Comparison of Chemical Composition and

Antimicrobial Activities of the Essential Oil of Taxus Media and Taxus

Chinensis Var. Mairei Leaves

Jing Zhang2, Ke Yuan1* and Yongchun Jin1

1A Nurturing Station for the State key Laboratory of Subtropical Silviculture,

Zhejiang Agriculture and Forestry University Lin’an, China,2College of Pharmacy,

Henan University of Traditional Chinese Medicine Zhengzhou, China

*Corresponding author: Ke Yuan

yuan_ke001@163.com

Keywords:Taxus media; Taxus chinensis var. Mairei; leaves; essential oil; GC-MS; antimicrobial activity

Abstract.The study was conceived to evaluate the difference in the chemical composition as well as the difference in the antimicrobial activities of the essential oils of Taxus media and Taxus chinensis var. mairei leaves. The chemical composition of essential oils which obtained byhydrodistillation method, were analysed by gas chromatography-mass spectrometry (GC-MS) system, and the relative amount of the chemical components of the essential oils were determined by the peak area normalization method. Remarkable differences were recorded between the percentages of a few constituents from the two essential oils. A total of 34 compounds were identified from the essential oil of Taxus media leaves, with the compounds of the dominant are Benzene propanenitrile (21.30%), 1, 4-Dioxane-2, 3-diol (20.13%), 3-Bromo-3-methyl-Butyric acid (17.92%) and 1-hydroxy-2-Butanone (9.85%). For the essential oil of Taxus chinensis var. Mairei leaves, 33 compounds were identified with the compounds of the dominant are Benzene propanenitrile (49.39%), 1-hydroxy-2-Butanone (12.72%), Acetic acid (5.39%), 1-octen-3-ol (4.28%) and (E)-3-(2-hydroxyphenyl)-2-Propenoic acid, (3.53 %). The filter paper disc agar diffusion method was used to test the antimicrobial activities of the essential oils, and diameter of antibacterial zones (DD) value and minimum inhibitory concentration (MIC) value were used to evaluate the antimicrobial activity. The result showed that the essential oil of Taxus media leaves possessed stronger antimicrobial activity than the essential oil of Taxus chinensis var. mairei leaves against the two tested bacteria.

Introduction

Essential oils which also known by the name volatile oils are widely present in the variety of plant, and their main compounds can be classified into three major kinds, such as aliphatic, aromatic, terpene as well as their oxy-derivatives, some nitrogen and sulfur compounds. According to the previous studies, it can be known that the pharmacological effect of essential oils included antimicrobial, anti-inflammatory, diaphoresis, antiviral and antitumor, analgesia, local anesthesia, strengthening stomach, antiasthmatic, cough relief, expectorant and deworming, et al. . Taxus belongs to Taxaceae and Taxus genus, and has 11 species all over the world. It is a genus of evergreen trees or shrubs, distributed in the Northern Hemisphere, i.e. Europe, North America, Northern India, China and Japan. Some species are grown in gardens for their graceful foliage and scarlet fruits. In addition, it is precious rare and endangered species of the world. Because of its huge medicinal values, all countries of the world always list it as “National Treasure” . Taxol firstly discovered from Taxus brevifolia, can be extracted from almost all parts (needles, bark, roots, seeds and heartwood) of Taxus. It is without obvious side-effects, and has become the top-quality antitumor medicine in the world . The majority researches have been carried out on the chemical investigation of Taxus, especially on the anticancer components of taxanes .

This paper took Taxus media and Taxus chinensis var. mairei as research objects, and used hydrodistillation method to obtain their leaves essential oils, and applied gas chromatography-mass spectrometry (GC-MS) to determine the chemical composition of the essential oils, and utilized the peak area normalization method to determine the relative amount of them. The filter paper disc agar diffusion method was used to evaluate the antimicrobial activities of the essential oils simultaneously. According to our best knowledge, there are some reports on the chemical composition of the essential oils from the branches and leaves, and the seeds of Taxus chinensis var. mairei in the literature . This paper aims to help us to further understand the volatile constituents of Taxus media and Taxus chinensis var. mairei leaves, and provide theory basis for more comprehensively developing the precious rare resource.

