Abstract
The chemical components of essential oil of Toona sinensis leaf blades and their petioles from China were extracted by simultaneous distillation solvent extraction (SDE) and were analyzed by GC-MS. The antimicrobial and DPPH scavenging activity of the essential oil were evaluated. The results showed that there were differences in chemical compositions and content among essential oils extracted from T. sinensis in different parts and different geographical areas in China, but the main components of essential oils were sesquiterpene and sesquiterpene oxygenated compounds, accounting for 90.1% (No. 1), 92.6% (No. 2), and 80.9% (No. 3) of the relative mass fraction, respectively. T. sinensis essential oil exhibited noticeable growth inhibitory activity against the tested microorganisms. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of different essential oils against microorganisms were different. For all essential oil samples, MIC and MBC against Escherichia coli and Bacillus subtiliswere less than 25 μg·mL−1, MIC and MBC against Penicillium citrinum were 200 and 400 μg·mL−1, respectively, and MIC and MBC against Colletotrichum gloeosporioides were 50 and 200 μg·mL−1, respectively. The IM50 of DPPH scavenging for T. sinensis essential oil was less than 0.3 g DPPH per g essential oil. The results indicated that T. sinensis essential oil may be a useful natural antiseptic source from forest products.
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Chemical Composition and Antimicrobial and DPPH Scavenging Activity of Essential Oil of Toona sinensis (A. Juss.) Roem from China
Congjin Chen,a,c,* Zhangfa Tong,a Dankui Liao,a Yue Li,a Guoen Yang,b and Mingfei Li c
The chemical components of essential oil of Toona sinensis leaf blades and their petioles from China were extracted by simultaneous distillation solvent extraction (SDE) and were analyzed by GC-MS. The antimicrobial and DPPH scavenging activity of the essential oil were evaluated. The results showed that there were differences in chemical compositions and content among essential oils extracted from T. sinensis in different parts and different geographical areas in China, but the main components of essential oils were sesquiterpene and sesquiterpene oxygenated compounds, accounting for 90.1% (No. 1), 92.6% (No. 2), and 80.9% (No. 3) of the relative mass fraction, respectively. T. sinensis essential oil exhibited noticeable growth inhibitory activity against the tested microorganisms. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of different essential oils against microorganisms were different. For all essential oil samples, MIC and MBC against Escherichia coli and Bacillus subtilis were less than 25 μg·mL−1, MIC and MBC against Penicillium citrinum were 200 and 400 μg·mL−1, respectively, and MIC and MBC against Colletotrichum gloeosporioides were 50 and 200 μg·mL−1, respectively. The IM50 of DPPH scavenging for T. sinensis essential oil was less than 0.3 g DPPH per g essential oil. The results indicated that T. sinensis essential oil may be a useful natural antiseptic source from forest products.
Keywords: Toona sinensis essential oil; Chemical components; GC-MS; Antimicrobial activity; DPPH scavenging activity
Contact information: a: School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China; b: Material Science and Engineering College, Central South University of Forestry and Technology, Changsha 410004, China; c: Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, China; * Corresponding author: gxdxccj@163.com
INTRODUCTION
Toona sinensis (A. Juss) Roem, a species of Toona, is widely distributed in North Korea south through most of eastern, central, and southwestern China to Nepal, northeastern India, Myanmar, etc. It is a perennial hardwood also called “Xiangchun” in Chinese and is a common plant in China. It is uniquely aromatic, and its young leaves and shoots can be used as a kind of vegetable called “xiangchun ya,” known as a “tree vegetable”. The leaves of T. sinensis are also used in Chinese traditional and herbal drugs for the treatment of diarrhea, chronic dysentery, bloody stools, seminal emissions, leucorrhoea, and metrorrhagia (Luo et al. 2001). Extracts from T. sinensis leaves can alter lipid metabolism (Hsu et al. 2003; Zhang et al. 2007), alleviate hyperglycemia (Wang et al. 2008), alleviate liver fibrosis (Fan et al. 2007), induce apoptosis of cancer cells (Yang et al. 2006), inhibit tumor growth (Chang et al. 2002; Chang et al. 2006), increase locomotor activity of human sperm (Wang et al. 2005), prevent H2O2-induced oxidative stress and DNA damage in cultured Madin-Darby canine kidney cells (Hsieh et al. 2004), and exhibit anti-oxidative activity in vitro (Chen et al. 2006; Hseu et al. 2008); they also exhibit antibacterial activity (Tian and Yang 2002; Ouyang et al. 2008).
