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Chemical composition, antimicrobial and antioxidant activities of essential oil from Gnaphlium affine
Food and Chemical Toxicology 49 (2011) 1322–1328
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Chemical composition, antimicrobial and antioxidant activities of essential oil from Gnaphlium affine Wei-Cai Zeng a, Rui-Xue Zhu a, Li-Rong Jia a, Hong Gao a,⇑, Yue Zheng b, Qun Sun c a
College of Light Industry, Textile and Food Engineering, Sichuan University, Chengdu 610065, PR China National Center for Food Safety and Technology, Illinois Institute of Technology, IL 60501-1957, USA c College of Life Sciences, Sichuan University, Chengdu 610064, PR China b
a r t i c l e
i n f o
Article history: Received 14 January 2011 Accepted 10 March 2011 Available online 15 March 2011 Keywords: Gnaphlium affine Chemical composition Antimicrobial Antioxidant
a b s t r a c t The chemical composition of the essential oil from Gnaphlium affine was determined, and its antimicrobial and antioxidant activities were evaluated. Twenty-four compounds, representing 94.95% of the amount of total oil, were identified by gas chromatography–mass spectrometry (GC–MS) analysis. Main constituents of the essential oil were found to be eugenol (18.24%), linalool (10.62%), trans-caryophyllene (8.86%), a-terpineol (5.97%), p-cymene (5.75%), hexadecanoic acid (5.63%), c-cadinene (4.98%), d-cadinene (4.22%), a-humulene (3.22%), and ()-b-elemene (3.15%). The essential oil revealed a remarkable antimicrobial effect against the tested food-borne microorganisms with the MIC and MBC values in the ranges of 0.2–1.56 lg/ml and 0.39–3.13 lg/ml, respectively. The essential oil showed a potent antioxidant activity in ABTS radical scavenging, lipid peroxidation and reducing power assay. It was suggested that the essential oil from G. affine may be a new potential source as natural antimicrobial and antioxidant agents applied in food systems. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Gnaphlium affine, normally named Cudweed or Ching Ming vegetable in China, belongs to Asteraceae and has been used as food and traditional medicinal plant (Lu et al., 2009). Every year after the traditional Ching Ming festival, it is extensively harvested nationally as a wild vegetable and then be processed into a variety of foods, such as drinks, canned products and frozen vegetables. G. affine is believed to be of high nutritional value since it has the eight essential amino acids for human body of a reasonable proportion, a high content of minerals, trace elements, and vitamins, thus to be developed into a functional food (Wang et al., 2005; Chen, 1999). Besides the nutritional value, G. affine was reported to possess many pharmacological activities. G. affine was observed to lower the blood pressure in human (Liu, 1965). A recent study showed that G. affine exhibited the protective effect for carbon tetrachloride-induced acute liver injury (Jiang et al., 2008). Furthermore, G. affine had been reported to be useful for the treatment of respiratory diseases (Tian, 1997). Microbial contamination and oxidation in foods not only result in food deterioration and shelf life reduction, but also lead to disease and economic losses. At present, the food industry is facing an enormous pressure coming from food deterioration caused by ⇑ Corresponding author. Tel.: +86 28 85405236; fax: +86 28 85405137. E-mail address: [email protected] (H. Gao). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.03.014
the two factors above. The growth of microorganisms in food products may cause intestinal disorders, vomiting and diarrhea (Friedman et al., 2002). It has been reported that in the global incidence, 1.8 million people died from diarrhea disease in 2005 alone. A great proportion of these can be attributed to microbial contamination in food and drinking water. It has been estimated that as many as 30% of people in industrialized countries suffer from a food-borne disease each year. With the environment pollution, more population will suffer from food-borne diseases (Loizzo et al., 2010). During the raw material storage, processing, heat treatment and further storage of final products, oxidation is another familiar processes causing rancidity in food products, leading to the degradation of lipids and proteins, and contributes to the deterioration in flavor, texture, and color of food products. The odors and flavors from oxidation can easily destroy the organoleptic and nutritional quality of processed foods (Karpin´ska et al., 2001). In addition, reactive oxygen species from the oxidative processes in food, such as superoxide radicals, hydroxyl radical, and peroxyl radicals, are involved in the onset of many diseases such as carcinogenesis, coronary heart disease, and many other health issues related to advancing age (Borneo et al., 2009). Essential oils (also called volatile oils) are natural volatile complex compounds characterized by a strong smell, and form in edible, medicinal and herbal plants as secondary metabolites (Burt, 2004). They can be obtained by compression, enfleurage or extraction but the method of steam distillation is most commonly
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used. For the various and remarkable biological properties already observed in nature, essential oils have been largely utilized in perfumes, make-up products and sanitary products, in food industry as food additives or food preservers, in dentistry as natural remedies and in agriculture as green pesticides. A variety of studies had been carried out to evaluate the antimicrobial and antioxidant activities of essential oils, and the results indicated that essential oil did have noticeable antimicrobial and antioxidant activities (Bakkali et al., 2008; Rahman and Kang, 2009). To the best of our knowledge and literary survey, there is no information available on the chemical composition analysis, antimicrobial and antioxidant activities of essential oil from G. affine. Therefore, efforts have been made to investigate the role of this essential oil for its antimicrobial and antioxidant potential. The aims of this work are to detect the chemical composition of essential oil from G. affine by gas chromatography–mass spectrometry (GC–MS) and evaluate its in vitro antimicrobial and antioxidant properties. 2. Materials and methods 2.1. Plant materials and chemicals The aerial parts (leaves and flowers) of G. affine were collected from Chengdu city area of China, in March 2010. The plant was initially identified by morphological features and the database was present in Department of Biology, Sichuan University. A voucher specimen was dried and preserved at the Key Laboratory of Food Science and Technology of Sichuan Province, Sichuan University, China. Ascorbic acid (Vc), 2,20 -azinobis-3-ethylbenzthiazoline-6-sulfonate (ABTS), sodium dodecyl sulfate, acetic acid, potassium persulfate, thiobarbituric acid, ferrous sulfate, anhydrous sodium sulfate, n-hexane, methanol, phosphate balanced solution (PBS), glutaraldehyde, osmium tetroxide, acetone, epoxy, 1-butanol, ethanol, potassium ferricyanide, trichloroacetic acid and ferric chloride were purchased from Sigma–Aldrich (St. Louis, MO). Gentamicin was obtained from Sichuan Changwei Pharmaceutical Co. Ltd., China. Agar, beef extract, sucrose and peptone were purchased from Bio-Ketai (Langsan Ketai Biological Products Co. Ltd., China). Potatoes were obtained from a local supermarket in Chengdu, China and stored at 4 °C until used for experiment. The solvents for GC–MS analysis were of HPLCgrade. All other reagents used were of analytical grade. 2.2. Preparation of essential oil The essential oil was extracted by the method of hydro-distillation. The airdried (25 °C, 15 d) plant samples (300 g) were crushed into powder (about 40 granularities) with a mixer (JYL-350, Jiuyang Co. Ltd., China) and subjected to hydro-distillation for 8 h, using a Clevenger-type apparatus (Shudu Co. Ltd., China). The oil was collected and dried over anhydrous sodium sulfate and then stored in sealed glass vials at 4 °C prior to further analysis. The yield based on dry weight of the sample was 0.86% (2.58 g/300 g dry samples). 2.3. GC–MS analysis GC–MS analysis of the essential oil was performed using a Thermo GC-MSD (Trace DSQ II, Thermo Fisher Corporation, USA) apparatus equipped with a TR-5 capillary column (30 m 0.25 mm internal diameter, 0.25 m film thickness). Helium gas was used as the carrier gas at a constant flow rate of 1 ml/min. For GC– MS detection, an electron ionization system was used with ionization energy set at 70 eV, and mass range at m/z 40–500. Injector and MS transfer line temperature were set at 250 °C and 280 °C, respectively. The sample of diluted essential oil solution (1:100, v/v, in n-hexane) of 1 ll was injected and analyzed with the column maintained initially at 50 °C for 1 min and then increased to 250 °C with a 5 °C/ min heating ramp and subsequently kept at 250 °C for 20 min. The relative percentages of individual components of the essential oil were expressed as percentages of the peak area relative to the total peak area. The compounds of essential oil were identified on the basis of comparison of their retention time and mass spectra with publish data (Adams, 2001) and computer matching with National Institute of Standards and Technology (NIST, 3.0) libraries provided with computer controlling the GC–MS system. 2.4. The tested microorganisms The following food spoilage and food-borne microorganisms including Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 21216, Bacillus cereus ATCC 10231, Bacillus laterosporus laubach ATCC 64, Salmonella typhimurium ATCC 14028, Saccharomyces cerevisiae ATCC 9763, Aspergillus niger
