Original Article

Anticholinesterase and Anti-inflammatory Activities of Essential Oils of Naturally Grown Daucus L. Species in Turkey


  • Betül DEMİRCİ

Received Date: 09.12.2021 Accepted Date: 21.02.2022 Bezmialem Science 2022;10(6):722-734


This present study was conducted to determine the interspecific chemical variability and evaluate the biological effects of the essential oils of Daucus L. species growing naturally in Turkey. The species were D. carota, D. broteri, D. guttatus, D. littoralis, D. involucratus and D. conchitae (endemic).


The essential oils were obtained from fruit samples by distillation method and were analyzed both by GC-FID and GC-MS. The anti-inflammatory and anticholinesterase effects of the essential oils were investigated. The anti-inflammatory effect was evaluated by in vitro LOX enzyme inhibition activity. The anticholinesterase effect was tested on AChE and BChE enzymes.


The components, ratios, and yields of D. carota essential oils differed depending on the locations where the samples were collected. The main components were detected as carotol (1-74.6%), β-bisabolen (0.9-62.4%), 11αH-himachal-4-en-1β-ol (0.3-49.4%), trans-methylisoeugenol (1-45.7%). The main volatile compounds were found in D. broteri, D. guttatus, D. involucratus, D. littoralis, D. conchitae as β-sinensal (30.4%), methyleugenol (30.5%), methyleugenol (40.9%), α-humulene (29.4%), methyleugenol (29.6%) respectively. The essential oils didn’t exhibit anti-inflammatory activity. Two essential oil samples of D. carota showed high anticholinesterase effects compared to the standard. The AChE IC50 was calculated as 6.04±0.30 μg/mL, 2.15±0.10 μg/mL (Galantamine IC50 1.13±0.02 μg/mL) and BChE IC50 11.32±0.20 μg/mL, 31.03±0.02 μg/mL (Galantamine IC50 12.15±0.36 μg/mL). These essential oils contained high levels of 11αH-himachal-4-en-1β-ol (25.04%, 49.42%).


Because of their anticholinesterase potential, some D. carote essential oils can be evaluated in the preparation of pharmaceutical or nutraceutical products as a complementary therapy for Alzheimer’s disease by standardizing their components.

Keywords: Daucus spp., essential oil, anti-inflammatory, anticholinesterase


Research on plants for protection of health and treatment of diseases has come for ages and increasingly continues. Wild plants are an important resource for new drug discoveries. Essential oils are mixtures of natural terpenes with a wide range of pharmacological activities and their preparation from various plant species has become increasingly popular in recent years. Antimicrobial, sedative, antispasmodic, anthelmintic, anti-inflammatory, expectorant and diuretic effects are some of them (1). Daucus L. belongs to the essential oil content rich Apiaceae family. In the Flora of Turkey and the East Islands, 6 species including D. carota L., D. broteri Ten., D. guttatus Sibth Sm., D. littoralis Sm., D. involucratus Sm. and D. conchitae W Greuter (endemic) are registered (2,3). D. carota L. (carrot) is the best-known species of the genus and is cultured widely around the world. Carrot root is used as food and essential oil derived from fruits is used in perfumery (4). D. carota fruits are used in folk medicine as stomachic, carminative, diuretic, anthelmintic, emmenagogue, contraceptive and aphrodisiac. Young aerial parts of wild types are also eaten as vegetables (5).

Phytochemical and biological activity studies on Daucus species other than D. carota are very limited. Several studies have described that essential oils and extracts obtained from D. carota show a wide range of biological activities, such as antifungal, antibacterial, antioxidant, anti-inflammatory, hepatoprotective, antihyperlipidemic and antitumour activities against human oesophageal cancer cell (6-14). D. guttatus essential oil showed significant antibacterial activity against Corynebacterium pyogenes (15). When examined for use in the treatment of tuberculosis, the essential oil obtained from the aerial parts of D. littoralis showed antibacterial effect against Mycobacterium tuberculosis (16).

