Characterization of Pimpinella anisum Germplasm: Diversity Available for Agronomic Performance and Essential Oil Content and Composition

Abstract

Anise (Pimpinella anisum L.) is one of the most important annual herbs of the Apiaceae family, widely cultivated in southern Spain. Their seeds are highly valued for culinary uses and for producing quality essential oils widely used in food and beverage products, as well as for industry, medicinal, and cosmetics applications. This study investigates the seed yield and essential oil content within a set of 50 anise accessions from worldwide origin, as well as their composition by GC–MS and GC–FID analysis. Accessions showed significant differences in the agronomic parameters measured, including plant height (cm), seed yield (kg ha⁻¹), and the Harvest Index (%), with accessions PA_87 (Spain), PA_47 (Greece), and PA_21 (unknown origin) being the most performant. Essential oil (EO) content varied between 0.8% and 5.7% across different genotypes, resulting in EO production values ranging from 0.1 to 300 kg ha⁻¹. Trans-anethole was identified as the dominant terpene, comprising 84.4% to 94.4% of the content, followed by eugenol (1.4% to 5.5%) and α-muurolene (1.4% to 7.2%). PCA analysis identified five distinct groups and one outlier, influenced by minor terpenes. Indeed, there was a strong negative correlation between estragole and pseudoisoeugenyl 2-methylbutyrate. This study underscores the significance of minor terpenes, which play crucial roles in defining unique aniseed chemotypes, allowing for the selection of cultivars optimized for specific uses in food, cosmetics, and pharmaceuticals. Additionally, these findings emphasize the impact of cultivar genetics on agronomic traits and EO profiles, suggesting the need for further research to optimize plant growth and yield and EO quality.

1. Introduction

The Apiaceae family includes a wide range of crops that are economically important due to their culinary use and/or applications in medicine. Among these, one of the oldest and most significant plants is Pimpinella anisum L., commonly known as anise. This annual herb is native to the western Mediterranean coast, Egypt, and Asia Minor [1,2,3], where it was first cultivated as a spice for its seeds (commercially called “aniseeds”). Evidence of its pharmaceutical properties, including its use as a diuretic, digestive aid, and analgesic, date back to 1500 BC [4]. Anise is widely cultivated in regions with warm climates throughout the growing season, with a total global production of about 2.9 million tons. India accounts for 68.5% of global production, followed by Turkey, Mexico, and Iran (http://Tridge.com). Spain is the European country with the largest area dedicated to its cultivation, with 6741 hectares in 2022. Of this, 82% is located in Andalusia, with the majority of cultivation being rainfed (5987 ha). The total national production is around 4600 tons, with average yields of approximately 610 kg/ha in rainfed areas, reaching up to 1300 kg/ha in irrigated areas (https://www.mapa.gob.es/).

Aniseed has a long history of use in both traditional and modern medicine. Its essential oil (EO), typically obtained with a yield range of 1.5–6.0%, is one of the most pharmacologically important substances, used in the food, medicine, cosmetics, pharmaceutical, liquor, and perfumery industries [3,4,5]. Essential oils are known for their carminative, expectorant, antispasmodic, and digestive properties, as well as their ability to help manage blood pressure and various hormonal issues in women [6]. Aniseed is also a rich source of essential B-complex vitamins, minerals like calcium and potassium, and antioxidant vitamins C and A [1]. The pharmacological properties of aniseed essential oil are being increasingly studied, particularly its effects on the central nervous system [7]. Recent studies have highlighted its potential as a green pesticide due to its antimicrobial, insecticidal, and antifungal effects [8,9,10]. Its efficacy against agricultural pests and insect vectors, along with its safety for non-target organisms such as beneficial insects and earthworms, makes it a promising alternative to conventional chemical pesticides [11].

The quality and the composition of essential oil extracted from aniseed are critical factors for its use in the above-mentioned industries. The EO primarily consists of volatile phenylpropanoids, such as trans-anethole, which accounts for approximately 75–90% of the composition [1]. It also contains smaller amounts of estragol, α-himachalene, α-zingiberene, and cis-anethole [1]. Seed yield and essential oil content and composition vary with the genotype [12,13] but are also affected by environmental factors [4,14]. Indeed, specific environmental conditions, such as moderate temperatures and adequate rainfall, have been linked to higher essential oil concentrations and better overall seed quality. In contrast, adverse weather conditions, such as drought and excessive heat, have been shown to reduce fruit yield, germination rates, and essential oil content, although stress conditions may increase the concentration of some secondary metabolites like trans-anethole [15]. Additionally, the essential oil content of aniseed varies significantly depending on the maturation stage, and the optimal harvesting periods should be carefully timed to maximize essential oil yield and quality [16,17,18].

