The Leaf Volatile Constituents of Isatis tinctoria by Solid-Phase Microextraction and Gas Chromatography/Mass Spectrometry

• Abstract
• Introduction
• Material and Methods
• Plant material
• SPME procedure
• GC/MS analysis
• Results and Discussion
• References

Abstract

The leaf volatile constituents of Isatis tinctoria L. (Brassicaceae) have been studied by Solid-Phase Microextraction and Gas chromatography/Mass Spectrometry (SPME/GC-MS). Seventy components were fully characterized by mass spectra, linear retention indices, and injection of standards; the average composition (ppm) as single components and classes of substances is reported. Aliphatic hydrocarbons, acids, alcohols, aldehydes and esters, aromatic aldehydes, esters and ethers, furans, isothiocyanates and thiocyanates, sulfurated compounds, nitriles, terpenes and sesquiterpenes were identified. Leaf volatiles in Isatis tinctoria L. were characterized by a high amount of isothiocyanates which accounted for about 40 % of the total volatile fraction. Isothiocyanates are important and characteristic flavour compounds in Brassica vegetables and the cancer chemo-protective attributes are recently responsible for their growing interest.

Introduction

Isatis tinctoria L. (woad, Brassicaceae) is an upright biennial herbaceous plant; although native to the grasslands of south-eastern Russia, it is spread widely across Europe, Asia and North-Africa. In Mediterranean countries, the plant was cultivated throughout centuries to produce the blue dye indigo that has been the most important blue dye for mankind since prehistoric times [1]. Isatis tinctoria L. has, moreover, a well documented history of use as a medicinal herb, and it is commonly used in traditional Chinese medicine; root extracts of the herb are known as Bang Lang Gen and leaf extracts as Da Qing Ye. Documented medicinal use of Isatis in the western tradition extends back to at least the first century A.D. [2]. Inflammatory conditions have been considered as the major indication for Isatis tinctoria L. leaf extracts; hence, the most recent researches on the active principles in woad deal with the presence of anti-inflammatory substances. In particular, the indolo[2,1-b]quinazoline alkaloid tryptanthrin has recently been identified as a pharmacologically active compound in Isatis tinctoria with potent dual inhibitory activity on prostaglandin and leukotriene synthesis [3], [4], [5], [6], [7], [8], [9]. The leaves also contain several derivatives of hydroxycinnamic acid, including ferulic acid and sinapic acid [10]; these agents are thought to be important in the anti-inflammatory and anti-allergic activities of Isatis tinctoria L. leaf preparations. The indigo precursors were studied too and a number of indoxyl derivates have been identified [11], [12], [13], [14], [15], [16]. These substances are thought to have a number of anti-cancer effects and may help explain the traditional use of Isatis tinctoria L. in the treatment of cancer [17].

The aim of the present paper is to study the volatile constituents of the leaf of Isatis tinctoria L, the composition of which, to our knowledge, has not been previously investigated. The potential presence of volatile isothiocyanates, which are typical constituents of Brassica vegetables with well-known cancer chemoprotective attributes [18], aroused our particular interest in Isatis tinctoria L. A headspace solid-phase microextraction (HS-SPME) method in combination with gas chromatography-mass spectrometry (GC-MS) has been used for the extraction, identification and quantification of the leaf volatile constituents.

Material and Methods

Plant material

The plants of Isatis tinctoria L. (Brassicaceae) were collected in Fiumefreddo (Catania, Sicily, Italy) at about 150 m above the sea level, in the period from April to May 2005. The fresh leaves were harvested manually from plants at the vegetative stage. Five leaf samples were analyzed by HS-SPME/GC-MS as described below, each sample in triplicate; each sample consisted of a batch of ground and homogenized fresh leaves from at least ten different plants. A voucher specimen, numbered 160/05, has been deposited at the Herbarium of the Pharmacobiological Department of the University of Messina (Italy).

