Abstract: To determine essential oil composition, antioxidant and antimicrobial features of the aerial parts of Chaerophyllum crinitum Boiss. The obtained oil of the plant analysed by GC and GC/MS. The analysis has led to the identification of 64 components comprising 85.5% of the oil. In addition, DPPH assay and disc diffusion method were used to evaluate antioxidant and antimicrobial properties, respectively. α-terpinolene (20.3%), β-cubebene (9.3%), α-terpineol (7.2%) and limonene (5.8%) were dedected as major components. Moreover, C. crinitum exhibited remerkable antimicrobial effect against most of used microorganisms. But it was inactive in the DPPH assay. The chemical composition, antioxidant and antimicrobial effects of the essential oil of aerial parts of C. crinitum were reported for the first time and obtained results show that it may be a good candidate for the future studies.
INTRODUCTION
Apiaceae (Umbelliferae) usually known as carrot or parsley family is a member of mostly aromatic plants with hollow stems (Simpson, 2006). Apiaceae is the 8th largest family in Turkey and the family comprising about 446 genus and 3540 species among which 33% are endemic in Turkey (Davis et al., 1988; Guner et al., 2000; Ozhatay et al., 2009). This family is rich in secondary metabolites and embodies numerous genera of high economic and medicinal value yielding flavonoids, terpens, coumarins and essential oils. It is well-known that origination of essential oils and oleoresins are characteristic properties of the family (Yilmaz and Tekin, 2013). The genus Chaerophyllum is the largest genus in the subtribe Scandicinae, belonging to Apiaceae family, comprised of about 110 species which includes annual and perennial herbal plants widely distributed in temperate and subtemperate zones of Asia, Africa and Europe. The genus is also represented in the Flora of Turkey by 15 species of which four are endemic (Guner et al., 2000).
These plants are also used in traditional medicine in a lot of countries, fresh leaves and stems are occationally added to salads, while tea made of dried leaves and roots are used as herbal remedy to soothe sore throat, cough and allergies (Shafaghat, 2013). Preceding phytochemical investigations of Chaerophyllum species have revealed the presence of secondary plant metabolites like lignans, phenylpropanoids and polyacetylenes, phenolic acids and related compounds flavonoid glycosides as reported by Mikaya et al. (1981), Dall’Acqua et al. (2004) and Gonnet (1986). Now a days, there is an increasing interest in usage of plant extracts and essential oils. Particularly, the antimicrobial and antioxidant activities of solvent extracts and essential oils as well as their potential anticancer activity have been investigated in recent date (Mimica-Dukic et al., 2004; Sylvestre et al., 2005).
As far as we know that there is no study about biological activity of Chaerophyllum criniticum’s essential oil. With this aim, we did not study only antioxidant properties but also antimicrobial features. Moreover, chemical composition of essential oil was determined in this study.
MATERIALS AND METHODS
Plant material: Chaerophyllum crinitum was collected from the rocky and plain areas with an altitude of 1,400-1,500 m in Nurs village, Bitlis in 2013. The taxonomic identification of the plant materials was confirmed by Dr. Hayta. Voucher specimens were kept at the Fırat University Herbarium (FUH-4822).
Extraction of the essential oils: Air-dried aerial parts of the plant samples (100 g) were subjected to hydrodistillation using a Clevenger-type apparatus for 3 h to yield.
Gas Chromatography (GC) analysis: The essential oil was analyzed using HP 6890 GC equipped with an FID detector and an HP-5 MS column (30 m×0.25 mm i.d., film tickness 0.25 μm capillary column). The column and analysis conditions were the same as in GC-MS. The percentage composition of the essential oil was computed based on the GC-FID peak areas without correction factors.