In this paper, the chemical compositions of the fruit essential oils of all Daucus species grown in Turkey were comparatively analyzed for the first time. The volatile compounds, extracted using hydrodistillation, were analyzed by gas chromatography/flame ionization detection (GC/FID) and GC-mass spectrometry (GC/MS). Also, in vitro anticholinesterase and anti-inflammatory activities of Daucus species’ essential oils were investigated. .

Examination of plants with ethnomedicinal use in drug research makes important contributions to the development of new drugs. Turkey has a rich floristic structure because experiences different geological periods and covers ecologically different regions. Many plants in Turkey have traditional medicinal uses for various diseases and one of them is D. carota. The purpose of this study was to evaluate the biological effects and to determine the interspecific chemical variability of the essential oils of Daucus species growing naturally in Turkey. Since D. carota is the most common species of the genus, samples taken from different locations were examined and compared. On the other hand, other species of the genus had limited growing areas, so they were collected from only one location. The essential oils, which gave the most effective results in biological activity studies, were divided into sub-fractions to determine the effective components that caused the activity. Content analysis and activity studies of the obtained fractions were performed again.


Plant Material

The following 6 species belonging to Daucus genera were collected by Betül Büyükkkılıç-Altınbaşak in the fruiting period from the Marmara, Aegean, and Mediterranean regions: D. carota, D. broteri, D. guttatus, D. littoralis, D. involucratus and D. conchitae (endemic). Plant samples were collected from İstanbul, Denizli, Muğla, and Antalya between June and September in 2016, 2017 and 2018 (Figure 1). Taxonomical determinations of the collected specimens were made using the Flora of Turkey and the Aegean Islands by Dr. Gülay Ecevit-Genç and Betül Büyükkılıç-Altınbaşak (3,17). The voucher specimens were kept at the Herbarium of the Faculty of Pharmacy, İstanbul University (ISTE). Nine samples of D. carota species were collected from different locations. Due to the large number of samples examined, a code was given to each sample to avoid confusion. This is presented in Table 1. These scientific names were verified for each of the species using the International Plant Name Index (18).

Extraction of Essential Oils

The dried fruits of Daucus species were cut into small pieces and immediately subjected to hydrodistillation in Clevenger apparatus for 3 hours. Essential oils, thus obtained were stored in sealed vials at +4 °C until analyzed and tested.

Gas Chromatography Analysis

The GC analysis was performed on the Agilent 6,890N GC system at an FID detector temperature of 300 °C. To achieve the same elution order as GC/MS, the simultaneous automatic injection was performed on a replicate of the same column, applying the same operating conditions. The relative percentage amounts of the separated compounds were calculated from the FID chromatograms. The analysis results are given in Table 2.

The GC/MS analysis was performed on an Agilent 5,975 GC/MSD system on an Innowax FSC column (60 m x0.25 mm, 0.25 µm film thickness) using helium (0.8 mL/min) as carrier gas. The GC oven temperature was held at 60 °C for 10 minutes and programmed at 220 °C at 4 °C/min, for 10 minutes, then 240 °C at 1 ratio. °C/min. The division ratio was set to 40:1 and the injector temperature was set to 250 °C. Mass spectra were recorded at 70 eV. The mass range was m/z 35 to 450.

Identification of the essential oil components were carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index to series of n-alkanes. Commercial (Wiley GC/MS Library, MassFinder Software 4.0) (19,20) and in-house libraries (“Başer Library of Essential Oil Constituents” which was built up by genuine compounds and components of known oils) were used.

Anti-inflammatory Activity

In the present study, the essential oils were evaluated for possible anti-inflammatory activity by in vitro soybean lipoxygenase (Soy LOX) (, Type I-B, 7.9 unit/mg) enzyme inhibition which was performed spectrophotometrically. Based on the study conducted by Baylac and Racine (21) in 2003, the method updated in microscale was applied (21,22). 1.94 mL of potassium phosphate buffer (100 mM; pH: 8.80), 40 µL essential oil samples at a concentration of 100 µg/mL and 20 µL lipoxygenase enzyme were incubated for 10 min at 25 °C. Three hundred µL of this mixture was added to each well of quartz microplate. The reaction was then initiated by the addition of 7.5 µL linoleic acid solution.  The experiments were carried out in 3 replicates. The results were calculated by recording the changing absorbance values every minute for 10 minutes in the ELISA reader (ELx808IU) at 234 nm. nordihydroguaiaretic acid (NDGA) was used as a positive control at 20, 12, 4, 3, 2 µg/mL concentrations.