SPME procedure

The volatile components were extracted by the HS-SPME (headspace solid-phase microextraction) method. SPME was performed with a commercially available fibre housed in its manual holder (Supelco; Bellefonte, PA, USA). All extractions were carried out using a DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethyl siloxane) fibre, 50/30 μm film thickness (Supelco; Bellefonte, PA, USA).

Using a 40-mL vial, about 3.5 g of each sample was exactly weighed and dissolved in 5 mL of water. The vial was equipped with a ”mininert” valve (Supelco; Bellefonte, PA, USA) that allowed the introduction of the fibre without piercing any septum. Extraction was performed in the headspace keeping the vial at 30 °C. The sample was equilibrated for 30 min; the extraction time was 25 min. During the extraction, the sample was continuously stirred with a magnetic stir bar on a stir plate revolving at 750 rpm. The fibre was carefully placed in the same location for each exposure to the headspace in order to obtain maximum repeatability. After sampling, the SPME fibre was introduced onto the GC/MS injector. The fibre was kept in the splitless injector, maintained at 260 °C, for 3 min for the thermal desorption of the analytes. In order to optimize the technique, the effects of various parameters, such as sample volume, sample headspace volume, sample heating temperature, extraction time, etc. were studied on the extraction efficiency. Each measurement was repeated three times. The criteria of the efficiency was the desorption peak area (total ion current chromatogram) and the coefficient of variation (CV %) of the measurements.

GC/MS analysis

A Varian 3800 gas chromatograph, directly interfaced with a Varian 2000 ion trap mass spectrometer (Varian Spa; Milan, Italy), was used to analyze the headspace components. Two different fused silica capillary columns were used: 1) Mega 5 MS, 60 m, 0.25 mm i. d., 0.25 μm film thickness (Mega; Legnano; Milan, Italy). GC oven temperature, 40 °C held for 2 min then increase to 240 °C at a rate of 3.0 °C/min; carrier gas helium at a constant pressure of 90 kPa; 2) CP-Wax 52 CB, 60 m, 0.25 mm i. d., 0.25 μm film thickness (Chrompack Italy; Milan, Italy); GC oven temperature, 45 °C held for 5 min, then increased to 80 °C at a rate of 10 °C/min, and to 240 °C at 2 °C/min; carrier gas helium at constant pressure of 90 kPa. For both columns: injector temperature, 260 °C; injection mode, splitless.

MS scan conditions: interface temperature, 250 °C; ionization technique, electronic impact (EI) at 70 eV; acquisition range, 30 - 200 m/z; scan rate, 1 μ per sec. Mass Spectra Library, NIST’98 (NIST/EPA/NIH, version 1.7 USA). The compound identification was based on comparison of linear retention indices (LRI) with those of authentic compounds, computer matching with mass spectral libraries and comparison with spectra of authentic samples or literature data; linear retention indices of the sample components were determined on the basis of homologous n-alkane hydrocarbons, analyzed under the same GC conditions; the linear retention indices (LRI) were calculated according to the Van der Dool and Kratz equation both on polar and apolar columns [19]. Quantitative results were obtained using the method of internal standard: aliquots of an aqueous solution of 1-butanol (1 mg/mL) were added in the slurry prior to extraction. The coefficient of variation (CV %) for the three replicates of the same sample was inferior to 12.0, for all the analyzed compounds.

Results and Discussion

Table [1] reports the average composition (ppm) as single components and classes of substances of all the identified components with their retention indices calculated both on CP-Wax 52 CB and DB-5 columns. The technique used made it possible to identify seventy components which accounted for about 99.6 % of the total volatile fraction.

Leaf volatiles in Isatis tinctoria L. were characterized by a high portion of isothiocyanates (X = 571.5 ppm) which accounted for about 40 % of the total volatile fraction; these compounds were also found to be predominant in Brussels sprouts [20]. Isothiocyanates are synthesized and stored as glucosinolates in plants and are released when the plant tissue is damaged; the conversion is catalyzed by myrosinase, an enzyme that in plants coexists with glucosinolates although it is physically separated. The kinetics of the myrosinase reaction differs widely from species to species, and multiple forms of the enzyme can exist even within the same plant [21]. However, under certain conditions hydrolysis of glucosinolates may lead to non-isothiocyanate products, including thiocyanates, nitriles, epithionitriles, indoles, and oxazolidine-2-thiones [22], [23].