Previous studies on T. sinensis have led to the isolation of flavones, triterpene, phenolic compounds, alkaloids, anthraquinone, and tannins (Chen et al. 2000; Wang et al. 2007). Many volatile compounds (Mu et al. 2007; Liu et al. 2008; Chen et al. 2009a, 2009b) with bioactivity are also present, such as β-caryophyllene, the main volatile compound of T. sinensisleaves (Chen et al. 2009b) is used as a fragrance chemical and has the effects of a local anesthetic, anti-inflammatory substance, insect repellant, and treatment for general anxiety neurosis and depression (Cho et al. 2007; Ghelardini et al. 2001; Liu et al. 2012; Skold et al. 2006). Caryophyllene oxide is an analgesic and has anti-inflammatory, antifungal, and cytotoxicity activities (Chavan et al. 2010; Monzote et al. 2009; Yang et al. 1999). Spathulenol, one of the main volatile compounds of T. sinensis essential oil, has shown the capacity to inhibit proliferation in lymphocytes and to induce apoptosis in these cells, possibly through a caspase-3 independent pathway (Ziaei et al. 2011). The essential oil compounds from plants grown in different geographical areas or different parts of the plants may be different either quantitatively or qualitatively (Maggi et al. 2009; Ouni et al. 2011; Saei-Dehkordi et al.2010; Youssef et al. 2011). 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical that has an unpaired valence electron at one atom of the nitrogen bridge. Scavenging of DPPH radicals is the basis of the popular DPPH antioxidant assay (Sharma and Bhat 2009). The DPPH assay has been used to predict the oxidative stability of edible oils (Lee et al. 2007; Scherer and Godoy 2009). However, there have been few published reports on the chemical composition, antimicrobial, and DPPH scavenging activity of the essential oil (Mu et al.2007; Liu et al. 2008; Chen et al. 2009a, 2009b) especially from different parts of T. sinensis, in different geographical areas in China.
The effect of the geographical origin in which plants grow and the parts of the plants on the yield, composition, and bioactivities of T. sinensis essential oils is of importance. The aim of the present work is to determine any volatile components and to carry out a comparative evaluation of yield, components, antimicrobial, and DPPH scavenging activity of the essential oil of T. sinensis leaf blades and petioles from different geographical areas in China.
EXPERIMENTAL
Materials
Plant material and chemicals
Samples of T. sinensis, grown in Nandan and Dongnan county, Guangxi Province, China, were collected in August, 2007. The plants were identified and authenticated by Professor He Taiping of the Forestry College, Guangxi University. The leaf blades and their petioles from T. sinensis were collected and air-dried. The petioles were cut into small pieces of about 5 mm. 2,2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich (USA). Rutin, quercetin, gallic acid, and other biochemical reagents were purchased from Shanghai Sinopharm Chemical Reagent Co. All other chemicals, including ascorbic acid, ethanol, ethyl ether, sodium sulfate, sodium chloride, dextrose, agar, peptone, beef extract, and potato extract, were made in China and were of analytical grade.
Microorganisms and media
The essential oil was tested against four microorganisms: bacteria (Escherichia coli, Bacillus subtilis) and fungi (Penicillium citrinum, Colletotrichum gloeosporioides). All microorganisms were provided by the microbiology laboratory of Central South University of Forestry and Technology.
Each microorganism was inoculated in test-tube slant medium. Bacteria were cultured for 24 h at 37 oC, and fungi were cultured for 72 h at 28 oC in an electro-thermal incubator. On a sterile workbench, inocula were suspended in sterile saline and the suspension was adjusted to contain approximately 105 to 106 colony forming units (CFU) per mL bacteria suspension or 105 to 106 spores per mL of fungi suspension using a blood cell counting chamber.
Beef extract peptone agar medium for bacteria consisted of 5 g·L−1 beef extract, 10 g·L−1 peptone, 20 g·L−1 agar, and 5 g·L−1 sodium chloride, with a pH value of 7.2 to 7.4. Potato dextrose agar medium for fungi consisted of 6 g·L−1 potato extract, 20 g·L−1 dextrose, and 20 g·L−1 agar, with a pH value of 5.6.