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ATCC 16404, Pencicillium citrinum ATCC 14994, Rhizopus oryzae ATCC 9363, and Aspergillus flavus ATCC 204304, were used as the tested microorganisms. All the microbial strains were maintained on culture medium at 4 °C and were sub-cultured every month in our laboratory. 2.5. In vitro antimicrobial assay 2.5.1. Inhibitory zone assay The essential oil was dissolved in 80% methanol (diluted with sterile water) to a final concentration of 50 mg/ml and sterilized by filtrating through 0.22 lm Millipore filters. Antimicrobial activity of the essential oil was determined by Oxford plate method (Beverlya et al., 2008). In brief, all the microbial cultures (sub-cultured before assay) were diluted with sterile water to obtain a bacterial suspension of 106 CFU/ml. Petri plates containing 20 ml of culture medium were inoculated with 200 ll of bacterial suspension and allowed to dry in sterile chamber. The Oxford plates (6 mm in diameter) were impregnated with 100 ll of 50 mg/ml sample and placed on the inoculated culture medium. The 80% methanol was also used as the negative control. Gentamicin (50 lg/disk) was used as positive control. The plates of bacteria inoculated were incubated at 37 °C for 24 h, while fungi at 28–32 °C for 72 h, and yeast at 28 °C for 48 h. The antimicrobial activity was evaluated by measuring the inhibition zone against the tested microorganisms. 2.5.2. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) The minimum inhibitory concentration (MIC) of the essential oil was evaluated for the bacterial strains which were determined by the method of broth dilution. An aliquot of 2 ml of nutrient broth was placed into each tube, and all tubes were autoclaved at 121 °C for 20 min. The essential oil (filtered, 0.22 lm) was first dissolved in methanol, and added to the tubes to keep the final concentrations of 0.2–50 lg/ml. The test bacteria suspension was added into to the inoculum size of 106 CFU/ml. Then, the inoculated tubes were incubated at 37.5 °C for 18–24 h. The final concentration of methanol in culture medium was maintained at 0.1% (v/v). Another culture medium without adding bacteria suspension was prepared as the negative control. The MIC was defined as the lowest concentration of the essential oil which inhibited the visible growth of tested microorganism. The minimum bactericidal concentration (MBC) of the essential oil was determined according to the MIC values. The samples showing no increases in turbidity were streaked on nutrient agar medium and incubated at 37.5 °C for 18–24 h. The lowest concentration of the essential oil, which did not show any viable bacteria, was determined as the MBC. 2.5.3. Influence on the growth of B. subtilis Influence of the essential oil on the growth of B. subtilis was evaluated using the method of growth curve. B. subtilis cultured to logarithmic growth phase was diluted with nutrient broth to the inoculum size of 106 CFU/ml. The essential oil (filtered, 0.22 lm) was first dissolved in methanol, and added to nutrient broth to keep the final concentrations as the MIC value of B. subtilis. Final concentration of methanol in nutrient broth was maintained at 0.1% (v/v). Then, the culture was incubated in incubation shaker (37.5 °C, 130 rpm). The growth of B. subtilis was evaluated by determining the optical density of culture at 600 nm per 2 h using the Lambda 25 UV–visible spectrophotometer (PerkinElmer Co. Ltd., USA), ending at 24 h. The culture without the essential oil was used as negative control. 2.5.4. Observation with transmission electron microscope (TEM) B. subtilis cultured in Section 2.5.3 was collected and prepared for TEM analysis. Briefly, the treated B. subtilis was centrifugal at 5000 rpm at 25 °C for 10 min, and the precipitation was washed three times with PBS (0.1 M, pH 7.4). An amount of 0.5% of glutaraldehyde was added to the precipitation keeping 15 min at 4 °C. Then, bacterial cells were collected by 20 min centrifugation at 15,000 rpm at 4 °C. After deal with 3% glutaraldehyde, 1% osmium tetroxide, acetone and epoxy, the sample was cut into thin sections using a microtome (Ultracut-E, Reichert-Jung, Austria). Then the sample was observed by TEM (H-600IV, Hitachi, Japan). 2.6. In vitro antioxidant assay 2.6.1. ABTS radical scavenging assay The ABTS radical scavenging assay was estimated according to the previously reported procedure with a slight modification (Re et al., 1999). The essential oil was first dissolved in ethanol at the ranges of 0.16–2.5 lg/ml. ABTS radical cation solution was prepared through the reaction of 7 mM ABTS and 2.45 mM potassium persulfate, after incubation at 23 °C in the dark for 16 h. The ABTS radical cation solution was then diluted with 80% methanol (dissolved with pure water) to obtain an absorbance of 0.700 ± 0.005 at 734 nm. ABTS radical cation solution (absorbance of 0.700 ± 0.005, 3.9 ml) was added to 0.1 ml of the test sample and mixed vigorously. The reaction mixture was allowed to stand at 23 °C for 6 min and the absorbance at 734 nm was immediately recorded by the Lambda 25 UV–visible spectrophotometer. Vc was used as a positive control. The ability to scavenge ABTS radical in percent was calculated as:
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Scavenging activity ð%Þ ¼ ð1 Asample 734 =Acontrol 734 Þ 100 where Acontrol
734
was the absorbance of control (80% methanol, instead of sample).
Table 1 The main chemical components of the essential oil from Gnaphlium affine. No.
2.6.2. Lipid peroxidation assay The lipid peroxidation assay was measured by the previously reported method with a minor modification (Dasgupta and De, 2004). Egg yolk homogenates were prepared as lipid-rich media. In brief, an amount of 0.1 ml of various concentrations (0.625, 1.25, 2.5, 5 and 10 lg/ml, respectively) of the essential oil in methanol were thoroughly mixed with 0.5 ml of egg yolk homogenate (10%, v/v, diluted with pure water) and made up to 1 ml with pure water. Ferrous sulfate (50 ll, 70 mM) was added to induce lipid peroxidation and the mixture incubated for 30 min at 37.5 °C. Then, 1.5 ml of 20% acetic acid (v/v, pH 3.5, diluted with pure water) and 1.5 ml of 0.8% (w/v) thiobarbituric acid in 1.1% sodium dodecyl sulfate (w/v, diluted with pure water) were added and the resulting mixture was vortexed and then heated at 95 °C for 60 min. After cooling, 5 ml of 1-butanol were added to each tube and centrifuged at 5000 rpm for 15 min. The organic upper layer was collected and the absorbance at 532 nm was immediately measured by the Lambda 25 UV–visible spectrophotometer. Vc was used as a positive control. The inhibition of lipid peroxidation was calculated as:
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Inhibition ð%Þ ¼ ð1 Asample 532 =Acontrol 532 Þ 100 where Acontrol
532
was the absorbance of control (methanol, instead of sample).
2.6.3. Reducing power assay The reducing power of the essential oil was determined according to the previously reported method with a minor modification (Yen and Chen, 1995). In brief, an amount of 1 ml various concentrations of samples (62.5, 125, 250, 500, 1000 and 2000 lg/ml, respectively) in methanol was mixed with 2.5 ml of PBS (0.2 M, pH 6.6) and 2.5 ml of potassium ferricyanide (1%, w/v). The mixture was incubated at 50 °C for 20 min. Then, trichloroacetic acid of 2 ml (10%, w/v) was added to the mixture to terminate the reaction, which was then centrifuged at 2000 rpm for 15 min. The upper layer of solution (2.5 ml) was mixed with pure water (2.5 ml) and ferric chloride (0.5 ml, 0.1%, w/v), and the absorbance was measured at 700 nm. Vc was used as a positive control in the assay. 2.7. Statistical analysis All analyses were conducted in triplicate and statistical analysis of the data was performed using SPSS 13.0 software (SPSS Inc., 223 South Wacker Drive, CO., USA). A probability value at p < 0.05 was considered statistically significant. Data were presented as mean value ± standard deviation (SD), which were calculated from triplicate determinations.
