Variations in Essential Oil Content and Compositions of Eight Zanthoxylum bungeanum Species

Hailan Zhu¹, Zhe Li¹, Wangwang Jia¹, Yonghong Liu¹, Keyou Li¹

Introduction

Zanthoxylum bungeanum is wildly distributed in most parts of China and some Southeast Asian countries. The fruits of this species are called ‘da hong pao’ (big red robe). The pericarps of the fruits of Z. bungeanum have been utilized as a pungent food stuff and traditional Chinese medicine for treatment of vomiting, toothache, stomach ache, abdominal pain, eczema and diarrhea [1-6]. Essential oils are volatile, natural, complex compounds characterized by a strong odour and are formed by aromatic plants as secondary metabolites. Essential oils have been widely used for bactericidal, virucidal, fungicidal, antiparasitical, insecticidal, medicinal and cosmetic applications, especially nowadays in pharmaceutical, sanitary, cosmetic, agricultural and food industries [7]. Previous Z. bungeanum essential oil (ZBEO), which is from the pericarps of Z. bungeanum, researching has focused on the essential oil extraction, chemical composition and its biological activities such as antioxidant [8-10], antitumor [11-13], anti-inflammatory [14], antimicrobial and insecticidal [15,16].

To the best of our knowledge, few information on comparsion of the chemical composition of ZBEO is available. Thus, this study investigated the chemical composition of essential oils from different Z. bungeanum species and illustrated the chemical polymorphism among different Z. bungeanum species based on the characteristics of essential oils. The study facilitates linking the oil compounds with the pharmacological activities of essential oils extracted from Z. bungeanum. These results can provide valuable reference information for resource assessment and the rational utilization of Z. bungeanum resources

Materials and Methods

Plant materials

Eight Z. bungeanum species: Chenjiawuci (CW), Dangcunwuci (DW), Fengjiao (FJ), Hanchengdahongpao (HD), Hanchengwuci (HW), Qin’anyihao (QY), Shizitou (SZ) and Xinongwuci (XW) were collected from different regions of China (Table 1). CW, DW, HD, HW, QY, SZ and XW were transplanted to Northwest A&F University FENGXIAN experiment and demonstration station of Chinese prickly ash (Shaanxi Province, China) for at least three years and FJ was cultivated in the station. All the species were harvested as mature products in August 2014. Every sample contained pericarps from multiple trees of the corresponding species. Identification of these eight species was confirmed by Prof. An-zhi Wei from College of Forestry, Northwest A&F University, China. A voucher specimen was deposited in the college of forestry, Northwest A&F University (Yangling, China). All the pericarps of the eight species were stored at 4°C for use.

Essential oil extraction

The air-dried pericarps of eight Z. bungeanum species were powdered using a laboratory mill and filtered to 50 meshes. One hundred g of each sample powder was soaked in 1L of deionized water. For hydrodistillation extraction, the sample mixtures were extracted under atmospheric distillation for 3 h in Clevenger type apparatus to produce essential oil. The obtained essential oil was dried over anhydrous sodium sulphate and then extracted with a 0.2μm pore size filtration paper. It was kept in a sealed opaque vial at -20°C for subsequent experiments. The essential oils yield of every species was conducted five times.

Gas Chromatography-Mass Spectrometry analysis

The chemical components of essential oil in this study were analyzed by a GC-MS system (Thermo SCIENTIFIC), equipped with an HP-5MS capillary column (30 m × 0.25 mm; 0.25μm film thickness). The setting of temperature programming was as follows:

Table 1: Zanthoxylum bungeanum species collected from different regions of China.

sample name Sources Growth years

Han Cheng, Shaanxi province, Chenjiawuci (CW) 3 China Han Cheng, Shaanxi province, Dangcunwuci (DW) 3 China Fengjiao (FJ) Feng Xian, Shaanxi province, China 4 Hanchengdahongpao Han Cheng, Shaanxi province, (HD) China Han Cheng, Shaanxi province, Hanchengwuci (HW) 3 China Qin’anyihao (QY) Qin An, Gansu province, China 4 Han Cheng, Shaanxi province, Shizitou (SZ) 3 China Xinongwuci (XW) She Xian, Hebei province, China 4 50°C (2min), 50-150°C at 3°C/min, 150-240°C at 8°C/min, 240°C (2 min). For the injection (split injector), 5μl sample was diluted in 620μl chloroform and then 1μl of the solution was injected. The injector temperature, MS transfer line temperature and ion source temperatures are maintained at 250, 280 and 250°C, respectively. The split ratio was 1:100, scan time was 3.5 min and helium (99.99%) was used as carrier gas at a flow rate of 1ml/min. The mass spectrometer was operated in EI mode at 70 eV in m/e range 45-500 amu.

