Journal of Ethnopharmacology 158 (2014) 43–48
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Chemical composition, antimicrobial and anti-inflammatory activities of the essential oil from Maqian (Zanthoxylum myriacanthum var. pubescens) in Xishuangbanna, SW China Ren Li a,b,1, Jing-jing Yang a,b,1, Yin-xian Shi a,b, Min Zhao a,b, Kai-long Ji a,b, Ping Zhang a, You-kai Xu a, Hua-bin Hu a,n a Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, Mengla, Yunnan 666303, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 25 February 2014 Received in revised form 29 May 2014 Accepted 7 October 2014 Available online 16 October 2014
Ethnopharmacological relevance: Maqian (Zanthoxylum myriacanthum var. pubescens Huang) is widely consumed as an indigenous remedy for digestive disorders, detoxification, detumescence and analgesia by the ethnic groups in Xishuangbanna, SW China. A related species, Huajiao (Zanthoxylum schinifolium Sieb. et Zucc.), has similar uses in traditional Chinese medicine. We aimed to scientifically validate the traditional uses by investigating and comparing the chemical composition, antimicrobial and antiinflammatory activities of the essential oils of Maqian and Huajiao. Materials and methods: Essential oils were collected from the fruits of Maqian and Huajiao by simultaneous distillation extraction and identified by gas chromatography–mass spectrometry (GC–MS) analysis. To assess antimicrobial activity, the minimum inhibitory and bactericidal concentrations (MIC and MBC) against 7 microbial strains, including 5 food-borne pathogens, were evaluated by serial dilution with a standardized microdilution broth methodology. For anti-inflammatory activity, the cell viability and nitric oxide (NO) production were determined on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells by MTS assay and the Griess reagent system, respectively. Results: The essential oil from Maqian is rich in limonene (67.06%) and has strong antimicrobial activity against the tested pathogens and spoilage organisms, with MIC ranging from 64 to1024 mg/ml and MBC ranging from 64 to 2048 mg/ml. It also showed anti-inflammatory activity by significantly inhibiting nitric oxide (NO) production induced by LPS in RAW 264.7 cells at 0.04‰ without effects on cell viability. Furthermore, it showed relatively stronger antimicrobial and anti-inflammatory activities than the essential oil from Huajiao. Conclusions: Our findings not only justify the use of Maqian as an indigenous remedy for digestive disorders, detoxification, detumescence and analgesia, but also suggest that it could be promoted as a preferred substitute for Huajiao. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Zanthoxylum myriacanthum var. pubescens Essential oil Antimicrobial Anti-inflammatory Xishuangbanna
1. Introduction The species in the genus Zanthoxylum (Rutaceae), which is distributed mostly in the tropical and temperate areas of East Asia and Northeast America, are shrubs, trees, scramblers or woody climbers, and contain aromatic volatile oils. There are more than 200 Zanthoxylum species, of which 41 species (25 endemic) are found in China. These include Zanthoxylum myriacanthum var. pubescens Huang (synonyms Zanthoxylum rhetsoides var. pubescens
n
Corresponding author. Tel.: þ 86 691 8715415; fax: þ86 691 8715070. E-mail address: [email protected] (H.-b. Hu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jep.2014.10.006 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
C. C. Huang and Z. utile C. C. Huang), which is a variety of Zanthoxylum myriacanthum Wall. ex Hook. f. distinguished by villous leaf rachises, petiolules, leaflet blades, and inflorescence rachises (Zhang et al., 2008). It is known as “Maqian” in the Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China. This area is rich in biodiversity and has a tropical monsoon climate characterized by a distinct rainy season (May–October), with peak rainfall in July– September. The people of this region are exposed to many tropical diseases, such as cholera, typhoid, malaria, plague and digestive disorders, and the indigenous people have developed plant remedies to treat them. These traditional remedies include Maqian, which is an important indigenous remedy consumed by Dai people in Xishuangbanna for digestive disorders, detoxification, and the relief of swelling and pain
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R. Li et al. / Journal of Ethnopharmacology 158 (2014) 43–48
(Lin et al., 2003; SATCM, 2005). Huajiao (Zanthoxylum schinifolium Sieb. et Zucc.), known as Chinese prickly ash, is a related species of Zanthoxylum commonly used in traditional Chinese medicine in the treatment of detoxification, vomiting and stomach ache (Chinese Pharmacopoeia Commission, 2005). However, our ethnobotanical research has found that Maqian is frequently used instead of Huajiao to treat these diseases by the ethnic groups in Xishuangbanna. Beside its medicinal usage, Maqian is also widely consumed as a spicy condiment, as is Huajiao. Specifically, Maqian is frequently used as a spice for roasting and salting meat, boiling fish and cooking vegetable soup in the ethnic villages of Xishuangbanna (Pei, 1985; Cheng, 1991; Xu et al., 2004). Many Zanthoxylum species are used as medicines and spices around the world, and have been reported to have antioxidant activity (Yamazaki et al., 2007; Yang et al., 2013), antimicrobial properties (Diao et al., 2013; Misra et al., 2013), antinociceptive effects (Pereira et al., 2010; Hu et al., 2013), modulate immune function (Seo et al., 2012), gastroprotective activity (Freitas et al., 2011), hepatoprotective activity (Ranawat et al., 2010), antiinflammatory and analgesic activities (Bastos et al., 2001; Tezuka et al., 2001; Lima et al., 2007). For example, the essential oil from Huajiao, of which main components were identified as linalool (28.2%), limonene (13.2%), and sabinene (12.1%), displayed strong antibacterial activity against tested Gram-positive bacteria, such as Staphylococcus epidermidis (Diao et al., 2013). The fruits of Zanthoxylum myriacanthum are used as a spice to cook sour fish by Miao people in Guizhou province, China, and the fruit essential oil, of which the main constituents were identified as sabinene (32.43%), limonene (28.91%) and 1,8-cineole (13.55%), exhibited no antibacterial activity against 7 bacterial strains (Zhu et al., 2007). While in the mountain regions of Northern and Central Vietnam, the dried fruit of Zanthoxylum myriacanthum was a preferred spice for beef and its essential oil, of which main constituents were identified as limonene (27.2%) p-Cymene (15.3%), and β-Phellandrene (11.9%), was also used for poisoning fish by villagers (Weyerstahl et al., 1999). The stem and root bark of Zanthoxylum myriacanthum were reported to be rich in triterpenes, lignans and alkaloids (Waterman, 1975; Liang et al., 1988; Sukari et al., 1999), and a 95% ethanol extract of its fruit, which were used as a traditional Thai medicine to treat different kinds of ailments, including microbial infections, showed antibacterial activity against Staphylococcus aureus and Escherichia coli (Chansakaow et al., 2005), and its leaf, stem and bark extracts showed antitumor activity (Bowen and Lewis, 1978). However, limited scientific and bioactivity information is available on the essential oil of Maqian. The aim of this study was therefore to provide scientific evidence to justify its traditional usage as an ethnic medicine and spice by investigating its chemical composition, antimicrobial and anti-inflammatory activities. In addition, parallel and comparative laboratory analysis of the essential oil of Huajiao was conducted in order to support the preference of the ethnic group for Maqian.
2. Material and methods
mill. The essential oils were collected by simultaneous distillation extraction and stored at 20 1C in the dark for further use. 2.2. Chemicals and reagents Dimethylsulfoxide (DMSO), L-glutamine, and Lipopolysaccharide (LPS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and penicillin–streptomycin were purchased from Thermo Scientific (Logan, UT, USA). The CellTiter 96sAQueous One Solution Reagent for MTS assay and the Griess reagent system for NO measurement were purchased from Promega Corporation (Madison, WI, USA). Standard Mueller–Hinton agar and broth (MHA and MHB) and Sabouraud agar and broth (SA and SB) were bought from Tianhe Microbial Agents Company (Hangzhou, China). All reagents were analytical standard. 2.3. GC–MS analysis The analysis of the essential oils was performed using an Agilent Technologies 7890 A GC, equipped with a HP-5 MS capillary column (30 m 0.25 mm; film thickness, 0.25 mm) and a mass spectrometer 5975C (Agilent Technologies, USA) as detector. MS were taken at 70 eV with a mass range of m/z 45–500. Helium was used as the carrier gas, at a flow rate of 1 ml/min. Injector and detector (MS transfer line) temperatures were both 250 1C. Column temperatures were gradually increased from 40 1C to 160 1C at 3 1C/min, and increased to 250 1C at 20 1C/min, then held for 10 min. 0.2 ml of the diluted sample was injected manually. 2.4. Identification of the essential components The components were identified by comparing calculated experiment GC retention indices, which were determined with reference to homologous series of n-alkanes C7–C30 under identical experimental condition, with the GC retention indices reported in NIST Standard Reference Database (NIST Chemistry WebBook, 2014), by matching their mass spectra with those recorded in the NIST 08 database (National Institute of Standards and Technology, Gaithersburg, MD, USA) and mass spectra with published data. The percentage composition of individual components was computed by the normalization method from the GC peak areas, assuming an identical mass response factor for all compounds. 2.5. Microbial strains and cultural medium The essential oils were individually tested against 7 microbial strains. Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Candida albicans (ATCC Y0109) were provided by the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, China); Aspergillus funigatus, Acinetobacter baumannii and Klebsiella pneumonia were provided by Kunming General Hospital. Standard Mueller–Hinton agar and broth (MHA and MHB), and Sabouraud agar and broth (SA and SB) were used as the bacterial and fungal culture media, respectively.
