Abstract
The aim of this study included the phytochemical screening of extracts and essential oil of Erodium cicutarium (L.) L’Hér. ex Aiton (Geraniaceae), a native traditional medicinal plant from Croatia, as well as a highly detailed micromorphological characterization of its aerial parts. The contents of total polyphenols and tannins (TP and T), total flavonoids (TF), and total hydroxycinnamic derivatives (THD) in plant extracts were quantified in plant material from 4 localities (Plitvice, Podvinje, Buzin, and Trešnjevka). The contents of TP, T, TF, and THD significantly varied depending on the collection site, and were in the range of 4.78%-12.85% (TP), 3.23%-5.80% (T), 0.42%-1.09% (TF), and 1.08%-2.59% (THD) of dry weight of plant material, with the Plitvice collection having the highest polyphenol content. GC-MS analysis of the essential oils showed a similarity in composition of the major compounds from all investigated populations. Fifty compounds were identified in all 4 investigated oils (90.4%-96.7% of total oil) and classified into 7 structural classes, with hydrocarbons (59.8%-65.7%) as the main class of constituents. Two types of trichomes, nonglandular and glandular, were observed on the calyces, leaves, and stems, including 3 capitate trichome types. The bioactive substances and micromorphological characterization of Croatian populations of E. cicutarium were investigated for the first time. In general, the scanning electron microscopy imaging of Erodium cicitarium trichomes has not been reported before.
The genus Erodium (family Geraniaceae) is widespread and contains 74 species all over the world, excluding Antarctica. 1 The biggest diversity of species is found in the Mediterranean region with 62 species in different habitats. 1 There are 34 species of Erodium in the flora of Europe 2 and, depending on references, in Croatia, 4 3 to 6 Erodium species 4 can be found. Erodium cicutarium (L.) L’Hér. ex Aiton is known by the common names stork’s bill, alfilaria, filaria, pin-clover, common stork’s bill (United Kingdom), and redstem filaree (United States). 5 It is a native species to the Mediterranean and has fern-like, pinnate leaves, 10-30 cm long with a “green” grassy aroma, arising from a rosette. 6 It is an annual or biennial with small, pink flowers with 5 petals. After flowering, the fruits, which consist of 5 mericarps joined together, grow in a large spine-like style. 7 The Erodium dispersal mechanism contributed to the global distribution of the genus, especially noted in the invasive example of E. cicutarium in the United States. 7
Many Erodium species are known for their use in traditional medicine and, therefore, much ethnopharmacological data can be found in the literature. 6,8-14 Erodium cicutarium has been used internally and externally for the treatment of dysentery, fever, wounds, and worm infections as a traditional medicine. 8 It was employed as an antihemorrhagic drug in gynecology to stop uterine bleeding and as a general haemostipticum. 8 Among other plants containing ellagitannins in the Erodium genera, it was used for preparing astringent and antiseptic teas used for stomatitis. 9 Other ethnopharmacological data suggest the use in dermatological diseases, 10 hepatitis, nephritis and bleeding from wounds, 11 stomach pain, heart problems, influenza, 12 sores and rashes, 13 and even as an abortifacient. 6
Essential oils are a source of wide chemical diversity, but there are not many studies exploring the process of essential oil production within trichomes. 15 Also, trichomes have an overall important role in taxonomic determination of plant species. The only known data available for trichome types and structures of E. cicutarium are from 1902. 16 In order to improve that scientific knowledge, a detailed micromorphological study of trichomes was undertaken, which we hope will contribute to the Croatian flora in general.
Despite a broad literature based on essential oil composition of other species of the family Geraniaceae, 17 -20 few similar investigations have focused on the chemical composition of E. cicutarium essential oil. To our knowledge, this oil has previously been reported from only 3 studies. 21 -23 In the first, 21 a leaf hexane extract showed isomenthone (11.2%), citronellol (15.4%), geraniol (16.7%), and methyl eugenol (10.6%) to be main components. In the other two, more recent studies, 22,23 essential oils were obtained by hydrodistillation in yields of 0.01% (w/w) and showed a significantly higher number of different chemical components, mainly aliphatic compounds and their derivatives. Radulović et al reported 177 components, mainly aliphatic compounds and their derivatives. 22 Fatty acids and fatty acid-derived compounds were the most common ones (51.3% in essential oil from the entire plant and 60.1% in essential oil from leaves and stems), followed by carotenoid-derived compounds (12.6% and 20.2%, respectively) and terpenoids (14.9% and 14.2%, respectively). Stojanović-Radić et al identified a similar chemical composition with a total of 162 different compounds in the essential oil of E. cicutarium. 23 Since the available data for the chemical composition of the essential oil of E. cicutarium are still limited, there is a need to explore different populations in order to compare them.