Identification of the essential oil components

The identification of the components was performed by matching their recorded mass spectra with the standard massspectra from the National Institute of Standards and Technology (NIST08. LIB) library data provided by the software of the GC–MS system, literature data and standards of the main components. The results were also confirmed by the comparison of the elution order of the compounds with their relative retention indices (determined with reference to a homologous series of normal alkanes) on the HP-5ms capillary column [17,18]. Quantitative analyses of each essential oil component (expressed as an area percentage) were carried out by a peak area normalization measurement, calculated as the mean value of injections from each sample.

Statistical analysis

Hierarchical cluster analysis (HCA) is a widely used classificatory technique in many areas of scientific knowledge. It is one of the most used classification methods in chemistry, being widely used in analytical chemistry, biochemistry, and multidisciplinary chemistry [19], which could discover the differences in species caused by multiple factors. By using HCA method on SPSS statistics 17.0 software (SPSS Inc., Chicago, IL, USA), and based on the content (%) of chemical constituents in essential oils, the relationship among the eight Z. bungeanum species were examined. For the same sample, and adopting different methods of system type is broadly in line with a result, the average clustering method is widely used so we sued the average method for clustering. The compositions above 1% were selected as the clustering variables.

Results and Discussion

The essential oils yield

Essential oils extracted from aromatic plants have a wide range of medicinal and industrial applications, such as antiseptic, preservatives, and antiphlogistic uses [20]. The total essential oil content of plants is generally very low and rarely exceeds 1% by mass. In the present study, the yield of essential oil strong varied for eight different Zanthoxylum bungeanum species. It fluctuated between 0.944 and 1.722% (v/w) and ranged from high to low: 1.722 ± 0.172% (CW), 1.314 ± 0.094% (FJ), 1.264 ± 0.093% (HD), 1.222 ± 0.050% (HW), 1.152 ± 0.093% (DW), 1.136 ± 0.062% (SZ), 0.951 ± 0.049% (QY), and 0.944 ± 0.045% (XW) (v/w). All the essential oils were colorless transparent and fragrant. There were many factors influenced the essential oil yields, such as species, different parts, harvesting time, developmental stages and environmental conditions [21,22]. Eight Zanthoxylum bungeanum species were cultivated in China under the four environmental conditions, including Han Cheng and Feng Xian, Shaanxi province, Qin An, Gansu province, She Xian, Hebei province (Table 1). Where are different from precipitation, temperature, solar radiation, and soil condition. Thus, the differences in essential oil yield and composition could mainly be attributed to the genotypes of Zanthoxylum bungeanum species and the environmental conditions.

Chemical composition of the essential oils

The detailed compositions of the essential oils identified by GC– MS displayed remarkable chemical polymorphism among the eight Zanthoxylum bungeanum species (Table 2). A total of 98 components were identified from all the samples including 17 components firstly reported in the essential oils of Zanthoxylum bungeanum species. Table 2 showed that 46, 52, 33, 41, 38, 49, 46, 62 compounds were identified from CW, DW, FJ, HD, HW, QY, SZ and XW, accounting for 100.0%, 99.12%, 97.92%, 99.55%, 99.50%, 99.43%, 99.44% and 98.81% of the total essential oils, respectively. According the previous study, a total of 35 and 39 essential compounds were analyzed in

ZBEO while more compounds were identified in present study [23,24]. Discrepancies of species, growth condition, environmental conditions may be cause of the difference. Forty-three compounds which existed in more than half of the species (≥4), while the contents of every compound were greatly different. Thirteen compounds were shared among the essential oils of the eight Z. bungeanum species while the contents of every compound were also great different. The major shared components were Terpinen-4-ol (11.81-31.44%), Linalool (5.08-28.71%), Terpinyl acetate (5.45-19.36%), Terpineol (1.41-18.67%), Cymene (0.48-10.08%), Eucalyptol (1.5-9.43%), Linalyl acetate (0.59-9.38%) (Figure 1).