Gas Chromatography/Mass Spectrometry (GC-MS) analysis: The oil was analyzed by GC-MS, using a Hewlett Packard system (HP-Agilent 5973 N GC-MS system with 6890 GC) at the Plant Products and Biotechnology Res. Lab. (BUBAL) of Fırat University. The HP-5 MS column (30 m×0.25 mm i.d., film thickness 0.25 μm) was used with Helium as the carrier gas. Injector temperature and split flow were 250°C and 1 mL min–1, respectively. The GC oven temperature was kept at 70°C for 2 min and programmed to 150°C at a rate of 10°C min–1. Then constant was kept at 150°C for 15 min to 240°C at a rate of 5°C min–1. Alkanes were used as reference points in the calculation of the Relative Retention Indices (RRI). The MS was taken at 70 eV with a mass range of 35-425. Component identification was carried out using spectrometric electronic libraries (Wiley, NIST). The identified constituents of the essential oil were listed in Table 1.
Antioxidant activity
DPPH assay: Different aliquots (1.5-40 μL) samples of pure essential oil of Chaerophyllum crinitum were mixed with 0.4 mL 0.5 mM DPPH in etanol. Final volume adjusted up to 2 mL with etanol. Mixtures were vorteksed and left 30 min at room temperature in dark. Ethanol was used as blank. One milliliter 0.5 mM DPPH diluted in 4 mL of ethanol (1:4), respectively and used as control. Absorbaces were determined at 517 nm (Cuendet et al., 1997). Inhibition percentages (I%) was calculated as follows:
where, I: Inhibition value, Acontrol: Absorbance of control, Asample: Absorbance of sample.
| Table 1: | Constituents of the essential oil Chaerophyllum crinitum |
| *RRI: Relative retention index | |
Antimicrobial activity
Microbial strains: The essential oil of C. crinitum was tested against a set of ten microorganisms include; Entorococcus faecium (ATCC 700229), Candida tropicalis (ATCC 13803), Citrobacter freundii (ATCC 8090), Escherichia coli (ATCC 35218), Proteus mirabilis (ATCC 7002), Enterococcus faecalis (ATCC 29212), Bacillus subtilis (ATCC 6633), Burkholderia cepacia (ATCC 25608), Staphlococcus aureus (ATCC 33862) and Acinetobacter baumannii (RSKK 02026). The used microorganisms are commersial standart strains. In this study, ATCC (American Type Culture Collection) numbers of strains are clear and there is no necessity to add isolates.
Disc diffusion assay: Antimicrobial activity of essential oil of Chaerophyllum crinitum was dedected with disc diffusion method (NCCLS., 1997). The microorganisms were cultured in brain heart infusion broth at 37°C for 24 h and standardized to number 0.5 of the McFarland Nephelometer that according to the order of 108 CFU mL–1 (Barry and Thornsberry, 1985). Hundred microliters of prepared cultures were spread on the surface of mueller-hinton agar. Twenty five microliters pure essential oil of the plant was impregnanted to 6 mm steril discs and placed on the culture medium (Barry and Thornsberry, 1985). Streptomiycine standart discs were used as positive control. The samples were inoculated at 37°C for 24 h and inhibition zone diameters were dedected.
RESULTS
In this study, chemical composition of essential oil of C. crinitum was investigated by GC and GC-MS. The composition, percentage and retention indices of components of the oil were listed in Table 1. The essential oil yield is 0.2 (v/w) of C. crinitum. Sixty four constituents were comprised the 85.5% of the total oil. The main constituents were determined as α-terpinolene (20.3%), β-cubebene (9.3%), α-terpineol (7.2%), limonene (5.8%), p-cymene (5.0%) and 3-cyclohexen-1-ol (4.3%).
In addition, antimicrobial activity of the essential oil was dedected with disc diffusion method and it showed remarkable antibiotic effect against most of used microorganisms.
| Table 2: | Antimicrobial activities of Chaerophyllum crinitum |
| EF: E. faecalis, EnF: Entorococcus faecium, BC: B. cepacia, SA: S. aureus, AB: A. baumannii, BS: B. spizemi, CF: C. freundii, EC: E. coli, PM: P. mirabilis, CT: C. tropicalis a: Streptomiycine | |
The inhbition zone diameters presented in Table 2. On the other hand, the essantial oil of the plant did not exhibit antioxidant effect. It’s inhibition percentage was 18%.