Although Spain is one of the largest producers of this essential oil globally and the demand for aniseed products has increased in recent decades, unfortunately, as is the case in most producing countries, there are few registered aniseed varieties. Anise production is still mainly carried out using landraces of uncertain origin, exchanged between farmers in different regions, which do not always offer standard quality due to the mixed nature of these populations [19]. In fact, a recent search (https://online.plantvarieties.eu/varieties (accessed on 15 July 2025)) shows only nine cultivars registered globally, with only one listed in Europe (cv. Budakalászi in the Hungarian National List, registered in 1978 and expiring in 2028). Therefore, breeding programs should be reinforced, focusing on selecting cultivars adapted to local conditions, with an emphasis on traits that allow for mechanical harvesting, as well as seed quality and essential oil composition. To address this gap, a diverse collection of Pimpinella anisum, comprising 50 landraces from various countries, was gathered and cultivated under field conditions in southern Spain, the largest anise-growing region in the country. Agronomic performance, essential oil content, and composition were recorded and compared.

2. Materials and Methods

2.1. General Experimental Procedure

Distilled water, xylene, and methanol were purchased from Panreac AppliChem (Barcelona, Spain); Sodium Sulphate Anhydrous and Clevenger apparatus were purchased from Merck (Darmstadt, Germany). The essential oil profile qualitative analysis is Bruker Instruments Scion model (Concord, MA, USA), while quantitative analysis is in a PerkinElmer Mod Clarus 500 Gas Chromatograph (Shelton, CT, USA).

2.2. Plant Material

A total of 50 Pimpinella anisum landraces from worldwide origin were sown under field conditions in Córdoba, Spain (37°87′ N and 4°78′ W, 97 m above sea level, vertisol, pH 7.2–7.8, mean air temperature: 18.6 °C, mean precipitation: 352 mm/year) during the 2023 growing season (Supplementary Table S1). These accessions were selected from a previous field study that included a total of 88 accessions [20], gathering among the best yielders. A randomized complete block design with three replications was used. The experimental unit consisted of 1 m² plot per accession, comprising four 1 m-long rows spaced 0.2 m apart, with 3 repetitions. Seed density was adjusted to 20 kg/ha. Sowing occurring by end-December, according to local practices. Hoeing and mechanical weeding were made regularly. Anise plants were irrigated three times 15 mm (15 May 2023), 20 mm (1 June 2023), and 20 mm (9 July 2023) according to irrigation requirements. Attention was paid to record presence and to quantify eventually naturally occurring pests and disease. Plant maturity was recorded following [21]. Plant harvesting took place from mid-July until early-August, depending on the genotype, when the primary umbel was matured completely. Plants were mechanically threshed, and seeds were collected for yield measurement and chemical analysis. Plant height (cm) was measured when the primary umbrella started maturing. The Harvest Index (HI%) was calculated as percentage ratio of grain weight (g) and total dry matter (g) per plant [22]. When the essential oil content per genotype was obtained (%EO) (see below), estimation of the production of EO per genotype was calculated (kg EO ha⁻¹).

2.3. Essential Oil (EO) Extraction

The oil from the seeds of each accession and repetition was obtained by hydro-distillation in all-glass Clevenger-type apparatus according to the method recommended by the European Pharmacopoeia [23]. Briefly, 10.0 g of dry aniseed were reduced to a coarse powder in a mortar immediately before the determination. A 250 mL round-bottomed flask was used, and 100 mL of water as the distillation liquid. A measurement of 0.50 mL of xylene was placed in the graduated tube of the Clevenger apparatus. The distillation was carried out at a rate of 2.5–3.5 mL/min for 2 h. The essential oil was dried over anhydrous sodium sulfate (Na₂SO₄) to remove water traces before GC–MS analysis.

