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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Chemical composition, antimicrobial and antioxidant activities of essential oil from Ampelopsis megalophylla a
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Xian-Fei Xie , Jian-Wu Wang , Han-Ping Zhang , Qi-Xiong Li & Bang-Yin Chen
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Department of Pharmacology, Basic Medical School, Wuhan University, Wuhan 430072, P.R. China b
College of Pharmacy, Huazhong University of Science and Technology, Wuhan 430030, P.R. China Published online: 13 Feb 2014.
To cite this article: Xian-Fei Xie, Jian-Wu Wang, Han-Ping Zhang, Qi-Xiong Li & Bang-Yin Chen , Natural Product Research (2014): Chemical composition, antimicrobial and antioxidant activities of essential oil from Ampelopsis megalophylla, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.886208 To link to this article: http://dx.doi.org/10.1080/14786419.2014.886208
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.886208
Chemical composition, antimicrobial and antioxidant activities of essential oil from Ampelopsis megalophylla Xian-Fei Xiea*, Jian-Wu Wangb, Han-Ping Zhangb, Qi-Xiong Lia and Bang-Yin Chenb a
Department of Pharmacology, Basic Medical School, Wuhan University, Wuhan 430072, P.R. China; College of Pharmacy, Huazhong University of Science and Technology, Wuhan 430030, P.R. China
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(Received 19 October 2013; final version received 19 January 2014) Chemical composition, antimicrobial and antioxidant activities of the essential oil of Ampelopsis megalophylla were evaluated in this research. GC– MS analysis of the essential oil revealed 42 compounds, representing 88.54% of the oil. The major compounds were borneol (10.81%), a-pinene (6.74%) and b-elemene (6.23%). The antimicrobial activity of the essential oil was evaluated against 13 micro-organisms using the disc diffusion and broth microdilution methods. Results demonstrated higher effects of this oil against Gram-positive bacteria than the other reference strains tested. The antioxidant effect of the essential oil was evaluated by using 1,1-Diphenyl-2picrylhydrazyl (DPPH) and 2,20-azinobis-3-ethylbenzthiazoline-6-sulfonate scavenging assays. The essential oil exhibited moderate antioxidant activity. Keywords: Ampelopsis megalophylla; essential oil; GC – MS; antimicrobial; antioxidant
1. Introduction Plant-derived natural chemicals, known as secondary metabolites, have been widely used in dietary regimens, food preservation, folk medicine and fragrance industry (Kalemba & Kunicka 2003; Huang et al. 2005). Application of plant materials as dietary regimens and preservatives is mainly due to their antimicrobial, antioxidant and other biological potentials. The essential oils and extracts of many plant species have become popular in recent years, and attempts to characterise their antimicrobial and antioxidant activity and their use in pharmaceutical or foods processing are recommended (Vardar-Unlu et al. 2003; Sokmen, Serkedjieva, et al. 2004). Ampelopsis megalophylla Diels et. Gilg is a medicinal plant of Ampelopsis, which is an original plant of the ethnic medicine used commonly in the western parts of Hubei Province in China and most often consumed as an herbal tea. Its consumption in this manner could be utilised in the treatment of hypertension in clinical practice with significant effect. Preliminary researches (Zhang, Shen, Chen, et al. 2008; Shen et al. 2010) indicate that flavonoids such as ampelopsin, myricetin and myricitrin are its main effective substances. Some reports have shown that A. megalophylla possesses antihypertensive, antivirus and antihyperglycaemic effects (Huang et al. 2006; Zhang, Shen, Chen, Chen, et al. 2008; Yang et al. 2011). To the best of our knowledge and literary survey, there is no information available on the chemical composition, antimicrobial and antioxidant activities of essential oil from A. megalophylla. Therefore, the aim of this study was to determine the chemical composition of the hydro-distilled essential oil from leaves of A. megalophylla by GC –MS and to evaluate its antimicrobial and antioxidant properties.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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2. Results and discussion 2.1. Chemical composition of the essential oil The essential oils were obtained by hydro-distillation from the dried leaves of A. megalophylla with the yield of 0.42% (v/w) on a dry-weight basis which was in the normal range of the amount we usually find (Xie et al. 2007; Abdollah et al. 2013). A total of 42 compounds (88.54% of the total oil) were identified by using two chromatographic techniques. Qualitative and quantitative analytical results by GC –MS are shown in Table 1. The components of the essential oil were separated into four classes: monoterpene hydrocarbons,
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Table 1. Main chemical composition of A. megalophylla essential oil. Compounds a-Thujene a-Pinene Camphene Sabinene 1-Octen-3-ol Myrcene a-Terpinene p-Cymene 1,8-Cineole g-Terpinene Linalool Camphor Pinocarvone Borneol Terpinene-4-ol a-Terpineol Myrtenol trans-Carveol cis-Piperitone oxide Carvone Bornyl acetate Thymol Carvacrol r-Menth-3-3-en-8-ol, acetate Eugenol b-Elemene Longifolene b-Caryophyllene a-Humulene Dodecanal Germacrene D b-Selinene (E,E)-a-Farnesene Germacrene A g-Cadinene d-Cadinene Spathulenol Caryophyllene oxide epi-a-Cadinol T-Cadinol b-Eudesmol a-Bisabolol a b
Retention indices on DB-5 capillary column. Retention time (in min).
