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Volatile constituents of micropropagated plants of Bupleurum fruticosum L.
Plant Science 167 (2004) 807–810
Volatile constituents of micropropagated plants of Bupleurum fruticosum L. Alessandra Bertoli a,∗ , Luisa Pistelli a , Ivano Morelli a , Daniele Fraternale b , Laura Giamperi b , Donata Ricci b a
Dipartimento di Chimica Bioorganica e Biofarmacia, Università degli Studi di Pisa Via Bonanno 33, 56100 Pisa, Italy b Istituto Botanico “Pierina Scaramella” Università degli Studi di Urbino, Via Bramante 28, 61029 Urbino, Italy Received 8 April 2004; received in revised form 14 May 2004; accepted 18 May 2004 Available online 8 June 2004
Abstract The essential oil and solid phase micro extraction (SPME) samples of Bupleurum fruticosum micropropagated plants were analysed by GC and GC–MS and compared with those obtained from the leaves and the stems of field-grown parent plants. The main constituents of the essential oil of the micropropagated plants were -phellandrene (61%), sabinene (13%), terpinen-4-ol, tricyclene and bicyclogermacrene (3%). Regarding to the field-grown B. fruticosum plants, the leaf essential oil showed the same major components detected in the micropropagated plants, while the stem oil showed ␥-terpinene (50%) and ␣-phellandrene (18%) as the most important constituents. Furthermore, SPME analyses were carried out in order to show for the first time a complete investigation on the volatile organic constituents of the micropopagated and field-grown B. fruticosum parent plants. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Bupleurum fruticosum L.; Micropropagated plants; -Phellandrene; Sabinene; GC–MS; SPME
1. Introduction Extracts and essential oils of Bupleurum genus plants have been largely used in traditional medicine for their anti-inflammatory and antiseptic activity [1]. Bupleurum fruticosum L. is a shrubby, perennial, evergreen plant, typical of many zones of Mediterranean area [2,3]. Regarding to phytochemical investigations, saponins and coumarins and phenylpropanoids were isolated and identified from B. fruticosum roots [4–6]. Several studies were carried out about the biological activity of this species especially related to the presence of saponins [7–10]. In Sardinia animals, except goats which eat the sprouts, are generally repelled by this shrub and that is probably due to the presence of the essential oils in the branches and leaves. Manunta et al. [11] identified 22 constituents in the oil obtained from both the branch and leaf oils of B. fruticosum, grown in the Botanical Garden of the University of Urbino. Giamperi et al. [12] analysed the composition ∗
Corresponding author. Tel.: +39 5022 19700; fax: +39 5022 19660. E-mail address: [email protected] (A. Bertoli).
of the essential oil from the epigean parts of B. fruticosum, harvested in Cirenaica (Libya). Recently, Dugo et al. [13] analysed the essential oil of Sicilian B. fruticosum, which was rich in ␣-pinene (21.7%), -phellandrene (21.3%) and -pinene (13.2%). Regarding to biological properties of the essential oil of B. fruticosum, anti-inflammatory activity was studied by Lorente et al. [14], while Horne et al. [15] tested its anti-microbial effects against Streptococcus pneumoniae. Also, Manunta et al. [16] studied also the anti-bacterial activity of the leaf and flower essential oils of B. fruticosum. The research and development in biotechnology for the production of secondary metabolites have received a major impetus from the very positive response of plant cells and tissues, cultured in vitro. Arguments in support of in vitro cultures stress the advantages of year-round availability of plant material, process isolation, and magnification of chemical reactions under growth control [17]. A micropropagation protocol from nodal explants of B. fruticosum plants, cultivated in the Botanical Garden of the University of Urbino, was developed by Fraternale et al. [18] in order to obtain micropropagated plants for the production of sec-
0168-9452/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2004.05.017
808
A. Bertoli et al. / Plant Science 167 (2004) 807–810
ondary metabolites. The aim of this work was to investigate, for the first time, the essential oil and the headspace of these micropropagated plants of B. fruticosum in comparison with their field-grown parent plants.
described above. All the volatile fractions were analyzed by GC and GC–MS.
2. Experimental
GC analyses were accomplished with a 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 of 0.5 l; the identification of the components was performed, for both the columns, by comparison of their retention indices (RI) relative to a series of n-alkanes (C8 –C26 ).