2.1. Plant materials 2.6. Determination of MIC and MBC The fruits of Maqian (Zanthoxylum myriacanthum var. pubescens Huang) and Huajiao (Zanthoxylum schinifolium Sieb. et Zucc.) were both collected form Mengwang township, Jinghong municipality, Xishuangbanna Dai Autonomous Prefecture, China in October 2012, and identified by Ms. Chun-fen Xiao from Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (no. 152673) for Maqian was deposited in the herbarium (HITBC). The fruits were air-dried and powdered by a laboratory
The MIC and MBC were determined against tested microbial strains according to the procedures reported previously (NCCLS, 1999; CLSI, 2006). The MICs and MBCs were both determined with starting inoculums of 1.0 106 CFU/ml for bacteria and 1.0 104 CFU/ ml for fungi, incubated at 35 1C for 24 h, and were examined for growth in daylight. The serial dilution method was used for the determination of the MICs of the samples. For the MIC assay, the
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essential oils was dissolved in dimethylsulfoxide (DMSO), and sterilized by filtration through 0.45 mm Millipore filters. 100 ml of an appropriate medium, essential oil solutions and inoculums were dispensed onto a 96-well plate. MIC values were the lowest concentration of a given extract that completely inhibited the visible microbial growth. For the MBC assay, 10 ml samples taken from the clear wells of the microbroth susceptibility studies were placed onto the surfaces of MHA or SA plates to determine MBC. The MBC was defined as the concentration of drug that resulted in 499.9% mortality of the bacterium relative to the concentration of bacterium that was present in test wells at 0 h (NCCLS, 1999). The MICs and MBCs of Amikacin, Fluconazole, Vancomycin, Tigecycline, Ciprofloxacin and Cefotaxime were also determined in parallel experiments as positive control. The culture medium and solvent (DMSO) were used as a negative control in parallel tests and their values were deducted to get the final results of activity. All experiments were performed in triplicate. 2.7. Cell culture The murine macrophage cell line RAW 264.7 was obtained from Kunming Institute of Zoology, Chinese Academy of Sciences (KCB200603YJ) and maintained in DMEM (Thermo Scientific, Logan, UT, USA) containing 10% fetal bovine serum, 1% penicillin–streptomycin and 1% L-glutamine (Sigma-Aldrich, St Louis, MO, USA) at 37 1C in a 5% CO2 incubator (Thermo Scientific, Forma371, Steri-cycle, USA) and were subcultured every 2 days. 2.8. Cell viability Cell viability was evaluated by MTS assay as previously reported (Chae et al., 2004). In the MTS assay, 100 μl samples of cell suspension (1 105 cells/well) were cultivated in 96-cell plates for 18 h as described. Then cells were pre-treated with various concentrations of the essential oils for 30 min before they were further incubated in the presence of 1 μg/ml LPS for 24 h. Finally, 20 ml of CellTiter 96sAQueous One Solution Reagent, prepared by MTS (3-[4,5, dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) in the presence of phenazine ethosulfate (PES), was added to each well and incubated for 1 h at 37 1C in the 5% CO2 incubator. The absorbance of each well was measured at 490 nm directly using a Multifunctional Microplate Reader (Thermo Scientific VarioSkan Flash, USA). The untreated cells, incubated in culture medium with 0.4% DMSO, were employed as blank controls in every experiment. Results were expressed as a percentage of the MTS production by untreated cells (control group). Values were presented as mean7standard deviation (SD) of three independent tests. 2.9. Measurement of NO production NO production by LPS-stimulated RAW 264.7 cells was measured by the accumulation of nitrite in the culture supernatants, using the Griess reagent system (Promega, Madison, WI, USA) and following the manufacturer's instructions. Cells were seeded in 96-well plates at 1 106 cells/well and then incubated with culture medium (control) for 18 h. Then cells were pretreated with various concentrations of the essential oils for 30 min before they were stimulated with 1 μg/ml LPS and further cultured for an additional 24 h. In brief, 50 μl of culture supernatants were collected and mixed with the Griess reagent system (1% sulfanilamide in 0.1 mol/l HCl and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride). Then the plates were incubated at 37 1C in the 5% CO2 incubator for 10 min. The absorbance was measured at 550 nm in a Multifunctional Microplate Reader (Thermo Scientific VarioSkan Flash, USA). Nitrite concentration was determined from a sodium nitrite standard curve. The untreated cells, incubated in culture medium with 0.4% DMSO, were employed as blank controls in
45
every experiment. Results were expressed as percentage of NO by LPStreated cells (LPS group). Values were presented as mean7standard deviation (SD) of three independent tests. 2.10. Statistical analysis All experiments were performed in triplicate and expressed as mean values 7standard deviation (SD). One-way ANOVA with a Dunnett's multiple comparison test was performed using SPSS 14.0 for windows (SPSS Inc. Chicago, USA). Significance of difference was accepted at P o0.05. 3. Results and discussion 3.1. Chemical composition of the essential oils The essential oil yields were 3.6% and 4.8% (w/w) on a dry weight basis for Maqian and Huajiao, respectively. Fifty components were identified in the essential oils (38 in Maqian and 25 in Huajiao) representing 94.86% and 98.29% of the respective oils. The main constituents of the oil from Maqian were limonene (67.06%), α-Pinene (6.49%), β-Myrcene (3.87%), and linalool (2.96%), while the main constituents from Huajiao were linalool (79.00%), limonene (4.97%), β-Thujene (4.29%) (Table 1). These components have been commonly found in Zanthoxylum species (Weyerstahl et al., 1999; Zhu et al., 2007; Yang, 2008; Misra et al., 2013), but Maqian has the highest reported content of limonene: 67.06%, compared with 4.97% from Huajiao in this study, and 13.2% and 14.3% from green Huajiao (Yang, 2008; Diao et al., 2013), 27.2% and 28.91% from Zanthoxylum myriacanthum in Vietnam and China (Weyerstahl et al., 1999; Zhu et al., 2007), or not found at all in the fruit essential oil of red and green Huajiao in some previous studies (Gong et al., 2009; Lee et al., 2009). The high content of limonene could explain why the ethnic groups insisted that Maqian was more fragrant than Huajiao. 3.2. Antimicrobial activity The essential oil from Huajiao showed antimicrobial activity against all seven tested microbial strains, with MIC ranging between 160 and 5120 mg/ml and MBC ranging from 160 to more than 5120 mg/ml (Table 2). At the concentrations tested in this study (up to 10 mg/ml), Maqian showed no antimicrobial activity against Escherichia coli but obvious activity against the other six strains, with MIC ranging between 64 and 1024 mg/ml and MBC ranging from 64 to 2048 mg/ml. Compared with Huajiao, Maqian showed stronger activity against some of the pathogens and spoilage organisms tested, including Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumonia, Aspergillus fumigates and Candida albicans. Compared with the positive control, Maqian showed better MIC and MBC values than Fluconazole against Aspergillus fumigates, and comparable MIC and MBC values to Amikacin against Klebsiella pneumonia. The antimicrobial activity of Maqian is probably due not only to the abundance of limonene, which has broad and strong antimicrobial capacities (LisBalchin et al., 1996; Neirotti et al., 1996; Bevilacqua et al., 2010), but also to the other constituents. This result matched with the traditional use of Maqian as a remedy to treat digestive disorders, which might be caused by the food-borne pathogens contained in fresh and uncooked meat and green vegetables. 3.3. Anti-inflammatory activity 3.3.1. Effect on LPS-induced RAW 264.7cell viability The effect of the essential oils on LPS-induced RAW 264.7 cells viability was determined by MTS assay. There was a slight but
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Table 1 Chemical compositions of the essential oils from Maqian and Huajiao. cal
926 935 949 976 978 996 1008 1013 1015 1023 1037 1043 1053 1058 1066 1072 1078 1092 1108 1117 1122 1135 1138 1147 1176 1184 1192 1194 1196 1198 1199 1212 1216 1229 1237 1241 1250 1256 1263 1278 1285 1291 1372 1391 1425 1460 1484 1488 1531 1592
