Antifungal and Antioxidant Activities of the Essential Oil from Angelica koreana Nakai

Purpose. The purpose of this study is to determine the antifungal and antioxidant activities of the essential oil from Angelica koreana. Methods. Essential oil was obtained from the dried roots of A. koreana by steam distillation, and its composition was identified by gas chromatography and mass spectrometry (GC-MS). The minimal inhibitory concentrations (MICs) of the oil fraction and its main components were determined by broth dilution assay using common pathogenic Aspergillus and Trichophyton species. The combined effects of the oils with itraconazole were evaluated using a checkerboard titer test. In addition, 1,1-diphenyl-2-picryl-hydrazil (DPPH) free radical scavenging, nitrite inhibition, and reducing power were determined to assess the antioxidant activity of this oil. Results. The essential oil fraction and its main components showed inhibitory activity against all of the tested fungi, with minimal inhibitory concentrations (MICs) of 250–1000 μg/mL. Furthermore, this oil exhibited synergism when combined with itraconazole. Conclusion. In the treatment of infections caused by Aspergillus and Trichophyton species, combining itraconazole with either A. koreana essential oil or its main components may reduce the minimum effective dose of itraconazole required and, thus, minimize its side effects. In addition, this oil is a promising source of natural antioxidant agents.


Introduction
The roots of Angelica koreana Nakai (Apiaceae) are used in oriental medicine for treatment of cold, especially for patients that present with symptoms such as chills, fever, headaches, body aches, and pains [1]. The acaricidal toxicity and hypopigmenting activity of the components of A. koreana have been reported [2,3]. Essential oils are one of the most promising sources for the development of new natural antimicrobial and antioxidant agents, even though these oils appear to have relatively mild activities compared to commercial, synthetic agents [4][5][6][7].
Aspergillus species cause a number of severe diseases, both in normal and immunocompromised hosts, encompassed under the name aspergillosis and including allergic disease, saprophytic disease, superficial infections, and invasive infections [8][9][10]. Among the Aspergillus species, A. fumigatus is the most common human infectious agent, followed by A. flavus, A. niger, and A. terreus [11][12][13]. A. versicolor produces many toxic compounds, which can cause severe symptoms in humans and animals infected through inhalation or other forms of contact with debris or spores [14,15].
Antioxidants are used as food additives to protect humans and food from undesirable oxidative reactions. The antioxidant activities of plant compounds have been increasingly investigated in recent decades for the development of new natural antioxidative agents [21,22]. Plant essential oils are a promising source of natural antioxidants [23,24].
Here, to specifically determine whether stable and safe antifungal agents could be developed from the essential oil of Angelica koreana, its inhibiting activity against common pathogenic fungi (three Aspergillus fungi and five Trichophyton species) was investigated using a microbroth dilution test. In addition, we determined the synergistic effects of A. koreana root essential oil fraction and its main components with itraconazole using checkerboard microtiter tests. To estimate the value of this oil as an antioxidant agent, 1,1-diphenyl-2-picryl-hydrazil (DPPH) free radical scavenging, nitrite inhibition, and reducing power, the corresponding experiments were performed.

Sample Preparation.
The essential oil was obtained by steam distillation of the dried roots of Angelica koreana, which was cultivated in Jinbu, Gangwon-do, Korea. The inocula suspensions of Trichophyton species were prepared using the method as described by the Clinical and Laboratory Standards Institute (CLSI) [25]. The microconidia were counted on a hemocytometer and diluted to a final concentration of ca. 0.5 × 10 4 CFU/mL.

Gas Chromatography-Mass Spectrometry (GC-MS)
Analysis. GC-MS analysis was carried out using a Hewlett Packard (HP) 5973-6890 system equipped with an HP-5MS capillary column. The oven was programmed with an initial temperature of 50 ∘ C and heated at a rate of 2 ∘ C/min to 170 ∘ C.