Percentage (v/w)
RIa
RTb
1.22 6.74 1.24 3.78 1.56 2.34 0.82 1.33 1.56 3.12 1.05 5.12 2.46 10.81 3.77 0.43 0.33 1.27 0.23 0.15 1.57 2.36 1.29 1.08 1.46 6.23 3.45 1.79 3.41 0.12 0.93 2.67 1.42 1.56 1.12 0.78 0.17 3.43 1.12 0.65 1.47 1.13
930 934 949 972 976 991 1016 1023 1031 1057 1100 1146 1163 1168 1178 1193 1197 1205 1232 1243 1283 1294 1298 1316 1348 1379 1405 1426 1453 1464 1484 1498 1503 1509 1518 1524 1576 1582 1634 1641 1652 1681
9.62 9.91 10.72 11.45 11.74 12.75 13.87 14.32 14.93 15.88 17.84 20.58 21.41 21.78 22.37 22.85 23.13 23.46 24.52 25.03 26.87 27.50 27.74 28.45 29.79 30.94 31.76 32.50 33.61 34.26 35.19 35.66 35.87 36.08 36.47 36.75 40.13 40.78 44.15 44.62 45.23 47.07
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oxygenated monoterpenes, sesquiterpene hydrocarbons and oxygenated sesquiterpenes, which were in the ratios of 22.15%, 34.94%, 23.48% and 7.97%, respectively. Borneol (10.81%), apinene (6.74%) and b-elemene (6.23%) were the main compounds of the essential oil, followed by camphor (5.12%), sabinene (3.78%) and terpinene-4-ol (3.77%).
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2.2. Antimicrobial activity The diameters of inhibition zones including disc diameter (DDs), minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the essential oils for the micro-organisms tested are shown in Table 2. Although the agar disc diffusion method is time-consuming and labour-intensive, it is still a reliable and effective technique which is most often used by many laboratories for antimicrobial screening. Therefore, this method was employed for the determination of the antimicrobial activity by us (Xie et al. 2007; Yu et al. 2007; Elena et al. 2013). Data obtained from the disc diffusion method indicated that Staphylococcus epidermidis was the most sensitive micro-organism to the essential oil with the strongest inhibition zone (29 mm), while Pseudomonas aeruginosa ATCC 27853 exhibited weak inhibition zones (12 mm). Although the results of MICs and MBCs varied between the organisms tested, in most cases the MIC was equivalent to the MBC, indicating a bactericidal activity of the oil. The results of MIC indicated that the oils inhibited all the micro-organisms tested. The Gram-positive bacteria were more sensitive to the oils than Gram-negative bacteria and yeast. S. epidermidis had the lowest MIC (0.39 mg mL21). The highest MIC was 25.00 mg mL21 for P. aeruginosa ATCC 27853 and Candida albicans ATCC 14053. The lowest MBC was 0.39 mg mL21 for S. epidermidis. P. aeruginosa ATCC 27853 had the highest MBC of 50.00 mg mL21. The antimicrobial activities of the essential oil could be attributed to the presence of high concentrations of borneol, a-pinene, camphor and terpinene-4-ol, which possess Table 2. Antimicrobial activities of A. megalophylla essential oil. Essential oil Micro-organisms Reference strains S. aureus ATCC 25923 E. coli ATCC 25922 P. aeruginosa ATCC 27853 C. albicans ATCC 14053 Clinically isolated strains S. epidermidis S. simulans S. heamolyticus S. saprophyticus S. marcescens E. faecalis E. cloacae P. mirabilis K. pneumoniae
Levofloxacin
DDa
MICb
MBCb
DDc
MICd
MBCd
27 ^ 1.3a 21 ^ 1.1b 12 ^ 0.5d 13 ^ 0.5d
0.78 3.13 25.00 25.00.
0.78 3.13 50.00 25.00
34 ^ 1.6a 30 ^ 1.4ab 22 ^ 1.1c NT
0.30 0.30 4.88 NT
0.30 0.61 4.88 NT
29 ^ 1.4a 24 ^ 1.2ab 25 ^ 1.3ab 22 ^ 1.1b 15 ^ 0.7cd 17 ^ 0.8c 19 ^ 0.8bc 14 ^ 0.6d 16 ^ 0.7c
0.39 3.13 1.57 6.25 12.50 6.25 6.25 12.50 12.50
0.39 3.13 1.57 6.25 12.50 12.50 6.25 12.50 12.50
32 ^ 1.4a 29 ^ 1.3ab 25 ^ 1.2bc 27 ^ 1.3b 17 ^ 0.7cd 22 ^ 1.0c 17 ^ 0.6cd 14 ^ 0.4d 24 ^ 1.2bc
0.61 1.22 1.22 2.44 4.88 4.88 9.76 19.52 4.88
0.61 1.22 1.22 2.44 9.76 4.88 9.76 19.52 4.88
Notes: DD, diameter of zone of inhibition (mm) including disc diameter of 6 mm; NT, not test; MIC, minimum inhibitory concentration, MBC, minimum bactericidal concentration. Different letters within a column indicate statistically significant differences between the means ( p , 0.05). a Tested at a concentration of 3 mg disc21. b Values given as mg mL21. c Tested at a concentration of 5 mg disc21. d Values given as mg mL21.
