Valorizing overlooked local crops in the era of globalization: the case of aniseed (Pimpinella anisum L.) from Castignano (central Italy)

Industrial Crops & Products 104 (2017) 99–110

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Valorizing overlooked local crops in the era of globalization: the case of aniseed (Pimpinella anisum L.) from Castignano (central Italy)

MARK

⁎

Romilde Iannarellia, , Giovanni Capriolia, Stefania Sutb, Stefano Dall’Acquab, Dennis Fiorinic, Sauro Vittoria, Filippo Maggia a b c

School of Pharmacy, University of Camerino, Camerino, Italy Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy School of Science and Technology, University of Camerino, Camerino, Italy

A R T I C L E I N F O

A B S T R A C T

Keywords: Pimpinella anisum Castignano Essential oil (E)-Anethole Anisi fructus Phenolic compounds

In the era of globalization, some local crops are at risk of extinction due to low competitiveness against products coming from foreign markets. This is the case of aniseed (Pimpinella anisum) from Castignano (Marche, central Italy), which was extensively cultivated in central Italy in the XIX century then abandoned after the global market favored products manufactured in Middle East countries. In order to support scientifically the recovery of this local crop, we performed a phytochemical investigation on the essential oil and polar compounds of aniseed cultivated in different fields of Castignano in the years 2013–2015 with the aim to demonstrate its high-quality value. For the purpose, the ‘Castignano ecotype’ samples were compared for essential oil yield, (E)-anethole levels and phenolic content with commercial samples of the Mediterranean area. Furthermore, several phenolic compounds were characterized by HPLC–MSn. Results showed that aniseed cultivated in Castignano contains higher content of essential oil and phenolic compounds than commercial samples, and provided the scientific rationale for its complete recovery and valorization.

1. Introduction

toothpaste), perfumery and herbal (e.g., digestive teas) products (Leung and Foster, 2003). In particular, the liqueurs manufactured with aniseed extracts and/or essential oil have a long tradition in the Mediterranean countries, namely the Italian ‘anisetta’, ‘sambuca’ and ‘mistrà’, the French ‘anisette’ and ‘pastis’, the Greek ‘ouzo’ and ‘mastik’, the Turkish ‘raki’ and the Lebaneese ‘arak’. On a pharmaceutical level, aniseed is used as a flavor enhancer of medical preparations. In this regard, European Pharmacopoeia reports the aniseed (Anisi fructus) epicarp of P. anisum which contains not less than 2% of essential oil (European Pharmacopoeia, 2005). The main constituent of aniseed essential oil is the phenylpropanoid (E)-anethole, present in concentrations of 75–95%, accompanied by minor amounts of methyl chavicol, p-anisaldehyde, γ-himachalene, α-zingiberene, (E)pseudoisoeugenyl 2-methylbutyrate and epoxy-pseudoisoeugenyl 2methylbutyrate (Boelens, 1991; Kubeczka and Ullmann, 1980; Tabanca et al., 2006). (E)-anethole is the key component assuring the aromatic and sweetener properties of aniseed and exerting stomachic, carminative, antispasmodic and expectorant effects (Kang et al., 2013). The best quality aniseed essential oils are considered those manufactured in Italy, Spain, Malta, France and Tunisia (Catizone et al., 1986). The estimated cost of manufacturing for essential oil is about 51 $/kg,

Pimpinella anisum L., also known as ‘aniseed’, is an annual herb belonging to the Apiaceae family and native to the eastern Mediterranean area and southwestern Asia (Pignatti, 1982). The plant possesses an erect stem, up to 50 cm high, rough and branched. The leaves are alternate and heteromorphic. The inflorescence is a terminal umbel composed of small hermaphrodite white flowers. The fruit, improperly called ‘seed’ for its small dimensions, is a greenish gray schizocarp, similar to rice grain, 4 mm in diameter, of pleasant smell, ripening in July-August, just one month after flowering. The plant is endowed with secretory channels and vittae containing the main secondary metabolites such as essential oil and phenolic compounds which are mainly concentrated into the fruit (Tabanca et al., 2006). Aniseed has been used in the Mediterranean folk medicine, like stomachic, digestive, carminative, expectorant, antitussive, anti-spasmodic, galactogogue, diuretic and diaphoretic agent (Shojaaii and Ford, 2012; Idolo et al., 2010; Leporatti and Ivancheva, 2003). Nowadays, aniseed is employed as flavouring of alcoholic (e.g., brandy and liquers) and non-alcoholic beverages and as ingredient in bakery (e.g., bread, donuts, cookies), confectionery (e.g., candies, cakes), oral hygiene (e.g.,

⁎

Corresponding author at: School of Pharmacy, University of Camerino, via S. Agostino 1, 62032, Camerino, Italy. E-mail address: [email protected] (R. Iannarelli).

http://dx.doi.org/10.1016/j.indcrop.2017.04.028 Received 3 February 2017; Received in revised form 24 March 2017; Accepted 17 April 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.

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whereas the selling price ranges from 86 $/kg in Egypt to 111 $/kg in Spain (Pereira and Meireles, 2007). Aniseed is an annual crop with a relatively simple growth cycle. In the agricultural traditional practice, the field is prepared in Autumn by plowing the ground and fertilizing with manure. The sowing is done on well-prepared soil in March-April. Sowing should not be anticipated in areas with colder climates because aniseed fears late frost (Ullah and Honermeier, 2013). The seeds take about one month to germinate. Vegetative growth proceeds very quickly after emergence of first leaves. Generally, irrigation is needed until the flowering stage which takes place in June-July. Also, the extirpation of weeds by hand is frequently needed. When umbels are not completely dry and just have a dark color (August-September), plants are collected in straight bundles and left to dry in a ventilated place in the shade for 7–8 days. They are then exposed to the sun for 1–2 h on a large towel. Finally, aniseeds are separated from the whole plants by shaking. In the past centuries, one of the most important regions for cultivation of aniseed in Italy was the Piceno area, Marche (central Italy). In particular, in the XIX century the town of Castignano (Ascoli Piceno, Italy) was a very famous center for the production of high quality aniseeds. The noteworthy organoleptic notes, together with the high yield and anethole content of the ‘Castignano ecotype’ were due to the particular exposition of fields, the favorable climate (subMediterranean) and the soil composition (Bellomaria, 1982). In particular, the area of Castignano lays down on a bedrock covered by clayey sediments and is characterized by erosive landforms due to running waters and gravity called ‘calanchi’ (Buccolini et al., 2010). This bedrock is favorable for the cultivation of aniseed because the plants enjoy well-drained soils. In the XIX century, most of production of aniseeds in Castignano, accounting for 8 tons per year, was devoted to the manufacture of local liqueurs such as anisette and mistrà, which were exported all over the world from the Piceno area (Bellomaria, 1982). After that, globalization caused a significant decrease of aniseed cultivation in the area because of the competition with the huge amounts produced and sold at lower prices in Middle East countries (Bhardwaj et al., 2011). Thus, cultivation of aniseed was significantly reduced in Castignano in the 90’s so that this ecotype was almost disappearing (Bellomaria, 1982). Thanks to the regional law for the conservation of local biodiversity and to the association of custodian farmers devoted to the preservation of the ‘Castignano ecotype’ germplasm (Marche Law no. 12/2003), recently aniseed cultivation recovery has been started in the Piceno area. In this scenario, the present work was aimed to provide a scientific basis supporting the recovery of this local crop. In particular, a comprehensive phytochemical analysis on essential oils and polar compounds of aniseed cultivated in Castignano during the years 2013–2015 was carried out, and comparison with twenty-two commercial samples of different geographic origin was made. Chemical data were then analyzed by principal component analysis (PCA) to highlight the peculiarity of the ‘Castignano ecotype’ samples.

Table 1 Main information on the aniseed samples cultivated in Castignano (central Italy) and on commercial samples of different geographic origin. Castignano samples

1 2 3 4 5

Farmer

Diamanti Galosi Corradetti Carboni Villa

Fields GPS coordinates

Altitude (m a.s.l.)