By using several solvents and extraction procedures, there have been reports describing E. cicutarium as a source of phenolic compounds, 24 including phenolic acids and depsides, flavonoids, tannins (T), saponins, sugars, amino acids, saturated and unsaturated fatty acids, and vitamins K and C. 8,9,25-30 Encouraged by the known ethnopharmacological data for E. cicutarium and its shown phytochemical potential, this research was designed to evaluate the phytochemical and micromorphological characteristics of wild growing populations of E. cicutarium, an insufficiently investigated native medicinal plant in the South East European flora.
The contents of total polyphenols (TP), T, total flavonoids (TF), and total hydroxycinnamic derivatives (THD) in aerial parts of the investigated populations of E. cicutarium significantly varied depending on the collection site, and were in the range of 4.78%-12.85% (TP), 3.23%-5.80% (T), 0.42%-1.09% (TF), and 1.08%-2.59% (THD) of the dry weight of plant material. The sample collected from Plitvice exhibited significantly higher content of TP (12.85 ± 0.33%), T (5.80 ± 0.34%), TF (1.09 ± 0.04%), and THD (2.59 ± 0.12%), in comparison with the other examined samples. It is followed by the Podvinje TP collection (9.95 ± 0.48%), T (5.35 ± 0.46%), TF (0.86 ± 0.15%), and THD (1.86 ± 0.13%), then Trešnjevka TP (8.04 ± 0.09%), T (3.81 ± 0.15%), TF (0.48 ± 0.01%), and THD (1.68 ± 0.17%), and Buzin with the lowest content of TP (4.78 ± 0.13%), T (3.23 ± 0.18%), TF (0.42 ± 0.04%), and THD (1.08 ± 0.05%). It is difficult to compare the obtained results of phenolic compounds contents with previously reported data, even within the Erodium genera, since the used approach and methodologies differ significantly. 8
Using similar methods, Sarikurkcu et al reported the TP and TF contents of ethanol extracts of E. cicutarium from Turkey to be 32.14 ± 1.18 mg gallic acid equivalents/g extract and 52.09 ± 0.45 mg rutin equivalents/g extract, respectively, while data of TP and TF in this study, as shown previously, are expressed as percentages of dry weight of plant material. 30 A reasonably high phenolic content in the examined Croatian populations is shown for the first time, which indicates their substantial antioxidant potential, and so it could be of benefit to explore their antioxidant activity and radical scavenging capacity in the future.
The essential oils of E. cicutarium from 4 Croatian populations were subjected to GC and GC/MS analyses in order to determine possible similarities and variability in chemical composition depending on locality. The specifically identified compounds and their percentages are shown in Table 1. The total yield, based on the dry mass of samples, ranged from 0.03% to 0.09%. In total, 50 compounds were identified in all 4 investigated oils (90.4%-96.7% of the total oil) and classified on the basis of their chemical structures into 7 classes (Table 1). Hydrocarbons (59.8%-65.7%) were the main class of constituents of all E. cicutarium populations, with hexadecanoic acid (41.5%-49.6%), followed by octadecanoic acid (3.8%-8.1%), octacosane (2.1%-4.8%), and heptadecanoic acid (2.3%-4.6%) as the major compounds. Radulović et al also identified hexadecanoic acid (22.8%) as the major compound. 22 According to Stojanović-Radić et al, hexahydrofarnesyl acetone was one of the main compounds of Erodium ciconium (15.5%), E. cicutarium (9.9%), and Erodium absinthoides (8.3%). 23 In the present study, we have not been able to identify this compound in the investigated E. cicutarium oils. Oxygenated monoterpenes were present in a percentage of 7.7-15.9 in the 4 investigated Erodium populations, with citronellol (2.3%-9.1%) as the predominant ingredient of this fraction. In a review article about the therapeutic potential of E. cicutarium, Al-Snafi alleges citronellol (11.6%, 15.4%) as the major compound of the essential oils of Pelargonium grossularioides and E. cicutarium. 31 Six oxygenated sesquiterpenes (8.5%-10.7%) were present in our oils, with caryophyllene oxide (6.8%-8.9%) as the major compound (Table 1). In contrast, the essential oil obtained from E. cicutarium collected in Serbia was characterized by less content of caryophyllene oxide, constituting 2.1%. 23 The diterpene fraction was constituted by only 1 compound—manool (1.7%-3.7%). Monoterpene hydrocarbons, sesquiterpene hydrocarbons, and carbonylic compounds were found in concentrations under 6% in all our Erodium oils, except the E-caryophyllene content of the Podvinje population with 14.5% (Table 1). Generally, these results represent the first insight into the essential oil composition of Croatian populations of E. cicutarium. The obtained data showed similarity in composition to the major compounds of the investigated essential oils from all 4 localities. Considering hexadecanoic acid as the main compound and its known anti-inflammatory effect, 33 further anti-inflammatory research of the investigated plant species may be a next research step in the future.