Our results revealed obvious differencesn the oil constituents of the eight Z. bungeanum species. The main components (above 3%) of the essential oils are as follows:

d 20.49 1180 Z-11-Hexadecenoic acid — 0.21 d 20.53 1146 Octyl acetate — — 21.01 d 1152 Isoascaridole 0.27 0.24 21.15 1153 cis-Geraniol — — d 21.29 1155 Citronellol — — 22.19 1235 Linalyl acetate 1.05 1.77 d 22.21 1302 Linalyl butyrate — — 22.33 1166 Piperitone 3.41 2.36 22.56 1168 Geraniol — — 23.67 1275 Bornyl acetate 0.37 0.53 24.22 1185 Piperitol acetate 0.44 0.57 24.24 1290 4-Terpinenyl acetate — — 24.98 1308 Terpineol acetate 0.56 0.5 d 25.40 1319 Myrtenyl acetate — 0.08 exo-2-Hydroxycineole d 26.08 1335 0.26 0.19 Acetate 2-Hydroxycineole 26.22 1338 — — acetate 26.42 1343 Terpinyl acetate 8.85 15.62 26.65 1348 Citronellyl acetate 4.17 4.97 27.05 1357 Nerol acetate 0.43 0.36 d 27.35 1364 (R)-Lavandulyl acetate 0.27 — 3,7-dimethyl-2,6- d 27.89 1376 — 0.39 octadien-1-yl Acetate 27.96 1219 Geranyl acetate 0.27 0.32 28.25 1349 Elemene 1.05 1.71 29.46 1389 Caryophyllene 0.9 1.71 30.93 1435 Humulene 0.39 0.54 d 31.13 1441 α-Longipinene — — 31.34 1447 (-)-δ-Elemene — — 31.61 1455 γ-Cadinene — — 31.79 1461 γ-Muurolene — — 32.01 1467 α-Copaene 0.35 0.95 32.12 1470 D-Germacrene — — 32.32 1476 Eudesmene — — 32.38 1478 Selinene — — 32.44 1480 β-Copaene — — 32.55 1483 α-Acorenol — — 32.61 1485 Bicyclogermacrene — — 32.76 1489 Muurolene 0.29 0.27 32.95 1495 Germacrene A — — 33.06 1498 β-Acorenol 0.31 0.16 33.12 1499 α-Bulnesene 0.48 — 33.25 1503 D-Guaiene — — 33.32 1505 δ-Muurolene 0.3 0.55 d 33.38 1507 γ-Amorphene — — 33.54 1511 Cadinene 0.56 1.47 34.77 1545 α-Elemol — 0.26 35.07 1553 (-)-Globulol — — — — — — — — — — — 0.15 — — — — — — — 0.15 0.65 — — — — — — — — — — 0.27 — — — — — 0.85 0.05 — — — — — 0.21 0.31 0.53 — 0.29 0.46 — — — 0.5 0.43 — 0.11 1.58 — 0.23 — — 0.11 — — — — — — 2.41 2.9 3.18 2.35 7.4 0.86 0.82 — 0.23 0.2 0.66 — — 0.18 — — — — — — — — — — 1.1 — 1.14 1.8 2.45 — 1.25 0.86 1.22 3.23 4.13 — 0.23 0.25 0.35 0.73 1.2 — — — — — 0.15 — — — 0.39 0.19 0.15 0.19 0.5 0.24 — — — — — — 1.08 0.92 1.69 — — — — — 0.09 — — — — — 0.12 — — — — — 0.09 — — — 0.16 — — — — — — — 0.62 — 0.22 — 0.22 0.46 0.71 — — — — — 1.08 0.7 0.41 0.15 — 0.39 — — — — — — — — — — 0.33 — — — — — — — 1.27 — 0.51 0.54 1.08 0.86 1.93 — 0.47 — 0.2 — 0.39 35.19 1557 Germacrene B — 0.22 35.38 1562 trans-Nerolidol — — 35.62 1568 Spathulenol 0.22 0.37 35.79 1573 (-)-Spathulenol — — 35.92 1576 Caryophyllene oxide 0.84 0.69 36.70 1597 Globulol — — d 36.85 1601 Humulene epoxide — — 37.38 1614 Cubenol — 0.14 37.52 1618 α-Guaiene — — 37.78 1616 Cadinol acetate — — 37.79 1624 Cadinol 0.62 0.64 d 37.83 1625 epi-α-Muurolol — 0.72 37.88 1627 Muurolol 0.71 — 37.95 1628 Torreyol — 0.25 38.12 1633 β-Cadinol 2.26 1.89 38.25 1636 α-Cadinol — — 38.74 1662 cis-Vaccenic acid — — 39.37 1663 Ledene oxide — — d 41.71 1825 trans-Geranylgeraniol — — d 41.95 1842 Cembrene — — 43.43 1945 Camphorene — — d 44.45 2014 Manoyloxide — — Total 100 99.12 a Retention indices on HP-5MS capillary column.