2.1. Plant material and in vitro culture methods Young branches were collected from plants of B. fruticosum L. growing in the Botanical Garden of the University of Urbino, Faculty of Pharmacy in the month of April 2000. Voucher specimens have been deposited in the Herbarium of the Faculty of Pharmacy, University of Urbino, Italy (voucher number B.F. 10). Explants were obtained using the apical portions of young branches with no more than 10 buds. The sterilization process was concluded in three steps, after dissection of leaves: washing for 20 min in distilled water plus a commercial solution of detergent; immersion in 30 ml of 2.5% (w/v) sodium hypoclorite plus 1 ml of Tween 20 for 20 min, under vacuum. The branches were then washed five times in sterile H2 O and dried with sterile filter paper. For micropropagation experiments of B. fruticosum, shoots were induced from nodal explants. Nodal explants of 7–10 mm with one bud were used to initiate cultures. Each explant was placed in a glass tube (20 cm long × 2 cm) containing MS medium [19] plus 30 g l−1 sucrose indole 3-acetic acid (IAA) and benzyladenine (BA), pH 5.8. The culture media were sterilized by autoclaving at 121 ◦ C, 1.2 bar for 20 min. Each glass tube contained 20 ml of culture medium. Growth conditions were 23 ◦ C and 16 h under fluorescent tubes at a light intensity of 65 mmol me−2 s−1 . Cultures were transferred to fresh culture medium every 4 weeks. Shoots from 12 weeks cultured explants were excised in order to initiate only the multiplication phase of micropropagation. Shoots were placed in 500 cm3 glass jars containing 100 cm3 of medium as described above. After 30 days of culture each shoot formed a cluster of plantlets (in media 15 for each shoot) that were collected and utilised for extraction. Micropropagation was carried on from shoots of 1 month grown in the same medium. 2.2. Isolation procedures The essential oils of three fresh field-grown B. fruticosum plant samples (70 g stems and leaves, vegetative state) were obtained separately by hydrodistillation (2 h, 400 ml water) at a flow of 1.3 ml/min using a Clevenger-type apparatus according to the Italian Pharmacopoeia [20]. Furthermore, the essential oils of three fresh micropropagated plant samples (72 g) of B. fruticosum were isolated and separately hydrodistillated using the same procedures
2.3. Gas chromatography
2.4. Gas chromatography–mass spectrometry GC–EIMS analyses were performed with a Varian CP-3800 gas chromatograph equipped with a DB-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 of 1 l (10% hexane solution); split ratio 1:30. Identification of the constituents was based on comparison of the retention times with those of authentic samples, comparing their linear retention indices relative to the series of n-hydrocarbons, and on computer matching against commercial (NIST 98 and ADAMS) and home-made library mass spectra built up from pure substances, components of known oils and MS literature data [21–26]. 2.5. SPME analyses Solid phase micro extraction (SPME) analyses were performed with Supelco SPME devices, coated with polydimethylsiloxane (PDMS, 100 m), in order to sample the headspace of three fresh samples (10 g) of B. fruticosum (both field-grown and micropropagated plants). Each plant aliquot was inserted separately into a 100 ml glass conic flask and allowed to equilibrate for 20 min. After the equilibration time, the fiber was exposed to the headspace for 15 min at room temperature. This sampling time was selected after three different experiments at 5, 10 and finally at 15 min, where a plateau of a maximum uptake was reached for the analytes. When the sampling was finished, the fiber was withdrawn into the needle and transferred to the injection port of the GC and GC–MS system, operating in the same conditions used as above both for the identification and the quantification of the constituents, apart from the splitless injection mode and the injector temperature (250 ◦ C).
A. Bertoli et al. / Plant Science 167 (2004) 807–810
3. Results and discussion The average yield of the essential oils obtained from three samples of fresh micropropagated plants of B. fruticosum L. was 0.1% (v/wfresh material ). Higher yields of 1.8 and 3.0% (v/wfresh material ) were obtained, respectively from the leaves and the stems of the field-grown parent plants. Moreover, we studied the volatile compounds in the headspace emitted without stress conditions from samples of the fresh field-grown and micropropagated plants. Tables 1 and 2 list the identified substances in the essential oil and the headspace samples, respectively. For each oil and headspace sample, the retention index and the peak area percentages were calculated as mean of two injections. The essential oils of B. fruticosum obtained separately from the stems and leaves of the field-grown parent plants showed a composition similar to literature data, confirming remarkable differences between the leaves and the stems Table 1 Composition of B. fruticosum essential oils from the field-grown parent plants and micropropagated plants Compounds
Tricyclene ␣-Thujene ␣-Pinene Sabinene -Pinene -Myrcene ␣-Phellandrene ␦-3-Carene ␣-Terpinene -Terpinene p-Cymene Limonene -Phellandrene -(Z)-Ocimene -(E)-Ocimene ␥-Terpinene ␣-Terpinolene Linalool Terpinen-4-ol Methyl chavicolc trans-Carveol Geraniol Citronellyl acetate Geranyl acetate ␣-Copaene Germacrene d Bicyclogermacrene ␦-Cadinene (E)-Nerolidol Total (%)
Ria
928 930 942 976 985 989 1003 1013 1017 1026 1030 1032 1035 1037 1050 1060 1088 1100 1176 1196 1210 1253 1335 1368 1377 1480 1495 1524 1563
Field-grown parent plantsb
Micropropagated plantsb
Stems
Leaves
t t 0.3 7.1 – t 18.3 – – 0.57 0.86 t 1.6 0.75 0.31 49.8 – 1.50 0.93 t 2.67 t 3.80 t t t 1.07 t –
t t 1.89 35.7 0.64 2.86 2.93 0.21 0.93 1.31 0.93 3.04 41.7 0.1 t 0.8 t t 2.25 t t t 0.3 0.3 t t 2.56 t –
2.8 0.1 0.1 12.8 0.5 2.1 2.6 0.4 0.8 t 2.9 t 60.9 0.8 – 1.4 0.4 t 3.0 0.4 t 0.2 0.8 0.7 0.9 1.0 3.2 0.3 0.2
90.0
98.4
99.3
t: traces (<0.1%). a Compounds are listed in order of their elution from DB5-apolar column (RI = retention index). b Percentage composition (%). c Tentative identification.