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well-documented antimicrobial potential (Dorman & Deans 2000; Tabanca et al. 2001; VardarUnlu et al. 2003; Lopes-Lutz et al. 2008). Apart from the major compounds, b-caryophyllene, 1,8-cineole and p-cymene as well as other minor constituents of the essential oils of A. megalophylla also possessed antimicrobial activity (So¨kmen, Gulluce, et al. 2004; Jirovetz et al. 2006; Rosato et al. 2007). In fact, the synergistic effects of the diversity of major and minor constituents present in the essential oils should be considered to account for their biological activity (Delamare et al. 2007). Thanks to their extra-protective outer membrane, Gram-negative bacteria were usually considerably more resistant to antibacterial agents than their Gram-positive counterparts (Saier 2009). In this regard, the essential oil of the A. megalophylla expressed its best antibacterial activity in the MIC test on Gram-positive bacteria tested. 2.3. Antioxidant activity To assess the antioxidant activity of the essential oil, we evaluated its radical-scavenging activity against DPPH and 2,20-azinobis-3-ethylbenzthiazoline-6-sulfonate (ABTS). The DPPH molecule, which contains a stable free radical, had been extensively used to evaluate the radical-scavenging ability of antioxidants (Nagai et al. 2003). As shown in Table 3, the essential oil exhibited a dose-dependent scavenging activity against DPPH radical, with an IC50 value of 0.48 mg mL21. ABTS assay was based on the antioxidant ability of the sample to react with ABTSþ (radical cation) generated in the assay system (Tel et al. 2010; C´avar et al. 2012). However, unlike scavenging of DPPH radical, essential oil exhibited increased scavenging activity towards ABTSþ (IC50 0.36 mg mL21) (Table 3). This was probably due to steric factors that were one of the major factors for reducing the stable DPPH radical (C´avar et al. 2012). It had been reported that free radical-scavenging activity is greatly influenced by the phenolic composition of the samples (Cheung et al. 2003). Phenolic compounds, generally considered as biologically active components, were the principal agents to donate hydrogen to free radicals. This high potential of phenolic compounds to scavenge radicals might be explained by their phenolic hydroxyl groups (Oke et al. 2009). Therefore, phenolic compounds, which were present in some proportion of the essential oil such as thymol, carvacrol and g-terpinene (Table 1), could be the main compounds responsible for the antioxidant activity. 3. Experimental 3.1. Plant material The leaves of A. megalophylla were collected in Hubei Province of China in July of 2009. A voucher specimen (No. 0315) has been deposited at the herbarium of School of Medicine, Wuhan University, China. The plant was identified in the Laboratory of Botany at the same institution. Table 3. Radical-scavenging activities of A. megalophylla essential oil.a Inhibition rate (%) Concentration (mg mL Essential oil 0.2 0.4 0.8 IC50 of oil
21
)
DPPH
ABTS
15.2 ^ 1.08c 45.3 ^ 1.85b 62.7 ^ 2.46a 0.48
21.3 ^ 1.24c 54.6 ^ 2.34b 71.2 ^ 2.87a 0.36
Note: Different letters within a column indicate statistically significant differences between the means ( p , 0.05). a Results are means of three replicates.
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3.2. Chemicals 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and ABTS were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of analytical grade.
3.3. Essential oil extraction and GC – MS analysis The leaves of A. megalophylla were dried in a dry shady place with well-ventilated air by spreading in a uniformly thin layer. When the moisture content was reduced to a minimum suitable for grinding, the plant materials were crushed into powder with a mixer, and then distilled for 3 h using a Clevenger-type apparatus. Anhydrous sodium sulphate was used to absorb the little water contained in the essential oil. The essential oil was subsequently stored at 2 108C until tested. GC – MS analyses were performed with a Thermo Finnigan TRACE GC (Thermo Finnigan, San Jose, CA, USA) coupled with a TRACE MS plus (EI 70 eV) from the same manufacturer. The analyses were carried out using two different fused silica capillary columns (30 m £ 0.25 mm i.d.; film thickness 0.25 mm) of different polarities [DB-5 and HP-Innowax from Agilent Company (Palo Alto, CA, USA)]. The oven temperature was programmed from 50 to 2508C at 38C min21 and held isothermal for 10 min. Injector and interface temperatures were 220 and 2508C, respectively. The carrier gas was helium at a flow rate of 1 mL min21. Diluted samples (1/10 in ether) of 1.0 mL were injected manually and the split ratio was adjusted to 40:1. The components were identified by comparing their mass spectra with those in the NIST98 GC – MS library and those in the literature (Adams 2001), as well as by comparing their retention indices with the literature data (Adams 2001). Retention indices of the components were determined relative to the retention times of a series of n-alkanes with linear interpolation.
