 0.25 0.23 0.03 0.02 tr 0.04
0.18 tr 0.12 4.94 0.03 2.21 32.99 0.83 0.92 0.92 0.17 3.04 – 1.25 14.13 0.35 32.19 2.84 0.03 tr 0.64 0.02 tr – – 0.01 tr tr 0.04
– – tr 7.16 0.02 2.51 72.64 tr tr tr tr 0.45 1.67 0.66 10.94 tr 0.17 0.10 tr – 1.34 tr tr 0.03 – 0.02 tr 0.01 0.05
– – tr 2.83 tr 0.82 82.87 tr tr – tr 1.21 1.31 0.63 7.52 tr 0.15 0.09 – – 1.87 0.01 tr tr – – – 0.01 0.08
Bold values are referred to the main constituents of the analysed samples. a LRI = Linear retention indeces on DB5-column; pdms = polydymethylsyloxane fibre; carb= carboxen fibre. b Relative percentage composition; tr = traces; <0.01%; (–) = not detected.
A. Bertoli et al. / Industrial Crops and Products 32 (2010) 329–337
2-Hexenal hexenol ␣-Thujene ␣-Pinene Camphene -Pinene Myrcene ␣-Phellandrene 3-Carene ␣-Terpinene p-Cymene Limonene -Phellandrene (Z)-ocimene (E)-ocimene ␥-Terpinene Terpinolene Caryophyllene ␥-Elemene ␣-Guaiene ␣-Humulene -Selinene Valencene ␣-Selinene ␣-Farnesene Cadin ␣-1,4-diene Selina 3,7 (11) diene Germacrene B Caryophyllene oxide
LRIa
Pop3
Table 3b Composition of static headspaces of the different fibre hemp varities. Red petiole 2005
Carmagnola 2006
LRIa
pdms
carb
2-Hexenal Hexenol ␣-Thujene ␣-Pinene Camphene -Pinene Myrcene ␣-Phellandrene 3-Carene ␣-Terpinene p-Cymene Limonene -Phellandrene (Z)-ocimene (E)-ocimene ␥-Terpinene Terpinolene Linalool ␣-Terpineol Caryophyllene ␥-Elemene (Z)--farnesene ␣-Humulene -Selinene Valencene ␣-Selinene ␣-Farnesene -Bisabolene Germacrene B Caryophyllene oxide
854 856 933 942 959 984 992 1010 1013 1022 1030 1035 1037 1040 1051 1064 1089 1101 1197 1420 1430 1454 1457 1489 1493 1496 1503 1506 1560 1584
0.49b 0.42 0.17 0.54 0.04 1.19 41.56 0.8 1.02 0.92 0.05 10.89 – – 0.58 0.42 34.86 tr 0.10 32.84 tr tr 11.03 0.97 tr 0.53 0.54 – 0.35 tr
0.48 0.12 – 0.13 tr 0.35 14.83 tr tr – tr 13.32 tr tr 0.23 0.15 14.94 – 0.13 40.67 0.52 – 13.35 1.13 0.24 0.73 0.87 0.15 tr 0.12
pdms – – tr 4.20 tr 4.60 46.23 0.9 0.87 0.75 – 8.66 – – 2.15 0.48 22.58 0.17 tr 15.30 0.34 1.74 4.22 – 0.10 – – – 0.09 0.23
2005
2006
CODI
2005
carb
pdms
carb
pdms
carb
– – – 2.10 tr 2.04 53.7 1.14 1.15 0.99 tr 8.55 tr tr 2.64 0.56 18.23 0.33 tr 20.01 0.23 1.82 5.12 tr 0.11 0.02 – – 0.05 0.02