RI
lit
926 937 954 976 979 992 1004 1011 1017 1020 1035 1043 1048 1060 1062 1073 1074 1094 1103 1115 1121 1131 1139 1146 1176 1182 1189 1197 1194 1206 1199 1206 1211 1229 1238 1246 1257 1257 1262 1272 1286 1289 1367 1385 1422 1456 1485 1490 1525 1590
Compound α-Thujene α-Pinene Camphene β-Thujene β-Pinene β-Myrcene α-Phellandrene 3-Carene α-Terpinene o-Cymene Limonene (E)-β-Ocimene α-Ocimene γ-Terpinene (E)-β-terpineol Linalool oxide Pentylcyclopropane α-Terpinolene Linalool β-Thujone (Z)-β-Terpineol Neo-allo-ocimene Limonene oxide (Z)-p-Mentha-2-en-1-ol Terpinen-4-ol L-4-terpineneol Cryptone Myrtenal p-Cymen-8-ol Estragole α-Terpineol Decanal (Z)-Piperitol (Z)-Carveol Citronellol (E)-Carveol D-Carvone Linalyl acetate Geraniol α-Citral Anethole Bornyl acetate Nerol acetate Geranyl acetate Caryophyllene Humulene 1-Dodecanol D-Germacrene δ-Cadinene Caryophyllene oxide
Total content of identified compounds (%)
Maqian (%)
6.49 0.03 0.69 0.11 3.87 1.78 0.46
67.06 1.17 2.32 0.07 0.22 0.23 2.96
Huajiao (%)
4.29 0.74 0.06 0.57 0.06 4.97 0.03 0.08 0.90 0.21 0.14
2.38
0.20 0.07 0.18 0.42
Positive controlb
Huajiao
MIC
MBC
MIC
MBC
MIC
MBC
512 256 nac 256 1024 64 128
2048 256 na 1024 2048 64 256
5120 2560 1280 2560 320 160 640
4 5120 2560 1280 5120 320 160 1280
0.25 0.5 0.05 256 0.25 512 0.5
0.5 1 0.25 512 1 1024 1
a
All tests were performed in triplicate. Positive control: Vancomycin for Staphylococcus aureus; Fluconazole for Acinetobacter baumannii and Candida albicans; Cefotaxime for Escherichia coli; Amikacin for Klebsiella pneumonia; Ciprofloxacin for Pseudomonas aeruginosa; Tigecycline for Aspergillus fumigatus. c na means not active. b
140
Cell viability of EOM
Cell viability of EOH
120 100 80 60 40 20 0 Control
0.11 0.27 0.05 0.73
94.86
Staphylococcus aureus Acinetobacter baumannii Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Aspergillus fumigatus Candida albicans
0.35 79.00 1.21 1.27
0.28 0.77
0.17 0.07 1.52 0.45 0.09 0.09 0.07 0.05 0.09
Maqian
0.11 0.19
0.04 0.32 0.08
1.33 0.51 0.73 0.08 0.07 0.12 0.09
Microbial strain
Cell viability (% of control)
RI
Table 2 Antimicrobial activities of the essential oils from Maqian and Huajiaoa (mg/ml).
120
98.29
RIca refers to the retention index experimentally calculated using C7–C30 alkanes. RIlit refers to the retention index taken from NIST.
obvious increased level of cell viability after the treatment of LPS (Fig. 1). Both oils increased the cell viability at 0.005‰, 0.01‰, 0.02‰ in the presence of 1 μg/ml LPS. Maqian reduced cell viability slightly at 0.04‰. However, this effect was not significantly different from the untreated control cells, as assessed by Dunnett's multiple comparison tests, indicating that the antiinflammatory activities of the essential oils up to 0.04‰ were not an effect of their toxicity. Therefore, we infer that concentrations up to 0.04‰ of the total essential oils could be safe for further development of Maqian and Huajiao in food and pharmaceutical applications.
3.3.2. Effect on NO production in LPS-induced RAW 264.7 cells The effect of the essential oils on NO production in LPS-induced RAW 264.7 cells was determined by the Griess reagent system. LPS
0.005‰
0.01‰
NO production of EOM
0.04
0.06
LPS
0.02‰
0.04‰
Fig. 1. Effect of the essential oils (EOM from Maqian, EOH from Huajiao) on LPSinduced RAW 264.7 cells viability by MTS assay. Results were expressed as a percentage of MTS production by untreated control cells. All values were performed in triplicate and expressed as means 7 SD. nPo 0.05, indicated significant difference with the untreated cells (control).
NO production (% of control)
46
100
# **
80
NO production of EOH
*
# ** ## **
60 40
## ***
20
*** *** 0 control
LPS
0.005‰
0.01‰
0.02‰
0.04‰
Fig. 2. Effect of the essential oils (EOM from Maqian, EOH from Huajiao) on NO production in LPS-induced RAW 264.7 cells by the Griess reagent system. Results were expressed as a percentage of NO production by the LPS-treated cells (LPS group). All values were performed in triplicate and expressed as means 7SD. n Po 0.05, nnPo 0.01 and nnnP o0.001 indicated significant difference with the LPStreated cells; #P o 0.05, ##P o 0.01 indicated significant difference with the same concentration and column of EOH group.
treatment significantly increased NO production compared with untreated cells (control) (Fig. 2). Both species showed a concentration-dependent inhibition of NO production. Huajiao decreased NO production 5.93% and 9.50% compared with LPS treated cells at 0.01‰ and 0.02‰ without significance, and it significantly inhibited
R. Li et al. / Journal of Ethnopharmacology 158 (2014) 43–48
NO release by 20.20% at 0.04‰. Maqian significantly reduced NO production by 18.50%, 30.46%, 50.48% and 83.79% compared with LPS treated cells at 0.005‰, 0.01‰, 0.02‰ and 0.04‰, respectively. Furthermore, the inhibition activity of Maqian was significantly higher than that of Huajiao at all tested concentrations, indicating it has far better anti-inflammatory activity. It is hypothesized that this differences reflects the higher content of limonene, which has been credited with anti-inflammatory effects (Hirota et al., 2010; Yoon et al., 2010; Chi et al., 2013). Previous studies have shown that nitric oxide (NO) is one of the key denominators in inflammation and carcinogenesis (Maeda and Akaike, 1998), so the significant NO inhibition effect supports the use of Maqian as an indigenous remedy for detoxification, detumescence and analgesia.
4. Conclusions In the present study, the chemical composition, and the antimicrobial and anti-inflammatory activity of Maqian (Zanthoxylum myriacanthum var. pubescens Huang) are reported for the first time. GC–MS analysis showed that the essential oil is rich in limonene. It showed stronger antimicrobial activity against five tested pathogens and spoilage organisms than Huajiao (Zanthoxylum schinifolium Sieb. et Zucc.), and comparable activity with the positive controls (Fluconazole and Amikacin) against Aspergillus fumigates and Klebsiella pneumonia. Maqian also showed better anti-inflammatory activity than Huajiao by significantly inhibiting nitric oxide production induced by LPS in RAW 264.7 cells at lower concentrations without a significant effect on cell viability. These antimicrobial and antiinflammatory activities support the recorded medicinal and culinary uses of Maqian by the ethnic villagers in Xishuangbanna. Moreover, based on its stronger antimicrobial and anti-inflammatory activities, our results suggest that Maqian could be promoted as a better alternative to Huajiao and that it has the potential for further exploration and application as a natural antimicrobial and antiinflammatory ingredient in the medical and functional food industries. Further studies are needed to discover the possible mechanisms of its significant anti-inflammatory effect.
Acknowledgment The authors gratefully acknowledge the financial support by the Special Grant for Basic Work of Science and Technology from the Ministry of Science and Technology, China (Grant no. 2012FY110300) and CAS 135 Program (XTBG-F02), and wish to thank Prof. Dr. Richard T. Corlett for his critical reading and language editing during the preparation of this manuscript. Special thanks are given to the Central Laboratory of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences for technical support of this study. References Bastos, J.K., Carvalho, J.C.T., de Souza, G.H.B., Pedrazzi, A.H.P., Sarti, S.J., 2001. Antiinflammatory activity of cubebin, a lignan from the leaves of Zanthoxyllum naranjillo Griseb. Journal of Ethnopharmacology 75, 279–282. Bevilacqua, A., Corbo, M.R., Sinigaglia, M., 2010. In vitro Evaluation of the antimicrobial activity of eugenol, limonene, and citrus extract against bacteria and yeasts, representative of the spoiling microflora of fruit Juices. Journal of Food Protection 73, 888–894. Bowen, I.H., Lewis, J.R., 1978. Rutaceous constituents. Planta Medica 34, 129–134. Chae, S.Y., Lee, M., Kim, S.W., Bae, Y.H., 2004. Protection of insulin secreting cells from nitric oxide induced cellular damage by crosslinked hemoglobin. Biomaterials 25, 843–850. Chansakaow, S., Leelapornpisid, P., Yosprasit, K., Tharavichitkul, P., 2005. Antibacterial activity of Thai medicinal plant extracts on the skin infectious microorganisms. In: Franz, C., Mathe, A. (Eds.), WOCMAP III: Targeted Screening of MAPs, Economics and Law, pp. 153–157.
