Chemical Composition and Antibacterial Activity of the Essential Oil

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Chemical Composition and Antibacterial Activity of the Essential Oil from Green Huajiao (Zanthoxylum schinifolium) against Selected Foodborne Pathogens Wen-Rui Diao, Qing-ping Hu, Sai-Sai Feng, Wei-Qin Li, and Jian-guo Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf4007856 • Publication Date (Web): 30 May 2013 Downloaded from http://pubs.acs.org on June 12, 2013

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Journal of Agricultural and Food Chemistry

Running Title: Antibacterial Activity of the Essential Oil from Green Huajiao Chemical Composition and Antibacterial Activity of the Essential Oil from Green Huajiao (Zanthoxylum schinifolium) against Selected Foodborne Pathogens Wen-Rui Diao, † Qing-Ping Hu, † Sai-Sai Feng, ‡ Wei-Qin Li, ‡ and Jian-Guo Xu, *, †, ‡ †

College of Life Sciences and

‡

College of Engineering, Shanxi Normal University,

Linfen City, 041004, China

1

ACS Paragon Plus Environment


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ABSTRACT:

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Green huajiao, which is the ripe pericarp of the fruit of Zanthoxylum schinifolium

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Sieb. et Zucc, is widely consumed in Asia as a spice. In this work, the chemical

4

composition of the essential oil from green huajiao was analyzed by gas

5

chromatography (GC) and GC-mass spectrometry (MS), and the majority of

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components were identified. Linalool (28.2%), limonene (13.2%), and sabinene

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(12.1%) were found to be the major components. The antibacterial activity, minimum

8

inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of

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essential oil were evaluated against selected bacteria, including food-borne pathogens.

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The results showed that the sensitivities to essential oil were different for different

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bacteria tested, and the susceptibility of Gram-positive bacteria tested was observed to

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be greater than that of Gram-negative bacteria. The antibacterial activity of essential

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oil was particularly strong against Staphylococcus epidermidis with the MIC and

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MBC values of 2.5 and 5.0 mg/mL, respectively. Postcontact effect (PCE) assay also

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confirmed the essential oil had a significant effect on the growth rate of surviving S.

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epidermidis. The antibacterial activity of the essential oil from green huajiao may be

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due to the increase in permeability of cell membranes, and the leakage of intracellular

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constituents based on cell constituents' release assay and electron microscopy

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observations.

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KEYWORDS: Green huajiao; zanthoxylum schinifolium Sieb. et Zucc; antibacterial

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activity; essential oil; foodborne pathogen


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INTRODUCTION

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In recent years, food spoilage and food poisoning are amongst the most important

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issues facing the food industry, 1, 2 and there has been a dramatic increase throughout

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the world in the number of reported cases of foodborne illness, even in developed

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countries. 3, 4 Many attempts, such as use of synthetic chemicals, have been made to

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control microbial growth and to reduce the incidence of food poisoning and spoilage.

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Recently, however, consumers have become increasingly concerned about the side

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effects of synthetic antimicrobial chemicals and want safer materials for preventing

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and controlling pathogenic microorganisms in foods.5, 6 Thus, development of safe

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and natural antibacterial compounds is becoming critically important.7 Essential oils

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are the odorous, volatile products of the secondary metabolism of aromatic plants. A

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great number of plant essential oils have been tested for their antimicrobial activities

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and some of them have been reported to possess strong antibacterial and antifungal

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effects.5, 6, 8-11 Unlike the action of chemical antibiotics, an important characteristic of

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essential oil components are their hydrophobicity, which enables them to partition

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into the lipids of the bacterial cell membrane, disturbing the cell structures and

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rendering them more permeable. The ability of essential oils to disrupt the

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permeability barrier of the cell membrane and the accompanying loss of

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chemiosmotic control is the most likely cause of their lethal action. 12, 13 In addition, it

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has been demonstrated that essential oils from various medicinal plants, spices and

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herbs possess high volatility, toxicity to stored-grain insect pests while have little

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harm to warm-blooded animals.

14, 15

Considering their excellent antimicrobial



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function, natural essential oils possess great potential as antimicrobial agents to

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increase the safety and shelf life of foods. However, it might also be noted that some

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essential oils may be toxic if ingested, depending on the type and concentration of oil.