0.3 0.24 0.14 17.35 tr 8.01 27.63 1.06 1.55 1.38 0.18 3.09 5.01 tr 2.99 0.66 27.27 0.16 tr 11.55 0.11 0.34 2.35 0.76 0.34 0.03 tr tr 0.37 0.09
0.39 – 0.02 9.01 0.17 4.92 21.54 0.44 0.67 0.57 0.09 1.47 3.22 tr 1.96 0.35 27.41 0.37 0.17 13.21 0.13 0.34 3.75 0.97 0.56 0.57 0.79 0.63 tr 0.17
– – – 7.43 0.23 3.47 76.56 – – – – 8.5 – – 0.86 tr 0.18 tr 0.09 1.56 0.09 0.25 0.31 0.87 0.34 0.76 0.45 0.23 tr 0.25
tr – – 3.81 0.1 1.87 82.36 0.02 – tr tr 8.41 tr tr 1.02 – 0.21 tr 0.05 1.36 0.05 0.03 0.26 0.34 0.36 0.78 0.13 0.09 tr –
pdms 0.28 tr 0.28 15.9 0.23 6.46 30.63 0.89 1.35 1.01 0.09 tr 3.63 1.84 8.71 0.44 23.31 0.21 0.07 2.59 0.04 0.02 4.72 0.09 0.12 0.99 tr 0.02 0.25 –
2006
Felina34
2005
2006
2005
2006
carb
pdms
carb
pdms
carb
pdms
carb
pdms
carb
0.23 tr – 6.98 tr 2.94 23.7 0.32 0.57 0.39 0.06 tr 1.55 0.76 5.84 0.18 18.07 0.3 0.05 22.54 0.16 0.13 7.59 0.71 0.46 0.46 1.25 0.15 tr 0.09
– tr 0.2 7.37 0.27 2.76 68.76 tr tr tr – 6.97 – – 8.46
– tr – 4.21 0.1 1.48 74.57 – tr – – 6.32 0.84 – 7.9 0.05 0.19 0.24 0.01 2.71 tr 0.74 0.73 0.09 0.07 0.04 tr – 0.2 0.23
0.5 – 0.51 17.53 0.25 5.38 2.12 tr 18.01 tr 0.1 1.54 0.37 4.45 36.93 0.27 0.63 0.28 tr 5.48 0.47 tr 1.13 0.16 – 0.13 tr tr 0.22 0.13
0.59 1.10 0.52 14.54 0.23 5.31 2.72 – 20.9 0.11 0.12 0.37 tr 4.85 36.57 0.3 0.62 0.25 tr 4.87 0.42 tr 1.01 tr – 0.12 tr tr tr tr
– – tr 12.69 0.15 5.84 10.13 0.27 – 0.23 tr 0.34 0.87 0.87 4.29 0.34 12.16 0.27 0.98 3.09 tr 0.15 0.33 0.46 0.03 0.12 0.09 tr 0.15 0.23
– – tr 10.12 0.07 7.21 9.13 0.23 1.03 0.13 – 0.62 0.77 0.67 6.09 0.25 13.47 0.11 0.14 2.05 0.11 0.13 0.35 0.98 – 0.17 0.23 0.11 0.15 0.12
tr tr tr 15.12 0.13 7.01 17.13 0.35 0.93 0.25 tr 0.61 0.56 0.58 5.35 0.15 16.41 0.13 0.14 15.16 0.11 0.13 8.12 0.50 – 0.17 0.23 0.11 Tr tr
tr tr tr 14.71 0.09 5.21 14.79 0.35 0.98 0.43 tr 0.57 0.24 0.87 4.98 tr 15.32 tr tr 17.18 tr tr 9.25 tr – – – – – 0.13
0.18 0.19 0.04 3.38 0.02 0.10 0.98 tr tr – – – tr 0.08
pdms 0.02 – 20.35 0.05 0.05 4.31 12.27 0.08 – 0.09 0.36 0.57 0.18 0.25 5.19 tr 13.18 0.24 0.09 21.13 – 0.01 4.16 0.03 tr tr tr tr – 0.10
carb 0.02 – 20.44 0.09 0.08 5.15 13.56 0.14 – tr 0.38 0.79 0.34 0.37 4.37 0.21 12.24 0.36 0.24 22.45 – 0.02 3.96 0.05 tr 0.11 0.05 – 0.96 0.09
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Compound
C.S.