Isothiocyanates have long been known for their fungicidal, bacteriocidal, nematocidal and allelophatic properties [24], [25], [26], and several recent epidemiological studies have already shown that the dietary intake is inversely correlated with cancer risk at several organ sites [18]. In our samples alkyl, alkenyl, ω-methylthioalkyl, β-hydroxy and aromatic isothiocyanates were identified. In I. tinctoria 3-butenyl isothiocyanate prevailed (X = 376.1 ppm); this agreed with Freichard et al. [27] who identified in seeds of I. tinctoria the glucosinolate gluconapin whose aglycone is the 3-butenyl isothiocyanate. The same authors identified the glucosinolates progoitrin and epiprogoitrin whose aglycone, 2-hydroxy-3-butenyl isothiocyanates, was present in our samples at high amount (X = 110.5 ppm). 3-Methylthiopropyl isothiocyanate was moreover present (X = 4.7 ppm); this component was identified by Cole [22] at a similar level in I. tinctoria. Allyl isothiocyanate is less represented in I. tinctoria leaf although it is the main compound in different cultivars of Brassica oleracea such as broccoli and cauliflower [28].

Among the sulfurated compounds a high amount of 2-ethylthiophene (X = 182.1 ppm) has been observed; the thiophenes are widely distributed in vegetables such as onion [29], their odour thresholds are very low and the flavour character very pronounced.

Following isothiocyantes, aldehydes constituted an important class of substances. Volatile leaf alcohols and aldehydes are known to be present in several plants families; among Brassicaceous vegetables, aldehydes were the most abundant cauliflower volatiles [20]. In our samples, aliphatic and aromatic aldehydes from C₄ to C₁₄ were identified; (Z)-2-hexenal, the typical leaf aldehyde which smells green and fresh, was the main component (X = 162.6 ppm); moreover, saturated and unsaturated alcohols from C₄ to C₁₇ were identified with tetradecanol as the main component; (E)-3-hexen-1-ol the typical leaf alcohol (green grassy) has been identified, too.

Monoterpenes and sequiterpenes constituted about the 7 % of the volatile fraction; ten monoterpenes (C₁₀) and two sesquiterpenes (C₁₅) were identified, with the monocyclic monoterpene limonene (X = 55.9 ppm) as the major one; monoterpenes and sesquiterpenes are well known components of plant essential oils, especially of Citrus species which are widely spread in Sicily [30]

The nitriles identified are breakdown products of glucosinolates, too; they could be considered as artifacts and come from isothiocyanates due to the loss of sulfur [22], [23]. Among the minor components we observed the presence of methyl salicylate previously identified by Hartleb and Seifert [10] in I. tinctoria.

In conclusion, the flavour profile of Isatis tinctoria L. has been investigated for the first time by the solvent-free HS-SPME extraction technique combined with the GC-MS analysis. I. tinctoria could be particularly interesting because of its remarkable amounts of substances - i. e. isothiocyanates - which have considerable therapeutic activity.

References

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• 29 Boelens M, De Valois P J, Wobben H J, Van der Gen A. Volatile flavor compounds from onion.  J Agric Food Chem. 1971;  19 984-91
  CrossrefPubMedSearch in Google Scholar
• 30 Dugo G, Cotroneo A, Verzera A, Bonaccorsi I. Composition of volatile fraction of cold-pressed citrus peel oils. In: Taylor and Francis Group, editors Citrus: essential oil and other products. London: England; 2002: p 201-317
  Search in Google Scholar