47
Cheng, B.Q., 1991. A study on edible spice plants used by minorities in Xishuangbanna. Flavour Fragrance Cosmetics 3, 27–31. Chi, G., Wei, M., Xie, X., Soromou, L.W., Liu, F., Zhao, S., 2013. Suppression of MAPK and NF-kappa B pathways by limonene contributes to attenuation of lipopolysaccharide-Induced inflammatory responses in acute lung injury. Inflammation 36, 501–511. Chinese Pharmacopoeia Commission, 2005. Chinese Pharmacopoeia. 1. Chemical industry press, Beijing p. 116. Clinical and Laboratory Standards Institute, 2006. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically (Approved Standard. Document M7. CLSI). seventh ed.vol. M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA. Diao, W.R., Hu, Q.P., Feng, S.S., Li, W.Q., Xu, J.G., 2013. Chemical composition and antibacterial activity of the essential Oil from green Huajiao (Zanthoxylum schinifolium) against selected foodborne pathogens. Journal of Agricultural and Food Chemistry 61, 6044–6049. Freitas, F.F.B.P., Fernandes, H.B., Piauilino, C.A., Pereira, S.S., Carvalho, K.I.M., Chaves, M.H., Soares, P.M.G., Miura, L.M.C.V., Leite, J.R.S.A., Oliveira, R.C.M., Oliveira, F.A., 2011. Gastroprotective activity of Zanthoxylum rhoifolium Lam. in animal models. Journal of Ethnopharmacology 137, 700–708. Gong, Y., Huang, Y., Zhou, L., Shi, X., Guo, Z., Wang, M., Jiang, W., 2009. Chemical composition and antifungal activity of the fruit Oil of Zanthoxylum bungeanum Maxim. (Rutaceae) from China. Journal of Essential Oil Research 21, 174–178. Hirota, R., Roger, N.N., Nakamura, H., Song, H.S., Sawamura, M., Suganuma, N., 2010. Anti-inflammatory effects of Limonene from Yuzu (Citrus junos Tanaka) essential oil on eosinophils. Journal of Food Science 75, H87–H92. Hu, J., Shi, X., Mao, X., Chen, J., Zhu, L., Zhao, Q., 2013. Antinociceptive activity of Rhoifoline A from the ethanol extract of Zanthoxylum nitidum in mice. Journal of Ethnopharmacology 150, 828–834. Lee, J.H., Chang, K.M., Kim, G.H., 2009. Composition and anti-inflammatory activities of Zanthoxylum schinifolium essential oil: suppression of inducible nitric oxide synthase, cyclooxygenase-2, cytokines and cellular adhesion. Journal of the Science of Food and Agriculture 89, 1762–1769. Liang, L., Gansun, L., Jibing, L., Chongren, Y., Jun, Z., 1988. Chemical compositions of Z. myriacanthum and chemotaxonomy of zanthoxyllum. Acta Botanica Yunnanica 10, 445–456. Lima, L.M., Perazzo, F.F., Carvalho, J.C.T., Bastos, J.K., 2007. Anti-inflammatory and analgesic activities of the ethanolic extracts from Zanthoxylum riedelianum (Rutaceae) leaves and stem bark. Journal of Pharmacy and Pharmacology 59, 1151–1158. Lin, Y.F., Yi, Z., Zhao, Y.H., 2003. Chinese Dai Medicine Colorful Illustrations. The Nationalities Publishing House of Yunnan, Kunming, China. LisBalchin, M., Ochocka, R.J., Deans, S., Asztemborska, M., Hart, S., 1996. Bioactivity of the enantiomers of limonene. Medical Science Research 24, 309–310. Maeda, H., Akaike, T., 1998. Reviews-nitric oxide and oxygen radicals in infection, inflammation, and cancer. Biochemistry 63, 854–865. Misra, L.N., Wouatsa, N.A.V., Kumar, S., Kumar, R.V., Tchoumbougnang, F., 2013. Antibacterial, cytotoxic activities and chemical composition of fruits of two Camerooriian Zanthoxylum species. Journal of Ethnopharmacology 148, 74–80. National Committee for Clinical and Laboratory Standards Institute, 1999. Methods for Determining Bactericidal Activity of Antimicrobial Agents (Approved Guideline. Document M26-A. CLSI (formerly NCCLS). . Neirotti, E., Moscatelli, M., Tiscornia, S., 1996. Antimicrobial activity of the limonene. Arquivos De Biologia E Tecnologia 39, 233–237. NIST Chemistry WebBook, 2014. NIST Standard Reference Database Number 69 http://webbook.nist.gov/chemistry/. Accessed on July 31, 2014. Pei, S.J., 1985. Preliminary study of ethnobotany in xishuang banna, people's republic of china. Journal of Ethnopharmacology 13, 121–137. Pereira, S.S., Lopes, L.S., Marques, R.B., Figueiredo, K.A., Costa, D.A., Chaves, M.H., Almeida, F.R.C., 2010. Antinociceptive effect of Zanthoxylum rhoifolium Lam. (Rutaceae) in models of acute pain in rodents. Journal of Ethnopharmacology 129, 227–231. Ranawat, L., Bhatt, J., Patel, J., 2010. Hepatoprotective activity of ethanolic extracts of bark of Zanthoxylum armatum DC in CCl4 induced hepatic damage in rats. Journal of Ethnopharmacology 127, 777–780. Seo, J.W., Park, D.H., Li, Y.C., Kang, H.J., De Xu, H., Kim, Y.J., Lee, J.H., Lee, M.S., Lee, I.C., Lee, Y.L., Ahn, J.B., Cho, S.C., Lee, M.J., 2012. The Zanthoxylum schinifolium seed oil modulates immune function under the biological safety level. Molecular & Cellular Toxicology 8, 179–185. State Adeministration of Traditional Chinese Medicine of the People' s Republic of China, 2005. Traditional Chinese Meteria Medica of Dai Nationality. Shanghai Science and Technology Press, Shanghai, China. Sukari, M., Salim, W., Ibrahim, N., Rahmani, M., Aimi, N., Kitajima, M., 1999. Phenantridine alkaloids from Zanthoxylum myriacanthum. Fitoterapia 70, 197–199. Tezuka, Y., Irikawa, S., Kaneko, T., Banskota, A.H., Nagaoka, T., Xiong, Q.B., Hase, K., Kadota, S., 2001. Screening of Chinese herbal drug extracts for inhibitory activity on nitric oxide production and identification of an active compound of Zanthoxylum bungeanum. Journal of Ethnopharmacology 77, 209–217. Waterman, P.G., 1975. Alkaloids from the root bark of Zanthoxylum myriacanthum. Phytochemistry 14, 2530. Weyerstahl, P., Marschall, H., Splittgerber, U., Son, P.T., Giang, P.M., Kaul, V., 1999. Constituents of the essential oil from the fruits of Zanthoxylum rhetsoides Drake from Vietnam and from the aerial parts of Zanthoxylum alatum Roxb. from India. Flavour and fragrance journal 14, 225–229.
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R. Li et al. / Journal of Ethnopharmacology 158 (2014) 43–48
Xu, Y.K., Tao, G.D., Liu, H.M., Yan, K.L., Dao, X.S., 2004. Wild vegetable resources and market survey in Xishuangbanna, southwest China. Economic Botany 58, 647–667. Yamazaki, E., Inagaki, M., Kurita, O., Inoue, T., 2007. Antioxidant activity of Japanese pepper (Zanthoxylum piperitum DC.) fruit. Food Chemistry 100, 171–177. Yang, L.C., Li, R., Tan, J., Jiang, Z.T., 2013. Polyphenolics composition of the leaves of Zanthoxylum bungeanum Maxim. grown in Hebei, China, and their radical scavenging activities. Journal of Agricultural and Food Chemistry 61, 1772–1778. Yang, X., 2008. Aroma constituents and alkylamides of red and green huajiao (Zanthoxylum bungeanum and Zanthoxylum schinifolium). Journal of Agricultural and Food Chemistry 56, 1689–1696.
Yoon, W.J., Lee, N.H., Hyun, C.-G., 2010. Limonene suppresses lipopolysaccharideinduced production of nitric oxide, prostaglandin E 2, and pro-inflammatory cytokines in RAW 264.7 macrophages. Journal of Oleo Science 59, 415–421. Zhang, D.X., Hartley, T.G., Mabberley, D.J., 2008. Rutaceae. http://flora.huh.harvard. edu.china/mss/volume11/Rutaceae.pdf. Accessed on March 31, 2014. Zhu, H.Y., Yang, F.M., Yang, X.S., Hao, X.J., 2007. Essential oils from Zanthoxylum rhetsoides Drake and their antibacterial activity. LiShiZhen Medicine and Materia Medica Research 18, 262–263.