b Not detected.

c The content of the compounds above 1% used the bold font.

d Firstly reported in the essential oils of Zanthoxylum bungeanum species.

Terpinen-4-ol (25.20%), Cymene (10.08%), Terpinyl acetate (8.85%), Linalool (8.06%), Limonene (7.12%), Terpieonl (5.95%), Citronellyl acetate(4.17%), Piperitone (3.41%) in CW; Terpinen-4- ol (21.17%), Terpinyl acetate (15.62%), Linalool (8.53%), Cymene (6.51%), Citronellyl acetate (4.97%), Terpinene (4.24%), Sylvestrene (3.17%) in DW; Terpinen-4-ol (18.67%), Terpinyl actate (14.43%), Eucalyptol (9.43%), Terpieonl (8.97%), Linalool (6.80%), Sylvestrene (5.20%), Linalyl acetate (4.45%), Piperitone (4.07%), Geranyl acetate (3.68%) in HD; Terpinen-4-ol (31.44%), Terpinyl acetate (9.43%), Linalool (8.13%), Limonene (8.10%), Eucalyptol (7.17%), Terpieonl (7.14%), Cymene (4.23%) in HW; Terpinen-4-ol (25.83%), Terpinyl acetate (10.18%), Linalool (8.62%), Terpieonl (7.14%), Cymene (7.10%), Eucalyptol (5.79%), Caryophyllene (3.23%) in SZ; Linalool (28.71%), Terpinen-4-ol (12.08%), Limonene (11.93%), Linalyl acetate (9.38%), Eucalyptol (6.40%), Terpinyl acetate (5.45%), Terpieonl (5.17%) in FJ; Terpinen-4-ol (21.95%), Terpinyl acetate (15.74%), Linalool (8.12%), Terpieonl (6.84%), Cymene (6.50%), Limonene (4.88%), Eucalyptol (4.67%), Citronellyl acetate (3.18%) in QY; Terpinyl acetate (19.36%), Terpinen-4-ol (13.05%), Citronellyl acetate (7.40%), Eucalyptol (5.32%), Linalool (5.08%), Caryophyllene (4.13%), β-Cadinol (3.46%) in XW.

Above these results showed some differences in the major composition with previous reports: D-Limonene (22.19%), β-yrcene (9.66%), trans-β-Ocimene (9.58%),Terpinen-4-ol (8.96%), γ-Terpinene (4.45%), cis-β-Ocimene (4.28%), Eucalyptol (3.92%), — — — 0.43 — 0.76 — 0.99 — 0.69 — — — 1.07 0.29 0.33 1.17 1.53 — — — — — 0.16 — — — — — 0.22 — — — — 0.16 0.29 — 0.2 — 0.13 — 0.31 — — — — — 1.23 — 0.51 0.27 0.5 0.83 — — 0.59 0.41 — — — — — — 0.62 0.86 1.4 — — — 0.14 0.23 0.39 — 1.94 1.16 — 2.3 3.46 — — — 1.81 — — — — — — — 0.1 — — — — — 0.16 — — — — — 0.26 — — — — — 0.45 — — — — — 0.22 β-Phellandrene (3.35%), Terpinyl actate (3.28%) and Linalool (3.15%) were reported by Li, et al. [23]; Terpinen-4-ol (18.42%), Eucalyptol (15.49%), limonene (7.47%), α-terpineol (5.79%), γ-terpinene (5.62%), terpinyl acetate (4.62%), and linalool (4.55%) were reported by Lan, et al. [6]. From Table 2, Terpinen-4-ol and Terpinyl acetate were found in essential oils of all Z. bungeanum species. They were the main components with contents in DW (21.17% and 15.62%), HD (18.67% and 14.43%), HW (31.44% and 9.43%), QY(21.95% and 15.74%), SZ (25.83% and 10.18%), XW (13.05% and 19.36%), respectively. On the other hand, Terpinen4-ol (25.20%) and Cymene (10.08%) were the main components of CW essential oil while Terpinen-4-ol (12.08%) and Linalool (28.71%) were the main components for FJ. The contents of other compounds were all less than 10%. They were different from the previous reports [6,24,25]. These changes in the essential oil composition may arise from environmental conditions and genetic difference and also show the essential oils have abundant diversity.