809
Table 2 Constituents of the headspaces from the micropropagated and field-grown parent plants of B. fruticosum Compounds
Tricyclene ␣-Thujene ␣-Pinene Sabinene -Pinene -Myrcene ␣-Phellandrene ␦-3-Carene ␣-Terpinene p-Cymene -Phellandrene -(Z)-Ocimene ␥-Terpinene ␣-Terpinolene Terpinen-4-ol ␣-Terpineol Methyl chavicol Geraniol Citronellyl acetate Geranyl acetate ␣-Copaene -Cubebene Germacrene d Bicyclogermacrene ␦-Cadinene (E)-Nerolidol Total (%)
RIa
928 930 942 976 985 986 1003 1013 1017 1030 1035 1037 1060 1088 1176 1189 1196 1253 1335 1368 1377 1390 1480 1495 1524 1563
Field-grown parent plantsb
Micropropagated plantsb
Stems
Leaves
t 0.1 0.5 7.6 t 0.1 18.6 0.4 0.8 1.0 1.8 0.8 48.4 0.4 1.5 0.2 0.4 0.2 0.8 0.7 0.9 t 1.5 3.2 0.3 0.2
– 0.1 0.9 17.3 0.7 0.8 5.4 2.1 3.4 9.3 39.1 t 1.1 1.6 0.5 1.0 1.4 1.4 0.3 0.2 1.4 t 0.5 0.6 1.2 0.8
– – 0.8 0.2 0.2 0.3 t t
89.5
91.1
99.0
0.8 0.2 0.6 22.0 0.5 1.2 1.3 0.7 0.3 1.5 66.4 – 1.2 0.3 0.5
t: traces (<0.1%). a Compounds are listed in order of their elution from DB5-apolar column (RI = retention index). b Percentage composition (%).
of the same plants [16]. The micropropagated plant essential oil showed -phellandrene (61%) and sabinene (13%) as the main constituents together with other monoterpenes as tricyclene (3%), -myrcene (2%), ␣-phellandrene (3%), ␥-terpinene (1%), terpinen-4-ol (3%). The leaf oil of the field-grown parent plants showed high amounts of sabinene and -phellandrene (36 and 42%, respectively) and a very low percentage of ␥-terpinene (0.8%), whereas the stem oil of parent plants was characterized especially by ␥-terpinene (50%). Therefore, the essential oil of the micropropagated plants (sabinene 13%, -phellandrene 60%, ␥-terpinene 1%), was more similar to the leaves than the branches of B. fruticosum parent plants. This fact could be due to the leaves/stems ratio in the biomass of the fresh micropropagated analysed plants which resulted higher than in B. fruticosum field-grown plants. Furthermore, Bupleurum genus is known for its widely secretory system, forming a continuos network between the roots, stems, leaves and umbels. No other independent secretory ducts are evident except for this network [11]. Regarding the sesquiterpene fraction in the essential oil of micropropagated plants (6%), it was very scarce in com-
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A. Bertoli et al. / Plant Science 167 (2004) 807–810
parison with the monoterpenes and it was characterized especially by bicyclogermacrene, germacrene d, ␣-copaene detected in percentages comparable with those of the essential oil of the B. fruticosum parent plants. Regarding minor constituents as monoterpene esters, citronellyl acetate (0.8%) and geranyl acetate (0.7%) were detected in the essential oil of the fresh micropropagated plants. A larger variability in the composition of these minor volatile constituents was reported in literature for the essential oil of B. fruticosum field-grown plants, harvested during flowering state [16]. In our case, the analysed micropropagated and parent plant samples were generated from the apical portions of young branches in the vegetative state and so the monoterpene esters could be typical of the aerial plant parts especially during the flowering state. Monoterpenes were predominant also in the headspace of the fresh micropropagated plants of B. fruticosum: -phellandrene (66%) and sabinene (22%) were the main constituents, while the other monoterpenes showed slightly higher amounts in comparison with the corresponding essential oils (Table 2). Therefore, SPME analyses confirmed the characteristic monoterpene composition observed in the corresponding essential oils besides the similarity on the volatile constituents between the micropropagated plants (-phellandrene 66%, sabinene 22%, ␥-terpinene 1%) and the leaves of the field-grown parent plants (-phellandrene 39%, sabinene 17%, ␥-terpinene 1%). In conclusion, our results showed that the fingerprint of the essential oil obtained from the fresh micropropagated plants of was comparable to the essential oil obtained from the leaves rather than the stems of the field-grown parent plants. This fact showed how it is sometimes important to consider which parent plant organ the essential oil was obtained from before comparing the metabolite production in the corresponding micropropagated plant. Furthermore, the technique solid phase micro extraction equipped with GC–MS system, gave us the possibility to investigate directly the headspaces of fresh samples in order to define the characteristic aromatic compounds of B. fruticosum micropropagated plants without any alteration in the vegetal material caused by hydrodistillation process.