N N N N N

487 375 412 355 496

42°56′31” 42°55′41” 42°56′09” 42°55′14” 42°56′10”

E E E E E

13°35′38” 13°38′52” 13°37′04” 13°36′44” 13°35′00”

Commercial samples

Description

Purchase

Geographic origin

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Pimpinella anisum Illicium verum Illicium verum

Supermarket Supermarket Supermarket Herbalist's shop Herbalist's shop Supermarket Herbalist's shop Pharmacy Supermarket Herbal factory Herbalist's shop Herbalist's shop Herbalist's shop Herbalist's shop Herbalist's shop Herbalist's shop Herbalist's shop Food store Herbalist's shop Herbalist's shop Herbal factory Herbalist's shop

Turkey Turkey Turkey Turkey Turkey Spain Spain Spain Spain Spain Greece Greece Malta Syria Syria Crete Italy Italy Italy Italy China Vietnam

in different stores (supermarket, herbalist’s shop, food store) in 2013 (Table 1). Their origin and authentication was certified by sellers. 2.2. Thermo-pluviometric data In order to evaluate the influence of climatic conditions on the aniseed oil content, monthly air temperatures and precipitations during three years (2013, 2014 and 2015) were recorded at the experimental station of Ascoli Piceno (10 km from Castignano). Data were provided by Centro di Ecologia e Climatologia of Osservatorio Geofisico Sperimentale, Macerata, central Italy. 2.3. Isolation of essential oils Forty grams of aniseed samples were hydrodistilled in a Clevenger type apparatus (2 L volume) using 800 mL of distilled water for 2 h. In order to avoid variability in essential oil content linked to the use of different extraction instruments, the same Clevenger apparatus as that reported in the European Pharmacopoeia was used for all samples. The obtained essential oils were stored in sealed vials protected from light at −20 °C before chemical analysis. The oil yields were estimated on a dry weight basis (n = 2), by calculating the residual water prior to the distillation by leaving plant material in a stove at 105 °C for 8 h.

2. Materials and methods 2.1. Plant material Schizocarps (improperly called seeds) of P. anisum were collected at maturity (August-Spetember) in different fields of Castignano (Ascoli Piceno, Marche, Central Italy, Table 1) in the growing seasons 2013, 2014 and 2015. The botanical identification was performed by checking against literature data (Pignatti, 1982). A voucher specimen was deposited in the Herbarium Universitatis Camerinensis (School of Biosciences and Veterinary Medicine, University of Camerino, Italy) under the codex CAME 28168. The latter was archived in the anArchive system for botanical data (anArchive system, http://www.anarchive.it). The commercial samples, having different geographical origin, were purchased

2.4. GC–MS analysis of essential oils GC–MS analysis was performed on an Agilent 6890N gas chromatograph coupled to a 5973N mass spectrometer using a HP-5 MS (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1 μm film thickness; J & W Scientific, Folsom) capillary column. The temperature program used was as follows: 5 min at 60 °C then 4 °C/min up to 220 °C, then 11 °C/min up to 280 °C, held for 15 min. Injector and transfer line 100

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temperatures were 280 °C; He was used as carrier gas, at a flow rate of 1 mL/min; split ratio, 1:50. Mass spectra were acquired in electronimpact (EI) mode with an ionization voltage of 70 eV; acquisition mass range, 29–400 m/z. Oil samples were diluted to 1:100 in n-hexane, and the volume injected was 2 μL. Data were analyzed by using MSD ChemStation software (Agilent, Version G1701DA D.01.00) and the NIST Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectral Library (Version 2.0 f, October 2008). The identification of the major essential oil constituents was by comparison of retention time, retention index and mass spectrum of peak with those of authentic standards purchased from Sigma-Aldrich (St. Louis, USA). In addition, the peak assignment was carried out according to the recommendations of the International Organization of the Flavor Industry (IOFI, http:// www.iofi.org/), i.e., by the interactive combination of chromatographic linear retention indices (AI, temperature-programmed arithmetic index) that were consistent with those reported in literature (Adams, 2007; FFNSC 2, 2012; NIST 08, 2008) for apolar stationary phases, and MS data consisting in the computer matching with the WILEY275, NIST 08 ADAMS, FFNSC 2 and homemade (based on the analyses of reference oils and commercially available standards) libraries. The percentages of identified compounds were computed by peak area normalization without using correction factors.

acid and trans-ferulic acid, chlorogenic acid, neochlorogenic acid and 3,5-di-O- caffeoylquinic acid. 2.6. HPLC-analysis of non-target metabolites Aniseeds were grinded and 100 mg of powder were extracted in 20 mL MeOH/H2O 50/50 with sonication for 20 min. HPLC–MSn were obtained on a Varian 212 chromatograph equipped with a Prostar 430 (Varian) autosampler and Ion trap Mass detector MS500 using Electrospray (ESI). Separations were obtained on an Agilent Eclipse XDB C-18 2.1 × 150 mm 3.5 μm column. For the analysis of polar constituents, the mobile phases were acetonitrile (A) and water with 1% of formic acid (B). The gradient started with 10% (A) and in 30 min reached 100% of (A). Re-equilibration time was 8 min. Flow rate was 200 μL/min. ESI parameters were: capillary voltage 80 V, needle voltage 5000 V, RF loading 100%, nebulizing gas pressure 35 psi, drying gas pressure 10 psi, drying gas temperature 350 °C. Mass range was 50–2000 Da. Fragmentation patterns of eluted compounds were obtained using the turbo detection data scanning (TDDS®) function of the instrument. The phytochemical analysis on the different samples of Aniseed showed the presence of apigenin and luteolin mostly Cglycosylated, those compounds are clearly detectable in MS/MS mode by the charachteristic loss of 90 and 120 Da related to the C-glycoside fragmentation (Martins et al., 2016; Ferreres et al., 2008). Furthermore, O-glycosides of apigenin and luteolin have been detected. Mono- and di-caffeoylquinic acids have also been identified showing the chemical diversity of phenolic compounds in Castigliano aniseed.

2.5. HPLC-analysis of target metabolites 2.5.1. Materials and chemicals Dry fruits of cultivated and commercial P. anisum samples, as well as one commercial I. verum sample, were grinded using liquid nitrogen. The finely powdered material (500 mg) was extracted with 5 mL of methanol by sonication (60 min, room temperature). After centrifugation at 5000 rpm for 10 min, the extracts were transferred to volumetric flask, which was filled up to 5 mL with extraction solvent. The sample solutions were filtered through a 0.45 μm pore size nylon membrane filter (Phenex, Phenomenex, Torrance, CA, USA) before injection into HPLC-DAD. All samples were stored in a refrigerator at the temperature of 4 °C until analysis. Each sample was analyzed in triplicate. The analytical standards of shikimic acid, gallic acid, caffeic acid, pcoumaric acid, trans-ferulic acid, chlorogenic acid, neochlorogenic acid, 3,5-di-O-caffeoylquinic acid, catechin, epicatechin, syringic acid were bought from Sigma-Aldrich (Milan, Italy). The stock standard solutions were prepared by dissolving each analyte in methanol to obtain a final concentration of approximately 1000 mg/L and the relative vials were stored at 4 °C in the dark. Standard working solutions of all compounds, at different concentrations, were prepared fresh every day by diluting the stock solution with methanol. HPLC-grade methanol was purchased from Sigma-Aldrich (Milan, Italy), while HPLC-grade formic acid 99–100% was bought from J.T. Baker B.V. (Deventer, Holland). For sample preparation and chromatographic analysis, deionized water ≥18 MΩ/cm resistivity purified with a Milli-Q system (Millipore, Bedford, USA) was used. All solvents and solutions were filtered through a 0.45-μm PTFE filter from Supelco (Bellefonte, PA, USA) before use.