Materials and Methods

A.Plant material and extraction of essential oils

Fresh leaves of Taxus media and Taxus chinensis var. mairei were three-years old, and were collected from Chang-shan County, GAP cultivation base of Taxus media Science Development Co.,Ltd (cultivated species) and Tong-lu County (wild species), Zhejiang Province, P. R. China in May 2010, respectively. The specimens were identified by Lu-huan Lou professor of Zhejiang Agriculture and Forestry University, and voucher specimens were deposited in our laboratory.

The fresh leaves were dried in an oven (40 ℃) for 30 h, then the dried leaves were powdered. 100 g powder were subjected to the steam distillation device for 7 h with 1400 ml distilled water. Later, the effluent liquid was extracted with 100 ml ether for 4 times, respectively, and then the extract liquid was dried over anhydrous sodium sulfate. After that, ether was recovered and light yellow essential oil with strong flavour was obtained, and then stored in sealed glass vial in a refrigerator at 4-5℃ prior to analysis. Yield based on dried weight of the sample was calculated.

B.Microorganisms

Two species bacteria (Gram-positive bacteria-Staphylococcus aureus and Gram-negative bacteria- Escherichia coli) were used for evaluating the antimicrobial activities of the essential oils of Taxus. They were supplied by Forestry Conservation Subject of the Institute for Forestry and Biotechnology of Zhejiang Agriculture and Forestry University.

C.Analysis of the essential oil

Gas Chromatography-Mass Spectrometry The analysis of the volatile constituents was run on a Varian CP3800 /1200L GC-MS system with a Fused Silica Capillary Column HP-5 (30 m×0.25 mm, 0.25 μm film thickness). The carried gas was helium, at a flow rate of 0.8 ml/min, with Splitless Injecting samples. Oven temperature was programmed (45 ℃ for 3 min, then 45-90 ℃ at 10 ℃/min, then 90-180℃ at 6 ℃/min, then 180-230 ℃ at 12℃/min, then 230-250 ℃ at 9 ℃/min and finally held at 250 ℃ for 9min). The injector and detector temperature were set at 250 ℃ and 280 ℃, resecitively. Mass spectrometer: The electron impact source was 70 eV, ion source temperature was 200 ℃, the mass range 33-450 amu and the scan rate was 0.5 s. The components of the essential oils were identified by comparing their mass spectral fragmentation patterns with those of similar compounds from databases (NIST and Willey Mass Spectral Libraries). For each compound on the gas chromatogram, the percentage of peak area relative to the total peak areas was determined as relative amount of that compound, without using correction factors.

D.Screening for antimicrobial activity

The antimicrobial activity was tested by the filter paper disc agar diffusion method and according to the diameter of antibacterial ring to evaluate the antimicrobial capacity of the essential oil. All bacteria were cultured in 10 ml inclined nutrient agar medium at 37 ℃ for 24 h. The microorganism suspensions were diluted with sterile distilled water and adjusted to approx 108 colony forming unit per millilitre (c. f. u. /ml) using standard curves at OD600 for use. Stock solutions of all tested substances were prepared and diluted to specified concentrations using ether, and then 3 sterile filter papers (diameter 5 mm) were added to the every diluted sample for 12 h. 0.1 ml of the diluted microorganism suspension was streaked onto the plate which with 15 ml nutrient agar medium, by using a sterile glass rod, and then the sterile filter paper impregnated with sample were placed uniformly on the surface of the media. The bacteria were incubated at 37 ℃for 24 h. After incubation period, the inhibition zone obtained around the disc was measured. A negative control, involving the presence of microorganisms without test material (blank) was used. The experiments were performed in triplicate.

E.Minimum inhibitory concentration (MIC)

MIC was established by adjusting the sample solutions to approximately concentrations. MIC was defined as the lowest concentration preventing visible microbial growth. In the present study, the tested concentrations of the essential oil of Taxus media are 10.00%, 9.00%, 8.00%, 7.00%, 6.00%, 5.00%, the tested concentrations of the essential oil of Taxus chinensis var. mairei are 100%, 90%, 80%, 70%, 60%, 50%.

