Biological Activities of Essential Oils from Angelica tenuissima Nakai

Abstract

The current study was conducted to evaluate the antibacterial and antioxidant activities of the essential oil fraction from the roots of Angelica tenuissima Nakai and its main components. We extracted the essential oil fraction from the roots of A. tenuissima using steam distillation and isolated its main components. Their antibacterial activities were determined by broth dilution test against food-borne pathogenic bacteria. Antioxidant activities were evaluated by DPPH-scavenging assay and reducing-power test. Also tested was their ability to inhibit the growth of two gastrointestinal cancer cell lines, Caco-2 and MKN-45. The A. tenuissima oil fraction and its main components, ligustilide and butylidene phthalide exhibited marked inhibitory effects against most of the tested antibiotic-susceptible and antibiotic-resistant bacterial strains with minimum inhibiting concentrations (MICs) from $0.21{\pm}0.08$ to $3.60{\pm}0.89mg/ml$. They also showed growth-inhibiting activity against Caco-2 and MKN-45 cells. The oil fraction showed significant antioxidant activities in DPPH radical scavenging assay and reducing-power test. Taken together, A. tenuissima essential oil could be used as a safe additive for preventing food contamination by pathogenic bacteria. Additionally, its antioxidative activity and the ability to inhibit gastrointestinal carcinoma cell lines could increase its value for functional foods and prevention of cancer.

Introduction

Increasing antimicrobial resistance is an important public health issue worldwide. The development of resistance in both human and animal bacterial pathogens has been associated with the extensive use of antimicrobials for therapeutic applications and as a feed additive for livestock growth promotion (Anderson et al., 2003; Devi et al., 2009; Wong et al., 2010; Haldar et al., 2011; Sakaridis et al., 2011; Dai et al., 2012; Da Rocha, 2012). Increased bacterial resistance is thought to have resulted largely from the consumption of processed foods and agricultural products that have been in contact with antibiotics (Roig et al., 2009; Kitiyodom et al., 2010; Kitaoka et al., 2011).

Antioxidants have been used as food additives to protect humans and food from undesirable oxidative reactions. The antioxidative activity of plant compounds has been increasingly investigated in recent decades for the development of new natural antioxidative agents (Hardin et al., 2010; Anthony et al., 2012; Prakash et al., 2012). Plant essential oils are a promising source of new natural antioxidants (Misharina et al., 2009; Mousavizadeh et al., 2011; Asensio et al., 2012).

Angelica tenuissima Nakai (Umbelliferae), a plant that grows in a deep valley area of Korea, is used in traditional medicine for pain, especially headaches from colds, rheumatic arthralgia, parietal headache and abdominal pain (Zhu, 1998; Jeong and Jung, 2002; Choi et al., 2010). The leaves and roots of the plant are edible and used in functional foods for anti-aging and in natural cosmetics.

In this study, we extracted a fraction from an essential oil from the dried roots of A. tenuissima using steam distillation. The essential oil was analyzed by gas chromatography-mass spectrometry (GC-MS), and the main components were isolated by column chromatography. The inhibitory activity of the oil was evaluated against ten antibiotic-susceptible and antibiotic-resistant food-borne pathogenic bacteria. In addition, the DPPH-scavenging and reducing activity of the A. tenuissima essential oil fraction and its main components were tested. The effects of A. tenuissima essential oils on the cell viability of Caco-2, a colon carcinoma cell line, and MKN-45, a human gastric cancer cell line, were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) test (Lee et al., 2006: Park et al., 2007; Lim et al., 2009: Natoli et al., 2012; Wang et al., 2013).

 

Experimental

Analysis of essential oil fraction by gas chromatography-mass spectrometry (GC-MS) − A. tenuissima was cultivated in Youngju, Korea and harvested in October, 2012. A voucher specimen was deposited at the herbarium of Duksung Women’s University (No. UMANTE1). An essential oil fraction (2.4 g) was obtained from the dried roots (1 kg) of A. tenuissima by steam distillation for 6 h using a spontaneous distillation and extraction apparatus and analyzed using an Hewlett-Packard 6890 GC (Agilent, USA) and an Agilent 5973 network mass selective detector (280 ℃) with a HP-5 MS capillary column (30 m × 250 μm × 0.25 μm). The injector was adjusted to 260℃ and the oven temperature was programmed as: initial temperature 50 ℃ for 5 min, heating 2 ℃/min to 180 ℃, heating 3 ℃/min to 280 ℃, and a final temperature of 260 ℃ for 10 min.

Isolation of essential oil fraction components − The essential oil fraction (1 g) was subjected to silica gel column chromatography and eluted with hexanedichloromethane (8 : 2). Rechromatography with a silica gel column with hexane-ethyl acetate (9 : 1 and 30 : 1) of fractions 14-17 and 35-73 yielded butylidene phthalide and ligustilide. Spectral data of the isolated compounds were identical to previously published data (Sim and Shin 2008).