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Chemical composition, antimicrobial and anti-inflammatory activities of the essential oil from Maqian (Zanthoxylum myriacanthum var. pubescens) in Xishuangbanna, SW China Ren Li a,b,1, Jing-jing Yang a,b,1, Yin-xian Shi a,b, Min Zhao a,b, Kai-long Ji a,b, Ping Zhang a, You-kai Xu a, Hua-bin Hu a,n a Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, Mengla, Yunnan 666303, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 25 February 2014 Received in revised form 29 May 2014 Accepted 7 October 2014 Available online 16 October 2014
Ethnopharmacological relevance: Maqian (Zanthoxylum myriacanthum var. pubescens Huang) is widely consumed as an indigenous remedy for digestive disorders, detoxification, detumescence and analgesia by the ethnic groups in Xishuangbanna, SW China. A related species, Huajiao (Zanthoxylum schinifolium Sieb. et Zucc.), has similar uses in traditional Chinese medicine. We aimed to scientifically validate the traditional uses by investigating and comparing the chemical composition, antimicrobial and antiinflammatory activities of the essential oils of Maqian and Huajiao. Materials and methods: Essential oils were collected from the fruits of Maqian and Huajiao by simultaneous distillation extraction and identified by gas chromatography–mass spectrometry (GC–MS) analysis. To assess antimicrobial activity, the minimum inhibitory and bactericidal concentrations (MIC and MBC) against 7 microbial strains, including 5 food-borne pathogens, were evaluated by serial dilution with a standardized microdilution broth methodology. For anti-inflammatory activity, the cell viability and nitric oxide (NO) production were determined on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells by MTS assay and the Griess reagent system, respectively. Results: The essential oil from Maqian is rich in limonene (67.06%) and has strong antimicrobial activity against the tested pathogens and spoilage organisms, with MIC ranging from 64 to1024 mg/ml and MBC ranging from 64 to 2048 mg/ml. It also showed anti-inflammatory activity by significantly inhibiting nitric oxide (NO) production induced by LPS in RAW 264.7 cells at 0.04‰ without effects on cell viability. Furthermore, it showed relatively stronger antimicrobial and anti-inflammatory activities than the essential oil from Huajiao. Conclusions: Our findings not only justify the use of Maqian as an indigenous remedy for digestive disorders, detoxification, detumescence and analgesia, but also suggest that it could be promoted as a preferred substitute for Huajiao. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Zanthoxylum myriacanthum var. pubescens Essential oil Antimicrobial Anti-inflammatory Xishuangbanna
1. Introduction The species in the genus Zanthoxylum (Rutaceae), which is distributed mostly in the tropical and temperate areas of East Asia and Northeast America, are shrubs, trees, scramblers or woody climbers, and contain aromatic volatile oils. There are more than 200 Zanthoxylum species, of which 41 species (25 endemic) are found in China. These include Zanthoxylum myriacanthum var. pubescens Huang (synonyms Zanthoxylum rhetsoides var. pubescens
n
Corresponding author. Tel.: þ 86 691 8715415; fax: þ86 691 8715070. E-mail address: [email protected] (H.-b. Hu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jep.2014.10.006 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
C. C. Huang and Z. utile C. C. Huang), which is a variety of Zanthoxylum myriacanthum Wall. ex Hook. f. distinguished by villous leaf rachises, petiolules, leaflet blades, and inflorescence rachises (Zhang et al., 2008). It is known as “Maqian” in the Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China. This area is rich in biodiversity and has a tropical monsoon climate characterized by a distinct rainy season (May–October), with peak rainfall in July– September. The people of this region are exposed to many tropical diseases, such as cholera, typhoid, malaria, plague and digestive disorders, and the indigenous people have developed plant remedies to treat them. These traditional remedies include Maqian, which is an important indigenous remedy consumed by Dai people in Xishuangbanna for digestive disorders, detoxification, and the relief of swelling and pain
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(Lin et al., 2003; SATCM, 2005). Huajiao (Zanthoxylum schinifolium Sieb. et Zucc.), known as Chinese prickly ash, is a related species of Zanthoxylum commonly used in traditional Chinese medicine in the treatment of detoxification, vomiting and stomach ache (Chinese Pharmacopoeia Commission, 2005). However, our ethnobotanical research has found that Maqian is frequently used instead of Huajiao to treat these diseases by the ethnic groups in Xishuangbanna. Beside its medicinal usage, Maqian is also widely consumed as a spicy condiment, as is Huajiao. Specifically, Maqian is frequently used as a spice for roasting and salting meat, boiling fish and cooking vegetable soup in the ethnic villages of Xishuangbanna (Pei, 1985; Cheng, 1991; Xu et al., 2004). Many Zanthoxylum species are used as medicines and spices around the world, and have been reported to have antioxidant activity (Yamazaki et al., 2007; Yang et al., 2013), antimicrobial properties (Diao et al., 2013; Misra et al., 2013), antinociceptive effects (Pereira et al., 2010; Hu et al., 2013), modulate immune function (Seo et al., 2012), gastroprotective activity (Freitas et al., 2011), hepatoprotective activity (Ranawat et al., 2010), antiinflammatory and analgesic activities (Bastos et al., 2001; Tezuka et al., 2001; Lima et al., 2007). For example, the essential oil from Huajiao, of which main components were identified as linalool (28.2%), limonene (13.2%), and sabinene (12.1%), displayed strong antibacterial activity against tested Gram-positive bacteria, such as Staphylococcus epidermidis (Diao et al., 2013). The fruits of Zanthoxylum myriacanthum are used as a spice to cook sour fish by Miao people in Guizhou province, China, and the fruit essential oil, of which the main constituents were identified as sabinene (32.43%), limonene (28.91%) and 1,8-cineole (13.55%), exhibited no antibacterial activity against 7 bacterial strains (Zhu et al., 2007). While in the mountain regions of Northern and Central Vietnam, the dried fruit of Zanthoxylum myriacanthum was a preferred spice for beef and its essential oil, of which main constituents were identified as limonene (27.2%) p-Cymene (15.3%), and β-Phellandrene (11.9%), was also used for poisoning fish by villagers (Weyerstahl et al., 1999). The stem and root bark of Zanthoxylum myriacanthum were reported to be rich in triterpenes, lignans and alkaloids (Waterman, 1975; Liang et al., 1988; Sukari et al., 1999), and a 95% ethanol extract of its fruit, which were used as a traditional Thai medicine to treat different kinds of ailments, including microbial infections, showed antibacterial activity against Staphylococcus aureus and Escherichia coli (Chansakaow et al., 2005), and its leaf, stem and bark extracts showed antitumor activity (Bowen and Lewis, 1978). However, limited scientific and bioactivity information is available on the essential oil of Maqian. The aim of this study was therefore to provide scientific evidence to justify its traditional usage as an ethnic medicine and spice by investigating its chemical composition, antimicrobial and anti-inflammatory activities. In addition, parallel and comparative laboratory analysis of the essential oil of Huajiao was conducted in order to support the preference of the ethnic group for Maqian.
2. Material and methods
mill. The essential oils were collected by simultaneous distillation extraction and stored at 20 1C in the dark for further use. 2.2. Chemicals and reagents Dimethylsulfoxide (DMSO), L-glutamine, and Lipopolysaccharide (LPS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and penicillin–streptomycin were purchased from Thermo Scientific (Logan, UT, USA). The CellTiter 96sAQueous One Solution Reagent for MTS assay and the Griess reagent system for NO measurement were purchased from Promega Corporation (Madison, WI, USA). Standard Mueller–Hinton agar and broth (MHA and MHB) and Sabouraud agar and broth (SA and SB) were bought from Tianhe Microbial Agents Company (Hangzhou, China). All reagents were analytical standard. 2.3. GC–MS analysis The analysis of the essential oils was performed using an Agilent Technologies 7890 A GC, equipped with a HP-5 MS capillary column (30 m 0.25 mm; film thickness, 0.25 mm) and a mass spectrometer 5975C (Agilent Technologies, USA) as detector. MS were taken at 70 eV with a mass range of m/z 45–500. Helium was used as the carrier gas, at a flow rate of 1 ml/min. Injector and detector (MS transfer line) temperatures were both 250 1C. Column temperatures were gradually increased from 40 1C to 160 1C at 3 1C/min, and increased to 250 1C at 20 1C/min, then held for 10 min. 0.2 ml of the diluted sample was injected manually. 2.4. Identification of the essential components The components were identified by comparing calculated experiment GC retention indices, which were determined with reference to homologous series of n-alkanes C7–C30 under identical experimental condition, with the GC retention indices reported in NIST Standard Reference Database (NIST Chemistry WebBook, 2014), by matching their mass spectra with those recorded in the NIST 08 database (National Institute of Standards and Technology, Gaithersburg, MD, USA) and mass spectra with published data. The percentage composition of individual components was computed by the normalization method from the GC peak areas, assuming an identical mass response factor for all compounds. 2.5. Microbial strains and cultural medium The essential oils were individually tested against 7 microbial strains. Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Candida albicans (ATCC Y0109) were provided by the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, China); Aspergillus funigatus, Acinetobacter baumannii and Klebsiella pneumonia were provided by Kunming General Hospital. Standard Mueller–Hinton agar and broth (MHA and MHB), and Sabouraud agar and broth (SA and SB) were used as the bacterial and fungal culture media, respectively.