2.7. HPLC quantification of non-target metabolites The quantitative analysis of polyphenols was performed on an Agilent 1260 series HPLC system equipped with autosampler and diode array detector (DAD detector series 1260). Chromatographic separation was performed on an Agilent Eclipse C18 column 4.6 × 150 mm, 5 μm. The mobile phase consisted of acetonitrile (A) and aqueous formic acid 0.1% (B). The solvent flow rate was 1 mL/min. The gradient used was as the gradient utilized for qualitative analysis. The column was thermostatted at 25 °C. The selected wavelengths were 280, 330 and 350 nm for gallic acid derivatives, caffeoylquinic derivatives and flavonoids, respectively. Spectra were acquired in the range 200–400 nm. As reference compounds chlorogenic acid and rutin were used at 4 levels of concentrations each, to build the calibration curves. For chlorogenic acid the concentrations were 18.1, 36.2, 90.5 and 181 μg/mL; for rutin 11.7, 23.4, 58.5 and 117 μg/mL. For quantitative analysis, a calibration curve for available phenolic standards was built based on the UV signal of rutin (y = 15.12x + 14.63; R2 = 0.999) and chlorogenic acid (y = 26.52 x + 1.568; R2 = 0.999). For the identified phenolic compounds for which a commercial standard was not available, the quantification was performed through the calibration curve of other compounds from the same phenolic group namely flavonoid (rutin), or caffeic acid derivatives (chlorogenic acid). The results were expressed in mg of analyte per 100 g of dried plant material. 2.8. Statistical analysis (PCA)

2.5.2. HPLC-DAD analysis HPLC-DAD studies were performed using a Hewlett-PackardHP1090 SeriesII (Palo Alto, CA, USA), equipped with a vacuum degasser, a binary pump, an autosampler and a model 1046A HP photodiode array detector (DAD). Chromatographic separation was accomplished on a Synergi Polar-RPC18 (4.6 × 150 mm, 4 μm) analytical column from Phenomenex (Chesire, UK). The column was preceded by a security cartridge. The mobile phase for HPLC-DAD analysis was a mixture of (A) water (v/v) and (B) methanol, flowing at 0.7 mL/ min in isocratic conditions: 60% A, 40% B. The injection volume was 5 μL. UV spectra were recorded in the range 210–350 nm for 11 compounds, where 210 nm was used for quantification of shikimic acid, gallic acid, catechin, epicatechin; 310 nm for p-coumaric acid; 325 nm for caffeic

To reveal the relationship among the different aniseed and star anise samples based on essential oil and phenolic compositions, and to identify the main constituents influencing the variability, the composition data matrix of 37 essential oils (35 P. anisum + 2 I. verum) and 33 methanolic extracts (32 P. anisum + 1 I. verum) was analyzed using principal component analysis (PCA) with STATISTICA 7.1 (Stat Soft Italia srl, 2005, www.statsoft.it). From all the essential oil constituents, those which did not show variance at all were excluded from the analysis. A total 1295 data (35 variables x 37 samples) for essential oils and 363 data (11 variables x 33 samples) for polar extracts were selected and subjected to PCA. Prior to the analysis, the variables were 101

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Fig. 1. a) Monthly average air temperature (°C) for the growing seasons 2013–2015. b) Monthly sum of precipitation (mm) for the growing seasons 2013–2015. Data are referred to the experimental station of Ascoli Piceno (10 km from Castignano) and were provided from Osservatorio Geofisico di Ecologia and Climatologia of Macerata (central Italy).

fruit essential oil yields in cultivated samples. As we can observe from Fig. 1, the three growing seasons were quite different in terms of monthly air temperatures and rainfall. The 2014 year was characterized by lower temperatures and higher precipitation during flowering, fruiting and ripening of aniseed. On the other hand, the years 2013 and 2015 were definitely warmer, especially during fruiting (July–August) when aniseed increases the content of essential oil (Ullah and Honermeier, 2013). Precipitations showed a different distribution in these two years, being more concentrated at sowing (March) and ripening (August) in 2015, and from sowing (March) to flowering (June) in 2013 (Fig. 1).

normalized: missing data were substituted for the purpose by 0.001%. Eigenvalues were calculated using a covariance matrix among 35 and 11 chemical compounds, respectively, as input, and the two-dimensional PCA biplots, including both P. anisum and I. verum samples and compounds, was generated. Furthermore, Pearson’s correlation coefficients (r), used to determine association between climatic parameters (i.e. average air temperature and precipitations) and oil yields and (E)anethole percentages obtained during the three cultivation seasons (2011–2013) in Castignano, were calculated by Microsoft Excel for Mac OS X.

3. Results and discussion 3.2. Essential oil content 3.1. Climatic data (years 2013–2015) The climatic trend recorded over three years during the growing seasons gave significant differences in the fruit oil content of the aniseed samples obtained by hydrodistillation (Table 2). According to European Pharmacopoeia (EP), P. anisum fruits must have an essential

In this study the thermo-pluviometric data recorded over a threeyears period (2013–2015) at the experimental station of Ascoli Piceno (near Castignano) were used to evaluate the effects of climate on the 102

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collected in 2013, was below this value (1.5%). This trend in essential oil content may be influenced by the thermo-pluviometric data recorded during the three growing seasons (Fig. 1). It has been reported that higher temperatures and lower precipitation occurring during the fruit formation stage (July) result in a higher yield of essential oil (Ullah and Honermeier, 2013). More in general, it is well-documented that under drought conditions the amount of essential oil is enhanced (Zehtab-Salmasi et al., 2001). Therefore, 2013 and 2015 growing seasons, being characterized by higher temperature and lower precipitation during formation and ripening of the fruit, gave samples richer in essential oil (Table 2). Notably, the highest values were reached in sample 5 of 2015 (5.5%) and sample 1 of 2013 (5.0%). On the other hand, all samples collected in 2014 gave generally lower oil yields. Pearson’s linear correlation showed that the oil yields were strongly correlated with the average air temperature values during the growing seasons showing a coefficient r = 0.86, whereas they were negatively correlated with the precipitations (r = −0.70). For comparative purposes, a total of 20 commercial aniseed samples and 2 commercial star anise samples were analyzed for the essential oil content and results are depicted in Table 2. Overall, the average percentage in all aniseed samples was 2%. However, significant differences were noticed between samples of different origin. This variability was also found in a previous work studying essential oil samples from various European countries (Orav et al., 2008). The

Table 2 Essential oil yields (min, max, mean and standard deviation values) obtained for aniseed samples ‘Castignano ecotype’ cultivated during the years 2013–2015; and for commercial aniseed and star anise samples of different geographic origin. Oil yields (%, w/w) Aniseed Samples

Min

Max

Mean

SD

Castignano ecotype 2013 2014 2015

2.0 1.5 2.8

5.0 4.0 5.5

3.4 2.9 3.9

1.1 1.0 1.1

1.0 1.0 1.0

5.6 2.3 1.7

1.9 0.5 0.5

2.0

2.3

1.5 2.8

4.3 4.7

2.4 1.7 1.4 1.0 2.2 1.0 2.6 3.8

Commercial samples Turkey Spain Greece Malta Syria Crete Italy Star anise

0.2 1.3 1.4

oil concentration higher than 2% (European Pharmacopoeia, 2005). In almost our ‘Castignano ecotype’ samples the essential oil content was higher than 2%, showing the following ranges: 2.0–5.0% in 2013, 1.5–4.0% in 2014 and 2.8–5.5% in 2015 (Table 2). Only one sample, Table 3 Essential oil composition of aniseed samples from ‘Castignano ecotype’. No.

Componenta

RI Exp.b

RI Lit.c

2013 (%)d 1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

p-cymene γ-terpinene linalool geijerene n-nonanol (2E)-nonen-1-al methyl salicylate methyl chavicol (Z)-anethole p-anisaldehyde (E)-anethole δ-elemene α-longipinene α-ylangene α-copaene anisyl methyl ketone methyl eugenol trans-α-bergamotene α-himachalene p-methoxypropiophenone γ-himachalene germacrene D allo-aromadendrene ar-curcumene β-himachalene (E)-methyl isoeugenol α-zingiberene δ-amorphene β-bisabolene (E)-pseudoisoeugenyl 2methylbutyrate epoxy-pseudoisoeugenyl 2methylbutyrate Total identified (%)

1022 1055 1100 1136 1160 1161 1190 1195 1250 1252 1288 1332 1341 1357 1364 1381 1396 1431 1437 1446 1468 1472 1474 1479 1484 1490 1492 1493 1505 1840

1020 1054 1095 1138 1165 1157 1190 1195 1249 1247 1282 1335 1350 1373 1374 1380 1403 1432 1449

1891

1895

1481 1484 1475 1479 1500 1491 1493 1511 1505 1841

2

2014 (%)d 3

4

5

1

2

2015 (%)d 3

4

f

tr tr tr

tr

tr

tr

tr

tr

tr

5 tr tr tr tr tr

1

tr

2

0,1 tr

IDe 3

4

5

tr 1,9

0,1 0,2 tr tr 2,8

RI,MS,Std RI,MS,Std RI,MS,Std RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS,Std RI,MS RI,MS RI,MS RI,MS,Std RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS,Std RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS RI,MS