References [1] M. Nose, S. Amagaya, Y. Ogihara, Corticosterone secretion-inducing activity of saikosaponin metabolites formed in the alimentary tract, Chem. Pharm. Bull. 37 (1989) 2736–2740. [2] M.C. Guinea, J. Parellada, G. Lacaille, M.A. Dubois, H. Wagner, Biologically active triterpene saponins from Bupleurum fruticosum, Planta Med. 60 (1994) 163–167. [3] A. Fiori, Nuova Flora Analitica d’Italia, Edagricole, Bologna, 1969. [4] L. Pistelli, A.R. Bilia, A. Marsili, N. De Tommasi, A. Manunta, Triterpenoid saponins from Bupleurum fruticosum roots, J. Nat. Prod. 56 (1993) 240–244.
[5] L. Pistelli, A.R. Bilia, A. Bertoli, A. Marsili, I. Morelli, Phenylpropanoids from Bupleurum fruticosum, J. Nat. Prod. 58 (1995) 112–116. [6] L. Pistelli, A. Bertoli, A.R. Bilia, I. Morelli, Minor constituents from Bupleurum fruticosum roots, Phytochemistry 41 (1996) 1579– 1582. [7] R. Scarpato, L. Pistelli, A. Bertoli, E. Nieri, L. Migliore, In Vitro genotoxicity and cytotoxicity of five new chemical compounds of plant origin by means of the human lymphocyte micronucleus assay, Toxicology 12 (1997) 153–161. [8] R. Scarpato, L. Pistelli, A. Bertoli, Different effects of newly isolated saponins on the mutagenicity and cytotoxicity of the anticancer drugs mitomycin C and bleomycin in human lymphocytes, Mut. Res. 420 (1998) 49–54. [9] M.C. Guinea, J. Parellada, G. Lacaille, M.A. Dubois, H. Wagner, Biologically active triterpene saponins from Bupleurum fruticosum, Planta Med. 60 (1994) 163–167. [10] L. Pistelli, A. Bertoli, I. Morelli, Scarpato R. Constituents of Bupleurum spp. from Italian flora: isolation, structure elucidation and their biological activity, Recent Res. Dev. Phytochem. 2 (1998) 463– 483. [11] A. Manunta, B. Tirillini, D. Fraternale, Anatomy of secretory tissue in Bupleurum fruticosum L., Studi Urbinati LXIII (32) (1990) 55–64. [12] L. Giamperi, D. Ricci, D. Fraternale, A. Manunta, R. Tabacchi, The essential oil from Bupleurum fruticosum L. of the Cyrenaica region of eastern Lybia and the problem of bupleurol, J. Essent. Oil Res. 10 (1998) 369–374. [13] G. Dugo, A. Trozzi, A. Verzera, A. Rapisarda, L’olio essenziale delle foglie di piante tipiche della flora mediterranea, Essenze, Derivati Agrumari 70 (2000) 201–205. [14] I. Lorente, M.A. Ocete, A. Zarzuelo, M.M. Cabo, J. Jimenez, Bioactivity of the essential oil of Bupleurum fruticosum, J. Nat. Prod. 52 (1989) 267–272. [15] D. Horne, M. Holm, C. Oberg, S. Chao, D.G. Young, Anti-microbial effects of essential oils on Streptococcus pneumoniae, J. Essent. Oil Res. 13 (2001) 387–392. [16] A. Manunta, B. Tirillini, D. Fraternale, Secretory tissues and essential oil composition of B. fruticosum L., J.Essent. Oil Res. 4 (1992) 461–466. [17] W. Kurz, F. Constabel, Production of secondary metabolites, in: A. Altman (Ed.), Agricultural Biotechnology, Marcel Dekker, New York, 1998. [18] D. Fraternale, L. Giamperi, D. Ricci, M.B.L. Rocchi, Micropropagation of Bupleurum fruticosum L.: the effect of triacontanol, Plant Cell Tissue Organ Cult. 69 (2002) 135–140. [19] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473–479. [20] Farmacopea Ufficiale della Repubblica Italiana IX ed., Istituto Poligrafico Zecca dello Stato, Roma, 1991. [21] R.P. Adams, Identification of essential oil components by gas chromatography–mass spectroscopy, Allured Publication Corporation Stream, Illinois, 1995. [22] A.A. Swigar, R.M. Silverstein, Monoterpenes, Aldrich Chemical Composition, Milwaukee, 1981. [23] Y. Massada, Analysis of Essential Oils by Gas Chromatography and Mass Spectrometry, Wiley, New York, 1976. [24] E. Stenhagen, S. Abrahamsson, F.W. McLafferty, Registry of Mass Spectral Data, Wiley, New York, 1974. [25] J.D. Connolly, R.A. Hill, Dictionary of Terpenoids, Chapman & Hall, London, 1991. [26] W. Jennings, T. Shibamoto, Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Gas Chromatography, Academic Press, New York, 1980.