Methods
Essential oil extraction and GC-MS analysis
The essential oil samples were isolated from the leaf blades and their petioles of T. sinensis by simultaneous-distillation and solvent-extraction (SDE) for 4 h in a Likens–Nickerson apparatus (Ansorena et al. 2001; Chung et al. 2002; Valette et al. 2003) using ethyl ether (50 mL) as the extracting solvent. The extraction was carried out at atmospheric pressure. The sample was kept at 105 ± 2 oC, and the solvent was kept at 50 ± 2 oC. The extracts were dried with anhydrous sodium sulfate and concentrated under vacuum at room temperature by a rotatory evaporator, until the solvent was evaporated. The collected oil was stored at 4 oC in the sealed brown vials until analysis.
GC-MS analysis was performed on a TRACE GC-POLARISQ MS (manufactured by Thermo Finnigan, America) using a DB-1 silica capillary column (i.d. = 0.25 mm, length = 30 m, and film thickness = 0.25 μm) and equipped with a flame ionization detector (FID). Helium was used as a carrier gas with a flow rate of 1 mL·min−1. The oven temperature was maintained at 80 oC for 2 min, raised to 240 oC at a rate of 5 oC·min−1, and finally kept for 20 min at 240 oC. Injector and detector temperatures were set at 250 oC and 290 oC, respectively. Diluted samples (1/1000 in acetone, v/v) of 10 μL were injected, not split. The MS detector was used in the electron impact ionization (EI) mode with an ionization voltage of 70 eV, electron multiplier voltage of 1.2 kV, and scan range of 40 to 400 amu. An FID was used for quantification of the constituents in the essential oil.
Identification of the components was based on the comparison of the mass spectrum of each compound with that of known compounds searched in the NIST mass spectral library data, and analysis after consulting correlation literature (Chen et al. 2009a, 2009b; Liu et al. 2008; Mu et al. 2007). Quantification was expressed as the percentage contribution of each compound to the total amount of material detected after peak area normalization.
Determination of the growth inhibition zone diameter of essential oil
The growth inhibition zone diameter of different essential oil samples against bacteria and fungi experiment was evaluated using cylinder plate assay methods (Jiao et al. 2007; Liu et al.2008a; Zhang et al. 2012; Zulfiya 2008). Ten milliliters of agar medium was poured into each test tube. After autoclaving, the agar solution in each tube was cooled to about 45 °C, then aseptically poured into a sterile Petri dish (90 × 15 mm) and allowed to solidify. After solidification, 0.1 mL of microbial suspension containing approximately 105 to 106 colony forming units (CFU) per mL bacteria or 105 to 106 spores per mL of fungi was swabbed on the top of the solidified medium and dried. Oxford cups (bore diameter, 6.0 ± 0.1 mm; outer diameter, 7.8 ± 0.1 mm; height, 10 ± 0.1 mm) were placed on each plate. Then, 100 μL of essential oils was dispensed into individual oxford cups and left for 30 min at room temperature to allow for diffusion. The plates were incubated for 24 h at 37 oC for bacteria and 72 h at 28 oC for fungi in an electro-thermal incubator. When essential oils in the oxford cups diffused completely, the cups were removed from the agar plate. The growth inhibition zone diameters were measured horizontally and vertically. The experiment was repeated twice, and the values were averaged.
Determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of essential oil
A serial agar macrodilution method (Arias et al. 2004; Buwa and van Staden 2006; Duraipandiyan and Ignacimuthu 2007) was used to assess the activity of different essential oil samples against bacteria and fungi. The initial test concentration of the various essential oil samples (0.8 mg·mL−1) was serially diluted two-fold with ethanol. Then, 1.0 mL serial two-fold dilution of each essential oil sample (400 μg·mL−1, 200 μg·mL−1, 100 μg·mL−1, 50 μg·mL−1, 25 μg·mL−1) was mixed with 9.0 mL of 50 to 60 oC sterile beef extract peptone agar for bacteria and sterile potato dextrose agar for fungi and put into Petri dishes (90 × 15 mm) as soon as possible. After the medium was solidified and cooled, each Petri plate was inoculated with spots of 10 μL of suspension containing 105 to 106 cfu·mL−1 of bacteria or 105 to 106 spore·mL−1 of fungi and incubated for 24 h at 37 oC for bacteria and 72 h at 28 oC for fungi (Ait-Ouazzou et al. 2012; Lv et al. 2011). Negative control experiments were conducted using ethanol and distilled water at the same time. Experiments were repeated twice at each concentration.