3. Results 3.1. Chemical composition of the essential oil The essential oil extracted from the air-dried aerial parts (leaves and flowers) of G. affine by hydro-distillation showed a color of light yellow. GC–MS analyses of the essential oil led to identification of 24 different compounds, representing 94.95% of the total amount of oil extracted. The identified compounds with their retention time and their percentage are listed in Table 1. All the compounds were arranged in the order of their retention time. The essential oil was a complex mixture mainly of oxygenated monoterpenes and sesquiterpenes, and hydrocarbons. It contained hydrocarbons (66.88%) and hydroxybenzene (18.24%). The major compounds in the essential oil were eugenol (18.24%), linalool (10.62%), trans-caryophyllene (8.86%), a-terpineol (5.97%), p-cymene (5.75%), hexadecanoic acid (5.63%), c-cadinene (4.98%), d-cadinene (4.22%), a-humulene (3.22%) and ()-b-elemene (3.15%). The structures of the major bioactive compounds are shown in Fig. 1. In the present study, elemol (0.76%), tetradecane (0.96%), a-cedrol (1.08%), nonadecane (1.44), 7-octen-4-ol (1.53%), [z,z]-9,12-octadecadienoic acid (1.38%) and tetradecanoic acid (1.59%) were also detected to be the minor components in the essential oil from G. affine. 3.2. In vitro antimicrobial activity The in vitro antimicrobial activity of the essential oil from G. affine against the tested microorganisms was qualitatively and quantitatively assessed by the inhibition zones. According to the
RTa
Compound
Composition (%)b
6.37 8.21 8.56 10.25 10.69 13.87 14.23 14.48 14.85 15.21 15.69 15.98 16.33 16.73 17.15 18.09 19.13 19.25 20.16 20.58 21.87 23.15 25.37 30.69
7-Octen-4-ol p-Cymene Linalool 2-Ethenyl-1,4-dimethylbenzene a-Terpineol Eugenol Tetradecane ()-b-Elemene a-Gurjunene trans-Caryophyllene a-Humulene c-Cadinene c-Gurjunene d-Cadinene Elemol a-Cedrol Heptadecane 2,6,10,14-Tetramethylpentadecane Tetradecanoic acid Octadecane Nonadecane Hexadecanoic acid [z,z]-9,12-Octadecadienoic acid Pentacosane
1.53 5.75 10.62 2.64 5.97 18.24 0.96 3.15 1.96 8.86 3.22 4.98 2.65 4.22 0.76 1.08 2.24 1.46 1.59 2.03 1.44 5.63 1.38 2.59
Total a b
94.95
RT, retention time. Composition (%), relative percentage based on peak area.
results shown in Table 2, the essential oil exhibited a potent inhibitory effect against all bacteria (E. coli, S. aureus, B. subtilis, B. cereus, B. laubach, and S. typhimurium), yeast (S. cerevisiae) and fungi (A. niger, P. citrinum, R. oryzae and A. flavus) with diameter of inhibition zones ranging from 15.43 to 24.73 mm, while it was 23.13–30.53 mm for the positive control. The essential oil showed a high antimicrobial effect on S. cerevisiae, A. flavus, R. oryzae and S. aureus with the diameter of inhibition zones of 24.73, 23.33, 22.60 and 21.20 mm, respectively. On the other hand, fungi were more sensitive than bacteria to this essential oil and S. cerevisiae was found to be the most susceptible one. Additionally, no inhibitory effect on the tested microorganisms was observed for the solvent (80% methanol) at the used concentration. 3.3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) MIC and MBC values of essential oil against the employed bacterial strains are shown in Table 2. These results demonstrated that this oil displayed remarkable antibacterial and bactericidal property. In general, the MIC values of the essential oil against the tested bacteria ranged from 0.2 lg/ml to 1.56 lg/ml and MBC from 0.39 lg/ml to 3.13 lg/ml, respectively. In addition, the MIC and MBC values of fungi were lower than that of bacteria. By considering the results of inhibition zone assay in Table 2, S. cerevisiae was the most sensitive fungi while S. aureus the most sensitive bacterium. Solvents did not inhibit the growth of any of tested microorganisms at the used concentration. 3.4. Effect of essential oil on the growth of B. subtilis In Fig. 2A, a prominent inhibitory zone (17.5 mm of diameter, 5 mg essential oil/dish) was present in the center of the petri dish. It was demonstrated that the essential oil from G. affine could inhibit the proliferation of B. subtilis in the nutrient agar medium. The growth curve of B. subtilis with the presence of the essential oil extracted is shown in Fig. 2B. Both the essential oil and the control
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Fig. 1. The chemical structures of major bioactive compounds of the essential oil from Gnaphlium affine. (a) eugenol; (b) linalool; (c) p-cymene; (d) a-terpineol; and (e) transcaryophyllene.
Table 2 Antimicrobial activity of the essential oil from Gnaphlium affine against food-borne and spoilage microorganisms. Microorganisms Strain
E. coli S. aureus B. subtilis B. cereus B. laubach S. typhimurium S. cerevisiae A. niger P. citrinum R. oryzae A. flavus a b c d e
ATCC 25922 ATCC 25923 ATCC 21216 ATCC 10231 ATCC 64 ATCC 14028 ATCC 9763 ATCC 16404 ATCC 14994 ATCC 9363 ATCC 204304
Inhibition zone (mm)
Essential oil (lg/ml)
Essential oila Positive controlb
MICc
MBCd
15.43 ± 0.89e 24.47 ± 0.97 1.56 ± 0.41 3.13 ± 0.52 21.20 ± 1.04 25.67 ± 0.79 0.39 ± 0.12 0.78 ± 0.21 17.50 ± 0.86 23.77 ± 0.78 0.78 ± 0.30 1.56 ± 0.15 17.70 ± 0.86 23.30 ± 1.31 0.78 ± 0.11 1.56 ± 0.24 16.43 ± 0.53 23.13 ± 0.83 0.78 ± 0.13 3.13 ± 0.18 17.50 ± 0.89 23.73 ± 0.69 0.78 ± 0.19 3.13 ± 0.25 24.73 ± 0.74 27.57 ± 0.78 0.20 ± 0.21 0.39 ± 0.16 19.47 ± 0.94 28.80 ± 0.94 0.20 ± 0.18 0.39 ± 0.14 18.80 ± 1.00 30.53 ± 0.92 0.20 ± 0.15 0.78 ± 0.20 22.60 ± 0.91 25.73 ± 0.69 0.20 ± 0.19 0.78 ± 0.11 23.33 ± 1.11 26.50 ± 1.02 0.20 ± 0.20 0.39 ± 0.16
The concentration of essential oil was 5 mg/disk. Gentamicin (50 lg/disk) was used as positive control. Minimum inhibitory concentrations. Minimum bactericidal concentrations. Each value is expressed as means ± SD (n = 3).
had a lag phase in first 6 h. From 6 h, the OD600 of control increased at a faster rate, and then kept stable until the end. However, the OD600 of essential oil treatment almost did not increase and kept
Fig. 2. Influence of the essential oil of Gnaphlium affine on the growth of B. subtilis. (A) Inhibitory effect of the essential oil (17.5 mm of inhibitory zone diameter against B. subtilis at the final concentration of 5 mg/ml) on B. subtilis; and (B) the growth curve of B. subtilis.
at a low value till the end. The difference of OD600 between the control and the essential oil group indicated that the growth of B. subtilis had been inhibited obviously under the presence of essential oil from G. affine.
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Fig. 3. External morphology of B. subtilis cells observed by transmission electron microscope. (A) and (B), untreated bacteria; (C) and (D) bacteria treated with the essential oil from Gnaphlium affine.
3.5. Morphological changes in B. subtilis after essential oil treatment
3.6. ABTS radical scavenging activity
As shown in Fig. 3, TEM analysis showed the change of external morphological features of B. subtilis treated by the essential oil. The untreated B. subtilis cells retained their connatural morphology (claviform and elliptical) and seemed to be normal (Fig. 3A). In contrast, B. subtilis cells treated with the essential oil appeared to undergo lysis, resulting in the release of their cellular contents into surrounding environment, and finally became empty (Fig. 3C). Compared with undamaged cells, it was easy to find cell debris around the damaged cells showing electron translucent cytoplasm. By observation at a greater magnification, the untreated cells of B. subtilis showed normal morphological characteristics and homogeneous electron density in cytoplasm. Their cell walls and membranes were intact, showing a well maintained peptidoglycan layer and cytoplasmic membrane (Fig. 3B). In contrast, significant morphological changes were observed in B. subtilis cells treated with the essential oil. They were lysed cells, and the cell walls and membranes were broken with decreases of heterogeneity in electron density in cytoplasm (Fig. 3D). The localized separation of cell membrane from cell wall could be discerned and the cellular degradation was also accompanied with the outflow of electrontranslucent cytoplasm in damaged cells.
ABTS assay is based on the antioxidant ability of sample to react with ABTS radical cation generated in the assay system. ABTS radical scavenging activity of the essential oil from G. affine at various concentrations is shown in Fig. 4A. At the concentrations of 0.16–2.5 lg/ml, scavenging ability of the essential oil on ABTS radical was in ranges of 21.01–100%. At the concentration of 2.5 lg/ml, the essential oil was observed to possess strong free radical scavenging activity against ABTS radical with a value of around 100%, while the scavenging activity of control (Vc) was only 5.89%. Moreover, as shown in Fig. 4A, a concentration-dependent ABTS radical scavenging activity was observed with the half-inhibitory concentration (IC50) being 0.32 ± 0.89 lg/ml. On the other hand, IC50 value of Vc was 24.06 ± 0.73 lg /ml. Evidently, ABTS radical scavenging activity of Vc was even weaker than essential oil in our antioxidant assay. 3.7. Lipid peroxidation Egg yolk lipids undergo rapid non-enzymatic peroxidation when incubated in the presence of ferrous sulfate. The effect of the essential oil from G. affine on non-enzymatic peroxidation is
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At the concentrations of 62.5, 125, 250, 500, 1000, and 2000 lg/ ml, reducing powers were around 0.283, 0.694, 0.852, 0.913, 0.966, and 0.983 for essential oil and 0.152, 0.496, 0.742, 0.835, 0.928, and 0.947 for positive control of Vc, respectively. The increase at absorbance of reaction mixture indicated the increase of reducing power. This phenomenon clearly showed that the essential oil had a stronger reducing power than Vc. Meanwhile, as also shown in Fig. 4C, reducing power of the essential oil and Vc was increasing with high concentration.
