Abstract: Daucus littoralis Smith subsp. hyrcanicus Rech.f. (Caspian carrot) is an endemic species in the north of Iran. The aim of this study was to report the separation and structural elucidation of constituents from the ethyl acetate extract of fruits, which have not been reported previously. A phenylpropanoid (myristicin), a sesquiterpenoid (acorenone B) and two fatty acids (palmitic and oleic acid) were identified by GC and GC/MS. Also acorenone B and triolein were isolated by different chromatographic methods and identified by 1H, 13C-NMR and MS spectra. Furthermore, the antifungal activity of the Fruits Ethyl Acetate (FEE) and Fruits Methanol Extracts (FME) were investigated against vulvovaginal isolates of Candida albicans. The results showed that both FEE and FME had antifungal activity for standard Candida (MIC 6.25 and 25 mg mL–1, respectively) as well as for the isolates with MIC ranged 25-100 mg mL–1.
INTRODUCTION
Daucus littoralis Smith subsp. hyrcanicus Rech.f. (Apiaceae), well known as Caspian carrot, is an annual or perennial plant growing exclusively in the north of Iran (Mazandaran and Guilan provinces) (Yousefbeyk et al., 2014b). The genus Daucus is distributed in Europe, Africa, West Asia, North America and Australia. Among the plants of this genus, the essential oil of D. carota has been used in traditional medicine as antibacterial and antifungal agents (Tavares et al., 2008). So far the antimicrobial activity of the essential oil from aerial parts of D. carota was investigated against Bacillus subtilis, S. aureus, E. coli, Pseudomonas aeruginosa, Candida albicans and Penicillium expansum (Maxia et al., 2009). Furthermore, the antifungal activity of D. carota L. subsp. carota and D. carota L. subsp. halophilus against yeasts, dermatophytes and Aspergillus strains were investigated (Maxia et al., 2009; Tavares et al., 2008). In addition, D. carota extract has a long history of usage in treating hepatic and renal insufficiency as well as skin disorders like burns and furunculous (Maxia et al., 2009). In other studies, anthelmintic, hepatoprotective, cytotoxic, antioxidant and iron-chelative activities have been reported for D. carota (Maxia et al., 2009).
Recently, composition of essential oils from leaves, fruits, flowers and roots of D. littoralis subsp. hyrcanicus has been investigated. Sesquiterpene hydrocarbons like germaceren D (22- 36%) and acorenone B (19.7-57.5%) were reported as major components. Also high amount of myristicin was identified in leaves/stems, roots and fruits essential oils (8.6, 9.5 and 15.2%, respectively) (Yousefbeyk et al., 2014b). Phytochemical investigation of fruits eventuated in identification of β-sitosterol, stigmasterol, caryophyllene oxide, β-amyrin, quercetin 3-O-β-glucoside, quercetin 3-O-β-galactoside and luteolin in this plant (Yousefbeyk et al., 2014a).
The aim of this study was to report the separation and structural elucidation of constituents from the fruits extract of D. littoralis subsp. hyrcanicus, which have not been reported previously. Furthermore, the antifungal activity of the fruits was investigated against vulvovaginal isolates of Candida albicans.
MATERIALS AND METHODS
General procedures: 1H and 13C-NMR spectra were acquired using a Bruker Avance TM500 DRX (500 MHz for 1H and 125 MHz for 13C) spectrometer with tetramethylsilane as an internal standard and chemical shifts are given in δ (ppm). Column chromatography was accomplished using silica gel (70-230 and 230-400 mesh) (Merck, Germany). Silica gel 60 F254 precoated plates (Merck, Germany) were used for TLC. The spots were detected by spraying anisaldehyde-H2SO4 (Sigma-Aldrich, Germany) reagent followed by heating. The HPLC separations were carried out on a Knauer system (Smart line system, Germany) connected to a photodiode array detector. The MPLC separations were carried out on a Buchi system. All the solvents, standards and reagents were obtained from Merck (Germany).