Generally, the major components determine the biological properties of the essential oil. For the essential oils of the eight Z. bungeanum species, Terpinen-4-ol was the main compound. So the biological activities of their essential oils were directly related with Terpinen-4-ol, which was in accordance with previous work that reported it as an effective antiinflammatory, antioxidant and antimicrobial [26-31], depressant and anticonvulsant [32], and insecticidal [33-38]. It has been reported that Terpinen-4-ol could enhance the transdermal permeation of the polar and non-polar drugs [39], Eucalyptol, terpineol [40], and limonene could also do [41], indicating these major oil contributors play a crucial role in the enhancement activity of ZBEO as a transdermal penetration enhancer [6]. Besides, there were many studies about the apoptosis induce activities of Terpinen-4-ol in human cells [27-30]. These conformed to the result in our previous study that ZBEO has an observable effect on HaCaT cell apoptosis [24]. Based on the high content of Terpinen4-ol. ZBEO are supposed to possess the aforementioned biological activities of Terpinen-4-ol.

Essential oils diversity

To evaluate the diversity of the ZBEO in the eight Z. bungeanum species the identified compounds were classified by their chemical structure (Table 3). Nine types of compounds were found in DW and XW, more than the compounds found in other six species. FJ has the least types of compounds. Alcohol (59.32%-33.07%), lipid (33.72%-15.31%) and olefin (25.86%-10.56%) are the most abundant three compositions of all eight species while other compositions are all below 10%. This phenomenon was caused by the highest content of Terpinen-4-ol and Terpinyl acetate, which were classified as alcohol and olefin, respectively. The result was similar to the previous study, in which the main compounds were oxygenated (41.65%) and non-oxygenated hydrocarbons (52.65%) [15]. Besides, the acid only existed in DW and XW while the cycloparaffin only existed in CW, DW and XW.

Relationship of the eight Zanthoxylum bungeanum species

The interspecies relationship of the eight Z. bungeanum species based on the 35 high content compositions (more than 1%, the bold font in Table 2) of their essential oils was calculated by hierarchical cluster analysis (HCA) in this study. HCA is a method used to measure the similarities among species by examining interpoint distances, representing all possible sample pairs in high dimensional space [42].

In the analysis, the chosen 35 high content compositions of eight

29 46.93(12) 41.03(15) 54.48(10) 43 21.36(18) 21.94(19) 22.41911) 17 16.67(10) 25.30(110 18.09910) species formed a matrix of 35 × 8. Analysis of the data was performed using the Statistics software SPSS. The results showed that the XW has a single cluster from other seven species while among this seven species, HD, FJ and WD have particular separate clusters. Based on the distance of the cluster, CW and HW have the closest relationship. QY and SZ also exhibited significant similarity but less than the CW and HW (Figure 2).

Conclusion

In this work, a direct comparison of chemical compositions in essential oils of eight Z. bungeanum species and their interspecies relationship were researched for the first time. Terpinen-4-ol and Terpinyl acetate were the major compounds of essential oil in DW, HD, HW, QY, SZ and XW. Terpinen-4-ol and Cymene were the main components of essential oil in CW while Terpinen-4-ol and Linalool were the main components of essential oil in FJ. CW and HW exhibited the highest similarity based on the high content compositions in their essential oils. The present study facilitates linking the oil compounds with the pharmacological activities of essential oils extracted from Z. bungeanum. These results can provide valuable reference information for resource assessment and the rational utilization of Z. bungeanum resources.