0,6

0,3

0,5

RI,MS

99,8

100,0

100,0

tr

tr

tr

1,5 0,2 0,2 92,5 tr tr tr

1,1 0,2 0,2 95,0 tr

2,8 0,1 0,1 92,1 tr

tr 1,6 0,1 97,5

1,8 0,2 0,7 92,2 tr

1,7 0,1

2,5 0,2

2,6 0,1

97,9

95,4 tr

94,8 tr

2,1 0,1 0,1 94,1 tr

1,8 0,1 tr 95,9

1,6 0,1 0,1 95,4 tr

1,2 0,2 0,1 96,1 tr

tr 1,8 0,4 1,0 91,6 tr

1,8 0,1 0,7 91,0 tr

1,6 0,6 1,2 89,5

tr

0,1

0,1

0,1 0,1 0,9 0,1 tr 0,1

0,1

tr

0,1

1,0 0,3

0,7 0,1 tr tr

0,9 0,2

tr 0,3

tr tr tr tr tr 0,1 0,4

2,0 0,2

0,1 0,3

0,1 0,1 0,1

0,4

100,0

0,1 2,2

99,9

tr

100,0

tr

tr

tr

1,2 0,1

0,5 tr

0,8 0,1

0,8 tr

tr 0,6 0,1

tr

tr

tr

tr

0,1

0,1

tr

0,2

0,1

0,1

0,1

0,2 0,1

0,1 0,2

1,3

tr 1,8

0,0 1,5

tr 1,6

tr 1,3

tr 3,0

0,1 0,2 tr 0,1 3,4

0,1 4,0

0,1 0,2 tr 0,1 2,8

0,2

0,1

0,1

tr

0,5

0,8

1,2

100,0

100,0

100,0

100,0

99,7

99,8

99,8

1,1 0,2

0,8 0,2

tr

tr

tr tr 0,2

100,0

tr

100,0

a

1,1 0,2

0,1

tr

Compounds are listed in order of their elution from a HP-5MS column. Linear retention index on HP-5MS column, experimentally determined using homologous series of C8–C30 alkanes. Linear retention index taken from Adams (2007) and/or NIST 08 (2008). d Relative percentage values are means of three determinations with a RSD% in all cases below 18%. e Identification methods: std, based on comparison with authentic compounds; MS, based on comparison with WILEY, ADAMS, FFNSC2 and NIST 08 MS databases; RI, based on comparison of calculated RI with those reported in ADAMS, FFNSC 2 and NIST 08. f Tr, % below 0.1%. b c

103

RI Exp.b

929 964 969 986 1000 1006 1012 1022 1024 1025 1055 1084 1100 1136 1167 1182 1195 1235 1235 1250 1252 1288 1332 1357 1364 1381 1383 1411 1431 1437 1446 1455 1467 1468 1472 1474 1479 1484 1485

Componenta

α-pinene sabinene β-pinene myrcene α-phellandrene δ-3-carene α-terpinene p-cymene limonene 1,8-cineole γ-terpinene terpinolene linalool geijerene terpinen-4-ol α-terpineol methyl chavicol cuminaldehyde carvone (Z)-anethole p-anis aldehyde (E)-anethole δ-elemene α-ylangene α-copaene anisyl methyl ketone β-elemene (E)-caryophyllene trans-α-bergamotene α-himachalene p-methoxypropiophenone 8,9-dehydro-neoisolongifolene 9-epi-(E)-caryophyllene γ-himachalene germacrene D allo-aromadendrene ar-curcumene β-himachalene 11-αH-himachala-1,4-diene

No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

104 1466 1481 1484 1475 1479 1500 1485

932 969 974 988 1002 1008 1014 1020 1024 1026 1054 1086 1095 1138 1174 1186 1195 1238 1239 1249 1247 1282 1335 1373 1374 1380 1389 1417 1432 1449

RI Lit.c

0.1 tr 1.2 0.1 tr tr

tr 0.1 1.9 0.2 0.1 tr

1.4 0.1

tr

0.2 0.1 95.0 tr

1.4

tr tr

tr

0.2

0.1

tr

0.2 0.3 92.2 0.1 tr

0.1 95.8 0.1

1.5 0.4

1.5

0.2

0.1

0.1

0.1

trf

3

0.1 0.1

1.6 0.2

tr

0.1

0.1 tr 94.5 0.1

1.5

tr

4

tr tr

tr tr 1.1

0.1

tr

0.4 0.3 0.3 94.9 0.1

2.6

tr tr

0.1

0.2

tr 0.1 2.3 0.1

0.2

0.1 tr 92.7 0.1 tr

1.3

tr

0.1

tr 0.1 2.3 0.1

0.1

92.7 0.1

0.2

1.3

7

8

9

10

15

Italy

0.1 0.2

0.1 0.1 2.8 0.1

0.2

tr

0.2 2.1 89.8 0.2 tr

1.1

0.1 0.1

2.7 0.2

tr

0.2

tr

0.3 0.1 91.6 0.1 tr

1.9

tr

tr

0.2

1.4 0.1

0.1

tr 0.1

tr tr

tr tr 1.1

tr

0.4 0.3 0.3 94.9 0.1

2.6

tr tr

tr

0.3 0.9 88.9 0.1 tr

1.0

tr

0.1

11

tr

1.9 0.2

0.1

0.1

95.2 0.1

0.1

1.4

12

tr

1.3 0.1

tr

0.1

0.2 0.2 93.6 tr

1.7

tr tr

13

0.1 0.1

1.7 0.3

0.1

95.6 0.1

0.1

1.1

14

0.1

tr 0.1 1.8 0.2

0.2

tr

0.2 0.2 93.4 0.1 tr

1.5

tr

tr

tr

0.6 tr

tr

0.2 1.3 92.5

1.6 tr

16

tr

0.8 0.1

tr

96.9 0.1

0.1

0.8

tr

17

18

19

20

1.4 0.1 tr tr tr

tr

0.1

0.2 0.1 94.9 0.1

1.6

0.1

tr

tr

0.7 tr

tr tr

0.1

0.9 1.8 93.9

0.8

tr

0.6 tr

95.6

0.1

1.9

tr

tr tr

tr

96.1

0.2

0.2 0.2 0.9

tr 0.1 0.2 tr tr 1.2 0.3 tr tr 0.4

0.2 tr

21

IDe

RI,MS,Std RI,MS,Std RI,MS,Std 0,1 RI,MS,Std 0.1 RI,MS,Std 0.3 RI,MS,Std 0.4 RI,MS,Std 0.1 RI,MS,Std 4.1 RI,MS,Std 0.8 RI,MS,Std 0.1 RI,MS,Std RI,MS,Std 0.3 RI,MS,Std RI,MS 0.2 RI,MS,Std 0.1 RI,MS,Std 0.2 RI,MS RI,MS RI,MS,Std RI,MS RI,MS 92.9 RI,MS,Std RI,MS RI,MS RI,MS,Std RI,MS RI,MS RI,MS,Std RI,MS RI,MS MS MS RI,MS RI,MS RI,MS RI,MS,Std RI,MS RI,MS RI,MS (continued on next page)

0.8 0.1

22

Vietnam

6

2

5

Crete

1

Syria

China

Malta

Spain

Turkey

Greece

Illicium verum (%)d

Pimpinella anisum (%)d

Table 4 Essential oil composition of commercial aniseed and star anise samples from different geographic areas.

R. Iannarelli et al.

Industrial Crops & Products 104 (2017) 99–110

Industrial Crops & Products 104 (2017) 99–110

97.3 99.9 99.8 99.9 99.9 99.9 99.9 99.9 Total identified (%)

Compounds are listed in order of their elution from a HP-5MS column. Linear retention index on HP-5MS column, experimentally determined using homologous series of C8–C30 alkanes. c Linear retention index taken from Adams (2007) and/or NIST 08 (2008). d Relative percentage values are means of three determinations with a RSD% in all cases below 18%. e Identification methods: std, based on comparison with authentic compounds; MS, based on comparison with WILEY, ADAMS, FFNSC2 and NIST 08 MS databases; RI, based on comparison of calculated RI with those reported in ADAMS, FFNSC 2 and NIST 08. f Tr, % below 0.1%.