MIC for microorganisms was determined as the lowest concentration of the essential oil sample at which no colony was observed after the bacteria were incubated for 24 h at 37 oC (fungi for 72 h at 28 oC). MBC for microorganisms was defined as the lowest concentration of the essential oil sample at which no colony was observed after the bacteria were incubated for 72 h at 37 oC (fungi for 120 h at 28 oC).
Determination of DPPH scavenging activity of the essential oil
The DPPH scavenging activity of the essential oil was determined by methods described in the literature (Chen et al. 2006). A series of essential oil ethanol solutions with varying concentrations was prepared by dissolving various masses of essential oil in 25 mL of ethanol. Then, 1 mL of essential oil ethanol solution was added to 4.0 mL of 51.54 mg·L−1 DPPH ethanol solution. After 50 min of incubation at room temperature, the optical absorbance was measured at 517 nm. The DPPH scavenging activity of essential oil was calculated by Eq. 1,
(1)
where Y is the DPPH scavenging effect (%), A0 is the absorbance of DPPH ethanol solution (mixture of 4.0 mL of 51.54 mg·L−1 DPPH ethanol solution and 1.0 mL ethanol), Ar is the absorbance of essential oil in the presence of the ethanol solution (mixture of 4.0 mL of ethanol and 1.0 mL essential oil ethanol solution), and As is the absorbance of essential oil ethanol solution after incubation (4.0 mL of 51.54 mg·L−1 DPPH ethanol solution and 1.0 mL essential oil ethanol solution). IC50 (the essential oil ethanol solution concentration that generated a 50% scavenging effect) values were also calculated according to Eq. 1, and IM50 (the removed DPPH mass when essential oil generated a 50% DPPH scavenging effect) values were calculated as follows:
(2)
MDPPH is 50% DPPH mass added when antioxidant sample generated a 50% DPPH scavenging effect, and Msample is sample mass added when antioxidant sample generated a 50% DPPH scavenging effect. The IM50 value is the removed DPPH mass when essential oil generated a 50 % DPPH scavenging effect. The higher the IM50 value is, the stronger the DPPH scavenging ability of the essential oil is. Rutin, quercetin, gallic acid, and ascorbic acid were used as positive controls.
RESULTS AND DISCUSSION
Yield and Character of the Essential Oils from Different T. sinensis Samples
The yield and characteristics of the essential oil from different T. sinensis samples are shown in Table 1.
Table 1. Yield and Character of the Essential Oils from Different T. sinensis Samples
All samples were from Guangxi Province, China, and collected in August, 2008
As can be seen in Table 1, the yield and appearance of the essential oils from different parts of T. sinensis in different geographical areas were different, and the yield of the essential oil from Guangxi Nandan county in China in August were the highest (7.33 mL·kg−1).
Chemical Composition of Essential Oils
The total ion chromatograms of essential oils from T. sinensis leaf blades and common petioles extracted by simultaneous distillation solvent extraction (SDE) are shown in Figs. 1 to 3. The components identified in these essential oils of T. sinensis are listed in Table 2 in order of their experimental retention time and retention indices. There were differences among the components and contents (relative mass fraction) of essential oils from T. sinensis in different geographical areas. A total of 63 compounds were identified in the essential oil of leaf blades grown in Nandan county in Guangxi province, China (No. 1), and 63 compounds in the essential oil of leaf blades (No. 2) and 72 compounds in the essential oil of common petioles (No. 3) grown in Dongnan county in Guangxi province, China, were also identified, accounting for 99.35%, 99.25%, and 96.78% (relative mass fraction), respectively. The relative mass fraction of each component is shown in Table 2.