4. Discussion
Fig. 4. Antioxidant activity of the essential oil from Gnaphlium affine. (A) Scavenging effect of the essential oil on ABTS radical; (B) inhibitory effect of the essential oil on lipid peroxidation; and (C) reducing power of the essential oil.
shown in Fig. 4B. In the experimental model of egg yolk homogenates, a significant inhibitory effect of the essential oil on lipid peroxidation was shown with IC50 value of 0.09 ± 0.75 lg/ml (IC50 of Vc = 6.73 ± 0.87 lg/ml). Moreover, as shown in Fig. 4B, at the concentrations of 0.625–5 lg/ml, the inhibition activity of the essential oil on lipid peroxidation ranged from 61.32% to 71.25%. At the concentration of 10 lg/ml, scavenging activity of Vc and the essential oil were 56.27% and 75.08%, respectively. Meanwhile, a concentration-dependent inhibitory effect on lipid peroxidation was observed for the essential oil. It was worth to note that the inhibition activity of Vc against lipid peroxidation was lower than essential oil used in this assay. 3.8. Reducing ability In reducing power assay, the presence of antioxidants in samples would result in the reducing of Fe3+–Fe2+ by donating an electron. An amount of Fe2+ complex can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm (Gholivand et al., 2010). Increasing absorbance at 700 nm indicated an increase in reductive ability. Reducing ability of the essential oil from G. affine is shown in Fig. 4C. As the antioxidant activities above, reducing ability of the essential oil correlated well with its concentration.
In spite of modern improvements in food production and food processing techniques, a high amount of food products are still perishable during their preparation, storage and distribution thus not to give them desired shelf-life. Although increasing use of chemical preservatives can effectively prevent the growth of most foodborne bacteria, safety problems related to chemical preservatives are receiving growing attention (Deba et al., 2008). Therefore, much effort has been expended in the search for new types of effective and nontoxic antimicrobial compounds from natural materials. One such possibility is the use of essential oils as antimicrobial additives (Holley and Patel, 2005). In the present study, the results of in vitro antimicrobial assay showed that the essential oil from G. affine had high antimicrobial activity against some representative food-borne spoilage pathogens, especially fungi. Antimicrobial activity of the essential oil from G. affine could be contributed to the presence of some major bioactive compounds contained, such as eugenol (Fig. 1a) and linalool (Fig. 1b). Eugenol, a major phenolic component of clove essential oil, has been widely used in medical and dental practice, due to its potent fungicidal, bactericidal, anesthetic, antioxidant and anti-inflammatory properties (He et al., 2007). It is used in the form of paste or mixture as dental cement, filler and restorative material. It belongs to the class of essential oils that is generally recognized as safe (GRAS) by the Food and Drug Administration (Hemaiswarya and Doble, 2009). As a well-known antimicrobial agent against food-borne pathogens, it has been reported to act primarily by disrupting the cytoplasmic membrane (Hemaiswarya and Doble, 2009). Linalool, a monoterpene alcohol, is the second abundant compound in essential oil and widely used in decorative cosmetics, fine fragrances, shampoos, toilet soaps and other toiletries (Letizia et al., 2003). It is usually present in essential oils of many medicinal and edible plants of a variety of biological activities, including antioxidant, antimicrobial, antiinflammatory, and antitumor properties (Letizia et al., 2003). Moreover, linalool also could enhance the permeability of a number of drugs through biological tissues like skin or mucus membranes (Peana et al., 2002). According to the previous studies, the other bioactive components in this essential oil, such as trans-caryophyllene (Fig. 1c), a-terpineol (Fig. 1d) and p-cymene (Fig. 1e), may also contribute to the antimicrobial activity (11). Our results suggested that phenolic compounds, monoterpenes, and sesquiterpenes in the essential oil from G. affine be the main bioactive constituents for the antimicrobial activity. Further, the results of growth curve and TEM image of B. subtilis revealed that the essential oil had a severe impact on the cell viability of B. subtilis. In the logarithmic growth phase, B. subtilis was inhibited obviously upon the presence of the essential oil (Fig. 2B). Observed under TEM, cell walls and membranes of B. subtilis treated by the essential oil were damaged, and the cytoplasm decreased, and finally the cells retained empty. These morphological changes may attribute to aromatic and phenolic compounds in essential oil. There is overwhelming consensus that the essential oil exerts its antimicrobial effect at cytoplasmic membrane by altering the structure and function of bacteria (Holley and Patel, 2005). A previous report
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showed that efflux of K+ is usually an early sign of damage (Walsh et al., 2003) and then often followed by efflux of cytoplasmic constituents including ATP (adenosine-triphosphate) (Brul and Coote, 1999). The loss of differential permeability character of the cytoplasmic membrane is frequently identified as the cause of cell death. As shown in Fig. 3D, cells of B. subtilis with an empty trunk were dead with an empty trunk. Furthermore, the dysfunction and disruption of the membrane, interference with the energy generation system in cell, and enzyme inhibition preventing substrate utilization for energy production may also lead to the death of bacterial cells (Holley and Patel, 2005). In addition, the essential oil possessing the strongest antimicrobial properties contains a high percentage of phenolic compounds, such as carvacrol, eugenol and thymol (Dorman and Deans, 2000). It seems reasonable to assume that the mechanism of their action would therefore be similar to that of other phenolics. This is generally considered to be the disturbance of the cytoplasmic membrane, resulting in disrupting the proton motive force, electron flow, active transport and coagulation of cell contents (Burt, 2004). Thus, the essential oil from G. affine in this study could be a potential source as inhibitory substances against some foodborne spoilage and pathogens and be candidates to be applied in foods or food-processing systems. Oxidation is an essential biological process for most organisms to produce energy. Meanwhile, fulsome or uncontrolled oxidations also happen. Uncontrolled production of superoxide anion free radicals is involved in the onset of many diseases such as cancer, atherosclerosis and degenerative processes with aging (Chen et al., 2005). Our results of various free radicals scavenging activity revealed that the essential oil from G. affine had a significant antioxidant activity even compared with Vc. As shown in Fig. 4, not only the free radical scavenging activity of the essential oil was better than Vc, but also the reducing power. The antioxidant activity may be contributed by different mechanisms, such as prevention of chain initiation, decomposition of peroxides, prevention of continued hydrogen abstraction, free radical scavenging, reducing capacity, and binding of transition metal ion catalysts (Jia et al., 2010). In present study, the remarkable antioxidant activity of essential oil may be contributed by the high content of phenolic compounds contained. Phenolic compounds, generally considered as biologically active components, are the main agents to donate hydrogen to free radicals and thus break the chain reaction of lipid oxidation at the first initiation step. This high potential of phenolic compounds to scavenge radicals may be explained by their phenolic hydroxyl groups (Oke et al., 2009). A number of studies about the health benefits of bioactive phenols have increased lately. Recent research suggested that phenolic compounds may exhibit beneficial health effects and manifold pharmaceutical functions for human (Cai et al., 2004). In conclusion, results of the present study suggested that the essential oil from G. affine can be useful for a new potential source as natural antimicrobial and antioxidant agent in food systems. Further studies are undergoing for the antimicrobial assessment of this essential oil in food models and the toxicological evaluation for the safety usage of the oil used in food products. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements We are grateful to Dr. Yi-Na Huang, Department of Public Health, Sichuan University, and Dr. Su Feng, College of Life Sciences, Sichuan University, for their helpful comments and suggestions. This work was financially supported by the National Natural