99.9 99.8 100.0

0.4 1895 1891 45

highest yields were obtained from aniseed from Turkey (5.6%, sample 1) and Italy (4.3%, sample 17), whereas twelve out of twenty samples gave values lower than 2% (Table 2). The latter concerned samples came from different countries such as Turkey, Spain, Greece, Malta, Crete and Italy. This high variability in essential oil content may be explained by the variable growing and storage conditions of aniseed before its final commercialization that can affect significantly the oil yield. As regards the star anise samples, the oil content ranged from 4.7% in sample from China to 2.8% in that from Vietnam Table 2. These values were comparable to those of ‘Castignano ecotype’ aniseed. In conclusion, our study showed that the ‘Castignano ecotype’ provides a good quality product according to Pharmacopoeia requirements to be used on an industrial level. 3.3. Essential oil composition of ‘Castignano ecotype’ The essential oil compositions of aniseed samples cultivated through the years 2013–2015 in five fields of Castignano (central Italy) are reported in Table 3. A total of 31 volatile components were identified by GC–MS in all samples analyzed, accounting for 99.7–100.0% of the total compositions. All samples displayed very high levels of (E)anethole (89.5–97.9%), with differences linked to the year of growth and farmer. Overall, the year 2013 allowed to obtain the highest amount of (E)-anethole (92.2–97.9%), followed by 2014 (91.6–96.1%) and 2015 (89.5–95.0%). This result confirms that anise enjoys warm climatic conditions throughout the growing season, being the 2013 a year particularly favorable for its cultivation in Castignano. Among the minor constituents occurring in the aniseed samples, the pseudoisoeugenols (E)-pseudoisoeugenyl 2-methylbutyrate (traces to 4.0%) and epoxy-pseudoisoeugenyl 2-methylbutyrate (traces to 1.2%), the propenylphenols methyl chavicol (1.1–2.8%), (Z)-anethole (0.1–0.7%) and panisaldehyde (traces to 0.7%), and the sesquiterpenes γ-himachalene (0.4–1.2%) and α-zingiberene (traces to 0.3%) were the most representative compounds. In particular, pseudoisoeugenols attained the highest levels during the 2015 year (Table 3). Pearson’s linear correlation was used to determine the relationship between the (E)-anethole content in Castignano aniseed samples and the climatic parameters (average air temperature and rainfall) during the three growing seasons. The coefficients obtained, in both cases close to 0 (r = −0.26), showed that the anethole content, unlike oil yield, seems to be not influenced by the thermopluviometric trend in the area. The levels of (E)-anethole found in the ‘Castignano ecotype’ samples were generally higher than those found in other European samples, such as France (76.9%), Hungary (91.3%), Russia (85.7–87.7%), Greece (90.2%), Scotland (93.6%), Lithuania (93.0%), Turkey (94.2%), Spain (87.9%), Germany (92.7–93.7%), Czech Republic (88.4) and Estonia (78.1%) (Orav et al., 2008; Tabanca et al., 2006). As concerns methyl chavicol (estragole), which is a restricted substance occurring in flavourings and food ingredients with flavouring properties (Cachet et al., 2014), the EP set up the optimal percentage range for this metabolite in the essential oil to 0.5–5% (European Pharmacopoeia, 2005). Thus, the concentration values for methyl chavicol in aniseed samples were all included in that range. For (Z)-anethole, the range reported by EP was of 0.1–0.4%. Thus, almost all aniseed samples contained amounts of this constituent in the range reported by the EP. Finally, we found also traces of some compounds such as anisyl methyl ketone and p-methoxypropiophenone, which are markers of oxidation of the oil. However, their presence was restricted to a few samples and at very scant levels so that it did not represent a serious risk of deterioration. 3.4. Essential oil composition of commercial samples A total of twenty aniseed samples and two star anise samples have been investigated. Overall, they came from different geographic areas. Overall, a total of forty-five volatile components were identified in all

b

100.0 100.0 100.0 100.0 99.9 100.0 99.9 99.9

0.1

tr 0.7 tr 2.3 0.8 tr 0.1 6.0 0.1 2.1 0.2 tr 3.0 0.1 2.4 tr 1.8 1.5

1491 1493 1511 1505 1841 1490 1492 1493 1505 1840

(E)-methyl isoeugenol α-zingiberene δ-amorphene β-bisabolene (E)-pseudoisoeugenyl 2methylbutyrate epoxy-pseudoisoeugenyl 2methylbutyrate

0.8

0.1 1.7

99.9

100.0

0.3 0.1 0.3

100.0

1.4 tr 1.8 1.2 1.2 3.3

tr tr 0.1 tr

0.4 tr 0.1 1.3 0.2 0.1 tr 0.1 tr 0.3 0.2 tr 0.3 0.1 0.1 0.1 0.3

4 3 2

40 41 42 43 44

a

RI,MS

22 17 16 14 13 12 11 6 1

RI Exp.b Componenta No.

Table 4 (continued)

RI Lit.c

Pimpinella anisum (%)d

5

Spain Turkey

7

8

9

10

Greece

Malta

Syria

15

Crete

Italy

18

19

20

21

Vietnam China

Illicium verum (%)d

IDe

RI,MS RI,MS RI,MS RI,MS RI,MS

R. Iannarelli et al.

105

Industrial Crops & Products 104 (2017) 99–110

R. Iannarelli et al.

3.5. HPLC-DAD analysis of target metabolites

essential oils by GC–MS, accounting for 97.3–100.0% of the total compositions (Table 4). In all commercial aniseed samples the predominant constituent was (E)-anethole (88.9–96.9%). The richest samples were those coming from Italy (93.9–96.9%), followed by Turkey (92.2–95.8%), Syria (93.4–95.6%), Greece (94.9–95.2%), Spain (88.9–92.7%), Malta (93.6%) and Crete (92.5%). Among the minor volatile components occurring in aniseeds the pseudoisoeugenols (E)pseudoisoeugenyl 2-methylbutyrate (traces to 2.4%) and epoxy-pseudoisoeugenyl 2-methylbutyrate (0.1-0.4%), the propenylphenols methyl chavicol (0.8–2.6%), p-anysaldehyde (0.1–1.8%) and (Z)-anethole (0.1–0.9%), and the sesquiterpene γ-himachalene (0.6–2.8%), were the most representative compounds. Notably, the samples from Spain were characterizied by the highest content of (E)-pseudoisoeugenyl 2-methylbutyrate (6.0%) (sample 10), γ-himachalene (2.8%) and p-anysaldehyde (2.1%) (sample 8) (Table 4). In the remaining samples the level of these components were in all cases below 3%. As regards methyl chavicol, the richest samples were those from Turkey (sample 5) and Greece (sample 11), both containing 2.6% of this component, whereas the lowest value was obtained in samples from Italy (0.8%, samples 17 and 19). The two star anise samples (samples 21 and 22) contained high levels of (E)-anethole (92.9–96.1%) (Table 4). Among the minor constituents, the monoterpenoids limonene (1.2–4.1%) and 1,8-cineole (0.3–0.8%), and the propenylphenol methyl chavicol (0.2–0.9%) were the most abundant. On the other hand, the pseudoisoeugenols (E)pseudoisoeugenyl 2-methylbutyrate and epoxy-pseudoisoeugenyl 2methylbutyrate were completely missing in star anise, confirming that the latters are chemotaxomic markers of the genus Pimpinella (Tabanca et al., 2006). These results are overall comparable with literature data except for limonene, p-anisaldheyde and (Z)-anethole that were reported in higher amounts (6.5, 1.8 and 0.2%, respectively) in the review by Wang et al. (2011). These differences may depend on the harvest time, seasonal factors, geographic origin and whether the plant is processed fresh or dry (Heath, 1981). In order to find possible correlations between ‘Castignano ecotype’ and commercial aniseed and star anise samples, all the essential oil compositions of Tables 3 and 4 were subjected to Principal Component Analysis (PCA). Results are depicted in Fig. 2a where the 2D graphical representation of PCA represents 83.84% of the total variance in the data set. Overall, most of aniseed samples clustered together in the upper part of the score plot where a clear separation between ‘Castignano ecotype’ and commercial aniseed samples was not observed (Fig. 2a, score plot). In this regard, the variability of data was generated mostly by the content of (E)-anethole (values of eigenvectors: −2.15; 0.33) and, to a minor extent, by (E)-pseudoisoeugenyl 2-methylbutyrate (values of eigenvectors: −1.03; 0.28) in the first PC and by (E)pseudoisoeugenyl 2-methylbutyrate and limonene (values of eigenvectors: −0.04; −0.48) in the second PC (Fig. 2a, loading plot). Most of ‘Castignano ecotype’ samples cultivated in 2013 and 2014, were situated in the upper right hand side of the score plot and that meant that they were correlated with high level of (E)-anethole. On the other hand, most of ‘Castignano ecotype’ samples cultivated in 2015 were on the upper left hand side of the same plot, being instead correlated with high level of (E)-pseudoisoeugenyl 2-methylbutyrate (Fig. 2a). This confirmed that the variable climatic conditions affected the final product obtained from the Castignano fields. As for the commercial aniseed samples, a clear separation based on their geographic origin was not possible. In fact, the samples situated on the upper right hand side of the score plot were those from Italy (5, 17, 20), Syria (14), Greece (11, 12) and Turkey (1) that resulted particularly rich in (E)anethole Fig. 2a. Finally, the two star anise samples (21 and 22) appeared to be correlated to a greater extent with high limonene content and that may be the element of differentiation with respect to aniseed samples.