Volatile constituents of micropropagated plants of Bupleurum fruticosum L. Alessandra Bertoli a,∗ , Luisa Pistelli a , Ivano Morelli a , Daniele Fraternale b , Laura Giamperi b , Donata Ricci b a
Dipartimento di Chimica Bioorganica e Biofarmacia, Università degli Studi di Pisa Via Bonanno 33, 56100 Pisa, Italy b Istituto Botanico “Pierina Scaramella” Università degli Studi di Urbino, Via Bramante 28, 61029 Urbino, Italy Received 8 April 2004; received in revised form 14 May 2004; accepted 18 May 2004 Available online 8 June 2004
Abstract The essential oil and solid phase micro extraction (SPME) samples of Bupleurum fruticosum micropropagated plants were analysed by GC and GC–MS and compared with those obtained from the leaves and the stems of field-grown parent plants. The main constituents of the essential oil of the micropropagated plants were -phellandrene (61%), sabinene (13%), terpinen-4-ol, tricyclene and bicyclogermacrene (3%). Regarding to the field-grown B. fruticosum plants, the leaf essential oil showed the same major components detected in the micropropagated plants, while the stem oil showed ␥-terpinene (50%) and ␣-phellandrene (18%) as the most important constituents. Furthermore, SPME analyses were carried out in order to show for the first time a complete investigation on the volatile organic constituents of the micropopagated and field-grown B. fruticosum parent plants. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Bupleurum fruticosum L.; Micropropagated plants; -Phellandrene; Sabinene; GC–MS; SPME
1. Introduction Extracts and essential oils of Bupleurum genus plants have been largely used in traditional medicine for their anti-inflammatory and antiseptic activity [1]. Bupleurum fruticosum L. is a shrubby, perennial, evergreen plant, typical of many zones of Mediterranean area [2,3]. Regarding to phytochemical investigations, saponins and coumarins and phenylpropanoids were isolated and identified from B. fruticosum roots [4–6]. Several studies were carried out about the biological activity of this species especially related to the presence of saponins [7–10]. In Sardinia animals, except goats which eat the sprouts, are generally repelled by this shrub and that is probably due to the presence of the essential oils in the branches and leaves. Manunta et al. [11] identified 22 constituents in the oil obtained from both the branch and leaf oils of B. fruticosum, grown in the Botanical Garden of the University of Urbino. Giamperi et al. [12] analysed the composition ∗
Corresponding author. Tel.: +39 5022 19700; fax: +39 5022 19660. E-mail address: [email protected] (A. Bertoli).
of the essential oil from the epigean parts of B. fruticosum, harvested in Cirenaica (Libya). Recently, Dugo et al. [13] analysed the essential oil of Sicilian B. fruticosum, which was rich in ␣-pinene (21.7%), -phellandrene (21.3%) and -pinene (13.2%). Regarding to biological properties of the essential oil of B. fruticosum, anti-inflammatory activity was studied by Lorente et al. [14], while Horne et al. [15] tested its anti-microbial effects against Streptococcus pneumoniae. Also, Manunta et al. [16] studied also the anti-bacterial activity of the leaf and flower essential oils of B. fruticosum. The research and development in biotechnology for the production of secondary metabolites have received a major impetus from the very positive response of plant cells and tissues, cultured in vitro. Arguments in support of in vitro cultures stress the advantages of year-round availability of plant material, process isolation, and magnification of chemical reactions under growth control [17]. A micropropagation protocol from nodal explants of B. fruticosum plants, cultivated in the Botanical Garden of the University of Urbino, was developed by Fraternale et al. [18] in order to obtain micropropagated plants for the production of sec-
0168-9452/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2004.05.017
808
A. Bertoli et al. / Plant Science 167 (2004) 807–810
ondary metabolites. The aim of this work was to investigate, for the first time, the essential oil and the headspace of these micropropagated plants of B. fruticosum in comparison with their field-grown parent plants.
described above. All the volatile fractions were analyzed by GC and GC–MS.
2. Experimental
GC analyses were accomplished with a 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 of 0.5 l; the identification of the components was performed, for both the columns, by comparison of their retention indices (RI) relative to a series of n-alkanes (C8 –C26 ).