3.4. Antimicrobial activity 3.4.1. Microbial strains The essential oil was tested against 13 micro-organisms. Reference strains were as follows: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, P. aeruginosa ATCC 27853, and C. albicans ATCC 14053; clinically isolated strains were as follows: S. epidermidis, Staphylococcus simulans, Staphylococcus heamolyticus, Staphylococcus saprophyticus, Serratia marcescens, Enterococcus faecalis, Enterobacter cloacae, Proteus mirabilis and Klebsiella pneumoniae.
3.4.2. Antimicrobial screening The agar disc diffusion method was employed for the determination of the antimicrobial activity of the essential oils (NCCLS 1997). Briefly, a suspension of the tested micro-organism (2 £ 108 CFU mL21) was spread on the solid media plates. Filter paper discs (6 mm in diameter) were individually impregnated with 15 mL of the diluted oil aliquots (200.00 mg mL21) and then placed on the incubated plates for 2 h at 48C, following which they were incubated at 378C for 24 h for bacteria and at 308C for 48 h for the yeasts. The DDs were measured and expressed in millimetres. Each test was performed in triplicate and repeated twice.
3.4.3. Determination of MIC and MBC A broth microdilution method was used to determine the MIC and MBC (NCCLS 1999; Xie et al. 2007). All tests were performed in Mueller –Hinton broth supplemented with Tween 80 at a
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final concentration of 0.5% (v/v). A serial doubling dilution of the oil was prepared in a 96-well microtitre plate over the range of 0.05 –100.00 mg mL21. Overnight broth cultures of each strain were prepared and the final concentration in each well was adjusted to 4 £ 104 CFU mL21. The plates were incubated at 378C for 24 h for bacteria and 308C for 48 h for the yeasts. The MIC is defined as the lowest concentration of the essential oil to which the micro-organism does not demonstrate visible growth. Microbial growth was indicated by the turbidity. To determine MBC, the broth was taken from each well and inoculated in Mueller –Hinton agar at 378C or 24 h for bacteria or in Sabouraud dextrose agar at 308C or 48 h for the yeasts. The MBC was defined as the lowest concentration of essential oil at which incubated micro-organism was completely killed. Each test was performed in triplicate and repeated twice. Levofloxacin served as positive control.
3.5. Antioxidant activity 3.5.1. DPPH radical-scavenging assay DPPH radical-scavenging activity was determined by using the method of Brand-Williams with some modifications (Yen & Duh 1994). Aliquots (200 mL) of various concentrations (0.2 – 1.0 mg mL21) of the essential oils were added to 3 mL of DPPH radical solution (6 £ 1025 mol L21) in absolute ethanol. The mixture was shaken vigorously and allowed to stand for 30 min; the absorbance of the resulting solution was measured at 517 nm with a spectrophotometer (Shimadzu UV-1601, Kyoto, Japan). Inhibition of free radical DPPH in percent (I%) was calculated as follows: I (%) ¼ [(A0 2 At)/A0] £ 100, where At is the absorbance value of the tested sample and A0 is the absorbance value of the blank sample. The sample concentration providing 50% inhibition (IC50) was calculated by plotting the inhibition percentages against the concentrations of the sample. All tests were carried out in triplicate and IC50 values were reported as means ^ SD of triplicate.
3.5.2. ABTS radical-scavenging assay The radical-scavenging activity of the essential oil was estimated according to the previously reported procedure with slight modification (Re et al. 1999). The essential oil was first dissolved in ethanol in the range of 0.2– 1.0 mg mL21. ABTSþ (radical cation) solution was prepared through the reaction of 7 mmol L21ABTSþ and 2.45 mmol L21 potassium persulfate, and incubating at room temperature in the dark for 16 h. The ABTSþ solution was diluted with 80% ethanol to an absorbance of 0.70 ^ 0.01 at 734 nm at the time of use. Each sample (0.1 mL) with various concentrations was added to 1 mL of ABTSþ solution and mixed vigorously. After reaction at room temperature for 6 min, the absorbance at 734 nm was measured. The ABTSþ scavenging effect was calculated by the following formula: I (%) ¼ [(A0 2 At)/A0] £ 100, where At is the absorbance value of the tested sample and A0 is the absorbance value of the blank sample. The sample concentration providing 50% inhibition (IC50) was calculated by plotting the inhibition percentages against the concentrations of the sample. All tests were carried out in triplicate and IC50 values were reported as means ^ SD of triplicate.
3.6. Statistical analysis The data were expressed as mean ^ SD. One-way analysis of variance and Duncan’s multiplerange tests were carried out to determine significant differences (p , 0.05) between the means. The analyses were carried out using SPSS package software (Version 19.0).
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4. Conclusion Owing to the undesirable problems and side effects arising from the consumption of artificial chemical compounds, the essential oils from various plant species, especially edible and medicinal plants have attained appreciable interest among the research community. This is the first study on the antimicrobial and antioxidant activity of the essential oil of A. megalophylla. Our data indicate that the essential oil shows a broad spectrum of antimicrobial activity against referenced strains and possesses a moderate antioxidant activity. These suggest that essential oil of A. megalophylla could be used as new medicinal resource for antimicrobial and antioxidant agents.
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Acknowledgements The authors wish to thank the National Natural Science Foundation of China (No. 81001617) for the financial support.