Antonella Verzera

Dipartimento di Chimica Organica e Biologica

Università di Messina

Salita Sperone 31

98168 Messina

Italy

Phone: +39-90-676-5240

Fax: +39-90-393-895

Email: averzera@pharma.unime.it

References

• 1 Guarino C, Casoria P, Menale B. Cultivation and use of Isatis tinctoria L. (Brassicaceae) in Southern Italy.  Econ Bot. 2000;  54 395-400
  PubMedSearch in Google Scholar
• 2 Isatis tinctoria Monograph. Altern Med Rev 2002 7: 523-4
  Search in Google Scholar
• 3 Danz H, Stoyanova S, Wippich P, Brattstrom A, Hamburger M. Identification and isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria .  Planta Med. 2001;  67 411-6
  Thieme ConnectPubMedSearch in Google Scholar
• 4 Danz H, Baumann D, Hamburger M. Quantitative determination of the dual COX-2/5-LOX inhibitor tryptanthrin in Isatis tinctoria by ESI-LC-MS.  Planta Med. 2002;  68 152-7
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Danz H, Stoyanova S, Thomet Olivier A R, Simon H -U, Dannhardt G, Ulbrich H. et al . Inhibitory activity of tryptanthrin on prostaglandin and leukotriene synthesis.  Planta Med. 2002;  68 875-80
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Hamburger M. Isatis tinctoria - from the rediscovery of an ancient medicinal plant towards a novel anti-inflammatory phytopharmaceutical.  Phytochem Rev. 2002;  1 333-44
  CrossrefPubMedSearch in Google Scholar
• 7 Oberthuer C, Heinemann C, Elsner P, Benfeldt E, Hamburger M. A comparative study on the skin penetration of pure tryptanthrin and tryptanthrin in Isatis tinctoria extract by dermal microdialysis coupled with isotope dilution ESI-LC-MS.  Planta Med. 2003;  69 385-9
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Heinemann C, Schliemann-Willers S, Oberthuer C, Hamburger M, Elsner P. Prevention of experimentally induced irritant contact dermatitis by extracts of Isatis tinctoria compared to pure tryptanthrin and its impact on UVB-induced erythema.  Planta Med. 2004;  70 385-90
  Thieme ConnectPubMedSearch in Google Scholar
• 9 Oberthuer C, Jaeggi R, Hamburger M. HPLC based activity profiling for 5-lipoxygenase inhibitory activity in Isatis tinctoria leaf extracts.  Fitoterapia. 2005;  76 324-32
  CrossrefPubMedSearch in Google Scholar
• 10 Hartleb I, Seifert K. Acid constituents from Isatis tinctoria .  Planta Med. 1995;  61 95-6
  PubMedSearch in Google Scholar
• 11 Gilbert K G, Hill D J, Crespo C, Mas A, Lewis M, Rudolph B. et al . Qualitative analysis of indigo precursors from woad by HPLC and HPLC-MS.  Phytochem Anal. 2000;  11 18-20
  CrossrefPubMedSearch in Google Scholar
• 12 Maugard T, Enaud E, Choisy P, Legoy M D. Identification of an indigo precursor from leaves of Isatis tinctoria (Woad).  Phytochemistry. 2001;  58 897-904
  CrossrefPubMedSearch in Google Scholar
• 13 Oberthuer C, Schneider B, Graf H, Hamburger M. The elusive indigo precursors in woad (Isatis tinctoria L.) - identification of the major indigo precursor, isatan A, and a structure revision of isatan B.  Chem Biodivers. 2004;  1 174-82
  PubMedSearch in Google Scholar
• 14 Oberthuer C, Graf H, Hamburger M. The content of indigo precursors in Isatis tinctoria leaves - a comparative study of selected accessions and post-harvest treatments.  Phytochemistry. 2004;  65 3261-8
  CrossrefPubMedSearch in Google Scholar