Plant material: The fruits were collected from Bandar-e-Anzaly sea coast, province of Guilan, north of Iran, June 2013. A voucher specimen of plant (6734-TEH) was deposited in Herbarium of Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
Extraction and isolation: The fruits (1 kg) were powdered and extracted successively with ethyl acetate and methanol by percolation method. The fruit ethyl acetate extract (FEE) (88 g) was subjected to column chromatography over a silica gel column (70-230 mesh, 10×3 cm), using a mixture of CHCl3: AcOEt (9:1) as mobile phase and afforded 10 fractions (A-J). Among these fractions, the fraction C was pale yellowish oil with characteristic odor. The fatty acids composition was determined as methyl esters following the procedures described by Metcalfe and Schmitz (1961). Briefly, 12 mL of methanol solution of boron trifluoride (BF3) 10% was added to sample under reflux condition and maintained for 2 min. The solution was extracted three times with 50 mL of hexane at room temperature. The hexane layer was separated and dried over anhydrous Na2SO4 (Metcalfe and Schmitz, 1961). The sample was analyzed by GC and GC/MS methods.
Furthermore, the fraction C subjected to MPLC (Buchi). The eluted ratio was adjusted with 50: 50 (hexane: CH3Cl) and delivered to the silica gel column (5 cm) for 60 min (flow-rate: 6 mL min–1, UV detector (λ: 254 nm) to give fractions Ca-Ci. The fraction Cc was selected for more purification by HPLC. The HPLC system consisted of a Knauer instrument (Smart line system, Germany) with a diode array detector. The column was a normal phase semi-preparative (250×18 mm i.d.) with a particle size of 5 nm (Eurospher). The eluents were hexane and CH3Cl with a flow rate of 3 mL min–1. The gradient condition was: 0-5 min, hexane 80%, CH3Cl 20%; 5-35 min, hexane 75%, CH3Cl 25%. The UV-vis spectra were recorded between 210 and 500 nm. The compounds 1 and 2 were purified with this method.
GC and GC/MS analysis of oily fraction of FEE extract: Oil sample analysis was performed on an Agilent 6890 gas chromatography equipped with Flame Ionization Detector (FID). A capillary column HP-5ms (30×0.25×0.25 μm film thickness) was used. The column temperature program was as follows: 5 min isothermal at 50°C, increased to 240°C at a rate of 3°C min–1, increased to 300°C at a rate of 15°C min–1 and finally held at this temperature for 3 min. Injection temperature was 290°C. Injection volume was 1.0 μL. Helium was the carrier gas and the split ratio was 25:1.
GC/MS analysis was performed with an Agilent 6890 gas chromatograph with a capillary column HP-5 ms (30×0.25×0.25 μm). Temperature program was as follows: 5 min at 50°C, increased to 240°C at a rate of 3°C min–1, then, increased to 300°C at a rate of 15°C min–1 and finally held at that temperature for 3 min. Injection temperature was 290°C. Injection volume was 1.0 μL. Helium was the carrier gas and the split ratio was 25:1. The MS was operated at 70 eV ionization energy and the interface temperature was 220°C. Mass range was 50-500 Da. Retention indices were calculated using standard hydrocarbons (C8-C30 n-alkanes, injected in the same conditions of the samples). Identification of each component was performed by comparison of their mass spectra with Wiley library or authentic compounds in our laboratory and those published in the references and confirmed by comparison of their calculated retention indices, with those of authentic compounds or data published in the literature (Adams, 2004). Quantitative data were obtained from the electronic integration of the FID peak areas.
Preparation of inoculum
Isolates: Seven clinical vulvovaginal isolates of C. albicans were investigated in this study. The isolates were kindly provided by Drug and Food Control Laboratory of Faculty of Pharmacy, Tehran University of Medical Sciences. All strains were stored at -80°C in the appropriate medium. Identification of the isolates was based on gross colony characteristics and microscopic morphology of their micro and macroconidia and accessory structures. Candida albicans ATCC 1023 was used as the control. Before performing antifungal susceptibility testing, each isolate was inoculated onto Sabouraud Dextrose Agar (SDA) for 24 h at 25°C to ensure optimal growth characteristics and purity.
Antifungal susceptibility test: Antifungal susceptibility tests for clotrimazole, ketoconazole and nystatin were used to determine the susceptibility of isolates and standard Candida albicans according to CLSI 2012 (Pakshir et al., 2011). The antifungal agents were obtained as standard powders and stock solutions were prepared by dissolving the powders in their specific solvents (DMSO, water and ethanol). After that, they were loaded into blank paper at the following concentrations: Ketoconazole 15 μg disc–1, clotrimazole 10 μg disc–1 and nystatin 100 μg disc–1. The inoculum was adjusted to 106 CFU mL–1 and spread on agar plate by a sterile swab.
| Table 1: | Criteria of susceptibility and resistance of antifungal discs |
| Table 2: | GC and GC/MS analysis of oily fraction obtained from fruit ethyl acetate extract of D. littoralis subsp. hyrcanicus |
| aRetention time, bRetention indices calculated for sample, cRetention indices in literature. Values (%) given are the mean of three replicated values | |
Four discs were placed onto each plate and incubated in 25°C. The inhibition zones were recorded after 48 h of incubation. Criteria of susceptibility and resistance of antifungal disks were measured according to following Table 1.