2.1. Plant material and in vitro culture methods Young branches were collected from plants of B. fruticosum L. growing in the Botanical Garden of the University of Urbino, Faculty of Pharmacy in the month of April 2000. Voucher specimens have been deposited in the Herbarium of the Faculty of Pharmacy, University of Urbino, Italy (voucher number B.F. 10). Explants were obtained using the apical portions of young branches with no more than 10 buds. The sterilization process was concluded in three steps, after dissection of leaves: washing for 20 min in distilled water plus a commercial solution of detergent; immersion in 30 ml of 2.5% (w/v) sodium hypoclorite plus 1 ml of Tween 20 for 20 min, under vacuum. The branches were then washed five times in sterile H2 O and dried with sterile filter paper. For micropropagation experiments of B. fruticosum, shoots were induced from nodal explants. Nodal explants of 7–10 mm with one bud were used to initiate cultures. Each explant was placed in a glass tube (20 cm long × 2 cm) containing MS medium [19] plus 30 g l−1 sucrose indole 3-acetic acid (IAA) and benzyladenine (BA), pH 5.8. The culture media were sterilized by autoclaving at 121 ◦ C, 1.2 bar for 20 min. Each glass tube contained 20 ml of culture medium. Growth conditions were 23 ◦ C and 16 h under fluorescent tubes at a light intensity of 65 mmol me−2 s−1 . Cultures were transferred to fresh culture medium every 4 weeks. Shoots from 12 weeks cultured explants were excised in order to initiate only the multiplication phase of micropropagation. Shoots were placed in 500 cm3 glass jars containing 100 cm3 of medium as described above. After 30 days of culture each shoot formed a cluster of plantlets (in media 15 for each shoot) that were collected and utilised for extraction. Micropropagation was carried on from shoots of 1 month grown in the same medium. 2.2. Isolation procedures The essential oils of three fresh field-grown B. fruticosum plant samples (70 g stems and leaves, vegetative state) were obtained separately by hydrodistillation (2 h, 400 ml water) at a flow of 1.3 ml/min using a Clevenger-type apparatus according to the Italian Pharmacopoeia [20]. Furthermore, the essential oils of three fresh micropropagated plant samples (72 g) of B. fruticosum were isolated and separately hydrodistillated using the same procedures
2.3. Gas chromatography
2.4. Gas chromatography–mass spectrometry GC–EIMS analyses were performed with a Varian CP-3800 gas chromatograph equipped with a DB-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 of 1 l (10% hexane solution); split ratio 1:30. Identification of the constituents was based on comparison of the retention times with those of authentic samples, comparing their linear retention indices relative to the series of n-hydrocarbons, and on computer matching against commercial (NIST 98 and ADAMS) and home-made library mass spectra built up from pure substances, components of known oils and MS literature data [21–26]. 2.5. SPME analyses Solid phase micro extraction (SPME) analyses were performed with Supelco SPME devices, coated with polydimethylsiloxane (PDMS, 100 m), in order to sample the headspace of three fresh samples (10 g) of B. fruticosum (both field-grown and micropropagated plants). Each plant aliquot was inserted separately into a 100 ml glass conic flask and allowed to equilibrate for 20 min. After the equilibration time, the fiber was exposed to the headspace for 15 min at room temperature. This sampling time was selected after three different experiments at 5, 10 and finally at 15 min, where a plateau of a maximum uptake was reached for the analytes. When the sampling was finished, the fiber was withdrawn into the needle and transferred to the injection port of the GC and GC–MS system, operating in the same conditions used as above both for the identification and the quantification of the constituents, apart from the splitless injection mode and the injector temperature (250 ◦ C).
A. Bertoli et al. / Plant Science 167 (2004) 807–810
3. Results and discussion The average yield of the essential oils obtained from three samples of fresh micropropagated plants of B. fruticosum L. was 0.1% (v/wfresh material ). Higher yields of 1.8 and 3.0% (v/wfresh material ) were obtained, respectively from the leaves and the stems of the field-grown parent plants. Moreover, we studied the volatile compounds in the headspace emitted without stress conditions from samples of the fresh field-grown and micropropagated plants. Tables 1 and 2 list the identified substances in the essential oil and the headspace samples, respectively. For each oil and headspace sample, the retention index and the peak area percentages were calculated as mean of two injections. The essential oils of B. fruticosum obtained separately from the stems and leaves of the field-grown parent plants showed a composition similar to literature data, confirming remarkable differences between the leaves and the stems Table 1 Composition of B. fruticosum essential oils from the field-grown parent plants and micropropagated plants Compounds
Tricyclene ␣-Thujene ␣-Pinene Sabinene -Pinene -Myrcene ␣-Phellandrene ␦-3-Carene ␣-Terpinene -Terpinene p-Cymene Limonene -Phellandrene -(Z)-Ocimene -(E)-Ocimene ␥-Terpinene ␣-Terpinolene Linalool Terpinen-4-ol Methyl chavicolc trans-Carveol Geraniol Citronellyl acetate Geranyl acetate ␣-Copaene Germacrene d Bicyclogermacrene ␦-Cadinene (E)-Nerolidol Total (%)
Ria
928 930 942 976 985 989 1003 1013 1017 1026 1030 1032 1035 1037 1050 1060 1088 1100 1176 1196 1210 1253 1335 1368 1377 1480 1495 1524 1563
Field-grown parent plantsb
Micropropagated plantsb
Stems
Leaves
t t 0.3 7.1 – t 18.3 – – 0.57 0.86 t 1.6 0.75 0.31 49.8 – 1.50 0.93 t 2.67 t 3.80 t t t 1.07 t –
t t 1.89 35.7 0.64 2.86 2.93 0.21 0.93 1.31 0.93 3.04 41.7 0.1 t 0.8 t t 2.25 t t t 0.3 0.3 t t 2.56 t –
2.8 0.1 0.1 12.8 0.5 2.1 2.6 0.4 0.8 t 2.9 t 60.9 0.8 – 1.4 0.4 t 3.0 0.4 t 0.2 0.8 0.7 0.9 1.0 3.2 0.3 0.2
90.0
98.4
99.3
t: traces (<0.1%). a Compounds are listed in order of their elution from DB5-apolar column (RI = retention index). b Percentage composition (%). c Tentative identification.