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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Chemical composition, antimicrobial and antioxidant activities of essential oil from Ampelopsis megalophylla a
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b
a
Xian-Fei Xie , Jian-Wu Wang , Han-Ping Zhang , Qi-Xiong Li & Bang-Yin Chen
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Department of Pharmacology, Basic Medical School, Wuhan University, Wuhan 430072, P.R. China b
College of Pharmacy, Huazhong University of Science and Technology, Wuhan 430030, P.R. China Published online: 13 Feb 2014.
To cite this article: Xian-Fei Xie, Jian-Wu Wang, Han-Ping Zhang, Qi-Xiong Li & Bang-Yin Chen , Natural Product Research (2014): Chemical composition, antimicrobial and antioxidant activities of essential oil from Ampelopsis megalophylla, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.886208 To link to this article: http://dx.doi.org/10.1080/14786419.2014.886208
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.886208
Chemical composition, antimicrobial and antioxidant activities of essential oil from Ampelopsis megalophylla Xian-Fei Xiea*, Jian-Wu Wangb, Han-Ping Zhangb, Qi-Xiong Lia and Bang-Yin Chenb a
Department of Pharmacology, Basic Medical School, Wuhan University, Wuhan 430072, P.R. China; College of Pharmacy, Huazhong University of Science and Technology, Wuhan 430030, P.R. China
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b
(Received 19 October 2013; final version received 19 January 2014) Chemical composition, antimicrobial and antioxidant activities of the essential oil of Ampelopsis megalophylla were evaluated in this research. GC– MS analysis of the essential oil revealed 42 compounds, representing 88.54% of the oil. The major compounds were borneol (10.81%), a-pinene (6.74%) and b-elemene (6.23%). The antimicrobial activity of the essential oil was evaluated against 13 micro-organisms using the disc diffusion and broth microdilution methods. Results demonstrated higher effects of this oil against Gram-positive bacteria than the other reference strains tested. The antioxidant effect of the essential oil was evaluated by using 1,1-Diphenyl-2picrylhydrazyl (DPPH) and 2,20-azinobis-3-ethylbenzthiazoline-6-sulfonate scavenging assays. The essential oil exhibited moderate antioxidant activity. Keywords: Ampelopsis megalophylla; essential oil; GC – MS; antimicrobial; antioxidant
1. Introduction Plant-derived natural chemicals, known as secondary metabolites, have been widely used in dietary regimens, food preservation, folk medicine and fragrance industry (Kalemba & Kunicka 2003; Huang et al. 2005). Application of plant materials as dietary regimens and preservatives is mainly due to their antimicrobial, antioxidant and other biological potentials. The essential oils and extracts of many plant species have become popular in recent years, and attempts to characterise their antimicrobial and antioxidant activity and their use in pharmaceutical or foods processing are recommended (Vardar-Unlu et al. 2003; Sokmen, Serkedjieva, et al. 2004). Ampelopsis megalophylla Diels et. Gilg is a medicinal plant of Ampelopsis, which is an original plant of the ethnic medicine used commonly in the western parts of Hubei Province in China and most often consumed as an herbal tea. Its consumption in this manner could be utilised in the treatment of hypertension in clinical practice with significant effect. Preliminary researches (Zhang, Shen, Chen, et al. 2008; Shen et al. 2010) indicate that flavonoids such as ampelopsin, myricetin and myricitrin are its main effective substances. Some reports have shown that A. megalophylla possesses antihypertensive, antivirus and antihyperglycaemic effects (Huang et al. 2006; Zhang, Shen, Chen, Chen, et al. 2008; Yang et al. 2011). To the best of our knowledge and literary survey, there is no information available on the chemical composition, antimicrobial and antioxidant activities of essential oil from A. megalophylla. Therefore, the aim of this study was to determine the chemical composition of the hydro-distilled essential oil from leaves of A. megalophylla by GC –MS and to evaluate its antimicrobial and antioxidant properties.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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2. Results and discussion 2.1. Chemical composition of the essential oil The essential oils were obtained by hydro-distillation from the dried leaves of A. megalophylla with the yield of 0.42% (v/w) on a dry-weight basis which was in the normal range of the amount we usually find (Xie et al. 2007; Abdollah et al. 2013). A total of 42 compounds (88.54% of the total oil) were identified by using two chromatographic techniques. Qualitative and quantitative analytical results by GC –MS are shown in Table 1. The components of the essential oil were separated into four classes: monoterpene hydrocarbons,
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Table 1. Main chemical composition of A. megalophylla essential oil. Compounds a-Thujene a-Pinene Camphene Sabinene 1-Octen-3-ol Myrcene a-Terpinene p-Cymene 1,8-Cineole g-Terpinene Linalool Camphor Pinocarvone Borneol Terpinene-4-ol a-Terpineol Myrtenol trans-Carveol cis-Piperitone oxide Carvone Bornyl acetate Thymol Carvacrol r-Menth-3-3-en-8-ol, acetate Eugenol b-Elemene Longifolene b-Caryophyllene a-Humulene Dodecanal Germacrene D b-Selinene (E,E)-a-Farnesene Germacrene A g-Cadinene d-Cadinene Spathulenol Caryophyllene oxide epi-a-Cadinol T-Cadinol b-Eudesmol a-Bisabolol a b
Retention indices on DB-5 capillary column. Retention time (in min).