Determination of AChE and BChE Inhibitory Activities

The essential oils’ anticholinesterase activity was determined by using in vitro AChE and BuChE enzymes inhibition assays. AChE and BuChE inhibition activities were determined using the method found by Ellman et al. (23). Galantamine is used as reference compound. The IC50 was determined by constructing an absorbance and/or inhibition (%) curve and examining the effect of seven different concentrations. Acetylthiocholine iodide and butyrylthiocholine iodide were used as substrates in the reaction, and 5,5’-dithio-bis(2-nitrobenzoic) acid (DTNB) was used as a reagent. Stock solutions of essential oils and galantamine were prepared with methanol at a 4,000 µg/mL concentration. Hundred and fifty  µL of 100 mM phosphate buffer (pH 8.0), 10 µL of sample solution, and 20 µL of AChE (2.476x10-4 U/µL) (or 3.1813x10-4 U/µL of BuChE) solution were mixed. It was incubated at 25 oC for 15 minutes. Ten µL of DTNB solution at a concentration of 2 mg/mL was added.The reaction was initiated by the addition of 10 µL acetylthiocholine iodide (or butyrylthiocholine iodide). In this method, the activity was measured by following the yellow color produced as a result of the presence of thio anion which was produced by the enzymatic hydrolysis of the substrate with DTNB. Also, methanol was used as a control solvent. The hydrolysis of the substrates was monitored using a BioTek Power Wave XS at 412 nm (24).

Fractionation by Column Chromatography

The essential oils effective in biological activity studies were divided into 2 sub-fractions by column chromatography to increase the proportion of their major components. Silica gel (7733; Merck) was used as column adsorbent in fractionation. It was first eluted with n-hexane and then with ethanol. Accumulated fractions were checked by TLC (thin layer chromatography) method (Figure 2). The fractions obtained were concentrated by rotavapor. After analyzed with GC/FID and GC/MS systems, biological activity studies were carried out.


Composition of the Essential Oils

The analysis of essential oils obtained by the hydrodistillation method were carried out with GC/FID and GC/MS systems. Comparative analysis results are given in Table 2.

The essential oil samples (D7, D8) belonging to D. carota species with high anticholinesterase effects were separated into 2 sub-fractions by column chromatography. As a result of GC/FID and GC/MS analysis, the ratios of the components belonging to n-hexane and ethanol fractions are given in Table 3.

LOX inhibition

The essential oils at 100 µg/mL concentration didn’t exhibit any anti-inflammatory activity while the positive control, NDGA showed strong anti-inflammatory activity. The IC50 value of NDGA was calculated as 9.00±0.01 µg/mL.

AChE and BChE Inhibition

An anticholinesterase effect was observed in three essential oil samples of D. carota species. With two of these, we achieved results close to the standard substance galantamine. Essential oils considered to be effective were divided into sub-fractions and cholinesterase inhibition was re-examined using the same method. Calculated IC50 values are given in Table 4 and Table 5.


The components, ratios, and yields of 9 essential oil samples of D. carota species collected from different locations differ according to the regions (Table 2). The main components of the examined D. carota essential oils were carotol (1-74.6%), b-bisabolen (0.9-62.4%), 11aH-himachal-4-en-1β-oil (0.3-49.4%) and trans-methylisoeugenol (1-45.7%). In previous studies, the main component of the essential oil found in D. carota fruits was determined as carotol (66.78%) (25). Also, the main component of essential oil obtained from the aerial parts of D. littoralis was determined as cis­chrysanthenyl acetate (46.8%) (16). In this study, the main component of the essential oil obtained from D. littoralis fruits was α-humulen (29.4%). In another study conducted in Turkey, the main components were found as carotol (27.7%), elemicin (18.1%), and limonene (16.0%) in aerial parts of D. carota essential oils’ (26). The reason for this is thought to be a feature of the Apiaceae family of which members contain different aromatic compounds in their different organs.