Results and Discussion

A.Chemical composition of Taxus media and Taxus chinensis var. mairei essential oil

The essential oils obtained by hydrodistillation method, were light yellow liquid at room temperature. The total yield of essential oils obtained from Taxus media and Taxus chinensis var. mairei leaves were 0.15%, 0.21%. In the present study, the chemical composition of the essential oils were characterized and compared. The essential oils were diluted 5 times with ether, and then 0.2 μl dilution sample was added to GC-MS system. The total ions chromatogram (TIC) of the essential oils was shown as Fig. 1. The composition of the essential oils was shown in Table Ⅰ with the relative content of them. It can be known that most of the chemical composition was the same in the two essential oils, except some unique components. The main differences of the essential oils were the relative content of these components. According to Table Ⅰ, the data showed that a total of 34 compounds were identified from the essential oil of Taxus media leaves (94.37% of the oil), with the dominant compounds being Benzene propanenitrile (21.30 %),1,4- Dioxane- 2,3-diol (20.13%), 3-Bromo-3-methyl-Butyric acid (17.92%), 1-hydroxy-2-Butanone (9.85%), and other Aromatic compounds (6.10%), Acids (9.78%), Alcohols (9.61%), Aldehyde (2.66%), Phenols (1.60%), Amine (0.51%), Esters (0.36%); For the essential oil of Taxus chinensis var. Mairei leaves, 34 compounds were also identified (95.57% of the oil) with Benzene propanenitrile (49.39%), 1-hydroxy-2- Butanone (12.72%), Acetic acid (5.39%), 1-Octen-3-ol (4.28%), (E)-3-(2-hydroxyphenyl)-2-Propenoic acid, (3.53%), and other Alcohols (10.12%), Acids (8.57%), Aldehyde (2.83%), Aromatic (2.10%), Phenols (0.69%), Ketone (0.37%), Esters(0.36%).

According to the previous studies, in China, Jun et al. (2006), found that the essential oils of Taxus chinensis var. mairei seeds had Benzaldehyde (0.61%) and Tetradecanoic acid (4.21%), Ting et al.(1995), found that the essential oils of Taxus chinensis var. mairei leaves had Benzaldehyde (2.31%), Tetradecanoic acid (2.15%), Penzyl-methanol (1.54%) and 2-Phenyl-ethanol (0.31%). In India, Merajuddin et al. (2006), found that the essential oils of Taxus wallichiana Zucc leaves had (Z)-3-Hexenol (4.10%), Benzaldehyde (1.30%), Hexanoic acid (5.50%) and Benzoic Acid (0.70%). These components were the same findings as the present study. The forming reseans of other divergences maybe due to the differences of the producing area, ages and species, et al.

B.Antimicrobial activities of Taxus media and Taxus chinensis var. mairei essential oil

The in vitro antimicrobial activities of the essential oils of Taxus media and Taxus chinensis var. mairei leaves were evaluated by the filter paper disc agar diffusion method and their potency were assessed qualitatively and quantitatively by diameter of antibacterial zones (DD) values and minimum inhibitory concentration (MIC) values [13]. The results are given in Table Ⅱ. From the Table Ⅱ, it can be known that the two essential oils both have antimicrobial activity against the tested bacteria, and the inhibitory activities increased with the increase of sample concentration. The two essential oils have bigger inhibitory activity on Escherichia coli than Staphylococcus Aureus. In addition, we can find that MIC for the essential oil of Taxus media on tested bacteria were both 5%, and MIC for the essential oil of Taxus chinensis var. mairei on Escherichia coli and Staphylococcus Aureus were 95% and 98%, respectively. At last, it can be concluded that antimicrobial activity of the essential oil of Taxus media leaves obviously was stronger than the essential oil of Taxus chinensis var. mairei leaves.

Conclusion

In conclusion, we noted that the chemical composition of the essential oils of Taxus media and Taxus chinensis var. mairei leaves had many same components, such as: Benzene propanenitrile, 1-hydroxy-2-Butanone, Acetic acid, Propanoic acid, but the relative content of them were different.

The antimicrobial activities of the essential oils obtained in the present study showed that Taxus media leaves possessed significant inhibitory activity on the tested bacteria, but the inhibitory activity of Taxus chinensis var. mairei leaves on the tested bacteria was very low. This may be related to the composition of essential oils, and the interaction between these components or the content of the key components, but the specific causes and other biological tests are problems which need further research.

Acknowledgment

We are grateful to the Zhejiang Provincial Key Laboratory for Modern Silvicultural Technology, Zhejiang Agriculture and Forestry University for performing microorganism experiment, and the Center of Analysis and Testing, Jiangnan University, for performing GC-MS.