Among the 63 kinds of compounds identified in essential oil (No. 1) were 25 kinds of sesquiterpenes (accounting for 44.30%), 20 kinds of sesquiterpene oxygenated compounds (accounting for 45.80%), and 16 kinds of non-terpenoid compounds (sulfur-containing compounds 0.2%, guaiazulene 1.5% among them). The main compounds in essential oil (No. 1) were (-)-spathulenol (13.24%), β-himachalenoxide (12.79%), α-copaene (7.35%), (±)-cadinene (5.07%), 1R,4S,7S,11R-2,2,4,8-tetramethyltricyclo[5.3.1.0(4,11)]undec-8-ene (5.07%), himachala-2,4-diene (3.59%), δ-cadinene (3.42%), cubenol (3.12%), 6-isopropenyl-4,8α-dimethyl-1,2,3,5,6,7,8,8α-octahydro-naphthalen-2-ol (3.08%), tau-cadinol (2.86%), cadala-1(10),3,8-triene (2.52%), longifolene-(V4)– (2.48%), 7-tetracyclo[6.2.1.0(3.8)0(3.9)]undecanol,4,4,11,11-tetramethyl- (2.24%), aristolene (2.01%), (+)-aromadendrene (1.88%), tricyclo[5.2.2.0(1,6)]undecan-3-ol, 2-methylene-6,8,8-trimethyl- (1.81%), isolongifolene,4,5,9,10-dehydro- (1.51%), guaiazulene (1.50%), in which guaiazulene was found in T. sinensis the first time. The mass spectrogram and structural formula of guaiazulene are shown in Fig. 4.
Among the 63 kinds of compounds identified in essential oil (No. 2) were one kind of monoterpene oxygenated compound (0.12%), 25 kinds of sesquiterpenes (accounting for 61.67%), 19 kinds of sesquiterpene oxygenated compounds (accounting for 30.97%), and 17 kinds of non-terpenoid compounds (sulfur-containing compounds 0.34%, guaiazulene 0.75% among them). The main compounds in essential oil (No. 2) were α-copaene (10.96%), himachala-2,4-diene (9.09%), α-himachalene (6.90%), α-guaiene (6.63%), 6-isopropenyl-4,8α-dimethyl-1,2,3,5,6,7,8,8α-octahydro-naphthalen-2-ol (6.29%), tau-cadinol (5.41%), (±)-cadinene (5.07%), (-)-spathulenol (4.56%), β-himachalene (4.17%), δ-cadinene (3.69%), 1R,4S,7S,11R-2,2,4,8-tetramethyltricyclo[5.3.1.0(4,11)]undec-8-ene (3.68%), cadala-1(10),3,8-triene (2.73%), aromadendrene oxide-(2.02%), tricyclo[5.2.2.0(1,6)]undecan-3-ol,2-methylene-6,8,8-trimethyl- (1.91%), cubenol (1.72%), β-caryophyllene (1.48%).
Among the 72 kinds of compounds identified in essential oil (No. 3) were 3 kinds of monoterpenes oxygenated compounds (accounting for 0.37%), 26 kinds of sesquiterpenes (accounting for 53.23%), 18 kinds of sesquiterpene oxygenated compounds (accounting for 27.64%), and 22 kinds of non-terpenoid compounds (sulfur-containing compounds 7.96%, guaiazulene 0.29% among them). The main compounds in essential oil (No. 3) were α-copaene (11.32%), methylthiirane (7.29%), α-copaen-11-ol (6.47%), δ-cadinene (5.66%), cubenol (4.85%), himachala-2,4-diene (4.2%), β-caryophyllene (4.11%), tau-cadinol (4.09%), (-)- spathulenol (4.00%), longifolene-(V4)– (3.8%), α-cubebene (3.74%), cadala-1(10),3,8-triene (2.99%), l-(+)-ascorbic acid 2,6-dihexadecanoate (2.74%), aromadendrene oxide-(1) (1.86%), humulen-(v1) (1.72%), (±)-cadinene (1.62%), aromadendrene, dehydro- (1.52%).
Fig. 1. Total ions chromatogram of chemical components in essential oil (No. 1) from T. sinensis leaves
Fig. 2. Total ion chromatogram of chemical components in essential oil (No. 2) from T. sinensis leaves
Fig. 3. Total ion chromatogram of chemical components in essential oil (No. 3) from T. sinensis common petioles
There were 57 shared chemical components in essential oils No. 1 and 2, including one kind of the same sulfur compounds: thiophene, 4-dimethyl-, one kind of the same azulenoids: guaiazulene, 24 kinds of the same sesquiterpene compounds, and 21 kinds of the same oxygenated sesquiterpene compound; however, the relative mass fraction of each components was different.