2.1. Plant materials 2.6. Determination of MIC and MBC The fruits of Maqian (Zanthoxylum myriacanthum var. pubescens Huang) and Huajiao (Zanthoxylum schinifolium Sieb. et Zucc.) were both collected form Mengwang township, Jinghong municipality, Xishuangbanna Dai Autonomous Prefecture, China in October 2012, and identified by Ms. Chun-fen Xiao from Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (no. 152673) for Maqian was deposited in the herbarium (HITBC). The fruits were air-dried and powdered by a laboratory
The MIC and MBC were determined against tested microbial strains according to the procedures reported previously (NCCLS, 1999; CLSI, 2006). The MICs and MBCs were both determined with starting inoculums of 1.0 106 CFU/ml for bacteria and 1.0 104 CFU/ ml for fungi, incubated at 35 1C for 24 h, and were examined for growth in daylight. The serial dilution method was used for the determination of the MICs of the samples. For the MIC assay, the
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essential oils was dissolved in dimethylsulfoxide (DMSO), and sterilized by filtration through 0.45 mm Millipore filters. 100 ml of an appropriate medium, essential oil solutions and inoculums were dispensed onto a 96-well plate. MIC values were the lowest concentration of a given extract that completely inhibited the visible microbial growth. For the MBC assay, 10 ml samples taken from the clear wells of the microbroth susceptibility studies were placed onto the surfaces of MHA or SA plates to determine MBC. The MBC was defined as the concentration of drug that resulted in 499.9% mortality of the bacterium relative to the concentration of bacterium that was present in test wells at 0 h (NCCLS, 1999). The MICs and MBCs of Amikacin, Fluconazole, Vancomycin, Tigecycline, Ciprofloxacin and Cefotaxime were also determined in parallel experiments as positive control. The culture medium and solvent (DMSO) were used as a negative control in parallel tests and their values were deducted to get the final results of activity. All experiments were performed in triplicate. 2.7. Cell culture The murine macrophage cell line RAW 264.7 was obtained from Kunming Institute of Zoology, Chinese Academy of Sciences (KCB200603YJ) and maintained in DMEM (Thermo Scientific, Logan, UT, USA) containing 10% fetal bovine serum, 1% penicillin–streptomycin and 1% L-glutamine (Sigma-Aldrich, St Louis, MO, USA) at 37 1C in a 5% CO2 incubator (Thermo Scientific, Forma371, Steri-cycle, USA) and were subcultured every 2 days. 2.8. Cell viability Cell viability was evaluated by MTS assay as previously reported (Chae et al., 2004). In the MTS assay, 100 μl samples of cell suspension (1 105 cells/well) were cultivated in 96-cell plates for 18 h as described. Then cells were pre-treated with various concentrations of the essential oils for 30 min before they were further incubated in the presence of 1 μg/ml LPS for 24 h. Finally, 20 ml of CellTiter 96sAQueous One Solution Reagent, prepared by MTS (3-[4,5, dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) in the presence of phenazine ethosulfate (PES), was added to each well and incubated for 1 h at 37 1C in the 5% CO2 incubator. The absorbance of each well was measured at 490 nm directly using a Multifunctional Microplate Reader (Thermo Scientific VarioSkan Flash, USA). The untreated cells, incubated in culture medium with 0.4% DMSO, were employed as blank controls in every experiment. Results were expressed as a percentage of the MTS production by untreated cells (control group). Values were presented as mean7standard deviation (SD) of three independent tests. 2.9. Measurement of NO production NO production by LPS-stimulated RAW 264.7 cells was measured by the accumulation of nitrite in the culture supernatants, using the Griess reagent system (Promega, Madison, WI, USA) and following the manufacturer's instructions. Cells were seeded in 96-well plates at 1 106 cells/well and then incubated with culture medium (control) for 18 h. Then cells were pretreated with various concentrations of the essential oils for 30 min before they were stimulated with 1 μg/ml LPS and further cultured for an additional 24 h. In brief, 50 μl of culture supernatants were collected and mixed with the Griess reagent system (1% sulfanilamide in 0.1 mol/l HCl and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride). Then the plates were incubated at 37 1C in the 5% CO2 incubator for 10 min. The absorbance was measured at 550 nm in a Multifunctional Microplate Reader (Thermo Scientific VarioSkan Flash, USA). Nitrite concentration was determined from a sodium nitrite standard curve. The untreated cells, incubated in culture medium with 0.4% DMSO, were employed as blank controls in
45
every experiment. Results were expressed as percentage of NO by LPStreated cells (LPS group). Values were presented as mean7standard deviation (SD) of three independent tests. 2.10. Statistical analysis All experiments were performed in triplicate and expressed as mean values 7standard deviation (SD). One-way ANOVA with a Dunnett's multiple comparison test was performed using SPSS 14.0 for windows (SPSS Inc. Chicago, USA). Significance of difference was accepted at P o0.05. 3. Results and discussion 3.1. Chemical composition of the essential oils The essential oil yields were 3.6% and 4.8% (w/w) on a dry weight basis for Maqian and Huajiao, respectively. Fifty components were identified in the essential oils (38 in Maqian and 25 in Huajiao) representing 94.86% and 98.29% of the respective oils. The main constituents of the oil from Maqian were limonene (67.06%), α-Pinene (6.49%), β-Myrcene (3.87%), and linalool (2.96%), while the main constituents from Huajiao were linalool (79.00%), limonene (4.97%), β-Thujene (4.29%) (Table 1). These components have been commonly found in Zanthoxylum species (Weyerstahl et al., 1999; Zhu et al., 2007; Yang, 2008; Misra et al., 2013), but Maqian has the highest reported content of limonene: 67.06%, compared with 4.97% from Huajiao in this study, and 13.2% and 14.3% from green Huajiao (Yang, 2008; Diao et al., 2013), 27.2% and 28.91% from Zanthoxylum myriacanthum in Vietnam and China (Weyerstahl et al., 1999; Zhu et al., 2007), or not found at all in the fruit essential oil of red and green Huajiao in some previous studies (Gong et al., 2009; Lee et al., 2009). The high content of limonene could explain why the ethnic groups insisted that Maqian was more fragrant than Huajiao. 3.2. Antimicrobial activity The essential oil from Huajiao showed antimicrobial activity against all seven tested microbial strains, with MIC ranging between 160 and 5120 mg/ml and MBC ranging from 160 to more than 5120 mg/ml (Table 2). At the concentrations tested in this study (up to 10 mg/ml), Maqian showed no antimicrobial activity against Escherichia coli but obvious activity against the other six strains, with MIC ranging between 64 and 1024 mg/ml and MBC ranging from 64 to 2048 mg/ml. Compared with Huajiao, Maqian showed stronger activity against some of the pathogens and spoilage organisms tested, including Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumonia, Aspergillus fumigates and Candida albicans. Compared with the positive control, Maqian showed better MIC and MBC values than Fluconazole against Aspergillus fumigates, and comparable MIC and MBC values to Amikacin against Klebsiella pneumonia. The antimicrobial activity of Maqian is probably due not only to the abundance of limonene, which has broad and strong antimicrobial capacities (LisBalchin et al., 1996; Neirotti et al., 1996; Bevilacqua et al., 2010), but also to the other constituents. This result matched with the traditional use of Maqian as a remedy to treat digestive disorders, which might be caused by the food-borne pathogens contained in fresh and uncooked meat and green vegetables. 3.3. Anti-inflammatory activity 3.3.1. Effect on LPS-induced RAW 264.7cell viability The effect of the essential oils on LPS-induced RAW 264.7 cells viability was determined by MTS assay. There was a slight but
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Table 1 Chemical compositions of the essential oils from Maqian and Huajiao. cal
926 935 949 976 978 996 1008 1013 1015 1023 1037 1043 1053 1058 1066 1072 1078 1092 1108 1117 1122 1135 1138 1147 1176 1184 1192 1194 1196 1198 1199 1212 1216 1229 1237 1241 1250 1256 1263 1278 1285 1291 1372 1391 1425 1460 1484 1488 1531 1592