Isolation of m-Cresol and Sabinene.
The essential oil fraction (10 g) of A. koreana was subjected to column chromatography over a silica gel and was eluted with the solvent, toluene-ethyl acetate (95 : 5). The fractions were combined based on thin layer chromatography (TLC) pattern in order to yield subfractions designated A1-A10. Subfraction A5 was further purified by column chromatography over a silica gel and was eluted with toluene-ethyl acetate to give 21 mg of mcresol (99.8%). Sabinene (91.5%) was obtained by repeated fractional distillation with the hydrocarbon fraction of this oil. The ultraviolet spectrometry (UV), mass spectrometry (MS), 1 H-nuclear magnetic resonance spectrometry (NMR), and 13 C-NMR results were identical to those of the authentic standard sample (Sigma, USA). Concentrations (MICs). The MICs of the antifungal agents against the various fungi were determined using a modified broth microdilution test as described by the CLSI. The essential oils of Angelica koreana, m-cresol and sabinene, were serially diluted with ethanol to obtain 0.0125∼16 mg/mL solutions, and 10 L/mL of Tween 80 was added to each solution. After shaking, 100-L aliquots of these solutions were added to the wells of a 96-well microtiter plate. Itraconazole was similarly diluted in dimethyl sulfoxide (DMSO) to generate a series of concentrations ranging from 128 to 0.25 g/mL per testing well. A 100-L suspension of each organism was adjusted to a final concentration of ca. 0.5 × 10 4 CFU/mL and then added to the individual wells and cultivated at 28 ∘ C. The MIC was defined as the lowest concentration that completely inhibited visible fungal growth after 48 h of incubation. Each organism was also cultured with a blank solution containing Tween 80, ethanol, and DMSO at concentrations equivalent to those in the test solutions in order to verify that these vehicles did not affect fungal growth. The tests were performed in triplicate.

Checkerboard Microtiter
Test. Ten serial, two-fold dilutions of A. koreana essential oil, m-cresol, sabinene, and itraconazole were, respectively, prepared with the same solvents used in the MIC tests. Then, 5-L aliquots of each dilution were added to the wells of a 96-well plate in the vertical orientation, and 5-L aliquots of each itraconazole dilution were added in the horizontal orientation so that the plate contained various concentration combinations of the two compounds. A 100-L suspension of each fungal strain (0.4×10 4 CFU/mL) was added to the individual wells, and the plate was incubated at 28 ∘ C for 72 h. Fractional inhibitory concentrations (FICs) were calculated as the MIC of the combination of essential oil/m-cresol/sabinene and itraconazole divided by the MIC of essential oil/m-cresol/sabinene or itraconazole alone. The FIC index (FICI) was calculated by summing both FICs and was interpreted as a synergistic effect when it was <0.5, as additive when it was 0.5 to 1.0, as indifferent when it was > 1.0 to 2.0, and as antagonistic when it was >2.0 [26].

DPPH Scavenging Effects of A. koreana Essential Oil.
A fresh solution of 0.1 mM DPPH and two-fold dilutions of an A. koreana essential oil fraction (or its main component) (3.2-0.05 mg/mL, final concentration) were prepared in ethanol, and 900 L of DPPH was mixed with 100 L of the oil sample. After vortexing for 10 sec, samples were added to five wells in 96-well plates and kept at room temperature for 30 min. A decrease in absorbance (ABS) was monitored at 540 nm. DPPH radical scavenging capacity was calculated using the following equation [27]: 2.8. Nitric Oxide Scavenging Activity. Nitric oxide scavenging activity was measured using the method of Kato et al. [28]. In brief, 10 L of sodium nitrite (2 mMol) and 10 L of each serial dilution of the oil sample were mixed with 80 L of citrate buffer (0.