Percentage (v/w)
RIa
RTb
1.22 6.74 1.24 3.78 1.56 2.34 0.82 1.33 1.56 3.12 1.05 5.12 2.46 10.81 3.77 0.43 0.33 1.27 0.23 0.15 1.57 2.36 1.29 1.08 1.46 6.23 3.45 1.79 3.41 0.12 0.93 2.67 1.42 1.56 1.12 0.78 0.17 3.43 1.12 0.65 1.47 1.13
930 934 949 972 976 991 1016 1023 1031 1057 1100 1146 1163 1168 1178 1193 1197 1205 1232 1243 1283 1294 1298 1316 1348 1379 1405 1426 1453 1464 1484 1498 1503 1509 1518 1524 1576 1582 1634 1641 1652 1681
9.62 9.91 10.72 11.45 11.74 12.75 13.87 14.32 14.93 15.88 17.84 20.58 21.41 21.78 22.37 22.85 23.13 23.46 24.52 25.03 26.87 27.50 27.74 28.45 29.79 30.94 31.76 32.50 33.61 34.26 35.19 35.66 35.87 36.08 36.47 36.75 40.13 40.78 44.15 44.62 45.23 47.07
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oxygenated monoterpenes, sesquiterpene hydrocarbons and oxygenated sesquiterpenes, which were in the ratios of 22.15%, 34.94%, 23.48% and 7.97%, respectively. Borneol (10.81%), apinene (6.74%) and b-elemene (6.23%) were the main compounds of the essential oil, followed by camphor (5.12%), sabinene (3.78%) and terpinene-4-ol (3.77%).
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2.2. Antimicrobial activity The diameters of inhibition zones including disc diameter (DDs), minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the essential oils for the micro-organisms tested are shown in Table 2. Although the agar disc diffusion method is time-consuming and labour-intensive, it is still a reliable and effective technique which is most often used by many laboratories for antimicrobial screening. Therefore, this method was employed for the determination of the antimicrobial activity by us (Xie et al. 2007; Yu et al. 2007; Elena et al. 2013). Data obtained from the disc diffusion method indicated that Staphylococcus epidermidis was the most sensitive micro-organism to the essential oil with the strongest inhibition zone (29 mm), while Pseudomonas aeruginosa ATCC 27853 exhibited weak inhibition zones (12 mm). Although the results of MICs and MBCs varied between the organisms tested, in most cases the MIC was equivalent to the MBC, indicating a bactericidal activity of the oil. The results of MIC indicated that the oils inhibited all the micro-organisms tested. The Gram-positive bacteria were more sensitive to the oils than Gram-negative bacteria and yeast. S. epidermidis had the lowest MIC (0.39 mg mL21). The highest MIC was 25.00 mg mL21 for P. aeruginosa ATCC 27853 and Candida albicans ATCC 14053. The lowest MBC was 0.39 mg mL21 for S. epidermidis. P. aeruginosa ATCC 27853 had the highest MBC of 50.00 mg mL21. The antimicrobial activities of the essential oil could be attributed to the presence of high concentrations of borneol, a-pinene, camphor and terpinene-4-ol, which possess Table 2. Antimicrobial activities of A. megalophylla essential oil. Essential oil Micro-organisms Reference strains S. aureus ATCC 25923 E. coli ATCC 25922 P. aeruginosa ATCC 27853 C. albicans ATCC 14053 Clinically isolated strains S. epidermidis S. simulans S. heamolyticus S. saprophyticus S. marcescens E. faecalis E. cloacae P. mirabilis K. pneumoniae
Levofloxacin
DDa
MICb
MBCb
DDc
MICd
MBCd
27 ^ 1.3a 21 ^ 1.1b 12 ^ 0.5d 13 ^ 0.5d
0.78 3.13 25.00 25.00.
0.78 3.13 50.00 25.00
34 ^ 1.6a 30 ^ 1.4ab 22 ^ 1.1c NT
0.30 0.30 4.88 NT
0.30 0.61 4.88 NT
29 ^ 1.4a 24 ^ 1.2ab 25 ^ 1.3ab 22 ^ 1.1b 15 ^ 0.7cd 17 ^ 0.8c 19 ^ 0.8bc 14 ^ 0.6d 16 ^ 0.7c
0.39 3.13 1.57 6.25 12.50 6.25 6.25 12.50 12.50
0.39 3.13 1.57 6.25 12.50 12.50 6.25 12.50 12.50
32 ^ 1.4a 29 ^ 1.3ab 25 ^ 1.2bc 27 ^ 1.3b 17 ^ 0.7cd 22 ^ 1.0c 17 ^ 0.6cd 14 ^ 0.4d 24 ^ 1.2bc
0.61 1.22 1.22 2.44 4.88 4.88 9.76 19.52 4.88
0.61 1.22 1.22 2.44 9.76 4.88 9.76 19.52 4.88
Notes: DD, diameter of zone of inhibition (mm) including disc diameter of 6 mm; NT, not test; MIC, minimum inhibitory concentration, MBC, minimum bactericidal concentration. Different letters within a column indicate statistically significant differences between the means ( p , 0.05). a Tested at a concentration of 3 mg disc21. b Values given as mg mL21. c Tested at a concentration of 5 mg disc21. d Values given as mg mL21.