RI
lit
926 937 954 976 979 992 1004 1011 1017 1020 1035 1043 1048 1060 1062 1073 1074 1094 1103 1115 1121 1131 1139 1146 1176 1182 1189 1197 1194 1206 1199 1206 1211 1229 1238 1246 1257 1257 1262 1272 1286 1289 1367 1385 1422 1456 1485 1490 1525 1590
Compound α-Thujene α-Pinene Camphene β-Thujene β-Pinene β-Myrcene α-Phellandrene 3-Carene α-Terpinene o-Cymene Limonene (E)-β-Ocimene α-Ocimene γ-Terpinene (E)-β-terpineol Linalool oxide Pentylcyclopropane α-Terpinolene Linalool β-Thujone (Z)-β-Terpineol Neo-allo-ocimene Limonene oxide (Z)-p-Mentha-2-en-1-ol Terpinen-4-ol L-4-terpineneol Cryptone Myrtenal p-Cymen-8-ol Estragole α-Terpineol Decanal (Z)-Piperitol (Z)-Carveol Citronellol (E)-Carveol D-Carvone Linalyl acetate Geraniol α-Citral Anethole Bornyl acetate Nerol acetate Geranyl acetate Caryophyllene Humulene 1-Dodecanol D-Germacrene δ-Cadinene Caryophyllene oxide
Total content of identified compounds (%)
Maqian (%)
6.49 0.03 0.69 0.11 3.87 1.78 0.46
67.06 1.17 2.32 0.07 0.22 0.23 2.96
Huajiao (%)
4.29 0.74 0.06 0.57 0.06 4.97 0.03 0.08 0.90 0.21 0.14
2.38
0.20 0.07 0.18 0.42
Positive controlb
Huajiao
MIC
MBC
MIC
MBC
MIC
MBC
512 256 nac 256 1024 64 128
2048 256 na 1024 2048 64 256
5120 2560 1280 2560 320 160 640
4 5120 2560 1280 5120 320 160 1280
0.25 0.5 0.05 256 0.25 512 0.5
0.5 1 0.25 512 1 1024 1
a
All tests were performed in triplicate. Positive control: Vancomycin for Staphylococcus aureus; Fluconazole for Acinetobacter baumannii and Candida albicans; Cefotaxime for Escherichia coli; Amikacin for Klebsiella pneumonia; Ciprofloxacin for Pseudomonas aeruginosa; Tigecycline for Aspergillus fumigatus. c na means not active. b
140
Cell viability of EOM
Cell viability of EOH
120 100 80 60 40 20 0 Control
0.11 0.27 0.05 0.73
94.86
Staphylococcus aureus Acinetobacter baumannii Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Aspergillus fumigatus Candida albicans
0.35 79.00 1.21 1.27
0.28 0.77
0.17 0.07 1.52 0.45 0.09 0.09 0.07 0.05 0.09
Maqian
0.11 0.19
0.04 0.32 0.08
1.33 0.51 0.73 0.08 0.07 0.12 0.09
Microbial strain
Cell viability (% of control)
RI
Table 2 Antimicrobial activities of the essential oils from Maqian and Huajiaoa (mg/ml).
120
98.29
RIca refers to the retention index experimentally calculated using C7–C30 alkanes. RIlit refers to the retention index taken from NIST.
obvious increased level of cell viability after the treatment of LPS (Fig. 1). Both oils increased the cell viability at 0.005‰, 0.01‰, 0.02‰ in the presence of 1 μg/ml LPS. Maqian reduced cell viability slightly at 0.04‰. However, this effect was not significantly different from the untreated control cells, as assessed by Dunnett's multiple comparison tests, indicating that the antiinflammatory activities of the essential oils up to 0.04‰ were not an effect of their toxicity. Therefore, we infer that concentrations up to 0.04‰ of the total essential oils could be safe for further development of Maqian and Huajiao in food and pharmaceutical applications.
3.3.2. Effect on NO production in LPS-induced RAW 264.7 cells The effect of the essential oils on NO production in LPS-induced RAW 264.7 cells was determined by the Griess reagent system. LPS
0.005‰
0.01‰
NO production of EOM
0.04
0.06
LPS
0.02‰
0.04‰
Fig. 1. Effect of the essential oils (EOM from Maqian, EOH from Huajiao) on LPSinduced RAW 264.7 cells viability by MTS assay. Results were expressed as a percentage of MTS production by untreated control cells. All values were performed in triplicate and expressed as means 7 SD. nPo 0.05, indicated significant difference with the untreated cells (control).
NO production (% of control)
46
100
# **
80
NO production of EOH
*
# ** ## **
60 40
## ***
20
*** *** 0 control
LPS
0.005‰
0.01‰
0.02‰
0.04‰
Fig. 2. Effect of the essential oils (EOM from Maqian, EOH from Huajiao) on NO production in LPS-induced RAW 264.7 cells by the Griess reagent system. Results were expressed as a percentage of NO production by the LPS-treated cells (LPS group). All values were performed in triplicate and expressed as means 7SD. n Po 0.05, nnPo 0.01 and nnnP o0.001 indicated significant difference with the LPStreated cells; #P o 0.05, ##P o 0.01 indicated significant difference with the same concentration and column of EOH group.
treatment significantly increased NO production compared with untreated cells (control) (Fig. 2). Both species showed a concentration-dependent inhibition of NO production. Huajiao decreased NO production 5.93% and 9.50% compared with LPS treated cells at 0.01‰ and 0.02‰ without significance, and it significantly inhibited
R. Li et al. / Journal of Ethnopharmacology 158 (2014) 43–48
NO release by 20.20% at 0.04‰. Maqian significantly reduced NO production by 18.50%, 30.46%, 50.48% and 83.79% compared with LPS treated cells at 0.005‰, 0.01‰, 0.02‰ and 0.04‰, respectively. Furthermore, the inhibition activity of Maqian was significantly higher than that of Huajiao at all tested concentrations, indicating it has far better anti-inflammatory activity. It is hypothesized that this differences reflects the higher content of limonene, which has been credited with anti-inflammatory effects (Hirota et al., 2010; Yoon et al., 2010; Chi et al., 2013). Previous studies have shown that nitric oxide (NO) is one of the key denominators in inflammation and carcinogenesis (Maeda and Akaike, 1998), so the significant NO inhibition effect supports the use of Maqian as an indigenous remedy for detoxification, detumescence and analgesia.
4. Conclusions In the present study, the chemical composition, and the antimicrobial and anti-inflammatory activity of Maqian (Zanthoxylum myriacanthum var. pubescens Huang) are reported for the first time. GC–MS analysis showed that the essential oil is rich in limonene. It showed stronger antimicrobial activity against five tested pathogens and spoilage organisms than Huajiao (Zanthoxylum schinifolium Sieb. et Zucc.), and comparable activity with the positive controls (Fluconazole and Amikacin) against Aspergillus fumigates and Klebsiella pneumonia. Maqian also showed better anti-inflammatory activity than Huajiao by significantly inhibiting nitric oxide production induced by LPS in RAW 264.7 cells at lower concentrations without a significant effect on cell viability. These antimicrobial and antiinflammatory activities support the recorded medicinal and culinary uses of Maqian by the ethnic villagers in Xishuangbanna. Moreover, based on its stronger antimicrobial and anti-inflammatory activities, our results suggest that Maqian could be promoted as a better alternative to Huajiao and that it has the potential for further exploration and application as a natural antimicrobial and antiinflammatory ingredient in the medical and functional food industries. Further studies are needed to discover the possible mechanisms of its significant anti-inflammatory effect.
Acknowledgment The authors gratefully acknowledge the financial support by the Special Grant for Basic Work of Science and Technology from the Ministry of Science and Technology, China (Grant no. 2012FY110300) and CAS 135 Program (XTBG-F02), and wish to thank Prof. Dr. Richard T. Corlett for his critical reading and language editing during the preparation of this manuscript. Special thanks are given to the Central Laboratory of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences for technical support of this study. References Bastos, J.K., Carvalho, J.C.T., de Souza, G.H.B., Pedrazzi, A.H.P., Sarti, S.J., 2001. Antiinflammatory activity of cubebin, a lignan from the leaves of Zanthoxyllum naranjillo Griseb. Journal of Ethnopharmacology 75, 279–282. Bevilacqua, A., Corbo, M.R., Sinigaglia, M., 2010. In vitro Evaluation of the antimicrobial activity of eugenol, limonene, and citrus extract against bacteria and yeasts, representative of the spoiling microflora of fruit Juices. Journal of Food Protection 73, 888–894. Bowen, I.H., Lewis, J.R., 1978. Rutaceous constituents. Planta Medica 34, 129–134. Chae, S.Y., Lee, M., Kim, S.W., Bae, Y.H., 2004. Protection of insulin secreting cells from nitric oxide induced cellular damage by crosslinked hemoglobin. Biomaterials 25, 843–850. Chansakaow, S., Leelapornpisid, P., Yosprasit, K., Tharavichitkul, P., 2005. Antibacterial activity of Thai medicinal plant extracts on the skin infectious microorganisms. In: Franz, C., Mathe, A. (Eds.), WOCMAP III: Targeted Screening of MAPs, Economics and Law, pp. 153–157.