Science Foundation of China (31071489) and Innovative Experiment of Food Science of Sichuan University (2010460). References Adams, R.P., 2001. Identification of essential oil components by gas chromatography/ quadrupole mass spectroscopy. Allured Publishing Corporation, Carol Stream, IL. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils – a review. Food Chem. Toxicol. 46, 446–475. Beverlya, R.L., Janes, M.E., Prinyawiwatkula, W., No, H.K., 2008. Edible chitosan films on ready-to-eat roast beef for the control of Listeria monocytogenes. Food Microbiol. 25, 534–537. Borneo, R., León, A.E., Aguirre, A., Ribotta, P., Cantero, J.J., 2009. Antioxidant capacity of medicinal plants from the Province of Córdoba (Argentina) and their in vitro testing in a model food system. Food Chem. 112, 664–670. Brul, S., Coote, P., 1999. Preservative agents in foods. Mode of action and microbial resistance mechanisms. Int. J. Food Microbiol. 50, 1–17. 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Antioxidant and antibacterial activities on food-borne pathogens of Artocarpus heterophyllus Lam. (Moraceae) leaves extracts. J. Food Sci. 75, M291– M295. Lu, Y., Li, J., Qiu, W.M., Tang, Y.Q., Zhou, X.F., Liu, Z.H., 2009. Separation and purification for flavones from Gnaphlium affine D.Don by macroporous resin. J. Hunan Agr. Univ. (Nat. Sci.) 35, 497–500. Oke, F., Aslim, B., Ozturk, S., Altundag, S., 2009. Essential oil composition, antimicrobial and antioxidant activities of Satureja cuneifolia Ten. Food Chem. 112, 874–879. Peana, A.T., Aquila, P.S.D’, Panin, F., Serra, G., Pippia, P., Moretti, M.D.L., 2002. Antiinflammatory activity of linalool and linalyl acetate constituents of essential oils. Phytomedicine 9, 721–726. Rahman, A., Kang, S.C., 2009. In vitro control of food-borne and food spoilage bacteria by essential oil and ethanol extracts of Lonicera japonica Thunb. Food Chem. 116, 670–675. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. 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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Chemical composition, antimicrobial and antioxidant activities of essential oil from Gnaphlium affine Wei-Cai Zeng a, Rui-Xue Zhu a, Li-Rong Jia a, Hong Gao a,⇑, Yue Zheng b, Qun Sun c a
College of Light Industry, Textile and Food Engineering, Sichuan University, Chengdu 610065, PR China National Center for Food Safety and Technology, Illinois Institute of Technology, IL 60501-1957, USA c College of Life Sciences, Sichuan University, Chengdu 610064, PR China b
a r t i c l e
i n f o
Article history: Received 14 January 2011 Accepted 10 March 2011 Available online 15 March 2011 Keywords: Gnaphlium affine Chemical composition Antimicrobial Antioxidant
a b s t r a c t The chemical composition of the essential oil from Gnaphlium affine was determined, and its antimicrobial and antioxidant activities were evaluated. Twenty-four compounds, representing 94.95% of the amount of total oil, were identified by gas chromatography–mass spectrometry (GC–MS) analysis. Main constituents of the essential oil were found to be eugenol (18.24%), linalool (10.62%), trans-caryophyllene (8.86%), a-terpineol (5.97%), p-cymene (5.75%), hexadecanoic acid (5.63%), c-cadinene (4.98%), d-cadinene (4.22%), a-humulene (3.22%), and ()-b-elemene (3.15%). The essential oil revealed a remarkable antimicrobial effect against the tested food-borne microorganisms with the MIC and MBC values in the ranges of 0.2–1.56 lg/ml and 0.39–3.13 lg/ml, respectively. The essential oil showed a potent antioxidant activity in ABTS radical scavenging, lipid peroxidation and reducing power assay. It was suggested that the essential oil from G. affine may be a new potential source as natural antimicrobial and antioxidant agents applied in food systems. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Gnaphlium affine, normally named Cudweed or Ching Ming vegetable in China, belongs to Asteraceae and has been used as food and traditional medicinal plant (Lu et al., 2009). Every year after the traditional Ching Ming festival, it is extensively harvested nationally as a wild vegetable and then be processed into a variety of foods, such as drinks, canned products and frozen vegetables. G. affine is believed to be of high nutritional value since it has the eight essential amino acids for human body of a reasonable proportion, a high content of minerals, trace elements, and vitamins, thus to be developed into a functional food (Wang et al., 2005; Chen, 1999). Besides the nutritional value, G. affine was reported to possess many pharmacological activities. G. affine was observed to lower the blood pressure in human (Liu, 1965). A recent study showed that G. affine exhibited the protective effect for carbon tetrachloride-induced acute liver injury (Jiang et al., 2008). Furthermore, G. affine had been reported to be useful for the treatment of respiratory diseases (Tian, 1997). Microbial contamination and oxidation in foods not only result in food deterioration and shelf life reduction, but also lead to disease and economic losses. At present, the food industry is facing an enormous pressure coming from food deterioration caused by ⇑ Corresponding author. Tel.: +86 28 85405236; fax: +86 28 85405137. E-mail address: [email protected] (H. Gao). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.03.014
the two factors above. The growth of microorganisms in food products may cause intestinal disorders, vomiting and diarrhea (Friedman et al., 2002). It has been reported that in the global incidence, 1.8 million people died from diarrhea disease in 2005 alone. A great proportion of these can be attributed to microbial contamination in food and drinking water. It has been estimated that as many as 30% of people in industrialized countries suffer from a food-borne disease each year. With the environment pollution, more population will suffer from food-borne diseases (Loizzo et al., 2010). During the raw material storage, processing, heat treatment and further storage of final products, oxidation is another familiar processes causing rancidity in food products, leading to the degradation of lipids and proteins, and contributes to the deterioration in flavor, texture, and color of food products. The odors and flavors from oxidation can easily destroy the organoleptic and nutritional quality of processed foods (Karpin´ska et al., 2001). In addition, reactive oxygen species from the oxidative processes in food, such as superoxide radicals, hydroxyl radical, and peroxyl radicals, are involved in the onset of many diseases such as carcinogenesis, coronary heart disease, and many other health issues related to advancing age (Borneo et al., 2009). Essential oils (also called volatile oils) are natural volatile complex compounds characterized by a strong smell, and form in edible, medicinal and herbal plants as secondary metabolites (Burt, 2004). They can be obtained by compression, enfleurage or extraction but the method of steam distillation is most commonly
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used. For the various and remarkable biological properties already observed in nature, essential oils have been largely utilized in perfumes, make-up products and sanitary products, in food industry as food additives or food preservers, in dentistry as natural remedies and in agriculture as green pesticides. A variety of studies had been carried out to evaluate the antimicrobial and antioxidant activities of essential oils, and the results indicated that essential oil did have noticeable antimicrobial and antioxidant activities (Bakkali et al., 2008; Rahman and Kang, 2009). To the best of our knowledge and literary survey, there is no information available on the chemical composition analysis, antimicrobial and antioxidant activities of essential oil from G. affine. Therefore, efforts have been made to investigate the role of this essential oil for its antimicrobial and antioxidant potential. The aims of this work are to detect the chemical composition of essential oil from G. affine by gas chromatography–mass spectrometry (GC–MS) and evaluate its in vitro antimicrobial and antioxidant properties. 2. Materials and methods 2.1. Plant materials and chemicals The aerial parts (leaves and flowers) of G. affine were collected from Chengdu city area of China, in March 2010. The plant was initially identified by morphological features and the database was present in Department of Biology, Sichuan University. A voucher specimen was dried and preserved at the Key Laboratory of Food Science and Technology of Sichuan Province, Sichuan University, China. Ascorbic acid (Vc), 2,20 -azinobis-3-ethylbenzthiazoline-6-sulfonate (ABTS), sodium dodecyl sulfate, acetic acid, potassium persulfate, thiobarbituric acid, ferrous sulfate, anhydrous sodium sulfate, n-hexane, methanol, phosphate balanced solution (PBS), glutaraldehyde, osmium tetroxide, acetone, epoxy, 1-butanol, ethanol, potassium ferricyanide, trichloroacetic acid and ferric chloride were purchased from Sigma–Aldrich (St. Louis, MO). Gentamicin was obtained from Sichuan Changwei Pharmaceutical Co. Ltd., China. Agar, beef extract, sucrose and peptone were purchased from Bio-Ketai (Langsan Ketai Biological Products Co. Ltd., China). Potatoes were obtained from a local supermarket in Chengdu, China and stored at 4 °C until used for experiment. The solvents for GC–MS analysis were of HPLCgrade. All other reagents used were of analytical grade. 2.2. Preparation of essential oil The essential oil was extracted by the method of hydro-distillation. The airdried (25 °C, 15 d) plant samples (300 g) were crushed into powder (about 40 granularities) with a mixer (JYL-350, Jiuyang Co. Ltd., China) and subjected to hydro-distillation for 8 h, using a Clevenger-type apparatus (Shudu Co. Ltd., China). The oil was collected and dried over anhydrous sodium sulfate and then stored in sealed glass vials at 4 °C prior to further analysis. The yield based on dry weight of the sample was 0.86% (2.58 g/300 g dry samples). 