MIC and MFC determination: Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of the extracts were evaluated by broth microdilution method with visible growth observed by using 96 U-shaped wells plates (Yousefbeyk et al., 2014b). A stock concentration of 200 mg mL–1 from each extract was prepared in DMSO. Then, two-fold serial dilution of the stock solution of each extract (100 mL) was prepared by using Sabouraud Dextrose Broth (SDB) (100 mL, each) in ten wells. The stock microbial suspension with two-fold test inoculum was prepared in SDB from 24 h old culture. Then, aliquot of 100 mL of twofold test strain inoculum was added to each well to reach the final inoculum size of 5×105 CFU mL–1. The MFC was determined by quantitative subculture of 100 mL from each clear well onto SDA agar plates. Plates were incubated at 25°C for 48 h. The MFC is defined as the lowest of extracts concentration that results in more than 99.9% killing of the bacteria being tested (Golfakhrabadi et al., 2014).
RESULTS AND DISCUSSION
In this study, the FEE of D. littoralis subsp. hyrcanicus was investigated for major components. The oily fraction obtained from FEE was analyzed by GC and GC/MS methods after preparing methyl ester derivations. The results demonstrated the presence of a phenylpropanoid, myristicin (1), a sesquiterpenoid, acorenone B (2) and two fatty acids, palmitic and oleic acid methyl esters (3-4) (Table 2).
Moreover, the FEE was used for isolation and purification of main components by different chromatography methods. The isolated compounds (Fig. 1) were identified as acorenone B (2) and triolein (5). Compounds 2 and 5 were identified by comparison of their spectroscopic data (1H-NMR, 13C-NMR and MS) with those in the literatures and authentic compounds from our laboratory (Vlahov et al., 2008; Wolf et al., 1976).
Acorenone B (2): 1H-NMR (500 MHz, CDCl3), δ (ppm): 0.78 (3H, d, J = 6.7 Hz, isopropyl methyl), 0.87 (3H, d, J = 6.7 Hz, isopropyl methyl), 0.97 (3H, d, J = 6.7 Hz, 4-CH3), 1.76 (3H, m, 8-CH3), 2.09 (1H, m, H-10), 2.23 (1H, d, J = 16.5 Hz, H-6), 2.29 (1H, m, H-10), 2.7 (1H, d, J = 16.5 Hz, H-6), 6.67 (1H, m, H-9).
| Fig. 1(a-b): | Structures of compounds 2 and 5 isolated from D. littoralis sub sp. hyrcanicus including, (a) Acorenone B (2) and (b) Triolein (5) |
Triolein (5): 1H-NMR (500 MHz, CDCl3): δ (ppm): 0.88 (9H, t, H-18), 1.64 (6H, m, H-3), 2.02 (12H, m, H-8, H-12), 2.33 (6H, m, H-3), 4.1 (2H, dd, J = 10 Hz, CH2O), 4.3 (2H, dd, J = 10 Hz, CH2O), 5.27 (1H, m, CHO), 5.36 (6H, m, H-9). 13C-NMR (125 MHz, CDCl3), δ (ppm): 14.1 (C-18), 22.17 (C-12 to C-17), 24.86 (C-3), 27.25 (C-8, C-11), 29.09 (C-4 to C-7), 29.75 (C-4 to C-7), 33.99 (C-2), 34.17 (C-2), 62.1 (CH2O), 68.88 (CHO), 128.91 (C-9), 130.54 (C-10), 173.13 (C-1).
Acorenone B is a sesquiterpenoid which is reported in the essential oil of plants like Bothriochloa intermedia (Zalkow et al., 1980), Angelica lucida (Lawrence and Morton, 1974), Euphorbia macrorrhiza (Lin et al., 2012), Levisticum persicum (Shafaghat, 2011) and Acorus calamus (Balakumbahan et al., 2010). This compound has been identified in the essential oil of D. littoralis subsp. hyrcanicus as a major compound (Yousefbeyk et al., 2014b). In a study, the antimicrobial, anti-inflammatory, antioxidant, antidiarrheal, antiulcer, antispasmodic, immunosuppressant and mitogen inhibitor activities of Acorus calamus are referred to acorenone and other constituents in the essential oil (Divya et al., 2011).