Acknowledgement

This work was supported by grants from the Research Achievement Transformation Program of the Northwest A&F University (No. Z222021310). We would like to acknowledge the Key Laboratory of Shaanxi Forestry Comprehensive at Northwest A&F University for providing access to the necessary equipment to complete this study.

Author Contributions

Keyou Li conceived and designed the project; Yonghong Liu collected material; Zhe Li and Wang-wang Jia hydrodistilled the essential oil; Hailan Zhu analyzed the data; Hailan Zhu and Keyou Li wrote the manuscript.

Conflict of interest

The authors declare no conflict of interest.

FJ (%) HD (%) HW (%) QY (%) SZ (%) XW (%) 51.26(15) 58.91(12) 47.45(15) 54.54(14) 32.68(16) 10.29(12) 19.04(13) 20.01(19) 18.40(17) 25.86(27) 29.91(10) 15.72(9) 24.15(11) 16.31(10) 34.11(11) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) 1.07 0.29 0.33 1.17 1.91 — (1) (1) (1) (1) (3) 0.15 — — — — — (1) 0.27 0.14 0.42 0.78 — — (1) (1) (1) (1) 0.10 — — — — — (1) Figures 1: Chemical structures of some major compounds of essential oils from eight Z. bungeanum species.

Figures 2: Relationship of the eight Zanthoxylu.