2.3. GC–MS analysis GC–MS analysis of the essential oil was performed using a Thermo GC-MSD (Trace DSQ II, Thermo Fisher Corporation, USA) apparatus equipped with a TR-5 capillary column (30 m 0.25 mm internal diameter, 0.25 m film thickness). Helium gas was used as the carrier gas at a constant flow rate of 1 ml/min. For GC– MS detection, an electron ionization system was used with ionization energy set at 70 eV, and mass range at m/z 40–500. Injector and MS transfer line temperature were set at 250 °C and 280 °C, respectively. The sample of diluted essential oil solution (1:100, v/v, in n-hexane) of 1 ll was injected and analyzed with the column maintained initially at 50 °C for 1 min and then increased to 250 °C with a 5 °C/ min heating ramp and subsequently kept at 250 °C for 20 min. The relative percentages of individual components of the essential oil were expressed as percentages of the peak area relative to the total peak area. The compounds of essential oil were identified on the basis of comparison of their retention time and mass spectra with publish data (Adams, 2001) and computer matching with National Institute of Standards and Technology (NIST, 3.0) libraries provided with computer controlling the GC–MS system. 2.4. The tested microorganisms The following food spoilage and food-borne microorganisms including Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 21216, Bacillus cereus ATCC 10231, Bacillus laterosporus laubach ATCC 64, Salmonella typhimurium ATCC 14028, Saccharomyces cerevisiae ATCC 9763, Aspergillus niger
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ATCC 16404, Pencicillium citrinum ATCC 14994, Rhizopus oryzae ATCC 9363, and Aspergillus flavus ATCC 204304, were used as the tested microorganisms. All the microbial strains were maintained on culture medium at 4 °C and were sub-cultured every month in our laboratory. 2.5. In vitro antimicrobial assay 2.5.1. Inhibitory zone assay The essential oil was dissolved in 80% methanol (diluted with sterile water) to a final concentration of 50 mg/ml and sterilized by filtrating through 0.22 lm Millipore filters. Antimicrobial activity of the essential oil was determined by Oxford plate method (Beverlya et al., 2008). In brief, all the microbial cultures (sub-cultured before assay) were diluted with sterile water to obtain a bacterial suspension of 106 CFU/ml. Petri plates containing 20 ml of culture medium were inoculated with 200 ll of bacterial suspension and allowed to dry in sterile chamber. The Oxford plates (6 mm in diameter) were impregnated with 100 ll of 50 mg/ml sample and placed on the inoculated culture medium. The 80% methanol was also used as the negative control. Gentamicin (50 lg/disk) was used as positive control. The plates of bacteria inoculated were incubated at 37 °C for 24 h, while fungi at 28–32 °C for 72 h, and yeast at 28 °C for 48 h. The antimicrobial activity was evaluated by measuring the inhibition zone against the tested microorganisms. 2.5.2. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) The minimum inhibitory concentration (MIC) of the essential oil was evaluated for the bacterial strains which were determined by the method of broth dilution. An aliquot of 2 ml of nutrient broth was placed into each tube, and all tubes were autoclaved at 121 °C for 20 min. The essential oil (filtered, 0.22 lm) was first dissolved in methanol, and added to the tubes to keep the final concentrations of 0.2–50 lg/ml. The test bacteria suspension was added into to the inoculum size of 106 CFU/ml. Then, the inoculated tubes were incubated at 37.5 °C for 18–24 h. The final concentration of methanol in culture medium was maintained at 0.1% (v/v). Another culture medium without adding bacteria suspension was prepared as the negative control. The MIC was defined as the lowest concentration of the essential oil which inhibited the visible growth of tested microorganism. The minimum bactericidal concentration (MBC) of the essential oil was determined according to the MIC values. The samples showing no increases in turbidity were streaked on nutrient agar medium and incubated at 37.5 °C for 18–24 h. The lowest concentration of the essential oil, which did not show any viable bacteria, was determined as the MBC. 2.5.3. Influence on the growth of B. subtilis Influence of the essential oil on the growth of B. subtilis was evaluated using the method of growth curve. B. subtilis cultured to logarithmic growth phase was diluted with nutrient broth to the inoculum size of 106 CFU/ml. The essential oil (filtered, 0.22 lm) was first dissolved in methanol, and added to nutrient broth to keep the final concentrations as the MIC value of B. subtilis. Final concentration of methanol in nutrient broth was maintained at 0.1% (v/v). Then, the culture was incubated in incubation shaker (37.5 °C, 130 rpm). The growth of B. subtilis was evaluated by determining the optical density of culture at 600 nm per 2 h using the Lambda 25 UV–visible spectrophotometer (PerkinElmer Co. Ltd., USA), ending at 24 h. The culture without the essential oil was used as negative control. 2.5.4. Observation with transmission electron microscope (TEM) B. subtilis cultured in Section 2.5.3 was collected and prepared for TEM analysis. Briefly, the treated B. subtilis was centrifugal at 5000 rpm at 25 °C for 10 min, and the precipitation was washed three times with PBS (0.1 M, pH 7.4). An amount of 0.5% of glutaraldehyde was added to the precipitation keeping 15 min at 4 °C. Then, bacterial cells were collected by 20 min centrifugation at 15,000 rpm at 4 °C. After deal with 3% glutaraldehyde, 1% osmium tetroxide, acetone and epoxy, the sample was cut into thin sections using a microtome (Ultracut-E, Reichert-Jung, Austria). Then the sample was observed by TEM (H-600IV, Hitachi, Japan). 2.6. In vitro antioxidant assay 2.6.1. ABTS radical scavenging assay The ABTS radical scavenging assay was estimated according to the previously reported procedure with a slight modification (Re et al., 1999). The essential oil was first dissolved in ethanol at the ranges of 0.16–2.5 lg/ml. ABTS radical cation solution was prepared through the reaction of 7 mM ABTS and 2.45 mM potassium persulfate, after incubation at 23 °C in the dark for 16 h. The ABTS radical cation solution was then diluted with 80% methanol (dissolved with pure water) to obtain an absorbance of 0.700 ± 0.005 at 734 nm. ABTS radical cation solution (absorbance of 0.700 ± 0.005, 3.9 ml) was added to 0.1 ml of the test sample and mixed vigorously. The reaction mixture was allowed to stand at 23 °C for 6 min and the absorbance at 734 nm was immediately recorded by the Lambda 25 UV–visible spectrophotometer. Vc was used as a positive control. The ability to scavenge ABTS radical in percent was calculated as:
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Scavenging activity ð%Þ ¼ ð1 Asample 734 =Acontrol 734 Þ 100 where Acontrol
734
was the absorbance of control (80% methanol, instead of sample).
Table 1 The main chemical components of the essential oil from Gnaphlium affine. No.
2.6.2. Lipid peroxidation assay The lipid peroxidation assay was measured by the previously reported method with a minor modification (Dasgupta and De, 2004). Egg yolk homogenates were prepared as lipid-rich media. In brief, an amount of 0.1 ml of various concentrations (0.625, 1.25, 2.5, 5 and 10 lg/ml, respectively) of the essential oil in methanol were thoroughly mixed with 0.5 ml of egg yolk homogenate (10%, v/v, diluted with pure water) and made up to 1 ml with pure water. Ferrous sulfate (50 ll, 70 mM) was added to induce lipid peroxidation and the mixture incubated for 30 min at 37.5 °C. Then, 1.5 ml of 20% acetic acid (v/v, pH 3.5, diluted with pure water) and 1.5 ml of 0.8% (w/v) thiobarbituric acid in 1.1% sodium dodecyl sulfate (w/v, diluted with pure water) were added and the resulting mixture was vortexed and then heated at 95 °C for 60 min. After cooling, 5 ml of 1-butanol were added to each tube and centrifuged at 5000 rpm for 15 min. The organic upper layer was collected and the absorbance at 532 nm was immediately measured by the Lambda 25 UV–visible spectrophotometer. Vc was used as a positive control. The inhibition of lipid peroxidation was calculated as:
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Inhibition ð%Þ ¼ ð1 Asample 532 =Acontrol 532 Þ 100 where Acontrol
532
was the absorbance of control (methanol, instead of sample).
2.6.3. Reducing power assay The reducing power of the essential oil was determined according to the previously reported method with a minor modification (Yen and Chen, 1995). In brief, an amount of 1 ml various concentrations of samples (62.5, 125, 250, 500, 1000 and 2000 lg/ml, respectively) in methanol was mixed with 2.5 ml of PBS (0.2 M, pH 6.6) and 2.5 ml of potassium ferricyanide (1%, w/v). The mixture was incubated at 50 °C for 20 min. Then, trichloroacetic acid of 2 ml (10%, w/v) was added to the mixture to terminate the reaction, which was then centrifuged at 2000 rpm for 15 min. The upper layer of solution (2.5 ml) was mixed with pure water (2.5 ml) and ferric chloride (0.5 ml, 0.1%, w/v), and the absorbance was measured at 700 nm. Vc was used as a positive control in the assay. 2.7. Statistical analysis All analyses were conducted in triplicate and statistical analysis of the data was performed using SPSS 13.0 software (SPSS Inc., 223 South Wacker Drive, CO., USA). A probability value at p < 0.05 was considered statistically significant. Data were presented as mean value ± standard deviation (SD), which were calculated from triplicate determinations.