DISCUSSION
Chaerophyllum crinitum has a high level of monoterpene groups as reported by Nematollahi et al. (2005) that chemical compositions of the essential oil of C. crinitum and C. macropodum. At the end of the study 98.5% (28 components) and 84.3% (11 components) were dedected from the oils of C. macropodum and C. crinitum, respectively. In the study, α-pinene (23.0%), β-pinene (17.3%), fenchyl acetate (13.8%), α-ocimene (8.6%), β-ocimene (6.5%), limonene (6.3%), myrcene (5.5%) and three oxygenated monoterpenes (2.4%) were determined as major components in C. macropodum oil. Besides, β-ocimene (50.5%), β-phellandrene (8.8%), p-cymene (7.1%) and ã-terpinene (6.5%) were determined as major components of C. crinitum oil (Nematollahi et al., 2005). In another study, the essential oil of C. macrospermum from Iran and they found β-ocimene (55.9%), terpinolene (9.8%) and α-pinene (7.5%) as major components (Sefidkon and Abdoli, 2005). Similarly, Mamedova (1994) reported that 33 and 28 components of the oil obtained from flowers and leaves-stems of C. macrospermum. In the work 1,8-cineole (7.2 and 1.4%), linalool (6.7 and 2.1%), δ-3-carene (4.4 and 5.0%), α-terpineol (4.7 and 1.5%) and eugenol (1.0 and 9.3%) were identified as major constituents when oxygenated monoterpenes were found as predominant constituents. The rate of monoterpene hydrocarbons of the essential oil of C. villosum leaf which collected from India-Uttarakhand is (91.34%) and γ-terpinene (74.93%), is the single major component. Furthermore, the antioxidant capacity of the essential oil was examined using an in vitro radical scavenging activity test (Joshi, 2013a). Chemical analysis of root essential oil of Chaerophyllum villosum from Uttarakhand, led to the identification of 31 constituents accounting for 91.49% of the total oil. The carvacrol methyl ether (31.12%), myristicin (19.06%), thymol methyl ether (18.60%), γ-terpinene (11.69%), were the principle components. The studies have shown that the essential oil composition of leaf and root was totally different. In another report, ã-terpinene and p-cymene were found as the major constituents of leaf oil and carvacrol methyl ether and thymol methyl ether were noticed as the major constituents in rhizome essential oil of C. villosum (Joshi, 2013b).
In the present work, antioxidant activity of essantial oil of C. crinitum was dedected with DPPH method. Essential oil of the aerial parts of C. crinitum did not show antioxidant activity (18%). In accordance with our study, essential oils from another Chaerophyllum species were also reported inactive in the DPPH assay (Ebrahimabadi et al., 2010). However, in a recent study, Joshi (2014) stated that the chelating power activity of essential oil of C. villosum and it was found as a good natural antioxidant.
On the other hand, the essential oil obtained from aerial parts of C. crinitum were tested against a set of ten microorganisms to estimate its antimicrobial activity. Chaerophyllum crinitum exhibited significant antibiotic activity against most of used microorganism. Results presented in Table 2. In parallel to findings by Kurkcuoglu et al. (2006) that essantial oils of C. libanoticum and C. byzantinum demonstrated antifungal and antimicrobial effect on some bacterial strains. Besides, Shafaghat (2009) indicated that flower, leaf and stem oils of C. macropodum showed antimicrobial acvtivity. Moreover, Ebrahimabadi et al. (2010) stated that the essential oils of C. macropodum exhibited considerable antibiotic activity against several microorganisms include Candida albicans. Furthermore, Lakusic et al. (2009) indicated that strong antimicrobial effects of C. aureum L. oil were found on gram-positive and gram negative bacteria. As a conclusion, according to our data C. crinitum may be a good candidate to use in several areas as a natural antimicrobial agent but it is need to further studies.