Bold values are referred to the main constituents of the analysed samples. a LRI = Linear retention indeces on DB5-column; pdms = polydymethylsyloxane fibre; carb = carboxen fibre. b Relative percentage composition; tr = traces; <0.01%; (–) = not detected.
333
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harvest was carried out at flowering, corresponding to the phenological codes 2103 and 2302 (Mediavilla et al., 1998) for dioecious and monoecius varieties, respectively. The harvesting dates went from the beginning of July to the end of August. Harvesting were hand accomplished and plants were cut at 4–5 cm from the soil. Production measurements (plant and stem yield for fibre production) were performed on a 5 m2 area excluding the plants on the outer rows. Plant density, biomass, stems and leaves fresh weight were measured then samples were subsequently allowed to dry into a ventilated oven, for dry weight determination. The stem height and the basal diameter on 20 plants for each plot were also determined. Afterwards, the fresh inflorescences were manually sampled from the same plants, cutting the 30 cm upper part of the stem, and weighed for fresh and dry weight (after ventilated oven drying at 50 ◦ C). The fresh inflorescences were further sampled directly in the field (by cutting the 30 cm upper part of the stem) from 10 to 20 plants per plot randomly chosen. They were collected in plastic bags, put into cool thermos, delivered to the analytical laboratory and hydrodistilled immediately.
2.2. Phytochemical analysis 2.2.1. Chemicals The linear hydrocarbons (C9 –C40 ) and the volatile compounds used as standards were commercial substances purchased from Fluka (Sigma–Aldrich) with 98–99% grade purity. The stock and working solutions were prepared using n-hexane (Carlo Erba, HPLC-grade)
2.2.4. Identification and quantitation The identification of the essential oil constituents was performed by the comparison of the retention times of their constituents with those of pure reference material and by mean of their Linear Retention Indices (L.R.I.) relative to a series of nhydrocarbons (C9 –C40 ) on the two different columns. In addition, it was used a computer matching of mass spectra with two commercial data base (ADAMS 1995; NIST 2000) and an experimental home-made library mass spectra built up from pure substances or known oils. Moreover, the molecular weights of the all identified substances were confirmed by GC/CIMS, using MeOH as CI ionizing gas. The composition of the different essential oils and headspaces were showed as relative percentage composition by internal peak area normalization, not inclusive of solvent peak and all relative response factors being taken as one. Repeatability of the measuring system showed variation coefficients under 5% for the identified components in the headspaces and essential oils (Tables 2, 3a and 3b). 2.2.5. Statistical analysis All measured and derived data were analysed by analysis of variance (ANOVA) using the CoStat software, version 6.2. In all cases, means were separated on the basis of least significant difference (LSD) only when the F-test of the ANOVA treatment was significant at the 0.05 or 0.01 probability level (Gomez and Gomez, 1984) 3. Results and discussion 3.1. Agronomic evaluation
2.2.2. Extraction procedures and sample preaparation The fresh inflorescences (150–200 g) were hydrodistilled (2 h, 2 l water distilled, flow 2.0 ml/min) by a Clevenger apparatus described in the Italian Pharmacopoeia F.U.I. XI Ed. The EOs yields are reported in Table 1. The essential oils were dissolved in Et2 O, dried over anhydrous MgSO4 , filtered and the solvent removed by evaporation on a water bath. All the essentials oil were diluted in n-hexane (HPLC solvent grade, 10%) and injected both in GC-FID (injection volume 1 ml, HPWAX and HP-5 capillary columns) and GC–MS (injection volume 0.1 ml, DB-5 capillary column). SPME analyses were performed with Supelco SPME devices, coated with polydimethylsiloxane (PDMS, 100 m) and with Carboxen (100 m) in order to sample the headspace of a fixed portion (10 g) of the fresh hemp fibre inflorescences at room temperature for 20 min.