There were 54 shared chemical components in essential oils No. 2 and 3, including 3 kinds of the same sulfur compounds: methylthiirane, thiophene,2,4-dimethyl-, 5-thiatricyclo[4.1.0.0(2,4)]heptanes, one kind of the same azulenoids: guaiazulene, 24 kinds of the same sesquiterpene compounds, and 19 kinds of the same oxygenated sesquiterpene compound; however, the relative mass fraction of each component was different.
Fig. 4. Mass spectrogram of guaiazulene
Table 2. Chemical Components and Relative Mass Fraction in the Essential Oil from T. sinensis
No. 1 is the essential oil derived from T. sinensis leaves grown in Guangxi, Nandan county in China, collected in August, 2008; No. 2 is the essential oil derived from T. sinensis leaves grown in Guangxi, Dongnan county in China, collected in August, 2008; and No. 3 is the essential oil derived from T. sinensis common petioles grown in Guangxi, Dongnan county in China, collected in August, 2008. M.W. is molecular weight, R.T. is retention time, and R.M.F. is relative mass fraction.
Antimicrobial Activity
Growth inhibition zone diameters of different essential oil samples against microorganisms
All essential oil samples of T. sinensis exhibited significant growth inhibitory activity against the tested microorganisms (Table 3). The growth inhibition zone diameters of oil samples against the tested bacteria and fungi were 26 ± 0.1 mm ~ 60 ± 0.1 mm and 11 ± 0.1 mm ~ 50 ± 0.1 mm, respectively. T. sinensis essential oil was found to exhibit growth inhibitory activity against C. gloeosporioides for the first time.
Table 3. Growth Inhibition Zone Diameters of the Essential Oils against Microorganisms (mm)
There were differences among the growth inhibitory activities of different essential oil samples against different microorganisms. The order of inhibitory activity of essential oil samples from strong to weak was, for essential oil No. 1: E. coli > B. subtilis > C. gloeosporioides > P. citrinum; for essential oil No. 2: E. coli = C. gloeosporioides > B. subtilis > P. citrinum; and for essential oil No. 3: C. gloeosporioides > B. subtilis > E. coli > P. citrinum.
Different microorganisms had different sensitivities to the same essential oil samples. E. coli exhibited the strongest sensitivity to essential oil No. 1, with a growth inhibition zone diameter of 60 ± 0.1 mm, and the weakest sensitivity to essential oil No. 3, with a growth inhibition zone diameter of 26 ± 0.1 mm. B. subtilis exhibited similar sensitivity to the three essential oils, with a growth inhibition zone diameter 35 ± 0.1 mm ~ 36 ± 0.1 mm. P. citrinum exhibited the strongest sensitivity to essential oil No. 1, with a growth inhibition zone diameter of 16 ± 0.1 mm, and the weakest sensitivity to essential oil No. 2, with a growth inhibition zone diameter of 11 ± 0.1 mm. C. gloeosporioides exhibited the same sensitivity to essential oils Nos. 2 and 3, with growth inhibition zone diameters of 50 ± 0.1 mm, greater than its sensitivity to essential oil No. 1, with a growth inhibition zone diameter of 31 ± 0.1 mm. The three essential oils exhibited noticeable growth inhibitory activity against C. gloeosporioides.
MIC and MBC of essential oils against microorganisms
MIC and MBC of different essential oils against microorganisms are shown in Table 4.
Table 4. MIC and MBC of Essential Oils against Microorganism (μg·mL−1)
All essential oils of T. sinensis exhibited noticeable growth inhibitory activity against the tested microorganisms. The MIC and MBC of different essential oils against microorganisms were different. The MIC and MBC against E. coli and B. subtilis of all essential oil samples were less than 25 μg·mL−1. The MIC against P. citrinum of all essential oil samples was 200 μg·mL−1, and the MBC against P. citrinum of all essential oil samples was 400 μg·mL−1. The MIC against C. gloeosporioides of all essential oils was 50 μg·mL−1, and the MBC against C. gloeosporioides of all essential oils was 200 μg·mL−1.