Bacterial strains − Antibiotic-susceptible and antibioticresistant bacteria were from the Korea Culture Center of Microorganisms (KCCM) and Culture Collection of Antibiotic Resistant Microbes (CCARM).

Minimum inhibiting concentrations − Minimum inhibiting concentrations (MICs) for oils were determined using a broth dilution method. Two-fold dilutions (16 - 0.125 mg/ml) of essential oils in medium containing 2% Tween-80 were prepared. A. tenuissima oil suspensions of 100 μl were added to 96-well plates and 100 μl prepared broth cultures (2 × 105 CFU/ml) of strains cultivated at 37 ℃ were added to each well. The MIC was determined by reading the turbidity of the wells after 24 h at 37 ℃ (Shin, 2009).

DPPH-scavenging effects of A. tenuissima essential oil − Following the method of Blois (1958), a fresh solution of 0.1 mM DPPH and two-fold dilutions of an A. tenuissima essential oil fraction (or its main component) (3.2 - 0.05 mg/ml, final concentration) were prepared in ethanol and 900 μl of DPPH was mixed with 100 μl of oil samples. After vortexing for 10 sec, samples were added to five wells in 96-well plates and kept at room temperature for 30 min. Decrease in absorbance was monitored at 540 nm. DPPH-radical scavenging capacity was calculated using the equation:

The IC50 was determined following the method of Blois (1958).

Determination of reducing power − Reducing power was determined according to the method of Elmastas et al. (2007). Various concentrations of A. tenuissima essential oil fraction were prepared as two-fold dilutions (12.5 - 200 μg/ml: final concentration) with methyl alcohol. Each sample (0.2 ml) was mixed with 0.6 ml of 0.2 M phosphate buffer (pH 6.6) and 0.6 ml of 1% potassium ferricyanide [K3Fe(CN)6]. The mixture was incubated at 50 ℃ for 20 min, and 0.6 ml of 10% trichloroacetic acid was added before 10 min of centrifugation at 1000 × g (MSE Mistral 2000, UK). The upper layer (1 ml) was mixed with FeCl3 (0.1 ml, 0.1%), and its absorbance was measured at 700 nm. Higher absorbance indicated higher reductive capability.

Effects of A. tenuissima essential oil fraction and main components on viability of Caco-2 and MKN-45 cells − Caco-2 and MKN-45 cells from the Korean Cell Line Bank were cultured in RPMI medium (Corning Cellgro, USA) supplemented with penicillin (10 U/ml), streptomycin (10 μg/ml), and 10% fetal bovine serum (FBS) at 37 ℃ in a humidified incubator with 5% CO2. Cells were subcultured for experiments, with 100 μl cell suspension seeded in 96-well plates in triplicate at 5 × 104 cells/well, determined using a hematocytometer. After a 24 h for cell adherence, 5 μl of medium was removed from each well. Cells were treated with 5 μl of A. tenuissima essential oil in RPMI with 10% FBS containing 2% Tween 80. After 2 h, MTT solution (25 μl, 5 mg/ml in PBS) was added, and cells were incubated for 4 h at 37 ℃ in 5% CO2. The medium was discarded, and isopropanol containing 0.04 M HCl was added to dissolve the produced formazan. Absorbance was measured at 570 nm with micro-plate reader. Results were expressed as percentage of viable cells compared to untreated control.

 

Results and Discussion

Analysis of the essential oil fraction from A. tenuissima roots - The composition of the essential oil fraction from A. tenuissima roots, determined by GC-MS analysis and GC, is in Table 1. Among 55 compounds identified in this oil the most prominent component (56.54%) was ligustilide. The next most common components were butylidene phthalide (12.61%) and spathulenol (10.29%). These results generally agreed with previous data of similar analysis of this oil qualitatively. (Kim and Chi, 1989; Park et al., 1997). However, the relative percentage of the individual components varies in different reports. As already known there are variations in the composition of metabolites in the same species of a plant resulted from several factors, such as chemical races, cultural conditions and etc. (Lee et al., 2008).

Table 1.RIa: retention indices calculated against C9 to C24 n-alkanes on a HP-5MS column. MSb: Mass Spectrum, GCc: Co-GC with a corresponding standard compound

The main components of the oil fraction, ligustilide (228 mg) and butylidene phthalide (4 mg), were isolated by silica gel column chromatography. Their structures were elucidated by UV, IR, 1H-NMR and 13C-NMR compared with those reported by Sim and Shin (2008).