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Zanthoxylum schinifolium Sieb. et Zucc, found in China, Japan and Korea, is an

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aromatic shrub with protective thorns, culinary and pharmacological properties which

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has been cultivated in the southern provinces of China.16-18 Green huajiao, which is

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the ripe pericarp of the fruits of Z. schinifolium, has strong anise, citrus, and pepper

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notes, and is widely consumed in Asia as a spice.18 The previous phytochemical

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studies indicated coumarins, alkaloids, triterpenoids, steroids, and flavonoids in the

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extract of Z. schinifolium.17-19 It has been reported that the essential oil components

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from Z. schinifolium showed various biological activities including antiplatelet

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aggregation, anti-inflammatory activity, 20 antioxidant

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and regulation of cardiac hormone secretion. 23 In addition, green huajiao possessed

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significant feeding deterrence against some insects (Tribolium castaneum and

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Sitophilus zeamais).16, 23 However, to the best of our knowledge, little information is

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available about the antibacterial effect and the mechanism of action of the essential oil

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from green huajiao on the growth and survival of food-borne pathogens. Therefore,

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the aim of the present study was to investigate the chemical composition and

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antibacterial activity of essential oil from green huajiao on several selected bacteria,

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including food-borne pathogens, and to further evaluate the possible mechanism of

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action of essential oil against sensitive strains by the postcontact effect assay, cell

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constituents' release test and scanning electron microscopy as well as transmission

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and anticancer activities,22, 23


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electron microscopy observations.

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MATERIALS AND METHODS

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Plant Materials and Chemicals/Reagents. Green huajiao, which grows in Jinyang

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County of Sichuan Province, was obtained as a commercial product in the market of

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Chengdu on 2012. The sample was identified as a single fruit and the dry and the ripe

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pericarp of Jiuye Green huajiao by Professor Xuezhi Wei. Dimethyl sulfoxide

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(DMSO) was purchased from Sigma (USA). Nutrient agar (NA), nutrient broth (NB)

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and tryptone soy agar were from Beijing Aoboxing Bio-tech Co. Ltd. (Beijing, China).

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Other chemicals used were all of analytical grade.

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Microorganisms and Culture. The antimicrobial activities of essential oil were

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tested against seven different bacteria. Three Gram-positive strains were

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Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 8799 and

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Bacillus subtilis ATCC 6051. Four Gram-negative strains were Salmonella

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typhimurium ATCC 19430, Pseudomonas aeruginosa ATCC 9027, Shigella

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dysenteriae CMCC (B) 51252 and Escherichia coli ATCC 25922. The strains were

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provided by the College of Life Science, Shanxi Normal University, and cultured at

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37 °C on NA or NB.

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Essential Oil Extraction. The green huajiao was again air-dried at 40 °C for 6 h

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and its moisture content was 8.92%. The dried green huajiao was ground with a micro

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plant grinding machine (FZ102; Tianjin Taisite Instruments, Tianjin, China) to a

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powder and then hydrodistilled for 4 h using a Clevenger-type apparatus. The oil was

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separated from water and dried over anhydrous sodium sulfate and stored in tightly



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closed dark vials at 4 °C until use. The essential oil was obtained as a light yellow

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transparent liquid and had distinct sharp odor with a 3.6% yield.

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GC-FID Analysis. The essential oil was analyzed using Hewlett-Packard 5890 II

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GC equipped with FID detector and HP-5 MS capillary column (30 m×0.25 mm; film

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thickness, 0.25 µm). Injector and detector temperatures were set at 250 and 200 °C,

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respectively. The oven temperature was programmed from 40 °C for 2 min, raised to

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80 °C at a rate of 3 °C/min, held isothermal for 2 min, and finally raised to 240 °C at

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5 °C/min for 10 min. Helium was the carrier gas, at a flow rate of 1 mL/min. A

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sample of 0.2 µL of essential oil was injected manually, and the GC split ratio used

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was 1:100. The percentages of the constituents were calculated by electronic

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integration of FID peak areas without the use of response factor correction.

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GC-MS Analysis. The analysis of the essential oil was performed using a

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Hewlett-Packard 5890 II GC, equipped with a HP-5 MS capillary column (30 m×0.25

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mm; film thickness, 0.25 µm) and a HP 5972 mass selective detector for the

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separation. The mass selective detector was operated in electron-impact ionization (EI)

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mode with a mass scan range from m/z 50 to 550 at 70 eV. Helium was the carrier gas

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at a flow rate of 1 mL/min. Injector and MS transfer line temperatures were set at 250

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and 200 °C, respectively. The oven temperature was programmed as in the GC-FID

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analysis. A sample of 0.2 µL of essential oil was injected manually using a 1:100 split

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ratio. The components were identified by comparing their GC retention indices, NIST

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mass spectral search program (version 2.0, National Institute of Standards and

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Technology), and mass spectra with published data.


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Antimicrobial Activity. The essential oil was first dissolved in DMSO to a

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concentration of 50% (v/v), and then sterilized by filtration through 0.22 µm Millipore

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filters. Antimicrobial tests were carried out by the Oxford cup method using 100 µL of

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suspension containing 108 CFU/mL of bacteria spread on nutrient agar (NA) medium.