From the analysis of fruits methanol extracts 11 polar compounds, i.e shikimic acid, gallic acid, catechin, epicatechin, p-coumaric acid, caffeic acid, trans-ferulic acid, chlorogenic acid, neochlorogenic acid and 3,5-di-O-caffeoylquinic acid were identified and quantified simultaneously (Table 5). Shikimic acid, gallic acid, chlorogenic acid, neochlorogenic acid were present in all samples analyzed. Neochlorogenic acid was the most abundant compound detected in all samples with concentrations ranging from 0.209 to 4.258 mg/g. The highest amounts were contained in sample 5 obtained in 2013 and 2015. Neochlorogenic acid is an isomer of chlorogenic acid, which is a caffeic acid derivative widespread in plants, fruits and vegetables, among which coffee beans are the main source. It has been proven that chlorogenic acids protect against oxidative stress, improve the glucose metabolism, reduce risk of cardiovascular disease, inhibit carcinogenesis and exhibit anti-obesity effects (Cho et al., 2010). 3,5-Dicaffeoylquinic acid was the second most abundant compound, showing a concentration of 2.369 mg/g in sample 5 obtained in 2015; its range was of 0.184–2.369 mg/g. Considerable amounts of gallic acid and chlorogenic acid were detected in Castignano aniseed samples, with ranges of 0.086–0.675 and 0.060–0.829 mg/ g, respectively. Shikimic acid was the only compound occurring in all samples in similar and high concentrations (0.635–0.885 mg/g). This compound is the byosinthetic precursor of aromatic amino acids and phenolic compounds and it is endowed with important biological effects such as anti-inflammatory, antiplatelet aggregation, antiviral, and antiischemic (Estevez and Estevez, 2012). The richest source of this compound is I. verum, an industrial source of the antiviral drug Tamiflu® (Gosh and Chisti, 2012). Ferulic acid was not present in all samples; it showed a range of 0.016–0,689 mg/g. Catechin was present in very low concentrations (0.020–0.052 mg/g). The content of phenolic compounds in the commercial aniseed samples is reported in Table 6. Seven out of eleven compounds were detected, namely shikimic acid, gallic acid, ferulic acid, chlorogenic acid, neochlorogenic acid and 3,5-di-O caffeoylquinic acid. The most abundant compound was shikimic acid that ranged from 0.042 mg/g in sample 17 (Italy) to 0.691 mg/g in sample 13 (Malta). The second most abundant compound was neochlorogenic acid detected in samples 1 (0.344 mg/g) and 4 (0.296 mg/g), both from Turkey. Chlorogenic acid was detected in very low amounts, ranging from 0.010 to 0.112 mg/g, meanwhile 3,5-di-O-dicaffeoylquinic acid was present with a range of 0.042–0.207 mg/g. Catechin and epicatechin were detected in very low concentration ranges (0.018–0.093 and 0.06–0.028 mg/g, respectively). In I. verum from Vietnam, shikimic acid, gallic acid, caffeic acid, 3,5dicaffeoylquinic acid, catechin and epicatechin were detected. As expected, the most abundant compound was shikimic acid (0.241 mg/g), followed by gallic acid (0.171 mg/g) and 3,5-di-Odicaffeoylquinic acid (0.113 mg/g). The lowest concentration was reported for catechin (0.028 mg/g). Phenolic compounds are commonly found in both edible and nonedible plants, and they have been reported to have multiple biological effects, including antioxidant activity. Crude extracts of fruits, herbs, vegetables, cereals, and other plant materials rich in phenolics (Manach et al., 2004) are increasingly of interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food (Kähkönen et al., 1999). The beneficial effects of polyphenols are mainly attributed to their antioxidant properties, since they can act as chain breakers or radical scavengers depending on their chemical structures (Rice-Evans, 2001). Polyphenols might also trigger changes in the signalling pathways and subsequent gene expression (Chen et al., 2002; Pfeilschifter et al., 2003). Overall, HPLC data evidenced that aniseed samples, especially the 106

Industrial Crops & Products 104 (2017) 99–110

R. Iannarelli et al.

Fig. 2. a) Left: Score plot (PCA) for main variation of volatile composition among aniseed and star anise samples. Right: The PCA loading plot for volatile constituents which explains 72.24% of the variation on horizontal axis (PC 1) and 11.60% on the vertical axis (PC 2). b) Left: Score plot (PCA) for main variation of phenolic compounds among aniseed and star anise samples. Right: The PCA loading plot for volatile constituents which explains 88.03% of the variation on horizontal axis (PC 1) and 7.37% on the vertical axis (PC 2). Numbers refer to samples reported in Table 1; ‘Castignano ecotype’ numbers and years of cultivation are reported in bold. Table 5 Concentration of shikimic acid and phenolic compounds in the aniseed samples from Castignano. Relative standard deviations (RSD%) were in the range 2–5% (n = 3). No.

1 2 3 4 5 6 7 8 9 10 11

Component

shikimic acid gallic acid caffeic acid coumaric acid ferulic acid chlorogenic acid neochlorogenic acid 3,5-dicaffeoylquinic acid catechin epicatechin syringic acid

2013 (mg/g)

2014 (mg/g)

2015 (mg/g)

1

2

3

4

5

1

2

3

4

5

1

2

3

4

5

0.812 0.177

1.164 0.663

0.635 0.264 0.01

0.861 0.086

0.812 0.441

0.851 0.252

0.777 0.397

0.824 0.152 0.007

0.750 0.165

0.885 0.332

0.752 0.148

0.716 0.186

0.857 0.246

0.817 0.675

0.267 0.481 0.295

0.689 0.504 2.18 2.113

0.016 0.111 0.661 0.5

0.174 0.060 0.267 0.586

0.802 0.541 2.51 1.65

0.352 0.24 1.078 0.928 0.037

0.399 0.219 1.110 1.047 0.023

0.842 0.258 0.022 0.020 0.426 0.348 1.59 1.114 0.026

0.020 0.140 0.060 0.342 0.395

0.392 0.224 1.137 1.108

0.096 0.466

0.039 0.209 0.184 0.037 0.033

0.829 4.258 2.369 0.052 0.606 0.174

‘Castignano ecotype’ ones, were richer in the target analytes, such as shikimic acid, gallic acid and 3,5-di-caffeoylquinic acid than commercial samples. The PCA score and loading plots on polar constituents detected in

0.174 0.060 0.267 0.022

0.020 0.056

0.199 0.092 0.459 0.445 0.042

the methanolic extracts of aniseed and star anise samples are reported in Fig. 2b where they represent 95.4% of the total variance in the data set. The variability of data was generated mostly by the content of neochlorogenic acid (values of eigenvectors: −0.87; −0.11) and 3,5107

Industrial Crops & Products 104 (2017) 99–110

R. Iannarelli et al.

Table 6 Concentration of shikimic acid and phenolic compounds in commercial aniseed and star anise samples. Relative standard deviations (RSD%) were in the range 2–5% (n = 3). No.