2.4. GC–MS and GC–FID Analysis

These analyses were performed at “Servicio Central de Apoyo a la Investigación” of Universidad de Córdoba, Spain. Sample preparation involved weighing 100 mg of essential oil and diluting it with 10 mL of hexane containing 1-chlorotetradecane as an internal standard. The resulting solution was filtered using a 0.22-micron filter. Gas chromatography–mass spectrometry (GC–MS) with electron impact ionization was conducted using a Bruker Instruments Scion model for qualitative analysis. For quantitative analysis, gas chromatography with flame ionization detection (GC–FID) was carried out using a Perkin Elmer Model 590 (PerkinElmer, Waltham, MA, USA). The analysis utilized an Elite-1 capillary column 30 m-long, with an internal diameter of 0.25 mm and a stationary phase thickness of 0.25 microns, composed of 5% phenyl silicone. The sample injection volume was set at one microliter, and injections were performed in split mode at a ratio of 1:25, using hydrogen as the carrier gas at a flow rate of 1 mL/min. The injector temperature was maintained at 265 °C. A temperature gradient was applied in the column oven, starting at 60 °C and increasing at a rate of 10 °C per minute until reaching 300 °C, which was then held constant for 10 min. The temperature of the FID detector was maintained at 300 °C. Identification of the constituents was based on comparison of the retention times with those of authentic samples comparing their linear retention indices (LRIs) [24], and on computer matching against commercial [25,26] and home-made library mass spectra built up from pure substances and components of known oils and the MS literature data [26].

2.5. Statistical Data Analysis

ANOVA analysis (threshold p < 0.05) was carried out using SPSS (IBM version 26) and post hoc analysis carried out using Tukey HSD. Multivariate statistical analysis, to highlight significant variation in fatty acid and polyphenol compositions, were carried out using Metaboanalyst 6.0 [27]. For multivariate statistical analysis, the data were mean centered, quantile normalized, and transformed using cube root transformation. Box plots in Figure 1 were generated by R version 4.4.1 (R Development Core Team, http://www.R-project.org).

3. Results and Discussion

In this study, a collection of 50 Pimpinella anisum accessions from diverse global origins (Table S1) were grown in Spain and analyzed for agronomic performance, essential oil (EO) content, and composition. The data presented in Table S1 reveal significant differences in the agronomic parameters measured. These parameters include plant height, seed yield, and the Harvest Index. Average plant height ranged from 35.7 to 74 cm, with accessions PA_42, PA_43, PA_67, and PA_70 being taller than 50 cm and accessions PA_30 and PA_34 shorter than 35 cm. Differences in plant stature were not associated with graphic origin (Table S2). Seed yields varied greatly with the accession ranging from 17 to 6060 kg ha⁻¹, with accessions PA_87 (from Spain), PA_47 (from Greece), PA_21 (unknown origin), and PA_75 (from Egypt) being the most yielders (with 6060, 5855, 3280, and 3040 kg ha⁻¹, respectively). The Harvest Index (HI) ranged from 0.4 to 31.7%, highlighting outstanding accessions PA_47, PA_65, PA_71, PA_75, and PA_87 with HI > 25%. Similarly, significant differences for grain yield and HI were observed among averages per country of origin, with the average grain yield being higher in accessions originating from Mediterranean countries (Croatia, Italy, Spain, and Greece) (Table S2). Pearson’s linear correlations between the agronomic components under field conditions are shown in Table S3. HI was highly positively correlated with seed yield (r = 0.77; p < 0.001) and negatively correlated with plant height (cm) (r = −0.37; p < 0.001).