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Oxford cups (6 mm in diameter) were placed on the inoculated agar, and 100 µL of

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essential oil was added. The diameter of zones of inhibition (ZOI) was measured after

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24 h of incubation at 37 °C. DMSO was used as negative control. Tests were

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performed in triplicate.

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Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal

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Concentration (MBC) Assay. MIC and MBC were determined according to the

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method described by Kubo et al.

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essential oil in DMSO as stock solutions was prepared. Two fold serial dilutions of

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essential oil were prepared in sterile NB medium ranging from 0.625 to 20 mg/mL.

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To each tube 100 µL of the exponentially growing bacterial cells was added to give a

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cell concentration of approximately 2×10

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performed containing inoculated broth supplemented with only DMSO. The

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maximum final concentration of DMSO in each medium was 2%, which did not affect

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the growth of the test strains. The tubes were incubated at 37 °C for 24 h and then

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examined for evidence of the growth. The MIC was determined as the lowest

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concentration of the essential oil that demonstrated no visible growth. The MBC was

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determined as follows. After the determination of the MIC, 100-fold dilutions with

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drug-free NB from each tube showing no turbidity were incubated at 37 °C for 48 h.

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with minor modifications. Briefly, 50% (v/v)

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CFU/mL. A control test was also



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The MBC was the lowest concentration of the essential oil that showed no visible

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growth in the drug-free cultivation. All experiments were performed in triplicate.

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Evaluation of Postcontact Effect (PCE). The PCE of the essential oil on tested

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strains was evaluated according to the method as described previously with some

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modifications.25 The bacterial cultures of the exponential phase (approximately 10

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CFU/mL) were exposed to 1× and 2×MIC of essential oils at 37 °C for 1 h. Exposed

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and control cultures were washed twice with PBS by centrifugation at 4000 rpm for

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20 min and then diluted in fresh broth before incubation at 37 °C. Viable counts were

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immediately measured in tryptone soy agar and 1-24 h (a time per hour) after

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incubation. PCE was determined using the equation PCE = t-c, where t was the time

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needed for a log increase in counts in treated cultures and c the time needed for a log

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increase in counts in untreated cultures. All experiments were performed in triplicate.

8

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Cell Constituents' Release. The release of cell constituents into supernatant was

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examined according to the method described by Rhayour et al.9 with some

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modifications. Cells from the 100 mL working culture of tested bacteria were

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collected by centrifugation at 5000 g for 15 min, washed three times with 0.1 M

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phosphate buffer solution (PBS, pH 7.4), and resuspended in 0.1 M PBS. One

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hundred milliliter of cell suspension was incubated at 37 °C under agitation for 1 h in

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the presence of essential oil at three different concentrations (control, MIC and

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2×MIC). Then, 5 mL of samples were collected and centrifuged at 11,000 g for 5 min.

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To determine the concentration of the constituents released, 2 mL supernatant was

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used to measure UV absorption at 260 nm. Correction was made for the absorption of


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the suspension with PBS containing the same concentration of essential oil after 2 min

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of contact with tested strains. The untreated cells (control) were corrected with pH 7.4

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PBS.

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Scanning Electron Microscope (SEM). To determine the efficacy of the essential

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oil and the morphological changes of the bacteria, SEM observation was performed

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on the tested bacteria according to the method as described previously.26 After 10 h

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incubation of a cell suspension of S. epidermidis of approximately 2×10

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37°C in NB, essential oil was added at either the MIC or 2 x MIC. The control culture

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was left untreated. All suspensions were incubated at 37 °C for 4 and 8 h respectively,

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and then centrifuged. The cells were washed twice with 0.1 M PBS (pH 7.4) and fixed

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with 2.5% (v/v) glutaraldehyde in 0.1 M PBS overnight at 4°C. After this, the cells

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were successively dehydrated using 30%, 50%, 70%, 90%, and 100% ethanol, and

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then the ethanol was replaced by tertiary butyl alcohol. Then, cells were dried at

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“critical point” in liquid CO2 under 95 bar pressure, and samples were gold-covered

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by cathodic spraying. Finally, morphology of the bacterial cell was observed with a

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scanning electronic microscope (SEM) (JSM-7500F, JEOL Ltd., Japan) operated at an

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accelerating voltage of 25–30 kV.

6

cfu/mL at

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Transmission Electron Microscope (TEM). The bacteria cells were incubated for

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10 h in NB at 37 °C, before addition of essential oil to give a final concentration of

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the MIC or 2× MIC. All treatments and controls were incubated at 37 °C for 4 and 8 h

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respectively, and then centrifuged. The cells were washed twice with 0.1 M PBS (pH

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7.4) and fixed with 2.5% (v/v) glutaraldehyde in 0.1 M PBS overnight at 4°C. Then



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the cells were postfixed with 1% (w/v) OsO4 in 0.1 M PBS for 2 h at room

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temperature and washed three times with the same buffer, then dehydrated by a

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graded series of ethanol solutions (30%, 50%, 70%, 90%, and 100%). Stained bacteria

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were viewed and photographed with a transmission electron microscope (H-600,

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HITACHIL Ltd., Japan) operated at 75 kV, and were analyzed with digital imaging

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software (MEGAVIEW G2).