• 15 Garcia-Macias P, John P. Formation of natural indigo derived from woad (Isatis tinctoria L.) in relation to product purity.  J Agric Food Chem. 2004;  52 7891-6
  PubMedSearch in Google Scholar
• 16 Gilbert K G, Maule H G, Rudolph B, Lewis M, Vandenburg H, Sales E. et al . Quantitative analysis of indigo and indigo precursors in leaves of Isatis spp. and Polygonum tinctorium .  Biotechnol Prog. 2004;  20 1289-92
  CrossrefPubMedSearch in Google Scholar
• 17 Spink B C, Hussain M M, Katz B H, Eisele L, Spink D C. Transient induction of cytochromes P450 1A1 and 1B1 in MCF-7 human breast cancer cells by indirubin.  Biochem Pharmacol. 2003;  66 2313-21
  CrossrefPubMedSearch in Google Scholar
• 18 Zhang Y. Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action.  Mutat Res. 2004;  555 173-90
  PubMedSearch in Google Scholar
• 19 Van den Dool H, Kratz P D. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography.  J Chromatogr. 1963;  11 463-71
  CrossrefPubMedSearch in Google Scholar
• 20 Van Langenhove H J, Cornelis C P, Schamp N M. Identification of volatiles emitted during the blanching process of brussels sprouts and cauliflower.  J Sci Food Agric. 1991;  56 483-7
  PubMedSearch in Google Scholar
• 21 Fahey J W, Zalcmann Amy T, Talalay P. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants.  Phytochemistry. 2001;  56 5-51
  CrossrefPubMedSearch in Google Scholar
• 22 Cole R A. Isothiocyanates, nitriles and thiocyanates as products of autolysis of glucosinolates in Cruciferae.  Phytochemistry. 1976;  15 759-62
  CrossrefPubMedSearch in Google Scholar
• 23 MacLeod A J, Panesar S S, Gil V. Thermal degradation of glucosinolates.  Phytochemistry. 1981;  20 977-80
  CrossrefPubMedSearch in Google Scholar
• 24 Vaughn S F, Boydston R A. Volatile allelochemicals released by crucifer green manures.  J Chem Ecol. 1997;  23 2107-16
  CrossrefPubMedSearch in Google Scholar
• 25 Smolinska U, Morra M J, Knudsen G R, James R L. Isothiocyanates produced by Brassicaceae species as inhibitors of Fusarium oxysporum .  Plant Disease. 2003;  87 407-12
  PubMedSearch in Google Scholar
• 26 Olivier C, Vaughn S F, Mizubuti E SG, Loria R. Variation in allyl isothiocyanate production within Brassica species and correlation with fungicidal activity.  J Chem Ecol. 1999;  25 2687-701
  CrossrefPubMedSearch in Google Scholar
• 27 Frechard A, Fabre N, Pean C, Montaut S, Fauvel M T, Rollin P. et al . Corrigendum to ”Novel indole-type glucosinolates from woad (Isatis tinctoria L.)”.  Tetrahedron Lett. 2002;  43 1591-2
  CrossrefPubMedSearch in Google Scholar
• 28 Hansen M, Moeller P, Soerensen H, De Trejo M C. Glucosinolates in broccoli stored under controlled atmosphere.  J Am Soc Hortic Sci. 1995;  120 069-74
  PubMedSearch in Google Scholar
• 29 Boelens M, De Valois P J, Wobben H J, Van der Gen A. Volatile flavor compounds from onion.  J Agric Food Chem. 1971;  19 984-91
  CrossrefPubMedSearch in Google Scholar
• 30 Dugo G, Cotroneo A, Verzera A, Bonaccorsi I. Composition of volatile fraction of cold-pressed citrus peel oils. In: Taylor and Francis Group, editors Citrus: essential oil and other products. London: England; 2002: p 201-317
  Search in Google Scholar

Antonella Verzera

Dipartimento di Chimica Organica e Biologica

Università di Messina

Salita Sperone 31

98168 Messina

Italy

Phone: +39-90-676-5240

Fax: +39-90-393-895

Email: averzera@pharma.unime.it