The essential oils were extracted following the European Pharmacopeia [23]. Along the collection studied, the EO content ranged from 0.8% to 5.7% (Table S1). Genotypic variation in EO content has earlier been reported [28,29,30,31]. Of the 50 genotypes, 12 exhibited high EO content (>5.0%), 25 had moderate EO content (between 3.0% and 4.9%), and 13 had low EO content (<3%). Notably, PA_1 (from Croatia) and PA_54 and PA_55 (both from Palestine) showed particularly high EO levels with a 5.7%. On average, analysis of variance (ANOVA) revealed statistically significant differences among the groups (p < 0.05), with anise from Bulgaria displaying the highest EO content (4.9% ± 0.7%). Aniseed from Spain also had a relatively high mean EO content (4.2% ± 1.2%). In contrast, seeds from Egypt had the lowest EO content, with a mean value of 2.6% ± 1.7%. EO % was significantly although weakly correlated with seed yield (r = 0.20; p < 0.05). Still, as the most valuable marketable product is EO, “EO yield” was expressed as kg of EO ha⁻¹, highlighting accessions PA_1, PA_21, PA_26, and PA_47 with >150 kg EO ha⁻¹. In order to better visualize and summarize these data, a heatmap was created showing the plant height, seed yield per hectare, and the Harvest Index and %EO of Pimpinella anisum cultivars grouped by native country (Figure 1).

Environmental conditions during the growing season aside, the content and composition of essential oils in aniseed are influenced by several agricultural practices and growing techniques, such as sowing time, inter-row spacing, seed density, harvesting time, use of manure, and irrigation [16,19]. We followed recommendations regarding sowing date and seed density to achieve the optimal balance between favorable competition conditions. This included shorter inter-row spacing that enhances seed yield and plant branching, while also maximizing EO seed content, as previously demonstrated [32,33,34]. Regarding harvesting timing, higher EO concentrations are obtained from fruits harvested at the waxy stage compared to those picked during the ripening stage [16]. A decrease in EO content in aniseed is reported when harvesting is delayed until the primary umbel turns brown and the main stem turns yellowish [17]. After this stage, the EO content tends to decrease and stabilize. In our experiment, harvesting was performed when the primary umbel was fully mature (showing green-yellow coloration). Future studies will focus on evaluating the impact of different harvesting times to identify potential differences among cultivars and possibly recommend the optimal harvest time for specific cultivars to be adopted by southern Spanish farmers.

The complete chemical composition of each essential oil is presented in Table S4, while an example of the GC–FID chromatogram is shown in Figure S1. The minimum and maximum content values of the identified compounds of P. anisum EO are summarized in

Table 1. Minimum and maximum content values of identified compounds of P. anisum EO.

Components	Minimum	Maximum	Variance
trans-Anethole	84.37	94.37	3.34
Eugenol	1.42	5.51	0.63
α-Muurolene	1.43	7.23	1.12
Estragole	0.03	2.94	0.33
Pseudoisoeugenyl 2-methylbutyrate	0.35	2.24	0.12
α-Zingiberene	0.11	0.75	0.02
α-Himachelene	0.11	0.55	0.01
γ-Elemene	0.02	0.48	0.01
β-Longipinene	0.06	0.39	0.00
cis-Anethole	0.03	0.16	0.00
α-Selinene	0.01	0.19	0.00
α-Ylangene	0.02	0.10	0.00
β-Farnesene	0.01	0.06	0.00
α-longipinene	0.01	0.06	0.00
Longicilene	0.01	0.03	0.00

. The primary compounds, each with a content exceeding 1% across all selected varieties, include trans-anethole, eugenol, and α-murulene. Notably, trans-anethole stands out as the most abundant component in all varieties, as expected. The highest concentration of this phenylpropanoid was found in PA_48, while the lowest was observed in PA_75. In contrast, eugenol reached its highest level in PA_67 and its lowest in cultivar PA_63. Similarly, α-murulene exhibited its highest level in PA_75 and its lowest in variety 48. Estragole and pseudoisoeugenyl 2-methylbutyrate concentrations exceed 1% in specific varieties. The highest estragole content was found in PA_87, while the lowest was in genotype PA_75. Pseudoisoeugenyl 2-methylbutyrate had its maximum content in PA_71 and its minimum in genotype PA_79.

The relationship between climate conditions and the quality of essential oils is complex and significantly influences the composition of aniseed’s EO [14], as well as the duration of hydrodistillation [4]. As this study is the first to examine the quality of EO from Pimpinella anisum grown in southern Spain, we followed the standard method outlined in the European Pharmacopoeia [23]. According to the European Pharmacopoeia, commercial anise oil should contain the following components within specified limits: linalool < 1.5%, estragole 0.5–5%, α-terpineol < 1.2%, cis-anethole 0.1–0.4%, trans-anethole 87–94%, anisaldehyde 0.1–1.4%, and pseudoisoeugenyl 2-methylbutyrate 0.3–2%. In our samples, linalool, α-terpineol, and anisaldehyde were not detected, while the levels of cis-anethole, trans-anethole, and estragole fell within the established limits. Therefore, all genotypes studied met the European Pharmacopoeia criteria, regardless of the percentage of extracted EO.