3. Results 3.1. Chemical composition of the essential oil The essential oil extracted from the air-dried aerial parts (leaves and flowers) of G. affine by hydro-distillation showed a color of light yellow. GC–MS analyses of the essential oil led to identification of 24 different compounds, representing 94.95% of the total amount of oil extracted. The identified compounds with their retention time and their percentage are listed in Table 1. All the compounds were arranged in the order of their retention time. The essential oil was a complex mixture mainly of oxygenated monoterpenes and sesquiterpenes, and hydrocarbons. It contained hydrocarbons (66.88%) and hydroxybenzene (18.24%). The major compounds in the essential oil were eugenol (18.24%), linalool (10.62%), trans-caryophyllene (8.86%), a-terpineol (5.97%), p-cymene (5.75%), hexadecanoic acid (5.63%), c-cadinene (4.98%), d-cadinene (4.22%), a-humulene (3.22%) and ()-b-elemene (3.15%). The structures of the major bioactive compounds are shown in Fig. 1. In the present study, elemol (0.76%), tetradecane (0.96%), a-cedrol (1.08%), nonadecane (1.44), 7-octen-4-ol (1.53%), [z,z]-9,12-octadecadienoic acid (1.38%) and tetradecanoic acid (1.59%) were also detected to be the minor components in the essential oil from G. affine. 3.2. In vitro antimicrobial activity The in vitro antimicrobial activity of the essential oil from G. affine against the tested microorganisms was qualitatively and quantitatively assessed by the inhibition zones. According to the
RTa
Compound
Composition (%)b
6.37 8.21 8.56 10.25 10.69 13.87 14.23 14.48 14.85 15.21 15.69 15.98 16.33 16.73 17.15 18.09 19.13 19.25 20.16 20.58 21.87 23.15 25.37 30.69
7-Octen-4-ol p-Cymene Linalool 2-Ethenyl-1,4-dimethylbenzene a-Terpineol Eugenol Tetradecane ()-b-Elemene a-Gurjunene trans-Caryophyllene a-Humulene c-Cadinene c-Gurjunene d-Cadinene Elemol a-Cedrol Heptadecane 2,6,10,14-Tetramethylpentadecane Tetradecanoic acid Octadecane Nonadecane Hexadecanoic acid [z,z]-9,12-Octadecadienoic acid Pentacosane
1.53 5.75 10.62 2.64 5.97 18.24 0.96 3.15 1.96 8.86 3.22 4.98 2.65 4.22 0.76 1.08 2.24 1.46 1.59 2.03 1.44 5.63 1.38 2.59
Total a b
94.95
RT, retention time. Composition (%), relative percentage based on peak area.
results shown in Table 2, the essential oil exhibited a potent inhibitory effect against all bacteria (E. coli, S. aureus, B. subtilis, B. cereus, B. laubach, and S. typhimurium), yeast (S. cerevisiae) and fungi (A. niger, P. citrinum, R. oryzae and A. flavus) with diameter of inhibition zones ranging from 15.43 to 24.73 mm, while it was 23.13–30.53 mm for the positive control. The essential oil showed a high antimicrobial effect on S. cerevisiae, A. flavus, R. oryzae and S. aureus with the diameter of inhibition zones of 24.73, 23.33, 22.60 and 21.20 mm, respectively. On the other hand, fungi were more sensitive than bacteria to this essential oil and S. cerevisiae was found to be the most susceptible one. Additionally, no inhibitory effect on the tested microorganisms was observed for the solvent (80% methanol) at the used concentration. 3.3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) MIC and MBC values of essential oil against the employed bacterial strains are shown in Table 2. These results demonstrated that this oil displayed remarkable antibacterial and bactericidal property. In general, the MIC values of the essential oil against the tested bacteria ranged from 0.2 lg/ml to 1.56 lg/ml and MBC from 0.39 lg/ml to 3.13 lg/ml, respectively. In addition, the MIC and MBC values of fungi were lower than that of bacteria. By considering the results of inhibition zone assay in Table 2, S. cerevisiae was the most sensitive fungi while S. aureus the most sensitive bacterium. Solvents did not inhibit the growth of any of tested microorganisms at the used concentration. 3.4. Effect of essential oil on the growth of B. subtilis In Fig. 2A, a prominent inhibitory zone (17.5 mm of diameter, 5 mg essential oil/dish) was present in the center of the petri dish. It was demonstrated that the essential oil from G. affine could inhibit the proliferation of B. subtilis in the nutrient agar medium. The growth curve of B. subtilis with the presence of the essential oil extracted is shown in Fig. 2B. Both the essential oil and the control
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Fig. 1. The chemical structures of major bioactive compounds of the essential oil from Gnaphlium affine. (a) eugenol; (b) linalool; (c) p-cymene; (d) a-terpineol; and (e) transcaryophyllene.
Table 2 Antimicrobial activity of the essential oil from Gnaphlium affine against food-borne and spoilage microorganisms. Microorganisms Strain
E. coli S. aureus B. subtilis B. cereus B. laubach S. typhimurium S. cerevisiae A. niger P. citrinum R. oryzae A. flavus a b c d e
ATCC 25922 ATCC 25923 ATCC 21216 ATCC 10231 ATCC 64 ATCC 14028 ATCC 9763 ATCC 16404 ATCC 14994 ATCC 9363 ATCC 204304
Inhibition zone (mm)
Essential oil (lg/ml)
Essential oila Positive controlb
MICc
MBCd
15.43 ± 0.89e 24.47 ± 0.97 1.56 ± 0.41 3.13 ± 0.52 21.20 ± 1.04 25.67 ± 0.79 0.39 ± 0.12 0.78 ± 0.21 17.50 ± 0.86 23.77 ± 0.78 0.78 ± 0.30 1.56 ± 0.15 17.70 ± 0.86 23.30 ± 1.31 0.78 ± 0.11 1.56 ± 0.24 16.43 ± 0.53 23.13 ± 0.83 0.78 ± 0.13 3.13 ± 0.18 17.50 ± 0.89 23.73 ± 0.69 0.78 ± 0.19 3.13 ± 0.25 24.73 ± 0.74 27.57 ± 0.78 0.20 ± 0.21 0.39 ± 0.16 19.47 ± 0.94 28.80 ± 0.94 0.20 ± 0.18 0.39 ± 0.14 18.80 ± 1.00 30.53 ± 0.92 0.20 ± 0.15 0.78 ± 0.20 22.60 ± 0.91 25.73 ± 0.69 0.20 ± 0.19 0.78 ± 0.11 23.33 ± 1.11 26.50 ± 1.02 0.20 ± 0.20 0.39 ± 0.16
The concentration of essential oil was 5 mg/disk. Gentamicin (50 lg/disk) was used as positive control. Minimum inhibitory concentrations. Minimum bactericidal concentrations. Each value is expressed as means ± SD (n = 3).
had a lag phase in first 6 h. From 6 h, the OD600 of control increased at a faster rate, and then kept stable until the end. However, the OD600 of essential oil treatment almost did not increase and kept
Fig. 2. Influence of the essential oil of Gnaphlium affine on the growth of B. subtilis. (A) Inhibitory effect of the essential oil (17.5 mm of inhibitory zone diameter against B. subtilis at the final concentration of 5 mg/ml) on B. subtilis; and (B) the growth curve of B. subtilis.
at a low value till the end. The difference of OD600 between the control and the essential oil group indicated that the growth of B. subtilis had been inhibited obviously under the presence of essential oil from G. affine.
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Fig. 3. External morphology of B. subtilis cells observed by transmission electron microscope. (A) and (B), untreated bacteria; (C) and (D) bacteria treated with the essential oil from Gnaphlium affine.
3.5. Morphological changes in B. subtilis after essential oil treatment
3.6. ABTS radical scavenging activity
As shown in Fig. 3, TEM analysis showed the change of external morphological features of B. subtilis treated by the essential oil. The untreated B. subtilis cells retained their connatural morphology (claviform and elliptical) and seemed to be normal (Fig. 3A). In contrast, B. subtilis cells treated with the essential oil appeared to undergo lysis, resulting in the release of their cellular contents into surrounding environment, and finally became empty (Fig. 3C). Compared with undamaged cells, it was easy to find cell debris around the damaged cells showing electron translucent cytoplasm. By observation at a greater magnification, the untreated cells of B. subtilis showed normal morphological characteristics and homogeneous electron density in cytoplasm. Their cell walls and membranes were intact, showing a well maintained peptidoglycan layer and cytoplasmic membrane (Fig. 3B). In contrast, significant morphological changes were observed in B. subtilis cells treated with the essential oil. They were lysed cells, and the cell walls and membranes were broken with decreases of heterogeneity in electron density in cytoplasm (Fig. 3D). The localized separation of cell membrane from cell wall could be discerned and the cellular degradation was also accompanied with the outflow of electrontranslucent cytoplasm in damaged cells.