2.2.3. Gas chromatographic analysis The GC-FID analyses of the standard compounds and EOs samples were accomplished by HP-5890 Series II instrument equipped with HP-WAX and HP-5 capillary columns (30 m × 0.25 mm, 0.25 m film thickness), working with the following temperature program: 60 ◦ C for 10 min, ramp of 5 ◦ C/min up to 220 ◦ C; injector and detector temperatures 250 ◦ C; carrier gas nitrogen (2 ml/min); detector dual FID; split ratio 1:30; injection volume of 1 ml (10% v/v n-hexane solution). The GC/EIMS analyses were performed by a Varian CP-3800 gas chromatograph equipped with a HP-5 capillary column (30 m × 0.25 mm; coating thickness 0.25 m) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions: injector and transfer line temperatures 220 and 240 ◦ C, respectively; oven temperature programmed from 60 to 240 ◦ C at 3 ◦ C/min; carrier gas helium at 1 ml/min; injection volume 0.1 ml (10% n-hexane solution); split ratio 1:30. The GCMS analysis was repeated three times for each sample.
3.1.1. Growth and biomass production The Italian dioeciuos (Carmagnola, Fibranova, C.S., Red Petiole, Pop1, Pop2, Pop3, Pop4, Pop5) cultivars showed biometric and phenological traits greatly different from the monoecious ones (Codimono and Felina 34). The monoecious varieties, developed through breeding and selection, show the staminate and pistillate flowers on the same plant, but this character is usually not completely stable. In fact Codimono, the Italian monoecious variety, proved to be still genetically unstable, as elevated percentages of male plants (10%) were present in the experimental plots. On the other hand, the French monoecious cultivars Felina 34, was rather stable with only 1% of male plants on the total plants present in each plot. These two monoecious cultivars showed greater earliness, reaching flowering roughly three/four weeks before the dioecious ones. The analysis of variance has outlined significant effect of type of cultivar (dioecious and monoecious), year and their interaction on the main biometric and productive characters. The monoecious cultivars were characterized by higher plant density, early growth, early flowering and a lower stem height. On the contrary, dioecious cultivars were characterized by lower plant density, late flowering and higher stem development (Table 1). In particular the dioecious cultivars showed a significant lower plant density than monoecious ones (121.7 and 136.6 plants m−2 ) but a greater plant height (2.89 and 1.92 m) (Table 1). Mean plant densities at harvest were 137.5 and 111.3 plants m−2 in the two years. In the 2006 growing season the unfavourable climatic conditions after sowing, due to heavy rains and low air temperatures, negatively affected the seed germination and consequently plant density both in dioecious and monoecious cultivars with lower values (−19 and −18%, respectively) than 2005 (Table 1). In 2005 Felina 34 achieved greatest density, although differences compared to Pop 1, Pop 2 were not statistically significant. In the second year, greatest density was recorded always for Felina 34 (135.3 plants m−2 ), while
A. Bertoli et al. / Industrial Crops and Products 32 (2010) 329–337
335
Fig. 1. Production of the essential oil target-compounds (relative composition %) in the analysed cultivars in the 2005 and 2006 growing seasons. For each year the mean values followed by the same letter are not statistically different at P ≤ 0.05 according to LSD test (for 2005 italic letters and for 2006 normal letter).