Our study identified trans-anethole, eugenol, and α-muurolene as the major components across all the cultivars examined. Trans-anethole, the primary essential oil (EO) constituent, accounted for 84.4% to 94.4% of the total composition. It belongs to the phenylpropanoid group of natural compounds, specifically the anisole class of organic compounds. Phenylpropanoid is a large group of organic compounds synthesized from the amino acid phenylalanine, and, to a lesser extent, tyrosine [35]. Trans-anethole is responsible for the sweet taste of aniseed and is widely used as a flavoring agent in foods and cosmetics [12]. In terms of pharmacological properties, trans-anethole is considered the most significant component due to its spasmolytic and expectorant activities, which help stimulate respiratory secretions [36]. Additionally, it exhibits estrogenic properties and acts as a local anesthetic. Recent research has shown that temperature and precipitation are linked to higher levels of trans-anethole [14], supporting the idea that drought stress before harvesting can enhance the production of bioactive compounds, particularly trans-anethole [30]. Eugenol, present in concentrations ranging from 1.4% to 5.5%, is also a phenylpropanoid synthesized through the coniferyl alcohol pathway. In this process, coniferyl alcohol is acylated with acetate (from acetyl-CoA) and then reductively cleaved by eugenol synthase [37]. Eugenol is well-known for its diverse pharmacological properties, including hypothermic, antioxidant, anti-inflammatory, and local anesthetic activities. It has long been used in traditional medicine to treat gastrointestinal disorders such as flatulence and chronic diarrhea [38]. α-Muurolene, detected in concentrations ranging from 1.4% to 7.2%, is a bicyclic sesquiterpene. Sesquiterpene is a diverse group of terpenoid compounds containing a 15-carbon skeleton. Their structure diversity and pharmacological activity make this group particularly interesting [38]. There is limited information on eugenol and α-muurolene in aniseed EO, with these compounds typically reported at very low concentrations [6,33]. In our study, the consistent presence of eugenol and α-muurolene above 1% across all cultivars may be attributed to specific environmental conditions. Given the significant pharmacological properties of eugenol, future research should focus on investigating how different environmental factors influence the concentration of these compounds in aniseed EO.

Estragole and pseudoisoeugenyl 2-methylbutyrate were found to exceed 1% only in specific cultivars. Estragole, a phenylpropanoid, is known for its characteristic sweet flavor and mild pharmacological effects, including antioxidant, antimicrobial, and anti-inflammatory properties. Traditionally, it has been used for its carminative and analgesic benefits, particularly for digestive health [1]. However, estragole has raised safety concerns, with studies indicating potential adverse effects on human and animal health. As a result, estragole was removed from the list of approved food flavorings [38]. In our study, estragole content ranged from 0.03% to 2.94% across cultivars, remaining within the upper limit of the European Pharmacopeia (6.0%) [23]. The variability in estragole content between cultivars is consistent with findings from previous research, which found that factors like genetics and environmental conditions influence its concentration. For instance, drought stress has been shown to cause significant changes in the oil’s constituents, including an increase in trans-anethole and a decrease in estragole levels [32]. Additionally, a significant negative correlation between estragole and trans-anethole was observed with early sowing, suggesting that genetic variability and sowing time influence estragole content [33].

Pseudoisoeugenyl 2-methylbutyrate is a phenol ester derived from isoeugenol. Although a minor component, it contributes sweet and spicy notes to essential oils (EOs) [1]. In our study, the content of pseudoisoeugenyl 2-methylbutyrate ranged from 0.35% to 2.24%. Cultivars from Italy had the highest average content at 0.93%, while those from Palestine had the lowest at 0.65%. However, ANOVA analysis revealed no statistically significant differences. Pharmacologically, pseudoisoeugenyl 2-methylbutyrate is relatively understudied. Given its structural similarity to other phenylpropanoids, it may possess mild antioxidant and antimicrobial properties, but further research is needed. Additionally, the literature is limited on the effect of environmental factors on the extraction process, highlighting the need for more studies in this area [4,12].