References

1. Gong, YW., Huang, YF., Zhou, LG., Shi, XY., Guo, ZJ., Wang, MG., et al. (2014) Chemical composition and antifungal activity of the fruit oil of Zanthoxylum bungeanum Maxim. (Rutaceae) from China. J Essent Oil Res, 21(2): 174–1
2. Tirillini, B., Manunta, A., Stoppini, AM. (1991) Constituents of the Essential Oil of the Fruits of Zanthoxylum bungeanum. Planta Med, 57(1): 90-91.
3. Yang, FX., Su, YQ., Li, XH., Zhang, Q., Sun, RC. (2008) Studies on the preparation of biodiesel from Zanthoxylum bungeanum Maxim Seed oil. J Agric Food Chem, 56(17): 7891–7896.
4. Huang, S., Zhao, L., Zhou, XL., Ying, M., Wang, CJ., Weng, J. (2012) New alkylamides from pericarps of Zanthoxylum bungeanum. Chin Chem Lett, 23(11): 1247.
5. Yang, LC., Li, R., Tan, J., Jiang, ZT. (2013) Polyphenolics composition of the leaves of Zanthoxylum bungeanum Maxim. grown in Hebei, China, and their Radical Scavenging activities. J Agric Food Chem, 61(8): 1772–1778.
6. Lan, Y., Wu, Q., Mao, YQ., Wang, YL., An, J., Chen, YY., et al. (2014) Cytotoxicity and enhancement activity of essential oil from Zanthoxylum bungeanum Maxim. as a natural transdermal penetration enhancer. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 15(2): 153–164.
7. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M. (2008) Biological effects of essential oils-A review. Food and Chemical Toxicology, 46(2): 446–475.
8. Wang, L., Wang, ZM., Li, XY., Zhang, HH., Zhou, X., Zhang, HQ. (2010) Analysis of volatile compounds in the pericarp of Zanthoxylum bungeanum Maxim by ultrasonic nebulization extraction coupled with headspace single-drop microextraction and GC-MS. Chromatographia, 71(5-6): 455−459.
9. Xia, L., You, JM., Li, GL., Sun, ZW., Suo, YR. (2011) Compositional and antioxidant activity analysis of Zanthoxylum bungeanum seed oil obtained by supercritical CO fluid extraction. J Am Oil Chem Soc, 88(1): 23−32.
10. Wei, SG., Zhang, HH., Wang, YQ., Wang, L., Li, XY., Wang, YH., et al. (2011) Ultrasonic nebulization extraction-heating gas flow transferheadspace single drop microextraction of essential oil from pericarp of Zanthoxylum bungeanum Maxim. Chromatogr, 1218(29): 4599−4605.
11. Yuan, TN., Wang, YL., Wang, JJ. (2008) The study of the anti-cervix cancer Hela cells effect of Zanthoxylum Maxim. Journal of Hubei University for Nationalities·Medical Edition, 25: 26–27.
12. Huang, HC., Wang, RY., Zhou, WM. (2010) Lethal effect of Zanthoxylum Maxim volatile oil on Pheochromocytoma cells. Heilongjiang Medicine Journal, 23: 514–515.
13. Lin, Z., Han, S., Jiang, J., Lin, Y., Chen, H., Yuan, Y. (2013) Antitumor compound identification from Zanthoxylum bungeanum essential oil based on composition-activity relationship. Chem Res Chin Unvi, 29(6): 1068–1071.
14. Tezuka, Y., Irikawa, S., Kaneka, T., Banskota, AH., Nagaoka, T., Xiong, QB., et al. (2001) Screening of Chinese herbal drug extracts for inhibitory activity on nitric oxide production and identification of an active compound of Zanthoxylum bungeanum. J. Ethnopharmacol, 77(2-3), 209-217.
15. Zhu, RX., Zhong, K., Zeng, WC., He, XY., Gu, XQ., Zhao, ZF., et al. (2011) Essential oil composition and anti-bacterial activity of Zanthoxylum bungeanum. African Journal of Microbiology Research, 5(26): 4631– 4637.
16. Wang, CF., Yang, K., Zhang, HM., Cao, J., Fang, R., Liu, ZL., et al. (2011) Components and insecticidal activity against the maize weevils of Zanthoxylum schinifolium fruits and leaves. Molecules, 16(4): 3077– 3088.
17. Adams, RP. (2001) Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy. Allured Publishing Corporation, Carol Stream, IL, USA.
18. Siani, AC., Ramos, MF., Menezes-de-Lima, OJ., Ribeiro-dos-Santos, R., Fernadez-Ferreira E., Soares, RO., et al. (1999) Evaluation of antiinflammatory related activity of essential oils from the leaves and resin of species of Protium. J. Ethnopharmacol, 66(1): 57–69.
19. Leal, W., Llanos, EJ., Restrepo, G., Suárez, CF., Patarroyo, ME. (2016) How frequently do clusters occur in hierarchical clustering analysis? A graph theoretical approach to studying ties in proximity. Journal of Cheminformatics, 4: 2–16.
20. Samejo, MQ., Memon, S., Bhanger, MI., Khan, KM. (2013) Essential oil constituents in fruit and stem of Calligonum polygonoides. Ind Crops Prod, 45: 293–295.
21. Baranauskiene, R., Venskutonis, PR., Dambrauskiene, E., Viskelis, P. Harvesting time influences the yield and oil composition of Origanum vulgareL. ssp. vulgare and ssp. hirtum. Ind Crops Prod, 49: 43–51.
22. Li, B., Zhang, C., Peng, L., Liang, Z., Yan, X., Zhu, Y., et al. (2015) Comparison of essential oil composition and phenolic acid content of selected Salvia species measured by GC–MS and HPLC methods. Ind Crops Prod, 69: 329–334.