Carmagnola and Red Petiole presented the lowest values (roughly 95 plants m−2 ) (Table 1). Plant height, similarly to plant density, was on average higher in the first year (2.89 versus 2.47 m). In the 2006 growing season the monoecious cultivar flowered about 2 weeks before than 2005. According to Amaducci et al. (2008) findings, once the flowering starts the dry matter accumulation drops rapidly and consequently
monoecious cultivars showed shorter stems (−28%) and lower above ground dry (−47%) and stem dry production (−51%) than the year before (Table 1). The earlier sowing time of 2006 (4th April instead 27th April) allowed the dioecious cultivars to prolong the growing season before flowering and to achieve higher plant and stem productions than 2005 (+9.4 and +4.6%). In these cultivars, selected with a longer critical photoperiod, the flowering began just
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one week later than 2005 and approx 4 weeks later than monoecious cultivars. Mean dry stem yields were 31.6 and 30.7 g plant−1 in the first and second year, respectively. Given the importance of this parameter, it is helpful to divide the trial cultivars into three groups for each of the years. Thus in 2005 C.S., Red Petiole, Carmagnola and Pop 5 were ranked in the top group, achieving the highest values with yields above 36.9 g plant−1 . The second group, composed of five cultivars, gave production ranging between 22.1 and 31.2 g plant−1 . The third group was composed of Felina 34 that showed a significantly lowest production compared to the other varieties. In 2006, the best performing cultivars were C.S., Red Petiole (as the year before), Pop 2, Pop 5, Pop 4, and Pop 1. In contrast to the previous year, lower dry stem yield was obtained with Carmagnola (26.65 g plant−1 ). Finally, the lower yields were recorded for the two monoecious cultivar Codimono and Felina 34, with values ranging from 11.86 to 10.5 g plant−1 . The development of the upper part of the plant, is greater in the monoecious cultivars, namely in Felina 34 than in the dioecious ones (Table 1). The inflorescence production of the female plants in the dioecious cultivars is greater in Red petiole, Pop 5 and Pop 1 in 2005 and in Pop 3, Pop 4 and Pop 1 in 2006. The lowest value was achieved in Carmagnola in 2005 (2.34 g plant−1 ) and in Pop 5 in 2006 (1.95 g plant−1 ). 3.2. Phytochemical investigation The essential oils (EOs) were obtained by hydrodistillation of inflorescences using the Clevenger apparatus described in the Italian Pharmacopoeia F.U.I. XI Ed. A preliminary screening of the headspaces (HSs) by SPME analysis were carried out on the fresh inflorescences of the different varieties to define the spontaneous aroma emitted from the plant material without artefacts due to the extraction process. Furthermore, only fresh upper parts were considered for the production of the essential oils in order to avoid any chemical variation during the drying process. The essential oil yields ranged from 0.10 to 0.25% (w/w) (Table 1) and they were similar or higher than literature data previous reported for C. sativa (Fournier and Paris, 1978; Hillig, 2004; Meijer and Mediavilla, 1998; Nissen et al., 2009; Novak et al., 2001; Novak and Franz, 2003). The EOs content in the monoecious upper inflorescences was lower in comparison with the dioecious ones, even if the Carmagnola cultivar (dioecious) showed the lowest yield (0.10%, w/w ±0.02). It is important to point out that the EO yield trend was opposite to the biomass inflorescence production as the upper part development was greater in the monoecious cultivars than in the dioecious ones. The development of the female inflorescences was significantly lower (2.98 and 3.87 g plant−1 dry weight), but the essential oil contained in them was higher (0.17 and 0.12%, w/w) (Table 1). Ten characteristic terpenes were chosen as marker compounds to monitor the EOs production in the plant material, collected in 2005–2006 (Fig. 1). ␣-Pinene (3.0–20.5%), -pinene (1.4–8.0%), myrcene (8.2–45.3%), limonene (0.3–6.4%), (E)-ocimene (0.7–10.3%), terpinolene (0.1–22.1%), -caryophyllene (7.3–28.0%), ␣-humulene (3.2–12.6%), caryophyllene oxide (1.9–5.7%) were the main constituents (Table 2 and Fig. 1). The gas chromatographic profiles of the corresponding headspaces showed significant qualitative and quantitative differences in comparison with the EOs, but these main constituents were confirmed apart from caryophyllene oxide (Tables 3a and 3b). However, the analysed monoecious and dioecious cultivars did not show menth-compounds such as menthone, menthol, and their acetate derivatives, which have been already reported for Cannabis cultivars (Tognolini et al., 2006). In the studied inflorescence EOs, ␣-pinene (7.9% ± 0.98, 2005; 11.4% ± 0.84, 2006), myrcene (20.2% ± 3.68, 2005; 15.6% ± 0.92, 2006), terpinolene (13.7% ± 0.61,
2005; 12.8% ± 0.97, 2006), and -caryophyllene (15.7% ± 0.61, 2005; 21.9% ± 0.67, 2006) were the main constituents in monoecious hemps, both in 2005 and 2006 (Fig. 1). The essential oils of dioecious cultivars produced significant percentages of myrcene (22.0% ± 1.4, 2005; 16.0% ± 1.1, 2006), terpinolene (10.3% ± 1.48, 2005; 11.7% ± 1.09, 2006) and -caryophyllene (15.2% ± 0.87, 2005; 22.9% ± 0.66, 2006) (Fig. 1). Particular attention was focused on ␣-pinene and -pinene, which showed much higher amounts in the monoecious cultivars (18.1% ± 1.0, year 2005; 17.3% ± 1.4, year 2006) than in dioecious ones (2.7% ± 0.1, year 2005; 4.5% ± 0.2, year 2006, Table 2 and Fig. 1). As ␣-pinene and -pinene were detected in such a huge amounts both in 2005 and 2006 collection, they might be useful markers to distinguish the monoecious Felina 34 and Codimono from the dioecious cultivars. A previous study on C. sativa cultivars, originated in different countries reported higher percentages of ␣-pinene (44–71%) in the essential oils in contrast to another monoterpene, terpinolene, which was found only around 1%. In the present work, the ten analysed cultivars, cultivated in Central Italy, showed much lower amounts of ␣-pinene than literature data (Hood and Barry, 1978), apart from the monoecious Felina 34 cultivar, which contained it in higher level (20.3% ± 1.4, 2005; 20.4% ± 0.9 2006) (Fig. 1). On the other hand, a significant presence of terpinolene was also observed in the EOs, especially in Felina 34 (19.0% ± 0.14, 2005; 19.0% ± 0.05) and in Codimono (20.1% ± 0.08, 2005; 15.5% ± 0.02, 2006) (Table 2 and Fig. 1). Therefore, significant production of ␣- and -pinenes as well as terpinolene generally characterized the EOs extracted from the monoecious cultivars (Fig. 1). Furthermore, terpinolene was another important constituent in many dioecious cultivars both in 2005 and 2006 (10.3% ± 1.5, 2005; 11.7% ± 1.1, 2006), except in the case of Carmagnola that showed the lowest yield (1.59% ± 0.6, 2005; 0.09% ± 0.05, 2006) (Fig. 1). Therefore, terpinolene was included in the pool of target monoterpenes to control the EOs production in the 2005–2006 hemp collections. Fewer hydrocarbon and oxygenated sesquiterpenes were generally present in high amounts in comparison with the monoterpenes (Fig. 1). -Caryophyllene (15.7% ± 0.61, 2005 average value; 21.9% ± 0.67, 2006), ␣-humulene (5.98% ± 0.61, 2005; 8.2% ± 0.31, 2006) and caryophyllene oxide (3.89% ± 0.61, 2005; 3.17% ± 0.17, 2006) were the main sesquiterpenes in all the analysed cultivars (Fig. 1). Their production was more constant than monoterpenes among the dioecious and monoecious cultivars in the two-year harvest (Fig. 1). Many other minor sesquiterpenes (around 1%) were identified such as -phellandrene, -selinene, germacrene B, trans-nerolidol, -bisabolol. These sequiterpenes have already reported in the literature as typical components of the Cannabis spp. essential oil (Hillig, 2004; Lemberkovics et al., 1981; Nissen et al., 2009), but in the present study, they were not considered as marker compounds because of their scarce and variable