T. sinensis essential oils were a complex mixture of components, and the difference in antimicrobial activity among the different samples resulted from different chemical compositions. There were differences in chemical compositions and contents among essential oils (No. 1, No. 2, and No. 3) extracted from T. sinensis from different parts and in different geographical areas in China, but the main components of essential oils were sesquiterpene and sesquiterpene oxygenated compounds, accounting for 90.10%, 92.64%, and 80.87% of the relative mass fraction, respectively. The primary compounds in essential oils are shown in Table 5.
The antimicrobial effect of the essential oils is the result of their chemical composition synergy. The major antimicrobial components and the antimicrobial chemical compositions’ antimicrobial action mechanisms should be subject to further study.
Table 5. Primary Compounds in Essential Oils Extracted from T. sinensis
DPPH Scavenging Activity of Essential Oil
The IC50 and IM50 values of essential oils scavenging DPPH calculated according to Eqs. 1 and 2 are shown in Table 6. Relationships between DPPH scavenging effects and the concentration of essential oil determined by regression analysis methods are also shown in Table 6. The IM50 values of scavenging DPPH of rutin, quercetin, gallic acid, and ascorbic acid were 3.300, 6.734, 4.730, and 13.506 g DPPH per g sample, respectively (Chen et al. 2006). It can be seen from Table 7 that the IM50 of scavenging DPPH of T. sinensis essential oil was low, less than 0.3 g DPPH per g essential oil, which may be related to the elevated temperature used in the simultaneous distillation and extraction. The weak DPPH scavenging activity of T. sinensis essential oil indicated its weak antioxidant capacity.
Table 6. Relationship between DPPH Scavenging Effect and the Concentration of T. sinensis Essential Oil
R is the correlation coefficient, C is the concentration of the essential oil (mg·L−1), and Y is the DPPH scavenging effect (%).
CONCLUSIONS
- A total of 63 compounds were identified in the essential oil of leaf blades grown in Nandan county in Guangxi province, China (No. 1), 63 in the essential oil of leaf blades (No. 2), and 72 in the essential oil of common petioles (No. 3) grown in Dongnan county in Guangxi province, China in August, accounting for 99.35%, 99.25% and 96.78% of the relative mass fraction, respectively. There were differences in chemical compositions and content among the essential oils extracted from different parts of Toona sinensis in different geographical areas in China, but the primary components of essential oils were sesquiterpene and sesquiterpene oxygenated compounds, accounting for 90.10%, 92.64%, and 80.87% of the relative mass fraction, respectively.
- All essential oils of T. sinensis exhibited noticeable growth inhibitory activity against the tested microorganisms. MIC and MBC of different essential oil samples against each microorganism were different. MIC and MBC against Escherichia coli and Bacillus subtilis of all essential oil samples were less than 25 μg·mL−1. MIC and MBC against Penicillium citrinum of all essential oil samples were 200 μg·mL−1 and 400 μg·mL−1, respectively. MIC and MBC against Colletotrichum gloeosporioides of all essential oil samples were 50 μg·mL−1 and 200 μg·mL−1, respectively. T. sinensis essential oil samples were a complex mixture of components, and the difference in antimicrobial activity among the different essential oil samples resulted from different chemical compositions. The major antimicrobial components and the antimicrobial chemical compositions’ antimicrobial action mechanisms are subjects for further study.
- The IM50 of scavenging DPPH of T. sinensis essential oil was low, less than 0.3 g DPPH per g essential oil. The weak DPPH scavenging activity of T. sinensis essential oil indicated its weak antioxidant capacity.
- The results indicated that T. sinensis essential oil may be a useful new source of natural antiseptic from forest products.
ACKNOWLEDGMENTS
The authors are grateful for the financial support from the Guangxi Natural Science Fund of China (No. 09236002), Guangxi Education Department Scientific Research Fund of China (No.2009LX003), and Guangxi University and Guangxi Education Department Education Reform in the 21st Century Research Fund of China (No. 2011JGA010).
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Article submitted: May 8, 2014; Peer review completed: Peer review completed: June 26, 2014; Revised version received and accepted: July 3, 2014; Published: July 17, 2014.