23. Diao, WR., Hu, QP., Feng, SS., Li, WQ., Xu, JG. (2013) Chemical composition and antibacterial activity of the essential oil from green huajiao (Zanthoxylum schinifolium) against selected foodborne pathogens. J Agric Food Chem, 61(25): 6044-604.
24. Li, KY., Zhou, R., Jia, WW., Li, Z., Li, JZ., Zhang, PF., et al. (2016) Zanthoxylum bungeanum essential oil induces apoptosis of HaCaT human keratinocytes. J Ethnopharmacol, 186: 351–361.
25. Wang, Z., Ding, L., Li, T., Zhou, X., Wang, L., Zhang, H., et al. (2006) Improved solvent-free microwave extraction of essential oil from dried Cuminum cyminum L. and Zanthoxylum bungeanum Maxim. J Chromatogr A, 1102(1-2): 11–17.
26. Hart, PH., Brand, C., Carson, CF., Ryley, TV., Prager, RH., Finlay-Jones, JJ. (2000) Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes. Inflamm Res, 49(11): 619-626.
27. Calcabrini, A., Stringaro, A., Toccacieli, L., Meschini, S., Marra, M., Colone, M., et al. (2004) Terpinen-4-ol, the main component of Melaleuca alternifolia (tea tree) oil inhibits the in vitro growth of human melanoma cells. J Invest Dermatol, 122(2): 349-360.
28. Khaw-on, P., Banjerdpongchai, R. (2012) Induction of intrinsic and extrinsic apoptosis pathways in the human leukemic molt-4 cell line by terpinen-4-ol. Asian Pac J Cancer Prev, 13(7): 3073-3076.
29. Wu, CS., Chen, YJ., Chen, JJ., Shien, JJ., Huang, CH., Liu, PS., et al. (2012) Terpinen-4-ol induces apoptosis in human nonsmall cell lung cancer in vitro and in vivo. Evid Based Complement Alternat Med.
30. Banjerdpongchai, R., Khaw-On, P. (2013) Terpinen-4-ol induces autophagic and apoptotic cell death in human leukemic HL-60 cells. Asian Pac J. Cancer Prev, 14(12): 7537-7542.
31. Nogueira, MNM., Rossa, JC. (2014) Terpinen-4-ol and alpha-terpineol (tea tree oil components) inhibit the production of IL-1b, IL-6 and IL- 10 on human macrophages. Inflamm Res, 63(9): 769–778.
32. Silva, LL., Garlet, QI., Benovit, SC., Doici, G., Mallmann, CA., Burger, ME., et al. (2013) Sedative and anesthetic activities of the essential oils of Hyptis mutabilis (Rich.) Briq. and their isolated components in silver catfish (Rhamdia quelen). Braz J Med Biol Res, 46(9): 771–779.
33. Lee, S., Tsao, R., Peterson, C., Coats, JR. (1997) Insecticidal activity of monoterpenoids to western corn rootworm (Coleoptera: Chrysomelidae), twospotted spider mite (Acari: Tetranychidae), and house fly (Diptera: Muscidae). J Econ Entomol, 90(4): 883–892.
34. Walton, SF., McKinnon, M., Pizzutto, S., Dougall, A., Williams, E., Currie, BJ. (2004) Acaricidal activity of Melaleuca alternifolia (tea tree) oil: in vitro sensitivity of sarcoptes scabiei var hominis to terpinen-4-ol. Arch Dermatol, 140(5): 563–566.
35. Kurekci, C., Padmanabha, J., Bishop-hurley, SL., Hassan, E., Jassim, RA., Mcsweeney, CS. (2013)
36. Antimicrobial activity of essential oils and five terpenoid compounds against Campylobacter jejuni in pure and mixed culture experiments. Int J Food Microbiol, 166(3): 450–457.
37. Yu, D., Wang, J., Shao, X., Xu, F., Wang, H. (2015) Antifungal modes of action of tea tree oil and its two characteristic components against Botrytis cinerea. J Appl Microbiol, 119(5): 1253–1262.
38. Wang, Y., You, CX., Yang, K., Wu, Y., Chen, R., Zhang, WJ., et al. (2015) Bioactivity of essential oil of zingiber purpureum rhizomes and its main compounds against two stored product insects. J Econ Entomol, 108(3): 925–932.
39. Grando, TH., Desa, MF., Baldissera, MD., Oliveira, CB., de Souza, ME., Raffin, RP., et al. (2016) In vitro activity of essential oils of free and nanostructured melaleuca alternifolia and of terpinen-4-ol on eggs and larvae of haemonchus contortus. J. Helminthol, 90(3): 377–382.
40. Magnusson, BM., Runn, P., Koskinen, LO. (1997) Terpene-enhanced transdermal permeation of water and ethanol in human epidermis. Acta Derm Venereol, 77(44): 264–267.
41. Narishetty, STK., Panchagnula, R. (2005) Effect of L-menthol and 1,8-cineole on phase behavior and molecular organization of SC lipids and skin permeation of zidovudine. J Control Release, 102(1): 59–70.
42. Okabe, H., Takayama, K., Ogura, A., Nagai, T. (1989) Effect of limonene and related compounds on the percutaneous absorption of indomethacin. Drug Des Deliv, 4(4): 313–321.
43. Lima, DC., Santos, AM., Araujo, GO. (2010) Principal component analysis and hierarchical cluster analysis for homogeneity evaluation during the preparation of a wheat flour laboratory reference material for inorganic analysis. Microchemical Journal, 95(2): 222-226.49.