Phytochemical Composition (%), Identification (%), and Major Groups of Chemical Components (%) of Essential Oil of Erodium cicutarium From Croatia.
Note. Retention indices determined relative to a series of n-alkanes (C8-C40) on a VF5-ms column (RIa) and CPWax 52 column (RIb), * average value from 2 columns, where possible; IM, Identification method: RI, comparison of Ris with those listed in a homemade library, reported in the literature, 32 and/or authentic samples; MS, comparison of mass spectra with those in the mass spectral libraries NIST02 and Wiley 9; S, co-injection with reference compounds; -, component not identified; tr, trace <0.1%.
Two types of trichomes, nonglandular and glandular, were observed on the calyces, leaves, and stems of E. cicutarium. In Figure 1(a), detailed characteristics of the trichomes are shown, whereas Figure 2 shows the distribution of the trichomes on the investigated plant parts. Nonglandular trichomes were unbranched, bicellular to multicellular, and uniseriate. They are not erect and stick out, but are more or less parallel to the surface (Figure 1a). The surface of these trichomes showed a warty appearance due to the occurrence of cuticular micropapillae. They could be found mainly along the veins, both on the adaxial and abaxial leaf sides, on the calyces and on stems (Figure 2). Nonglandular trichomes are also present in some other Geraniaceae species. 15,17,34
Scanning electron microscopy (a-f) and light microscopy (g-i) micrographs with different types of trichomes of Erodium cicutarium. Nonglandular trichomes—NG, type 1 capitate trichomes—C1, type 2 capitate trichomes—C2, and type 3 capitate trichomes—C3 on calyx (a, b, e, f), on adaxial (c) and abaxial (d, g, i) leaf sides, and stem (h).
Scanning electron microscopy micrographs showing distribution of trichomes on calyx (a-c), adaxial (d, e), and abaxial (f, g) leaf surfaces, and stem (h, i) of Erodium cicutarium.
Three types of capitate glandular trichomes were observed on E. cicutarium plant parts. Type 1 capitate trichomes (C1) were small and composed of 1 basal epidermal cell, 1 stalk cell, and 1 small head cell with a subcuticular space (Figure 1b). These trichomes were not upright but could be described as clinging to the surface. Similar trichomes were found in Geranium robertianum L. 19 Trichomes comparable with these C1 trichomes but composed of 1 basal cell and a more elliptically formed head cell are also known from various Lamiaceae species, such as Thymus capitatus (L.) Hoffmanns, Majorana syriaca (L.) Rafin., Satureja thymbra L., 35 and Clinopodium L. species. 36
Type 2 capitate trichomes (C2) are erect, small, and composed of 1 basal epidermal cell, 2 stalk cells, and 1 bigger head cell with a subcuticular space (Figure 1c, d). This type was observed in some Pelargonium L’Herit species. 34 Trichomes comparable with C2 trichomes, but with 3 stalk cells and a larger, more elongated head cell are known from Pelargonium × fragrans (Poir.) Willd. “Mabel Gray.” 15 Additionally, type 2 capitate trichomes are also known in Lamiaceae species, such as Salvia fruticosa Mill. 35 and Satureja L. species. 37 Type 3 capitate trichomes (C3) are upright and composed of 1 basal epidermal cell, 2 to several stalk cells, and a rounded single-celled head with a subcuticular space (Figure 1e, f). These types of trichomes were observed in G. robertianum, 19 Pelargonium spp., 34 and Pelargonium × fragrans “Mabel Gray.” 15 Trichomes comparable with C3 trichomes were also found in Salvia officinalis L. 35 and in several Stachys L. species. 38
In conclusion, quantification of phenolic compounds and contents of essential oils, as well as micromorphological traits of Croatian populations of E. cicutarium, were investigated for the first time. Among the 4 investigated localities, the population from Plitvice showed the highest polyphenol content regarding TP, T, TF, and THD. GC-MS analysis identified 50 compounds in all 4 investigated oils. The compounds were classified into 7 classes with hydrocarbons as the main class of constituents (59.8%-65.7%), with hexadecanoic acid (41.5%-49.6%) as the major compound. To our best knowledge, this is the first highly detailed micromorphological data obtained by scanning electron microscopy (SEM) imaging of E. cicitarium trichomes. The described nonglandular and glandular trichomes, including 3 capitate trichomes types, were observed on the calyces, leaves, and stems. The results are a scientific contribution to knowledge about the phytochemical potential of E. cicutarium, a poorly investigated plant species of the South East European flora, especially in relation to bioactive substances in the essential oil and the content of polyphenols, and therefore, give a strong impetus for further in vitro and in vivo studies.