The main volatile compound was β-sinensal (30.4%) in D. broteri, methyleugenol (30.5%) in D. guttatus, methyleugenol (40.9%) in D. involucratus, α-humulene (29.4%) in D. littoralis, methyleugenol (29.6%) in D. conchitaeas. In the literature, the main component of D. guttatus fruit essential oil was indicated as β-pinene (18.8%) (27). Such differences are thought to be due to subspecies, because the Daucus genus includes systematically problematic species due to its high hybridization rate (28). More studies are needed on its taxonomic status.

The fruit morphology of Daucus species is generally similar. Differences in the chemical composition of essential oils support the identification of Daucus species.

The chemical composition and biological activity of essential oils can be affected by many factors, such as harvest time and which part of the plant will be used for the essential oil. Significant differences are also found, especially in the composition of D. carota fruit essential oils, depending on geographical origin (8).

Our results reinforce previous data on the variability in fruit essential oils, depending on the geographical origin of the samples. Differences were observed in essential oils components and ratios of samples collected from different locations. Two samples collected from nearby locations showed strong anticholinesterase activity. Unlike other samples, high levels of 11aH-himachal-4-en-1β-ol (25.04%, 49.42 %) were found in the content of these essential oils.

These essential oils withhighest anticholinesterase activity were divided into sub-fractions to determine the effect of the main component on the activity. The ratios of 11aH-himachal-4-en-1β-ol in the sub-fractions were measured as 72.2% and 52.0%. However, it was observed that the anticholinesterase effect of the fractions decreased. Therefore, the activity is thought to be due to the synergistic effect of the components in the essential oil.

In the literature, it has been reported that the extract prepared with petroleum ether and ethanol from D. carota fruits significantly reduces brain acetylcholinesterase activity and cholesterol levels in young and old mice (29). Ethanol extract of D. carota seeds has been shown to have a memory-enhancing effect on rats. (30). It has been stated that essential oils can be developed as nutraceuticals in the prevention and improvement of neurodegenerative diseases such as Alzheimer’s disease since they have the ability to cross the blood-brain barrier and reach the central nervous system (12). Our findings are in agreement with these results. The essential oil of D. carota characterized by high amounts of 11aH-himachal-4-en-1β-ol can be a natural anticholinesterase agent and can be regarded for the management of Alzheimer’s disease.

Study Limitations

The fact that D. carota essential oil compositions and anticholinesterase effects are different increases the possibility that the samples belong to different subspecies. However, there was no taxonomic study on D. carota subspecies in Turkey, which limited the chemotaxonomic evaluation of the results. Since different methods were not tested in the anti-inflammatory activity study, the evaluation of this effect was limited.


In this study, the chemical composition of the fruit essential oils of Daucus species grown in Turkey was comparatively analyzed for the first time. The anti-inflammatory and anticholinesterase effects of the essential oils were investigated. Although there wasn’t any anti-inflammatory activity in the samples, the anticholinesterase effect was observed in three samples of D. carota species’ essential oils in our study. According to the results, it was seen that if the main components of the essential oil were standardized, it could be used in the preparation of potential pharmaceuticals and nutraceuticals. It is thought to be useful for complementary therapy, especially in neurodegenerative diseases.


Ethics Committee Approval: Since there is no study related to teeth, ethics committee approval is not required.

Peer-review: Externally peer reviewed.

Authorship Contributions

Concept: B.B.A., G.E.G., B.Z.K., B.D., Design: B.B.A., G.E.G., B.Z.K., B.D., Data Collection or Processing: B.B.A., G.E.G., B.Z.K., B.D., Analysis or Interpretation: B.B.A., G.E.G., B.Z.K., B.D., Literature Search: B.B.A., G.E.G., B.Z.K., B.D., Writing: B.B.A., G.E.G., B.Z.K., B.D.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: This study was supported by Anadolu University Scientific Research Projects Commission under the grant no: 1603S114 and Scientific Research Projects Coordination Unit of İstanbul University (project number: 22180).

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