Triolein is a triglyceride derived from a glycerol and three unites of mono-unsaturated fatty acid oleic acid (18:1n-9). This compound represents 4-30% of olive oil (Thomas, 2002; Shubert and Leyba, 2013). Oleic acid has been showed several beneficial effects, such as prevention of ischemic cardiovascular disease, acting as a potent inhibitor of Platelet Aggregating Factor (PAF) (Massimo et al., 2009), endothelial vasomodulatory and antioxidant activities (Visioli et al., 2005). Also, studies showed that oleic acid had antibacterial activity against Staphylococcus aureus, Micrococcus kristinae, Bacillus megaterium and Pseudomonas phaseolicola (Dilika et al., 2000) as well as antifungal activity against Crinipellis pernicosa and Pythium ultimum (Pohl et al., 2011). In a study, antifungal activities of four essential oils of D. carota L. subsp. carota in different vegetative phases of the plants collected in Portugal (sample 1 and 2) and in Italy (sample 3 and 4) were assessed against yeasts, dermatophyte and Aspergillus strains. The results showed that the essential oils had more sensibility to Cryptococcus neoformans and dermatophyte strains (MIC 0.16-0.64 μL mL–1) compared with Candida and Aspergillus strains.
| Table 3: | Antifungal susceptibility test of standard and vaginal isolates of Candida albicans to antifungal drugs including clotrimazole, ketoconazole and nystatin |
| aMean of three assays, bSusceptibility: S, Sensitive, DD: Dose dependent, R: Resistant | |
| Table 4: | Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of ethyl acetate and methanol extracts of D. littoralis subsp. hyrcanicus against standard and vaginal isolates of Candida albicans |
| MIC and MBC were determined by broth micro dilution method and expressed in mg mL–1 (W/V), Key to extracts employed: FEE: Fruits ethyl acetate extract, FME: Fruits methanol extract, MFC: Minimum fungicidal concentration, MIC: Minimum inhibitory concentration | |
In this study, antifungal activity of FEE and FME of D. littoralis subsp. hyrcanicus was investigated against some vulvovaginal isolates of C. albicans. Criteria for susceptibility to used antifungal drugs have been summarized in Table 1. Antifungal susceptibility tests of isolates and standard C. albicans for clotrimazole, ketoconazole and nystatin showed in Table 3. The results of antifungal test demonstrated that both FEE and FME had antifungal activity against standard Candida (MIC 6.25 and 25 mg mL–1, respectively) as well as the isolates with MIC ranged 25-100 mg mL–1. Both methanol and ethyl acetate extracts showed better antifungal activity against standard C. albicans than isolated strains. In addition, isolates C2 was more sensitive to FEE and FME (MIC 25 mg mL–1) than other isolates (Table 4).
Our previous study revealed the presence of β-Amyrin in the FEE (Yousefbeyk et al., 2014b). It has reported that β-Amyrin had moderate anti-microbial activity against S. aureus and C. albicans (MIC: 0.5 and 1.02 mg mL–1, respectively) (Rivero-Cruz et al., 2009). The antifungal activity of the FEE can be due to the presence of this compound. Also high amount of phenolic compounds and flavonoids like quercetin 3-O-β-glucoside, quercetin 3-O-β-galactoside and luteolin has been reported in FME. The presence of these compounds explains the antifungal activity of FME.
CONCLUSION
The phytochemical investigation of D. littoralis subsp. hyrcanicus fruits ethyl acetate extract resulted in isolation of two compounds including a sesquiterpenoid, acorenone B and a triglyceride, triolein. Also myristicin, palmitic and oleic acid were isolated and identified by GC/MS. The results of antifungal assays showed that both FEE and FME had antifungal activity against standard Candida (MIC 6.25 and 25 mg mL–1, respectively) as well as the isolates with MIC ranged 25-100 mg mL–1.
ACKNOWLEDGMENT
This investigation granted by research chancellor of Tehran University of Medical Sciences. We acknowledge Mr. Amir Yousefbeyk for his assistance in collecting plants.