Experimental
Plant Material
Randomly selected samples of wild growing plants of E. cicutarium L’Hér. ex Aiton were collected from 4 localities in Croatia during the blooming period in 2012 (Trešnjevka, Buzin, Mukinje, and Podvinje). Plant identification was performed at the Department of Pharmaceutical Botany with Pharmaceutical Botanical Garden “Fran Kušan,” Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia and voucher specimens of the herbal material were deposited in the Herbarium “Fran Kušan” (HFK-HR) (Table 2). For micromorphological investigations of trichomes, samples of leaves, flowers, and stems of 7 plants per locality were fixed in FAA (formalin/96% ethanol/acetic acid/water, 5:70:5:20, v/v). After a few days, samples were transferred into 70% ethanol. For the investigation of essential oil content and polyphenol analysis, the aboveground parts were harvested from mature plants on a dry day. Samples were air-dried single-layered for 10 days in a well-ventilated room at room temperature (22°C) and 60% relative humidity, protected from direct sunlight.
Details on Origin and Collection Data of 4 Investigated Wild Populations of Erodium cicutarium.
Determination of Phenolic Compounds
Total Polyphenol and Tannin Analysis
Total polyphenol and tannin analysis (Folin-Ciocalteu’s procedure, FCR procedure) is based on a reaction with Folin-Ciocalteu’s phenol reagent (FCR) and spectrophotometric determination of TP and T (indirect analysis, after precipitation with casein) at 720 nm. 39 Briefly, 0.250 g of powdered plant material (the aboveground parts) was extracted with 80 mL of 30% (v/v) methanol (70°C, water bath, 15 minutes). After cooling and filtration, each extract was diluted to 100.0 mL with 30% methanol (basic sample solution, BSS). BSS (2.0 mL) was mixed with 8.0 mL of water and 10.0 mL of acetate buffer (solution 1, S1). S1 (10.0 mL) was shaken with 50.0 mg of casein during 45 minutes to allow adsorption of T, and then filtered (solution 2, S2). S1 (1.0 mL) was mixed with 0.5 mL of FCR and diluted to 10.0 mL with 33% Na2CO3 × 10 H2O. The same procedure was performed with S2. After filtration, the absorbance at 720 nm of the final blue solution was measured. Absorbance values obtained for S1 correspond to TP content. The difference between the absorbance of S1 and S2 corresponds to the concentration of casein-adsorbed T in plant samples. The contents of TP and T were evaluated in 3 independent analyses and were expressed as the percentages of dry weight of plant material (% DW). T was used as the standard.
Total Flavonoid Analysis (TF Procedure)
Total flavonoid analysis (TF procedure) includes hydrolysis of glycosides (quercetin type), extraction of TF aglycones with ethyl acetate, and complex formation with AlCl3. 40 Powdered aboveground parts of the plant material (0.20 g) were extracted with 20 mL of acetone, 2 mL of 25% HCl, and 1 mL of 0.5% hexamethylenetetramine (boiling water bath, 30 minutes). Each extract was filtered and extraction of the same herbal material was repeated 3 times with 20 mL of acetone (boiling water bath, 10 minutes). After cooling and filtration, each extract was made up to 100.0 mL with acetone (BSS). Twenty milliliters of BSS was mixed with 20 mL of water and then extracted with ethyl acetate (first with 15 mL and then 3 times with 10 mL). Ethyl acetate extracts were rinsed 2 times with water, then filtered and made up to 50.0 mL with ethyl acetate (Solution 1, S1). In 10 mL of S1, 0.5 mL of 0.5% solution of sodium citrate and 2 mL of AlCl3 (2 g AlCl3 in 100 mL 5% acetic acid in methanol) were added and then made up to 25.0 mL with 5% methanolic solution of acetic acid (sample solution, SS). After 45 minutes, the yellow solutions were filtered and the absorbance of the developed complex was measured at 425 nm. Concentration was calculated as quercetin, using the following expression: % =A × 0.772/b where A is the absorbance, b is the mass of the dry plant material (g), and 0.772 represents the conversion factor related to specific absorbance of quercetin at 425 nm (ie, 810). TF concentration was measured in 3 independent analyses and expressed as percentage of dry weight of plant material (% DW).