47
Cheng, B.Q., 1991. A study on edible spice plants used by minorities in Xishuangbanna. Flavour Fragrance Cosmetics 3, 27–31. Chi, G., Wei, M., Xie, X., Soromou, L.W., Liu, F., Zhao, S., 2013. Suppression of MAPK and NF-kappa B pathways by limonene contributes to attenuation of lipopolysaccharide-Induced inflammatory responses in acute lung injury. Inflammation 36, 501–511. Chinese Pharmacopoeia Commission, 2005. Chinese Pharmacopoeia. 1. Chemical industry press, Beijing p. 116. Clinical and Laboratory Standards Institute, 2006. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically (Approved Standard. Document M7. CLSI). seventh ed.vol. M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA. Diao, W.R., Hu, Q.P., Feng, S.S., Li, W.Q., Xu, J.G., 2013. Chemical composition and antibacterial activity of the essential Oil from green Huajiao (Zanthoxylum schinifolium) against selected foodborne pathogens. Journal of Agricultural and Food Chemistry 61, 6044–6049. Freitas, F.F.B.P., Fernandes, H.B., Piauilino, C.A., Pereira, S.S., Carvalho, K.I.M., Chaves, M.H., Soares, P.M.G., Miura, L.M.C.V., Leite, J.R.S.A., Oliveira, R.C.M., Oliveira, F.A., 2011. Gastroprotective activity of Zanthoxylum rhoifolium Lam. in animal models. Journal of Ethnopharmacology 137, 700–708. Gong, Y., Huang, Y., Zhou, L., Shi, X., Guo, Z., Wang, M., Jiang, W., 2009. Chemical composition and antifungal activity of the fruit Oil of Zanthoxylum bungeanum Maxim. (Rutaceae) from China. Journal of Essential Oil Research 21, 174–178. Hirota, R., Roger, N.N., Nakamura, H., Song, H.S., Sawamura, M., Suganuma, N., 2010. Anti-inflammatory effects of Limonene from Yuzu (Citrus junos Tanaka) essential oil on eosinophils. Journal of Food Science 75, H87–H92. Hu, J., Shi, X., Mao, X., Chen, J., Zhu, L., Zhao, Q., 2013. Antinociceptive activity of Rhoifoline A from the ethanol extract of Zanthoxylum nitidum in mice. Journal of Ethnopharmacology 150, 828–834. Lee, J.H., Chang, K.M., Kim, G.H., 2009. Composition and anti-inflammatory activities of Zanthoxylum schinifolium essential oil: suppression of inducible nitric oxide synthase, cyclooxygenase-2, cytokines and cellular adhesion. Journal of the Science of Food and Agriculture 89, 1762–1769. Liang, L., Gansun, L., Jibing, L., Chongren, Y., Jun, Z., 1988. Chemical compositions of Z. myriacanthum and chemotaxonomy of zanthoxyllum. Acta Botanica Yunnanica 10, 445–456. Lima, L.M., Perazzo, F.F., Carvalho, J.C.T., Bastos, J.K., 2007. Anti-inflammatory and analgesic activities of the ethanolic extracts from Zanthoxylum riedelianum (Rutaceae) leaves and stem bark. Journal of Pharmacy and Pharmacology 59, 1151–1158. Lin, Y.F., Yi, Z., Zhao, Y.H., 2003. Chinese Dai Medicine Colorful Illustrations. The Nationalities Publishing House of Yunnan, Kunming, China. LisBalchin, M., Ochocka, R.J., Deans, S., Asztemborska, M., Hart, S., 1996. Bioactivity of the enantiomers of limonene. Medical Science Research 24, 309–310. Maeda, H., Akaike, T., 1998. Reviews-nitric oxide and oxygen radicals in infection, inflammation, and cancer. Biochemistry 63, 854–865. Misra, L.N., Wouatsa, N.A.V., Kumar, S., Kumar, R.V., Tchoumbougnang, F., 2013. Antibacterial, cytotoxic activities and chemical composition of fruits of two Camerooriian Zanthoxylum species. Journal of Ethnopharmacology 148, 74–80. National Committee for Clinical and Laboratory Standards Institute, 1999. Methods for Determining Bactericidal Activity of Antimicrobial Agents (Approved Guideline. Document M26-A. CLSI (formerly NCCLS). . Neirotti, E., Moscatelli, M., Tiscornia, S., 1996. Antimicrobial activity of the limonene. Arquivos De Biologia E Tecnologia 39, 233–237. NIST Chemistry WebBook, 2014. NIST Standard Reference Database Number 69 http://webbook.nist.gov/chemistry/. Accessed on July 31, 2014. Pei, S.J., 1985. Preliminary study of ethnobotany in xishuang banna, people's republic of china. Journal of Ethnopharmacology 13, 121–137. Pereira, S.S., Lopes, L.S., Marques, R.B., Figueiredo, K.A., Costa, D.A., Chaves, M.H., Almeida, F.R.C., 2010. Antinociceptive effect of Zanthoxylum rhoifolium Lam. (Rutaceae) in models of acute pain in rodents. Journal of Ethnopharmacology 129, 227–231. Ranawat, L., Bhatt, J., Patel, J., 2010. Hepatoprotective activity of ethanolic extracts of bark of Zanthoxylum armatum DC in CCl4 induced hepatic damage in rats. Journal of Ethnopharmacology 127, 777–780. Seo, J.W., Park, D.H., Li, Y.C., Kang, H.J., De Xu, H., Kim, Y.J., Lee, J.H., Lee, M.S., Lee, I.C., Lee, Y.L., Ahn, J.B., Cho, S.C., Lee, M.J., 2012. The Zanthoxylum schinifolium seed oil modulates immune function under the biological safety level. Molecular & Cellular Toxicology 8, 179–185. State Adeministration of Traditional Chinese Medicine of the People' s Republic of China, 2005. Traditional Chinese Meteria Medica of Dai Nationality. Shanghai Science and Technology Press, Shanghai, China. Sukari, M., Salim, W., Ibrahim, N., Rahmani, M., Aimi, N., Kitajima, M., 1999. Phenantridine alkaloids from Zanthoxylum myriacanthum. Fitoterapia 70, 197–199. Tezuka, Y., Irikawa, S., Kaneko, T., Banskota, A.H., Nagaoka, T., Xiong, Q.B., Hase, K., Kadota, S., 2001. Screening of Chinese herbal drug extracts for inhibitory activity on nitric oxide production and identification of an active compound of Zanthoxylum bungeanum. Journal of Ethnopharmacology 77, 209–217. Waterman, P.G., 1975. Alkaloids from the root bark of Zanthoxylum myriacanthum. Phytochemistry 14, 2530. Weyerstahl, P., Marschall, H., Splittgerber, U., Son, P.T., Giang, P.M., Kaul, V., 1999. Constituents of the essential oil from the fruits of Zanthoxylum rhetsoides Drake from Vietnam and from the aerial parts of Zanthoxylum alatum Roxb. from India. Flavour and fragrance journal 14, 225–229.
48
R. Li et al. / Journal of Ethnopharmacology 158 (2014) 43–48
Xu, Y.K., Tao, G.D., Liu, H.M., Yan, K.L., Dao, X.S., 2004. Wild vegetable resources and market survey in Xishuangbanna, southwest China. Economic Botany 58, 647–667. Yamazaki, E., Inagaki, M., Kurita, O., Inoue, T., 2007. Antioxidant activity of Japanese pepper (Zanthoxylum piperitum DC.) fruit. Food Chemistry 100, 171–177. Yang, L.C., Li, R., Tan, J., Jiang, Z.T., 2013. Polyphenolics composition of the leaves of Zanthoxylum bungeanum Maxim. grown in Hebei, China, and their radical scavenging activities. Journal of Agricultural and Food Chemistry 61, 1772–1778. Yang, X., 2008. Aroma constituents and alkylamides of red and green huajiao (Zanthoxylum bungeanum and Zanthoxylum schinifolium). Journal of Agricultural and Food Chemistry 56, 1689–1696.
Yoon, W.J., Lee, N.H., Hyun, C.-G., 2010. Limonene suppresses lipopolysaccharideinduced production of nitric oxide, prostaglandin E 2, and pro-inflammatory cytokines in RAW 264.7 macrophages. Journal of Oleo Science 59, 415–421. Zhang, D.X., Hartley, T.G., Mabberley, D.J., 2008. Rutaceae. http://flora.huh.harvard. edu.china/mss/volume11/Rutaceae.pdf. Accessed on March 31, 2014. Zhu, H.Y., Yang, F.M., Yang, X.S., Hao, X.J., 2007. Essential oils from Zanthoxylum rhetsoides Drake and their antibacterial activity. LiShiZhen Medicine and Materia Medica Research 18, 262–263.