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Table 2 Constituents of the headspaces from the micropropagated and field-grown parent plants of B. fruticosum Compounds
Tricyclene ␣-Thujene ␣-Pinene Sabinene -Pinene -Myrcene ␣-Phellandrene ␦-3-Carene ␣-Terpinene p-Cymene -Phellandrene -(Z)-Ocimene ␥-Terpinene ␣-Terpinolene Terpinen-4-ol ␣-Terpineol Methyl chavicol Geraniol Citronellyl acetate Geranyl acetate ␣-Copaene -Cubebene Germacrene d Bicyclogermacrene ␦-Cadinene (E)-Nerolidol Total (%)
RIa
928 930 942 976 985 986 1003 1013 1017 1030 1035 1037 1060 1088 1176 1189 1196 1253 1335 1368 1377 1390 1480 1495 1524 1563
Field-grown parent plantsb
Micropropagated plantsb
Stems
Leaves
t 0.1 0.5 7.6 t 0.1 18.6 0.4 0.8 1.0 1.8 0.8 48.4 0.4 1.5 0.2 0.4 0.2 0.8 0.7 0.9 t 1.5 3.2 0.3 0.2
– 0.1 0.9 17.3 0.7 0.8 5.4 2.1 3.4 9.3 39.1 t 1.1 1.6 0.5 1.0 1.4 1.4 0.3 0.2 1.4 t 0.5 0.6 1.2 0.8
– – 0.8 0.2 0.2 0.3 t t
89.5
91.1
99.0
0.8 0.2 0.6 22.0 0.5 1.2 1.3 0.7 0.3 1.5 66.4 – 1.2 0.3 0.5
t: traces (<0.1%). a Compounds are listed in order of their elution from DB5-apolar column (RI = retention index). b Percentage composition (%).
of the same plants [16]. The micropropagated plant essential oil showed -phellandrene (61%) and sabinene (13%) as the main constituents together with other monoterpenes as tricyclene (3%), -myrcene (2%), ␣-phellandrene (3%), ␥-terpinene (1%), terpinen-4-ol (3%). The leaf oil of the field-grown parent plants showed high amounts of sabinene and -phellandrene (36 and 42%, respectively) and a very low percentage of ␥-terpinene (0.8%), whereas the stem oil of parent plants was characterized especially by ␥-terpinene (50%). Therefore, the essential oil of the micropropagated plants (sabinene 13%, -phellandrene 60%, ␥-terpinene 1%), was more similar to the leaves than the branches of B. fruticosum parent plants. This fact could be due to the leaves/stems ratio in the biomass of the fresh micropropagated analysed plants which resulted higher than in B. fruticosum field-grown plants. Furthermore, Bupleurum genus is known for its widely secretory system, forming a continuos network between the roots, stems, leaves and umbels. No other independent secretory ducts are evident except for this network [11]. Regarding the sesquiterpene fraction in the essential oil of micropropagated plants (6%), it was very scarce in com-
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parison with the monoterpenes and it was characterized especially by bicyclogermacrene, germacrene d, ␣-copaene detected in percentages comparable with those of the essential oil of the B. fruticosum parent plants. Regarding minor constituents as monoterpene esters, citronellyl acetate (0.8%) and geranyl acetate (0.7%) were detected in the essential oil of the fresh micropropagated plants. A larger variability in the composition of these minor volatile constituents was reported in literature for the essential oil of B. fruticosum field-grown plants, harvested during flowering state [16]. In our case, the analysed micropropagated and parent plant samples were generated from the apical portions of young branches in the vegetative state and so the monoterpene esters could be typical of the aerial plant parts especially during the flowering state. Monoterpenes were predominant also in the headspace of the fresh micropropagated plants of B. fruticosum: -phellandrene (66%) and sabinene (22%) were the main constituents, while the other monoterpenes showed slightly higher amounts in comparison with the corresponding essential oils (Table 2). Therefore, SPME analyses confirmed the characteristic monoterpene composition observed in the corresponding essential oils besides the similarity on the volatile constituents between the micropropagated plants (-phellandrene 66%, sabinene 22%, ␥-terpinene 1%) and the leaves of the field-grown parent plants (-phellandrene 39%, sabinene 17%, ␥-terpinene 1%). In conclusion, our results showed that the fingerprint of the essential oil obtained from the fresh micropropagated plants of was comparable to the essential oil obtained from the leaves rather than the stems of the field-grown parent plants. This fact showed how it is sometimes important to consider which parent plant organ the essential oil was obtained from before comparing the metabolite production in the corresponding micropropagated plant. Furthermore, the technique solid phase micro extraction equipped with GC–MS system, gave us the possibility to investigate directly the headspaces of fresh samples in order to define the characteristic aromatic compounds of B. fruticosum micropropagated plants without any alteration in the vegetal material caused by hydrodistillation process.