Determination of Total Phenolic Acids (TPA Procedure)
Determination of total phenolic acids (TPA procedure) was performed according to the monograph of Rosmarini folium. 41 The extraction was performed as follows: to 0.200 g of the powdered drug, 80 mL of ethanol (50%, v/v) was added and boiled in a water bath under a reflux condenser for 30 minutes. After cooling and filtration, the filter was rinsed with 10 mL of ethanol (50%, v/v). The filtrate and the rinsings were combined in a volumetric flask and diluted to 100.0 mL with ethanol (50%, v/v). TPA (hydroxycinnamic derivatives) in these extracts was measured spectrophotometrically at 505 nm (3 independent analyses), using the nitrite-molybdate reagent of Arnow, in a sodium hydroxide solution, and the percent of their content, expressed as rosmarinic acid, was calculated from the expression: A × 2.5/m where A is the absorbance, m is the mass of the dry plant material (g), and 2.5 represents the conversion factor related to specific absorbance of rosmarinic acid at 505 nm (ie, 400).
GC-MS Analysis of Essential Oils
Dried aerial parts of E. cicutarium (100 g) from 4 Croatian localities were subjected to hydrodistillation for 3 hours in a Clevenger apparatus. Analysis of the obtained essential oils was carried out using a gas chromatograph (model 3900; Varian Inc., Lake Forest, CA, USA), supplied with a flame ionization detector (FID), mass spectrometer (model 2100 T; Varian Inc.) in 2 columns: a nonpolar capillary VF-5ms (30 m × 0.25 mm i.d., coating thickness 0.25 µm, Varian Inc.), and polar CP Wax 52 (30 m × 0.25 mm i.d., coating thickness 0.25 µm, Varian Inc.). The chromatographic conditions are as follows: helium as a carrier gas at 1 mL/min and injector temperature 250°C, FID temperature 300°C. Both columns were programmed as follows: VF-5ms column was held at 60°C isothermal for 3 minutes, and then increased to 246°C at a rate of 3 °C/min, and held isothermal for 25 minutes. CP Wax 52 column temperature was programmed at 70°C isothermal for 5 minutes, and then increased to 240°C at a rate of 3°C/min and held isothermal for 25 minutes. 36 The injected volume was 1 µL, the split ratio was 1:20, and the mass spectrometry (MS) conditions were ionization voltage 70 eV, ion source temperature 200°C, and mass scan range: 40-350 mass units. The individual peaks were identified by comparison of their retention indices (relative to C8-C40 n-alkanes for both columns) to those from a homemade library, literature 32 and with some authentic samples, as well as by comparing their mass spectra with literature, Wiley 9 MS (Wiley, New York, NY, USA) and NIST02 (Gaithersburg, MD, USA) mass spectral databases. The component percentages for each essential oil were calculated as mean values from the GC-FID and GC-MS peak areas using the normalization method (without correction factors, for nonpolar column, VF-5ms). The analyses were carried out in triplicate and the component percentages were expressed as mean values. 36
Micromorphological Investigation
For SEM imaging of the trichomes, the plant samples (stems, leaves, and calyces) were transferred from 70% ethanol into 70% acetone, then further dehydrated (90%, and 100% acetone), and subjected to critical point drying using liquid CO2 (CPD030; Baltec). Samples were mounted on aluminum tapes using a carbon-impregnated double-sided tube and were sputter coated with gold (Sputter Coater, AGAR). SEM analysis was carried out using a XL30 ESEM (FEI) at 20 kV acceleration voltage in the high vacuum mode. 42 Light microscopy of samples stored in 70% ethanol was performed on transverse sections of samples using a ZEISS Axioplan 2 microscope.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was financially supported by two short-term scientific research fundings from the University of Zagreb, Republic of Croatia.