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well-documented antimicrobial potential (Dorman & Deans 2000; Tabanca et al. 2001; VardarUnlu et al. 2003; Lopes-Lutz et al. 2008). Apart from the major compounds, b-caryophyllene, 1,8-cineole and p-cymene as well as other minor constituents of the essential oils of A. megalophylla also possessed antimicrobial activity (So¨kmen, Gulluce, et al. 2004; Jirovetz et al. 2006; Rosato et al. 2007). In fact, the synergistic effects of the diversity of major and minor constituents present in the essential oils should be considered to account for their biological activity (Delamare et al. 2007). Thanks to their extra-protective outer membrane, Gram-negative bacteria were usually considerably more resistant to antibacterial agents than their Gram-positive counterparts (Saier 2009). In this regard, the essential oil of the A. megalophylla expressed its best antibacterial activity in the MIC test on Gram-positive bacteria tested. 2.3. Antioxidant activity To assess the antioxidant activity of the essential oil, we evaluated its radical-scavenging activity against DPPH and 2,20-azinobis-3-ethylbenzthiazoline-6-sulfonate (ABTS). The DPPH molecule, which contains a stable free radical, had been extensively used to evaluate the radical-scavenging ability of antioxidants (Nagai et al. 2003). As shown in Table 3, the essential oil exhibited a dose-dependent scavenging activity against DPPH radical, with an IC50 value of 0.48 mg mL21. ABTS assay was based on the antioxidant ability of the sample to react with ABTSþ (radical cation) generated in the assay system (Tel et al. 2010; C´avar et al. 2012). However, unlike scavenging of DPPH radical, essential oil exhibited increased scavenging activity towards ABTSþ (IC50 0.36 mg mL21) (Table 3). This was probably due to steric factors that were one of the major factors for reducing the stable DPPH radical (C´avar et al. 2012). It had been reported that free radical-scavenging activity is greatly influenced by the phenolic composition of the samples (Cheung et al. 2003). Phenolic compounds, generally considered as biologically active components, were the principal agents to donate hydrogen to free radicals. This high potential of phenolic compounds to scavenge radicals might be explained by their phenolic hydroxyl groups (Oke et al. 2009). Therefore, phenolic compounds, which were present in some proportion of the essential oil such as thymol, carvacrol and g-terpinene (Table 1), could be the main compounds responsible for the antioxidant activity. 3. Experimental 3.1. Plant material The leaves of A. megalophylla were collected in Hubei Province of China in July of 2009. A voucher specimen (No. 0315) has been deposited at the herbarium of School of Medicine, Wuhan University, China. The plant was identified in the Laboratory of Botany at the same institution. Table 3. Radical-scavenging activities of A. megalophylla essential oil.a Inhibition rate (%) Concentration (mg mL Essential oil 0.2 0.4 0.8 IC50 of oil
21
)
DPPH
ABTS
15.2 ^ 1.08c 45.3 ^ 1.85b 62.7 ^ 2.46a 0.48
21.3 ^ 1.24c 54.6 ^ 2.34b 71.2 ^ 2.87a 0.36
Note: Different letters within a column indicate statistically significant differences between the means ( p , 0.05). a Results are means of three replicates.
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3.2. Chemicals 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and ABTS were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of analytical grade.
3.3. Essential oil extraction and GC – MS analysis The leaves of A. megalophylla were dried in a dry shady place with well-ventilated air by spreading in a uniformly thin layer. When the moisture content was reduced to a minimum suitable for grinding, the plant materials were crushed into powder with a mixer, and then distilled for 3 h using a Clevenger-type apparatus. Anhydrous sodium sulphate was used to absorb the little water contained in the essential oil. The essential oil was subsequently stored at 2 108C until tested. GC – MS analyses were performed with a Thermo Finnigan TRACE GC (Thermo Finnigan, San Jose, CA, USA) coupled with a TRACE MS plus (EI 70 eV) from the same manufacturer. The analyses were carried out using two different fused silica capillary columns (30 m £ 0.25 mm i.d.; film thickness 0.25 mm) of different polarities [DB-5 and HP-Innowax from Agilent Company (Palo Alto, CA, USA)]. The oven temperature was programmed from 50 to 2508C at 38C min21 and held isothermal for 10 min. Injector and interface temperatures were 220 and 2508C, respectively. The carrier gas was helium at a flow rate of 1 mL min21. Diluted samples (1/10 in ether) of 1.0 mL were injected manually and the split ratio was adjusted to 40:1. The components were identified by comparing their mass spectra with those in the NIST98 GC – MS library and those in the literature (Adams 2001), as well as by comparing their retention indices with the literature data (Adams 2001). Retention indices of the components were determined relative to the retention times of a series of n-alkanes with linear interpolation.