To further explore variation in the chemical composition and identify potential differences among the cultivars, PCA was carried out. We attempted to classify the cultivars according to the content of EO (%, high, medium, and low) or to native country. The score plots and the biplots of the analysis are reported in Figure 2. As a result, it was not possible to classify the variety neither according to the EO% nor the native country. However, in both score plots, five distinct groups and one cultivar as an outlier could be spotted (Figure 2A,C). The outlier was PA_75, which had a unique terpene profile compared to the rest of the collection.. The variations within these groups along PC1 are primarily due to differences in the concentrations of estragole, pseudoisoeugenyl 2-methylbutyrate, and γ-elemene. Additionally, there is a strong negative correlation between estragole and pseudoisoeugenyl 2-methylbutyrate: the higher the estragole content is, the lower the content of pseudoisoeugenyl 2-methylbutyrate is. Along PC2, the compounds mainly responsible for the variance and grouping of the cultivars are eugenol, cis-anethole, α-muurolene, and α-himachelene. Eugenol and cis-anethole exhibit negative correlations with α-muurolene and α-himachelene.

Limited information is available in the literature regarding correlation analysis and PCA of aniseed essential oil (EO), primarily highlighting a negative correlation between estragole and trans-anethole with factors like sowing date, sowing rate, and location [30]. A more in-depth study investigating the effect of climatic conditions on EO quality, using cluster analysis and PCA, emphasizes the significant influence of the year and, consequently, weather conditions on the chemical composition of cultivars [14]. A limitation of this approach is that PCA was performed on scaled percentage data without log-ratio transformation. While scaling mitigates some compositional data issues, it does not fully eliminate spurious correlations induced by the constant-sum constraint [39,40]. However, for exploratory analysis of essential oil composition, this approach remains commonly used and provides insights into the main sources of variation. Nevertheless, future studies should consider compositional data analysis techniques, such as centered log-ratio transformation. In our study, attempts to classify cultivars based on EO percentage (categorized as high, medium, or low) or country of origin were unsuccessful. This suggests that the diversity in chemical composition among aniseed cultivars may be influenced by factors beyond these two parameters, including environmental conditions or agricultural practices. Thus, it is essential to investigate specific cultivar genetics. Despite the lack of a definitive classification based on EO content or origin, PCA revealed five distinguishable groups and one distinct outlier, cultivar PA_75 (from Egypt). The unique terpene profile of this outlier may indicate specific genetic or environmental adaptations that warrant further investigation. This outlier behavior highlights the potential for within-species variation, which is often overlooked in EO studies, where minor components can contribute unique characteristics to the overall profile. Furthermore, clustering was primarily influenced by estragole, pseudoisoeugenyl 2-methylbutyrate, γ-elemene, eugenol, cis-anethole, α-muurolene, and α-himachalene. Notably, there is a strong negative correlation between estragole and pseudoisoeugenyl 2-methylbutyrate along PC1, which we believe is the first study to report such a correlation. Additionally, on PC2, eugenol and cis-anethole exhibited negative correlations with α-muurolene and α-himachalene. These differences could stem from genetic variations among the cultivars, activating specific biosynthetic pathways despite being grown in the same environment, suggesting the presence of multiple chemotypes within the aniseed population. In conclusion, these findings indicate that even minor constituents, often overlooked, can play significant roles in shaping the overall EO profile and may contribute distinct pharmacological or aromatic properties.

4. Conclusions

Aniseed demonstrates considerable potential due to its diverse phytochemical composition, medicinal properties, and broad industrial applications. The essential oil derived from aniseed serves as a rich source of bioactive compounds, notably trans-anethole, which contributes substantially to its health benefits and distinctive aroma. This study underscores the significance of minor terpenes, which—though often overlooked—play crucial roles in defining unique aniseed chemotypes, allowing for the selection of cultivars optimized for specific uses in food, cosmetics, and pharmaceuticals. Additionally, the findings emphasize the impact of cultivar genetics on adaptability to different environments, underscoring the importance of targeted selection for optimal growth and yield. These insights provide a robust foundation for future research focused on enhancing aniseed cultivation practices and increasing its commercial viability, particularly in Spain.