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Statistical Analysis. One-way analysis of variance (ANOVA) and Duncan’s

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multiple range tests were carried out to determine significant differences (p < 0.05)

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between the means by Data Processing System (DPS, version 7.05) and EXCEL

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program.

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RESULTS

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Chemical Composition of the Essential Oil. The essential oil was obtained by

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hydrodistillation of air-dried sample with a yield of 3.6% yield (v/w), which was

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consistent with most previous reports of yields of 3-4% of essential oil for the dried

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huajiao pericarp.27 The chemical composition of essential oil was analyzed by GC and

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GC-MS and the result was listed in Table 1. In total, 56 components were identified,

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representing 94.63% of the total amount. Results showed that linalool (28.2%),

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limonene (13.2%), and sabinene (12.1%) were found to be the major compounds in

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the essential oil of green huajiao, followed by myrcene (6.12%), linalyl acetate

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(3.90%), 4-terpinenol (3.72%), β-phellandrene (3.38%). Besides, other components

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were also found and to be lower content (0.04%-2.48%) in the essential oil of green

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huajiao in the present study (Table 1).


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[Table 1 about here]

198 199

ZOI, MIC and MBC of the Essential Oil. The ZOI, MIC, and MBC values of the

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essential oil from green huajiao against different bacteria are presented in Table 2.

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The results showed that the essential oil of green huajiao had certain antibacterial

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effects on all of the tested bacteria (Table 2). The ZOI, MIC and MBC values for

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Gram-positive bacterial strains were in the range of 13.8-18.2mm, 2.5-10 mg/mL and

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5-10 mg/mL, respectively; while they were in the range of 8.5-11.6 mm, 10-20

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mg/mL and greater than or equal to 20 mg/mL for Gram-negative bacterial strains,

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respectively. Of these bacteria, the essential oil of green huajiao had the greatest

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antibacterial effect against S. epidermidis, with both the lowest MIC of 2.5 mg/mL

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and MBC of 5.0 mg/mL. The MIC and MBC values of the essential oil for Salmonella

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typhimurium and Shigella dysenteriae strains were greater than 20mg/mL which was

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the highest concentration of essential oil tested in this study. [Table 2 about here]

211 212

Postcontact Effect (PCE) of Essential Oil. The PCEs of the essential oil from

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green huajiao on S. epidermidis were evaluated at 1×MIC and 2×MIC concentrations

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(Table 3). The PCE increased significantly with the increase of the concentration of

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essential oil (p < 0.05). At 1×MIC concentration, the PCE was 5.2 h, and increased to

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9.2 h at 2×MIC concentration (p < 0.05), which confirmed the antibacterial activity of

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essential oil from green huajiao and showed a severe effect on recovery of surviving S.

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epidermidis after treatment.

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[Table 3 about here]



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Cell Constituents' Release. The cell constituents' release was determined by the

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measurement of the absorbance at 260 nm of the supernatant of tested S. epidermidis

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strains. Table 3 showed the results when S. epidermidis were treated with different

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concentrations essential oil for 1 h, respectively. The results showed that after adding

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the corresponding essential oil to strains, the cell constituents' release increased with

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the increased concentration of the essential oil compared to the control group (p
20

NT b

P. aeruginosa

10.6±0.6 c

20

>20

S. dysenteriae

8.5±0.8 d

>20

NT

E. coli

11.6±0.5 c

10

20

Bacteria Gram-positive

Gram-negative

484

a

485

Different letters within a column indicate statistically significant differences between

486

the means (p < 0.05) for ZOI.

Values represent means of three independent replicates ± SD.

487


b

NT, not tested.


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Table 3. Postcontact Effect (PCE) and Effect of the Essential Oil on Cell

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Constituents' Release of Tested S. epidermidis. Concentrations (mg/mL)

PCE (h)

Cell constituents' release (OD260nm)

control

0

0.065±0.006 c

1×MIC

5.5±0.2 b

0.153±0.011 b

2×MIC

9.2±0.3 a

0.292±0.014 a

490

Values represent means of three independent replicates ± SD. Different letters within

491

a column indicate statistically significant differences between the means (p < 0.05).

492


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Figure 1.

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Figure 2.

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TOC Graphic


Chemical Composition and Antibacterial Activity of the Essential Oil

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