Antimicrobial activities of the A. tenuissima essential oil fraction and its main components − The activities of the A. tenuissima essential oil fraction and its main components, ligustilide and butylidene phthalide, to inhibit the growth of ten food-borne pathogenic bacteria were determined as MICs (Table 2). The susceptibilities of the tested bacterial strains to antibiotics are also listed in the table. Most of the bacteria were resistant to amphicillin or oxacillin but susceptible to norfloxacin or trimethoprim/sulfamethoxazole (T/S, 1 : 19). However, Listeria monocytogenes strains were resistant to T/S. The activities of the essential oil fraction of A. tenuissima (EOAT) depended on the bacterial species, with MICs from 0.21 ± 0.08 to 3.60 ± 0.89 mg/ml. The oil fraction showed the strongest inhibition against Shiegella sonnei strains. Ligustilide showed similar or stronger activity than the total essential oil fraction against most of the tested bacterial strains except Escherichia coli. These results suggested that the first main component, ligustilide might significantly affect the antibacterial activity of the essential oil fraction with contributions from other, minor compounds. The MICs of butylidene phthalide were higher than MICs for EOAT or ligustilide.

Table 2.EOAT: Essential oil fraction of A. tenuissima, T/S: trimethoprim/sulfamethoxazole. n: number of tested strains. Values are means ± SD of triplicate tests. Statistical analysis were by Student’s t-test.

Antioxidant activities − A. tenuissima roots has been used in functional health foods and in functional natural cosmetics for anti-aging. Anti-aging activity is closely related to antioxidative components. Ka et al, (2005) reported the antioxidant activity of A. tenuissima volatile extracts using an aldehyde/carboxylic acid assay, and the determination of the potential to protect against H2O2-induced cytotoxicity and lipid peroxidation. However, many antioxidative mechanisms are known and therefore various other methods are also needed to fully determine the antioxidative activity of A. tenuissima oil.

To evaluate A. tenuissima oil as an antioxidant, DPPH free radical-scavenging activity and reducing power were studied. As shown in Fig. 1, A. tenuissima essential oil showed significant, dose-dependent DPPH-scavenging activity in serially diluted samples (100 - 1600 μg/ml). However, the results showed lower scavenging activity than the control, α-tocopherol. When compared at a final concentration of 1.6 mg/ml, the DPPH-scavenging activity of the oil was 73.7% the activity of α-tocopherol.

Fig. 1.DPPH Free-radical scavenging activity of essential oil fractions from A.tenuissima roots and tocopherol (control). Values are means ± SD of triplicate measurements.

In reducing-power assays, the A. tenuissima essential oil fraction exhibited higher activity than α-tocopherol at concentrations of 12.5 - 200 μg/ml (Fig. 2). Effects of the oil fraction and α-tocopherol were dose dependent. At 50 μg/ml, A. tenuissima essential oils had 1.8-times higher reducing activity than α-tocopherol.

Fig. 2.Reducing power of essential oil fractions from A. tenuissima roots and tocopherol (control). Values are means ± SD of triplicate measurements.

Activities on Caco-2 and MKN-45 carcinoma cell lines- Caco-2 is a colon carcinoma cell line and MKN-45 is a gastric carcinoma cell line. We determined the effects of A. tenuissima essential oil on the viability of these human gastrointestinal cancer cell lines by MTT test. Fig. 3 shows that the essential oil fraction of A. tenuissima and ligstilide and butylidene phthalide dose-dependently inhibited Caco-2 cell growth. The essential oil fraction of A. tenuissima and its two main components showed similar inhibition of cell proliferation. At 80 μg/ml of the oil fraction or its main components, Caco-2 cell growth was more than 50% inhibited compared to untreated cells. However, as shown in Fig. 4, the viability of MKN-45 cells was most strongly decreased by treatment with the essential oil fraction at 160 μg/ml compared to the individual components, resulting in a 27.82% ± 4.87% of cell survival rate. Butylidene phthalide treatment resulted in relatively little growth inhibition.

Fig. 3.Effects of essential oil fraction from A. tenuissima roots, ligustilide and butylidene phthalide on viability of Caco-2 cells. Values are means ± SD of triplicate measurements.

Fig. 4.Effects of essential oil fraction from A. tenuissima roots, ligustilide and butylidene phthalide on viability of MKN-45 cells. Values are means ± SD of triplicate measurements.

 

Conclusion

The results of this study indicated that A. tenuissima essential oil and its main components, ligustilide and butylidene phthalide, could be useful agents for treating food-borne pathogenic bacterial infections. A. tenuissima essential oil could also be used as a safe additive for preventing food contamination by pathogenic bacteria. The antioxidative activity of A. tenuissima essential oil and its ability to inhibit gastrointestinal carcinoma cell lines could increase its value for functional foods and prevention of cancer. However, further studies are required.