amounts in the period 2005–2006. In addition to terpenes, other secondary metabolites typical of C. sativa EO such as cannabinoids, are extremely important for the pharmacological activity (Segelman et al., 1974). The ratio (THC + CBN)/CBD has been proposed to classify Cannabis plant as drug- or fibre-type (Bocsa and Karus, 1998). The cannabinoid amount is generally very low in the EOs of C. sativa (Lemberkovics et al., 1981; Malingrè et al., 1975), but their content is strictly dependant on the state, age, number of secretory glands in the plant material (Lanyon et al., 1981; Meijer and Mediavilla, 1998) as well as the extraction condition (de Meijer, 1998). Furthermore, the differences in cannabinoids content may reflect procedural variation in sampling rather than external environmental effects (Hemphill et al., 1980; Lanyon et al., 1981; Turner et al., 1977). Therefore, particular attention was focused on the plant material sampling in the present study as
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described before. The production of the hallucinogenous tetrahydrocannabinol (TCH) and the non-hallucinogenous cannabidiol (CBD) were investigated together with the main target terpenes in the essential oils of the analysed fibre hemp cultivars (Table 2 and Fig. 1). Tetrahydrocannabinol (TCH) was detected in traces (less than 0.01%) only in the two monoecious collected in 2005 (Codimono and Felina 34), while the non-hallucinogenous cannabidiol (CBD) was detected in the all analysed EOs samples, exception given for the two dioecious cultivars Pop4 and C.S., collected in 2005 (Fig. 1). In general, CBD content was higher in dioecious cultivars (4.4% ± 0.3, 2005; 2.7% ± 0.3, 2006) than in monoecious (0.9% ± 0.08, 2005; 1.2% ± 0.3, 2006) in both harvesting years (Table 2 and Fig. 1). The 2005 dioecious collection showed the highest CBD yields, especially for Pop1 (13.9% ± 0.03) and Pop5 (11.0% ± 0.01), even if this compound was not present in the EO of Pop4, Carmagnola and C.S. harvested in 2005. The monoecious cultivars showed a much more stable CBD production in the two years. In particular, Felina 34 EO showed much higher amount of CBD (1.7% ± 0.11, 2005; 2.0% ± 0.03, 2006) than Codimono (0.2% ± 0.02, 2005; 0.4% ± 0.38, 2006) (Fig. 1). 4. Conclusions This study investigated the biomass and EO production of ten fibre-hemp cultivars, cultivated in Central Italy in two-year field trial. Their fresh inflorescences yielded significant amounts of essential oils which were characterized by the typical C. sativa terpenes and a legal and safe cannabinoid content. The EOs and HSs profiles of the different inflorescences were suitable to confirm the fibre-type classification of these cultivars which showed specific volatile constituents. In addition, significant quali–quantitative differences in the production of some target terpenes such as ␣-pinene, -pinene, and terpinolene allowed to distinguish monoecious from dioecious hemp cultivars. Interest in industrial hemp has gained momentum world wide, suggesting that demand for natural fibres will continue to increase. Market segmentation for ethically produced goods and growing demand for biodegradable and natural products has led to a wide range of new industrial hemp products being developed. A wide variety of hemp-based products are already available on the market, but the hemp essential oil is still considered a nicheproduct. The present study confirm the hypothesis to grow industrial fibre hemp as a multi-use crop through a complete utilization of the plant material using stems for fibre industry and inflorescences to produce essential oils as alternative flavour and fragrance additives. References Amaducci, S., Colauzzi, M., Zatta, A., Venturi, G., 2008. Flowering dynamics in monoecious and dioecious hemp genotypes. J. Ind. Hemp 13 (1), 5–19. Bocsa, J., Karus, M., 1998. In: Hemp Tech (Ed.), The Cultivation of Hemp, Sebastopol, California.
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