Component

Pimpinella anisum (mg/g) Turkey

1 2 3 4 5 6 7 8 9 10 11

shikimc acid gallic acid caffeic acid coumaric acid ferulic acid chlorogenic acid neochlorogenic acid 3,5-dicaffeoylqionic acid catechin epicatechin syringic acid

Spain

Syria

Crete

Italy

2

3

4

5

7

8

9

10

11

13

14

15

16

17

19

20

22

0.085 0.099

0.061 0.070

0.066 0.078

0.382 0.571

0.091 0.084

0.089

0.063 0.032

0.086 0.108

0.054 0.072

0.099 0.076

0.691 0.201

0.079 0.040

0.079 0.104

0.080 0.046

0.042 0.078

0.760 0.091

0.079 0.078

0.241 0.171

0.108 0.068 0.296 0.201

0.087 0.059 0.213 0.207

0.071 0.031 0.13 0.178

0.081 0.025 0.096 0.126

0.085 0.050 0.247 0.175

0.038

0.096 0.084 0.337 0.302

0.039

0.035 0.178

0.094 0.062 0.290

0.047 0.311 0.157

0.074 0.010 0.048 0.115

0.073 0.058 0.225 0.234

0.01 0.107 0.076 0.344

0.021 0.010

0.025

0.029

0.021

Total Chlorogenic acid derivatives mg/g

Gallic acid derivatives mg/g

1 2 3 4 5

2.03 1.90 0.77 1.96 2.61

0.56 0.48 0.58 0.44 1.85

4.88 2.51 6.58 6.92 9.19

± ± ± ± ±

0.04 0.05 0.07 0.03 0.04

± ± ± ± ±

0.055 0.048

0.055 0.042

0.033 0.06

0.113 0.093 0.028

4. Conclusions The aim of this research was to provide a scientific basis for the valorisation and economic recovery of aniseed cultivation in Castignano, central Italy, after it was marginalized as a consequence of the globalization process. In a global context dominated almost exclusively by a quantitative approach for the competitiveness of goods, local products are endangered to be marginalized or even disappearing. Given this situation, results of this work supported scientifically the quality of a product, which corresponds to the quality of a territory. Chemical analyses performed on ‘Castignano ecotype’ samples showed that they were richer in essential oil and phenolic compounds than commercial samples coming from different countries of the Mediterranean basin. Aniseed cultivated in Castignano meets the demand of the European Pharmacopoeia in terms of high quality Anisi aetheroleum characterized by high yields and levels of (E)-anethole to be used in pharmaceutics, food and agriculture. Same situation was observed for polar constituents, which were more abundant in the ‘Castignano ecotype’ samples. Noteworthy, aniseed proved to be significantly richer in shikimic acid than star anise, thus having the possibility to find application on an industrial level as a source of this important precursor compound. These results, which were influenced by the peculiar soil and climate of the area, make Castignano an ideal place for the cultivation of high quality aniseed. Thus, it is desirable the implementation of this traditional agricultural practice in order to improve the economy of marginalized areas. Among the most important actions to relaunch this local crop, we believe that labelling the aniseed samples and its derivatives (e.g., liquors, confectionery, etc.) reporting the Castignano ecotype certified quality together with proposal for nutritional and/or health claim could be of great importance in improving the appeal of this product to the consumers. At the same time, the advertising and reinforcement of custodian farmers network (e.g., brochure, magazine, website, special events, congresses) may contribute to spread the image of the product in central Italy.

Table 7 Content of phenolic groups in the aniseed samples from Castignano. Total rutin derivatives mg/g

0.112 0.572 0.514

compounds, mainly caffeic acid esters, apigenin, isoorientin, and luteolin derivatives. Their identification was based on the comparison of MS fragmentation with those reported in literature (Martins et al., 2016; Ferreres et al., 2008) (Table 8). Among them, dicaffeoylquinic acid (27.7–175.8 mg/100 g), apigenin 2-O-pentosyl-6-C-hexoside (21.9–72.1 mg/100 g), isoorientin (8.5–53.2 mg/100 g), isoorientin 2” O arabinoside (5.9–25.2 mg/100 g), apigenin-6-C-glucoside (6.6–21.1 mg/100 g) and luteolin (10.0–20.9 mg/100 g) were the most abundant compounds (Table 9). These metabolites were particularly abundant in sample 5, followed in order of concentration by samples 1, 4, 2 and 3.

In order to understand the whole phenolic profile of the ‘Castignano ecotype’ samples, HPLC-DAD and HPLC–MS analyses were conducted. For the purpose, five ‘Castignano ecotype’ samples collected in 2015 (Table 1) were analyzed. Overall, the phenolic compounds identified in aniseeds may be conducted to three groups, namely chlorogenic acid, gallic acid and rutin derivatives. Their total content in the aniseed methanolic extracts are reported in Table 7. The most abundant metabolites were those derived from gallic acid, showing a range of concentrations from 2.51 to 9.19 mg/g, whereas rutin and chlorogenic acid derivatives gave a minor contribution, with concentration ranges of 0.77–2.61 and 0.44–1.85 mg/g, respectively. Many reports mentioned that gallic acid is the most important phenolic acid in tea, which exhibits antioxidant property, anticarcinogenic effect and antifungal activity (Sánchez-Moreno et al., 1999; Fukumoto and Mazza, 2000). Furthermore, HPLC–MS analysis allowed to identify 18 phenolic

Sample No.

0.039 0.211

0.018

3.6. HPLC-DAD and HPLC–MS analysis of non-target metabolites

0.03 0.08 0.04 0.10 0.11

Malta

1

dicaffeoylquinic acid (values of eigenvectors: −0.60; 0.04), in the first PC and by shikimic acid (values of eigenvectors: −0.23; 0.28) in the second PC. Overall, samples clustered in two main groups on the basis of the content of polar constituents. Interestingly, the ‘Castignano ecotype’ samples were in the left hand side of the score plot, being positively correlated with variable affording the highest contribution to the variability of data and taking place in the same zone of the loading plot, namely neochlorogenic acid, 3,5-dicaffeoylquinic acid and shikimic acid (Fig. 2b). Conversely, all commercial samples, including the star anise samples, were in the right hand side of the score plot and exhibited lower levels of polar constituents. It is worthy to note that star anise (sample 22), well known as a rich source of shikimic acid, was overcome by most of aniseed samples, most of them coming from the ‘Castignano ecotype’. These results, proved that aniseed has the potential to be exploited on an industrial level as a source of this metabolite (Gosh and Chisti, 2012). In conclusion, PCA analysis confirmed the chemical data highlighting the higher quality of extracts obtained from the ‘Castignano ecotype’ samples.

± ± ± ± ±

Greece

Illicium verum (mg/g) Vietnam

0.12 0.07 0.09 0.11 0.10

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Table 8 Phenolic compounds and their main fragmentations tentatively identified in the aniseed samples from Castignano. RT (min)

Component

[M−H]−

Fragment

Reference

11.8 12.3 12.5 12.5 12.6 12.8 13.0 13.3 13.6 13.8 14.0 14.4 14.4 15.4 15.6 16.8 18.7 20.6

caffeoylquinic acid apigenin-6,8-di-C-hexoside apigenin-O–pentoside Aa luteolin-6-C-hexoside-7-O- hexoside luteolin −2”-O-hexoside −6-C-hexoside luteolin −2”-O-pentosyl −6-C-hexoside luteolin-6-C-hexoside apigenin −2”-O-hexoside −6-C-hexoside apigenin −2”-O-pentosyl-6-C-hexoside methyl-luteolin 2”-O-pentosyl-6-C-hexoside apigenin-6-C-hexoside luteolin-7 O-hexoside apigenin-O-pentoside Ba apigenin-O-hexoside dicaffeoylquinic acid feruloylquinic acid luteolin apigenin