3.4. Antimicrobial activity 3.4.1. Microbial strains The essential oil was tested against 13 micro-organisms. Reference strains were as follows: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, P. aeruginosa ATCC 27853, and C. albicans ATCC 14053; clinically isolated strains were as follows: S. epidermidis, Staphylococcus simulans, Staphylococcus heamolyticus, Staphylococcus saprophyticus, Serratia marcescens, Enterococcus faecalis, Enterobacter cloacae, Proteus mirabilis and Klebsiella pneumoniae.
3.4.2. Antimicrobial screening The agar disc diffusion method was employed for the determination of the antimicrobial activity of the essential oils (NCCLS 1997). Briefly, a suspension of the tested micro-organism (2 £ 108 CFU mL21) was spread on the solid media plates. Filter paper discs (6 mm in diameter) were individually impregnated with 15 mL of the diluted oil aliquots (200.00 mg mL21) and then placed on the incubated plates for 2 h at 48C, following which they were incubated at 378C for 24 h for bacteria and at 308C for 48 h for the yeasts. The DDs were measured and expressed in millimetres. Each test was performed in triplicate and repeated twice.
3.4.3. Determination of MIC and MBC A broth microdilution method was used to determine the MIC and MBC (NCCLS 1999; Xie et al. 2007). All tests were performed in Mueller –Hinton broth supplemented with Tween 80 at a
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final concentration of 0.5% (v/v). A serial doubling dilution of the oil was prepared in a 96-well microtitre plate over the range of 0.05 –100.00 mg mL21. Overnight broth cultures of each strain were prepared and the final concentration in each well was adjusted to 4 £ 104 CFU mL21. The plates were incubated at 378C for 24 h for bacteria and 308C for 48 h for the yeasts. The MIC is defined as the lowest concentration of the essential oil to which the micro-organism does not demonstrate visible growth. Microbial growth was indicated by the turbidity. To determine MBC, the broth was taken from each well and inoculated in Mueller –Hinton agar at 378C or 24 h for bacteria or in Sabouraud dextrose agar at 308C or 48 h for the yeasts. The MBC was defined as the lowest concentration of essential oil at which incubated micro-organism was completely killed. Each test was performed in triplicate and repeated twice. Levofloxacin served as positive control.
3.5. Antioxidant activity 3.5.1. DPPH radical-scavenging assay DPPH radical-scavenging activity was determined by using the method of Brand-Williams with some modifications (Yen & Duh 1994). Aliquots (200 mL) of various concentrations (0.2 – 1.0 mg mL21) of the essential oils were added to 3 mL of DPPH radical solution (6 £ 1025 mol L21) in absolute ethanol. The mixture was shaken vigorously and allowed to stand for 30 min; the absorbance of the resulting solution was measured at 517 nm with a spectrophotometer (Shimadzu UV-1601, Kyoto, Japan). Inhibition of free radical DPPH in percent (I%) was calculated as follows: I (%) ¼ [(A0 2 At)/A0] £ 100, where At is the absorbance value of the tested sample and A0 is the absorbance value of the blank sample. The sample concentration providing 50% inhibition (IC50) was calculated by plotting the inhibition percentages against the concentrations of the sample. All tests were carried out in triplicate and IC50 values were reported as means ^ SD of triplicate.
3.5.2. ABTS radical-scavenging assay The radical-scavenging activity of the essential oil was estimated according to the previously reported procedure with slight modification (Re et al. 1999). The essential oil was first dissolved in ethanol in the range of 0.2– 1.0 mg mL21. ABTSþ (radical cation) solution was prepared through the reaction of 7 mmol L21ABTSþ and 2.45 mmol L21 potassium persulfate, and incubating at room temperature in the dark for 16 h. The ABTSþ solution was diluted with 80% ethanol to an absorbance of 0.70 ^ 0.01 at 734 nm at the time of use. Each sample (0.1 mL) with various concentrations was added to 1 mL of ABTSþ solution and mixed vigorously. After reaction at room temperature for 6 min, the absorbance at 734 nm was measured. The ABTSþ scavenging effect was calculated by the following formula: I (%) ¼ [(A0 2 At)/A0] £ 100, where At is the absorbance value of the tested sample and A0 is the absorbance value of the blank sample. The sample concentration providing 50% inhibition (IC50) was calculated by plotting the inhibition percentages against the concentrations of the sample. All tests were carried out in triplicate and IC50 values were reported as means ^ SD of triplicate.
3.6. Statistical analysis The data were expressed as mean ^ SD. One-way analysis of variance and Duncan’s multiplerange tests were carried out to determine significant differences (p , 0.05) between the means. The analyses were carried out using SPSS package software (Version 19.0).
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4. Conclusion Owing to the undesirable problems and side effects arising from the consumption of artificial chemical compounds, the essential oils from various plant species, especially edible and medicinal plants have attained appreciable interest among the research community. This is the first study on the antimicrobial and antioxidant activity of the essential oil of A. megalophylla. Our data indicate that the essential oil shows a broad spectrum of antimicrobial activity against referenced strains and possesses a moderate antioxidant activity. These suggest that essential oil of A. megalophylla could be used as new medicinal resource for antimicrobial and antioxidant agents.
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Acknowledgements The authors wish to thank the National Natural Science Foundation of China (No. 81001617) for the financial support.
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