References [1] M. Nose, S. Amagaya, Y. Ogihara, Corticosterone secretion-inducing activity of saikosaponin metabolites formed in the alimentary tract, Chem. Pharm. Bull. 37 (1989) 2736–2740. [2] M.C. Guinea, J. Parellada, G. Lacaille, M.A. Dubois, H. Wagner, Biologically active triterpene saponins from Bupleurum fruticosum, Planta Med. 60 (1994) 163–167. [3] A. Fiori, Nuova Flora Analitica d’Italia, Edagricole, Bologna, 1969. [4] L. Pistelli, A.R. Bilia, A. Marsili, N. De Tommasi, A. Manunta, Triterpenoid saponins from Bupleurum fruticosum roots, J. Nat. Prod. 56 (1993) 240–244.
[5] L. Pistelli, A.R. Bilia, A. Bertoli, A. Marsili, I. Morelli, Phenylpropanoids from Bupleurum fruticosum, J. Nat. Prod. 58 (1995) 112–116. [6] L. Pistelli, A. Bertoli, A.R. Bilia, I. Morelli, Minor constituents from Bupleurum fruticosum roots, Phytochemistry 41 (1996) 1579– 1582. [7] R. Scarpato, L. Pistelli, A. Bertoli, E. Nieri, L. Migliore, In Vitro genotoxicity and cytotoxicity of five new chemical compounds of plant origin by means of the human lymphocyte micronucleus assay, Toxicology 12 (1997) 153–161. [8] R. Scarpato, L. Pistelli, A. Bertoli, Different effects of newly isolated saponins on the mutagenicity and cytotoxicity of the anticancer drugs mitomycin C and bleomycin in human lymphocytes, Mut. Res. 420 (1998) 49–54. [9] M.C. Guinea, J. Parellada, G. Lacaille, M.A. Dubois, H. Wagner, Biologically active triterpene saponins from Bupleurum fruticosum, Planta Med. 60 (1994) 163–167. [10] L. Pistelli, A. Bertoli, I. Morelli, Scarpato R. Constituents of Bupleurum spp. from Italian flora: isolation, structure elucidation and their biological activity, Recent Res. Dev. Phytochem. 2 (1998) 463– 483. [11] A. Manunta, B. Tirillini, D. Fraternale, Anatomy of secretory tissue in Bupleurum fruticosum L., Studi Urbinati LXIII (32) (1990) 55–64. [12] L. Giamperi, D. Ricci, D. Fraternale, A. Manunta, R. Tabacchi, The essential oil from Bupleurum fruticosum L. of the Cyrenaica region of eastern Lybia and the problem of bupleurol, J. Essent. Oil Res. 10 (1998) 369–374. [13] G. Dugo, A. Trozzi, A. Verzera, A. Rapisarda, L’olio essenziale delle foglie di piante tipiche della flora mediterranea, Essenze, Derivati Agrumari 70 (2000) 201–205. [14] I. Lorente, M.A. Ocete, A. Zarzuelo, M.M. Cabo, J. Jimenez, Bioactivity of the essential oil of Bupleurum fruticosum, J. Nat. Prod. 52 (1989) 267–272. [15] D. Horne, M. Holm, C. Oberg, S. Chao, D.G. Young, Anti-microbial effects of essential oils on Streptococcus pneumoniae, J. Essent. Oil Res. 13 (2001) 387–392. [16] A. Manunta, B. Tirillini, D. Fraternale, Secretory tissues and essential oil composition of B. fruticosum L., J.Essent. Oil Res. 4 (1992) 461–466. [17] W. Kurz, F. Constabel, Production of secondary metabolites, in: A. Altman (Ed.), Agricultural Biotechnology, Marcel Dekker, New York, 1998. [18] D. Fraternale, L. Giamperi, D. Ricci, M.B.L. Rocchi, Micropropagation of Bupleurum fruticosum L.: the effect of triacontanol, Plant Cell Tissue Organ Cult. 69 (2002) 135–140. [19] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473–479. [20] Farmacopea Ufficiale della Repubblica Italiana IX ed., Istituto Poligrafico Zecca dello Stato, Roma, 1991. [21] R.P. Adams, Identification of essential oil components by gas chromatography–mass spectroscopy, Allured Publication Corporation Stream, Illinois, 1995. [22] A.A. Swigar, R.M. Silverstein, Monoterpenes, Aldrich Chemical Composition, Milwaukee, 1981. [23] Y. Massada, Analysis of Essential Oils by Gas Chromatography and Mass Spectrometry, Wiley, New York, 1976. [24] E. Stenhagen, S. Abrahamsson, F.W. McLafferty, Registry of Mass Spectral Data, Wiley, New York, 1974. [25] J.D. Connolly, R.A. Hill, Dictionary of Terpenoids, Chapman & Hall, London, 1991. [26] W. Jennings, T. Shibamoto, Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Gas Chromatography, Academic Press, New York, 1980.