353 593 401 609 609 579 447 593 563 593 431 447 401 431 515 529 285 269

191 503 269 447 489 459 357 413 413 443 311 285 269 269 353 353 217 225

Clifford et al. (2005) Ferreres et al. (2008) Fabre et al. (2001) Ferreres et al. (2008) Martins et al. (2016) Martins et al. (2016) Martins et al. (2016) Martins et al. (2016) Martins et al. (2016) Martins et al. (2016) Martins et al. (2016) Martins et al. (2016) Fabre et al. (2001) Fabre et al. (2001) Clifford et al. (2005) Kuhnert et al. (2010) Fabre et al. (2001) Fabre et al. (2001)

a

327 429 429 327 293 293 323 283

309 357 327 299 249 249 308 293

199 175 149

Different isomers are indicated with letters. Italy). Geomorphology 116, 145–161. Cachet, T., Brevard, H., Cantergiani, E., Chaintreau, A., Demyttenaere, J., French, L., Gassenmeier, K., Joulain, D., Koenig, T., Leijs, H., Liddle, P., Loesing, G., Marchant, M., Saito, K., Scanlan, F., Schippa, C., Scotti, A., Sekiya, F., Sherlock, A., Smith, T., 2014. Determination of volatile ‘restricted substances’ in flavourings and their volatile raw materials by GC–MS. Flavour Fragr. J. 30, 160–164. Catizone, P., Marotti, M., Toderi, G., Tétényi, P., 1986. Coltivazione delle piante medicinali e aromatiche. Pàtron editore, Quarto inferiore, Bologna, pp. 109–113. Chen, P.C., Wheeler, D.S., Malhotra, V., Odoms, K., Denenberg, A.G., Wong, H.R., 2002. A green tea-derived polyphenol, epigallocatechin-3-gallate, inhibits IkappaB kinase activation and IL-8 gene expression in respiratory epithelium. Inflammation 26, 233–241. Cho, A.-S., Jeon, S.-M., Kim, M.-J., Yeo, J., Seo, K.-I., Choi, M.-S., Lee, M.-K., 2010. Chlorogenic acid exhibits anti-obesity property and improves lipid metabolism in high-fat diet-induced-obese mice. Food Chem. Toxicol. 48, 937–943. Clifford, M.N., Johnston, K.L., Knight, S., Kuhnert, N.A., 2005. Discriminating between the six isomers of dicaffeoylquinic acid by LC–MSn. J. Agric. Food Chem. 53, 3821–3832. Estevez, A.M., Estevez, R.J., 2012. A short overview on the medicinal chemistry of (−)-shikimic acid. Mini Rev. Med. Chem. 12, 1443–1454. European Pharmacopoeia, 2005. 5th edn. European Pharmacopoeia, vol. 2 Council of Europe, Strasburg. FFNSC 2, 2012. Flavors and Fragrances of Natural and Synthetic Compounds. Mass Spectral Database. Shimadzu Corps, Kyoto. Fabre, N., Rustan, I., De Hoffmann, E., Quetin-Leclercq, J., 2001. Determination of flavone, flavonol, and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry. J. Am. Soc. Mass Spectrom. 12, 707–715. Ferreres, F., Andrade, P.B., Valentao, P., Gil-Izquierdo, A., 2008. Further knowledge on barley (Hordeum vulgare L.) leaves O-glycosyl-C-glycosyl flavones by liquid chromatography-UV diode-array detection-electrospray ionisation mass spectrometry. J. Chromatogr. A 1182, 56–64. Fukumoto, L.R., Mazza, G., 2000. Assessing antioxidant and prooxidant activities of phenolic compounds. J. Agric. Food Chem. 48, 3597–3604. Gosh, S., Chisti, Y., 2012. Banerjee Production of shikimic acid. Biotechnol. Adv. 30, 1425–1431. Heath, H.B., 1981. Source Book of Flavours. The AVI Publishing Company, Westport, Connecticut, USA, pp. 221–222. Idolo, M., Motti, R., Mazzoleni, S., 2010. Ethnobotanical and phytomedicinal knowledge in a long-history protected area, the Abruzzo, Lazio and Molise National Park (Italian Apennines). J. Ethnopharmacol. 127, 379–395. Kähkönen, M.P., Hopia, A.I., Vuorel, H.J., Rauha, J.P., Pihlaja, K., Kujala, S.T., Heinonen, M., 1999. Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem. 47, 3954–3962. Kang, P., Kim, K.P., Lee, H.S., Min, S.S., Seol, G.H., 2013. Anti-inflammatory effects of anethole in lipopolysaccharide-induced acute injury in mice. Life Sci. 93, 955–961. Kubeczka, K.H., Ullmann, I., 1980. Occurrence of 1,5-dimethylcyclodeca-1,5,7-triene (Pregeijerene) in Pimpinella species and chemosystematic implications. Biochem. Syst. Ecol. 8, 39–41. Kuhnert, N., Jaiswal, R., Matei, M.F., Sovdat, T., Deshpande, S., 2010. How to distinguish between feruloyl quinic acids and isoferuloyl quinic acids by liquid chromatography/ tandem mass spectrometry. Rapid Commun. Mass Spectrom. 24, 1575–1582. Leporatti, M.C., Ivancheva, S., 2003. Preliminary comparative analysis of medicinal plants used in the traditional medicine of Bulgaria and Italy. J. Ethnopharmacol. 87, 123–142. Leung, A.Y., Foster, S., 2003. Encyclopedia of Common Natural Ingredients Used in Food, Drugs, and Cosmetics. John Wiley & Sons, New York, USA. L.R. 12/2003. Tutela delle risorse genetiche animali e vegetali del territorio marchigiano. B.u.r. 12 giugno 2003, n. 51.

Table 9 Content of phenolic compounds in the aniseed samples from Castignano. Compound

apigenin-6,8-di-C-hexoside apigenin-O–pentoside Aa luteolin-6-C-hexoside-7-O- hexoside luteolin −2”-O-hexoside −6-C-hexoside luteolin −2”-O-pentosyl −6-C-hexoside luteolin-6-C-hexoside apigenin −2”-O-hexoside −6-C-hexoside apigenin −2”-O-pentosyl-6-C-hexoside methyl-luteolin 2”-O-pentosyl-6-C-hexoside apigenin-6-C-hexoside luteolin-7 O-hexoside apigenin-O-pentoside Ba apigenin-O-hexoside luteolin apigenin caffeoylquinic acid dicaffeoylquinic acid feruloylquinic acid a

127 473 353

Sample No. 1

2

3

4

5

2.5 3.1 1.0 3.5 23.1 38.5 7.1 56.8 7.1 20.5 12.7 0.7 4.0 17.5 4.9 12.9 34.6 8.5

1.4 4.6 0.0 2.6 22.1 37.7 6.3 46.6 15.6 18.2 7.6 1.2 1.7 19.2 5.1 1.7 39.7 6.6

0.9 4.0 0.8 1.5 5.9 8.5 2.9 21.9 4.3 6.6 3.9 0.5 2.2 10.0 3.3 17.6 32.5 7.9

2.2 15.9 1.3 4.1 14.9 23.8 7.8 57.0 9.1 13.8 16.3 1.3 10.3 11.7 6.6 7.5 27.7 8.8

1.6 4.6 2.3 4.5 25.2 53.2 10.8 72.1 18.2 21.1 17.1 0.7 4.4 20.9 4.2 6.7 175.8 2.5

Different isomers are indicated with letters.

Acknowledgements The authors would like to thank Mr. Luigi Contisciani, President of BIM Tronto (Bacino Imbrifero Montano, Ascoli Piceno) for financial support, and Mr. Sergio Corradetti and Castignano custodian farmers for kindly providing ‘Castignano’ ecotype samples. In addition, authors thank the University of Camerino (FAR2014/15, Fondo di Ateneo per la Ricerca, FPI 000044). References Adams, R., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry, 4th ed. Allured Publishing Corp., Carol Stream, IL, USA. Bellomaria, B., 1982. La coltivazione dell’anice verde a Castignano (Ascoli Piceno). Natura e Montagna, vol. 4. Pàtron editore, Bologna, pp. 87–90. Bhardwaj, R.K., Sikka, B.K., Singh, A., Sharma, M.L., Singh, N.K., Arya, R., 2011. Challenges and constraints of marketing and export of indian spices in India. International Conference on Technology and Business Management 739–749. Boelens, M.A., 1991. Spices and condiments II. In: Maarse, H. (Ed.), Volatile Compounds in Food And Beverages. Marcel Dekker, Inc, New York, Basel, Hong Kong, pp. 449–482. Buccolini, M., Gentili, B., Materazzi, M., Piacentini, T., 2010. Late Quaternary geomorphological evolution and erosion rates in the clayey peri-adriatic belt (central

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