Results and Discussion
The essential oil fraction was obtained by repeated steam distillation of the dried roots of A. koreana. The content of essential oil in A. koreana roots was 0.28%. The composition of the essential oil fraction was analyzed by GC-MS. As listed in Table 1, 48 compounds were identified in this oil, which account for 98.72% of the total composition. Sabinene (31.85%) was the predominant component of this oil, followed by m-cresol (4.46%) and -pinene (4.00%), -bisabolol (3.63%) and -bornyl acetate. The MICs of the essential oil fraction, its main components, sabinene and m-cresol, and the antibiotics amphotericin B and itraconazole are presented in Table 2. In the three samples of the oil, m-cresol showed the strongest growth inhibition against most of the tested fungi, with minimum inhibitory concentrations (MICs) between 125 and 1000 g/mL. Sabinene exhibited the lowest activity, with MICs ranging from 500 to >4000 g/mL. In most of the experiments, the Trichophyton species showed relatively higher sensitivity to the tested Angelica koreana oil fraction and its two main components compared to the Aspergillus species. The oil fraction showed the highest potency against  T. mentagrophytes, with MICs between 250 and 500 g/mL. However, the MICs of the oil were much higher than those of the antibiotics amphotericin B and itraconazole (MICs = 0.25-8.00 g/mL), which were used as controls. In most cases, the antibiotic activities of plant essential oils are weaker than those of synthetic antibiotics used commonly in therapy.
For this reason, their application in the therapy of fungal infections is very limited. Nevertheless, in consideration of the side effects of the antibiotics, essential oils have been considered as a source for the development of new natural antibiotics. Amphotericin B is highly toxic in its conventional form and expensive in its lipidic form [31]. Itraconazole is a relatively well-tolerated drug, but it has relatively low bioavailability after oral administration and produces a range of adverse effects similar to those of other azole antifungals [26]. Checkerboard titer tests were performed in this study in order to determine the synergism created by the combination of these two groups of compounds with regard to overcoming the weak activity of the oil and to minimize the side effects and toxicity of antibiotics by lowering the dosage. The FICs and FICIs results from these checkerboard tests are listed in Tables 3 and 4. There was significant synergism and additive effects between itraconazole and A. koreana oil fraction/m-cresol, with FICI values ranging from 0.26 to 1.00 against the Aspergillus and Trichophyton species. Among the tested fungi in this study, A. fumigatus and A. terreus showed the most distinctive synergism with the combination of A. koreana oil fraction with itraconazole, resulting in an FICI of 0.37 for both Aspergillus species. The FICIs between 0.65 and 1.00 in the tests with Trichophyton species indicated additive effects between itraconazole and A. koreana oil fraction/m-cresol. In particular, this combination against T. rubrum resulted in an eight-fold decrease in the MIC with itraconazole (FIC = 0.12) compared to that before combination with the essential oil fraction. Thus, A. koreana essential oil might be useful in antifungal therapy when combined with itraconazole, especially against Aspergillus and Trichophyton species.
To evaluate A. koreana oil as an antioxidant, DPPH free radical scavenging activity, nitrite formation inhibition, and reducing power procedures were performed with the oil fraction and its two main components using butylated hydroxyl anisole (BHA, Sigma-Aldrich, USA). As demonstrated in Figure 1, the essential oil of A. koreana and its main components showed significant dosedependent DPPH scavenging activities. One of the main components of the Angelicaessential oil fraction, m-cresol (56.12 ± 1.36%), showed greater inhibition of DPPH than did the oil fraction or sabinene (19.31 ± 1.02% and 4.45 ± 1.06%, resp.) at concentrations of 16 mg/mL. However, the results showed weaker scavenging activity than the control. At 1 mg/mL, the activity of BHA was more than 20 times higher than those of all three of the tested oil samples.
In nitric oxide scavenging tests, the Angelica oil fraction and its main components, m-cresol and sabinene, showed Evidence-Based Complementary and Alternative Medicine 5   significant scavenging activities dose-dependently ( Figure 2). Sabinene, exhibited the higher activity than the oil fraction or m-cresol. The results of reducing power assays are demonstrated in Figure 3. The A. koreana essential oil exhibited significant activity at concentrations of 0.25-4.00 mg/mL. Sabinene showed the strongest reducing power at 2.00 and 4.00 mg/mL. At 4.00 mg/mL, both the oil and sabinene resulted in high absorbance (0.90 ± 0.09 and 1.16 ± 1.06, resp.), indicating strong reducing power, while m-cresol showed relatively weaker activity. The absorbance tested with BHA was 1.23 ± 0.00 at the concentration of 0.25 mg/mL. This indicates that the concentration of Angelica oil needs to be five times greater than that of BHA in order to produce the same reducing power of BHA. As these results indicated, the antioxidant activity of the A. koreana essential oil appeared weaker than that of BHA, although its use would avoid the toxicity issues of BHA [32].
In the single dose oral toxicity study with male SPF-ICR mice, there was no death of any animal in the group administered 500 mg/kg of essential oil fraction during the experimental period. No clinical signs or body weight changes were found in relation to the tested samples. The LD 50 s of the sample were 1437.5 mg/kg for male mice. On the basis of the LD 50 and MICs, toxicity could be excluded in the use of A. koreana essential oil as an anti-Aspergillus and anti-Trichophyton agent, especially in combinatorial therapy with antibiotics.

Conclusion
For the treatment of infections caused by Aspergillus and Trichophyton species, combining itraconazole with either A. koreana essential oil or its main components, sabinene and m-cresol, may reduce the minimum effective dose of itraconazole required and thus minimize the side effects. In addition, A. koreana essential oil is a promising source of new natural antioxidants. However, further studies are required to assess the potential for clinical application.