ABTS assay is based on the antioxidant ability of sample to react with ABTS radical cation generated in the assay system. ABTS radical scavenging activity of the essential oil from G. affine at various concentrations is shown in Fig. 4A. At the concentrations of 0.16–2.5 lg/ml, scavenging ability of the essential oil on ABTS radical was in ranges of 21.01–100%. At the concentration of 2.5 lg/ml, the essential oil was observed to possess strong free radical scavenging activity against ABTS radical with a value of around 100%, while the scavenging activity of control (Vc) was only 5.89%. Moreover, as shown in Fig. 4A, a concentration-dependent ABTS radical scavenging activity was observed with the half-inhibitory concentration (IC50) being 0.32 ± 0.89 lg/ml. On the other hand, IC50 value of Vc was 24.06 ± 0.73 lg /ml. Evidently, ABTS radical scavenging activity of Vc was even weaker than essential oil in our antioxidant assay. 3.7. Lipid peroxidation Egg yolk lipids undergo rapid non-enzymatic peroxidation when incubated in the presence of ferrous sulfate. The effect of the essential oil from G. affine on non-enzymatic peroxidation is
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At the concentrations of 62.5, 125, 250, 500, 1000, and 2000 lg/ ml, reducing powers were around 0.283, 0.694, 0.852, 0.913, 0.966, and 0.983 for essential oil and 0.152, 0.496, 0.742, 0.835, 0.928, and 0.947 for positive control of Vc, respectively. The increase at absorbance of reaction mixture indicated the increase of reducing power. This phenomenon clearly showed that the essential oil had a stronger reducing power than Vc. Meanwhile, as also shown in Fig. 4C, reducing power of the essential oil and Vc was increasing with high concentration.
4. Discussion
Fig. 4. Antioxidant activity of the essential oil from Gnaphlium affine. (A) Scavenging effect of the essential oil on ABTS radical; (B) inhibitory effect of the essential oil on lipid peroxidation; and (C) reducing power of the essential oil.
shown in Fig. 4B. In the experimental model of egg yolk homogenates, a significant inhibitory effect of the essential oil on lipid peroxidation was shown with IC50 value of 0.09 ± 0.75 lg/ml (IC50 of Vc = 6.73 ± 0.87 lg/ml). Moreover, as shown in Fig. 4B, at the concentrations of 0.625–5 lg/ml, the inhibition activity of the essential oil on lipid peroxidation ranged from 61.32% to 71.25%. At the concentration of 10 lg/ml, scavenging activity of Vc and the essential oil were 56.27% and 75.08%, respectively. Meanwhile, a concentration-dependent inhibitory effect on lipid peroxidation was observed for the essential oil. It was worth to note that the inhibition activity of Vc against lipid peroxidation was lower than essential oil used in this assay. 3.8. Reducing ability In reducing power assay, the presence of antioxidants in samples would result in the reducing of Fe3+–Fe2+ by donating an electron. An amount of Fe2+ complex can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm (Gholivand et al., 2010). Increasing absorbance at 700 nm indicated an increase in reductive ability. Reducing ability of the essential oil from G. affine is shown in Fig. 4C. As the antioxidant activities above, reducing ability of the essential oil correlated well with its concentration.
In spite of modern improvements in food production and food processing techniques, a high amount of food products are still perishable during their preparation, storage and distribution thus not to give them desired shelf-life. Although increasing use of chemical preservatives can effectively prevent the growth of most foodborne bacteria, safety problems related to chemical preservatives are receiving growing attention (Deba et al., 2008). Therefore, much effort has been expended in the search for new types of effective and nontoxic antimicrobial compounds from natural materials. One such possibility is the use of essential oils as antimicrobial additives (Holley and Patel, 2005). In the present study, the results of in vitro antimicrobial assay showed that the essential oil from G. affine had high antimicrobial activity against some representative food-borne spoilage pathogens, especially fungi. Antimicrobial activity of the essential oil from G. affine could be contributed to the presence of some major bioactive compounds contained, such as eugenol (Fig. 1a) and linalool (Fig. 1b). Eugenol, a major phenolic component of clove essential oil, has been widely used in medical and dental practice, due to its potent fungicidal, bactericidal, anesthetic, antioxidant and anti-inflammatory properties (He et al., 2007). It is used in the form of paste or mixture as dental cement, filler and restorative material. It belongs to the class of essential oils that is generally recognized as safe (GRAS) by the Food and Drug Administration (Hemaiswarya and Doble, 2009). As a well-known antimicrobial agent against food-borne pathogens, it has been reported to act primarily by disrupting the cytoplasmic membrane (Hemaiswarya and Doble, 2009). Linalool, a monoterpene alcohol, is the second abundant compound in essential oil and widely used in decorative cosmetics, fine fragrances, shampoos, toilet soaps and other toiletries (Letizia et al., 2003). It is usually present in essential oils of many medicinal and edible plants of a variety of biological activities, including antioxidant, antimicrobial, antiinflammatory, and antitumor properties (Letizia et al., 2003). Moreover, linalool also could enhance the permeability of a number of drugs through biological tissues like skin or mucus membranes (Peana et al., 2002). According to the previous studies, the other bioactive components in this essential oil, such as trans-caryophyllene (Fig. 1c), a-terpineol (Fig. 1d) and p-cymene (Fig. 1e), may also contribute to the antimicrobial activity (11). Our results suggested that phenolic compounds, monoterpenes, and sesquiterpenes in the essential oil from G. affine be the main bioactive constituents for the antimicrobial activity. Further, the results of growth curve and TEM image of B. subtilis revealed that the essential oil had a severe impact on the cell viability of B. subtilis. In the logarithmic growth phase, B. subtilis was inhibited obviously upon the presence of the essential oil (Fig. 2B). Observed under TEM, cell walls and membranes of B. subtilis treated by the essential oil were damaged, and the cytoplasm decreased, and finally the cells retained empty. These morphological changes may attribute to aromatic and phenolic compounds in essential oil. There is overwhelming consensus that the essential oil exerts its antimicrobial effect at cytoplasmic membrane by altering the structure and function of bacteria (Holley and Patel, 2005). A previous report
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showed that efflux of K+ is usually an early sign of damage (Walsh et al., 2003) and then often followed by efflux of cytoplasmic constituents including ATP (adenosine-triphosphate) (Brul and Coote, 1999). The loss of differential permeability character of the cytoplasmic membrane is frequently identified as the cause of cell death. As shown in Fig. 3D, cells of B. subtilis with an empty trunk were dead with an empty trunk. Furthermore, the dysfunction and disruption of the membrane, interference with the energy generation system in cell, and enzyme inhibition preventing substrate utilization for energy production may also lead to the death of bacterial cells (Holley and Patel, 2005). In addition, the essential oil possessing the strongest antimicrobial properties contains a high percentage of phenolic compounds, such as carvacrol, eugenol and thymol (Dorman and Deans, 2000). It seems reasonable to assume that the mechanism of their action would therefore be similar to that of other phenolics. This is generally considered to be the disturbance of the cytoplasmic membrane, resulting in disrupting the proton motive force, electron flow, active transport and coagulation of cell contents (Burt, 2004). Thus, the essential oil from G. affine in this study could be a potential source as inhibitory substances against some foodborne spoilage and pathogens and be candidates to be applied in foods or food-processing systems. Oxidation is an essential biological process for most organisms to produce energy. Meanwhile, fulsome or uncontrolled oxidations also happen. Uncontrolled production of superoxide anion free radicals is involved in the onset of many diseases such as cancer, atherosclerosis and degenerative processes with aging (Chen et al., 2005). Our results of various free radicals scavenging activity revealed that the essential oil from G. affine had a significant antioxidant activity even compared with Vc. As shown in Fig. 4, not only the free radical scavenging activity of the essential oil was better than Vc, but also the reducing power. The antioxidant activity may be contributed by different mechanisms, such as prevention of chain initiation, decomposition of peroxides, prevention of continued hydrogen abstraction, free radical scavenging, reducing capacity, and binding of transition metal ion catalysts (Jia et al., 2010). In present study, the remarkable antioxidant activity of essential oil may be contributed by the high content of phenolic compounds contained. Phenolic compounds, generally considered as biologically active components, are the main agents to donate hydrogen to free radicals and thus break the chain reaction of lipid oxidation at the first initiation step. This high potential of phenolic compounds to scavenge radicals may be explained by their phenolic hydroxyl groups (Oke et al., 2009). A number of studies about the health benefits of bioactive phenols have increased lately. Recent research suggested that phenolic compounds may exhibit beneficial health effects and manifold pharmaceutical functions for human (Cai et al., 2004). In conclusion, results of the present study suggested that the essential oil from G. affine can be useful for a new potential source as natural antimicrobial and antioxidant agent in food systems. Further studies are undergoing for the antimicrobial assessment of this essential oil in food models and the toxicological evaluation for the safety usage of the oil used in food products. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements We are grateful to Dr. Yi-Na Huang, Department of Public Health, Sichuan University, and Dr. Su Feng, College of Life Sciences, Sichuan University, for their helpful comments and suggestions. This work was financially supported by the National Natural
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