Journal of Evidence-Based Complementary & Alternative Medicine http://chp.sagepub.com/
Trachyspermum ammi (L.) Sprague: Chemical Composition of Essential Oil and Antimicrobial Activities of Respective Fractions Mahmoodreza R. Moein, Kamiar Zomorodian, Keyvan Pakshir, Farnoosh Yavari, Marjan Motamedi and Mohammad M. Zarshenas Journal of Evidence-Based Complementary & Alternative Medicine published online 10 October 2014 DOI: 10.1177/2156587214553302 The online version of this article can be found at: http://chp.sagepub.com/content/early/2014/10/09/2156587214553302
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Trachyspermum ammi (L.) Sprague: Chemical Composition of Essential Oil and Antimicrobial Activities of Respective Fractions
Journal of Evidence-Based Complementary & Alternative Medicine 1-7 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/2156587214553302 cam.sagepub.com
Mahmoodreza R. Moein, PhD1,2, Kamiar Zomorodian, PhD3, Keyvan Pakshir, PhD3, Farnoosh Yavari, PharmD1, Marjan Motamedi, MS1, and Mohammad M. Zarshenas, PhD1,4
Abstract Resistance to antibacterial agents has become a serious problem for global health. The current study evaluated the antimicrobial activities of essential oil and respective fractions of Trachyspermum ammi (L.) Sprague. Seeds of the essential oil were extracted and fractionated using column chromatography. All fractions were then analyzed by gas chromatography/mass spectrometry. Antifungal and antibacterial activities of the oil and its fractions were assessed using microdilution method. Compounds g-terpinene (48.07%), r-cymene (33.73%), and thymol (17.41%) were determined as major constituents. The effect of fraction II was better than total essential oil, fraction I, and standard thymol. The greater effect of fraction II compared to standard thymol showed the synergistic effects of the ingredients in this fraction. As this fraction and also total oil were effective on the studied microorganism, the combination of these products with current antimicrobial agents could be considered as new antimicrobial compounds in further investigations. Keywords antimicrobial activity, essential oil, nosocomial infection Received June 11, 2014. Accepted for publication August 14, 2014.
Introduction Despite the extensive investigation in the field of systemic fungal and bacterial infections and profound challenges to eliminate such infectious diseases, prevalence of bacterial infection is further increasing. Additionally, resistance to antibacterial agents has become a serious problem for global health. These days, improper redress for hospital-resistant infections has resulted in higher mortality rates and treatment expenditures, particularly in developing countries. At the present time, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Enterococcus, and Candida albicans have been propounded as the main agents of hospital-acquired infections.1 In consideration of limitations in accessibility to different sources of antibiotics and also medicinal resistance within numerous microorganisms, identification of new natural products–based antimicrobial and antifungal medicaments could be beneficial. Examples of potential increase of specific resistance to antimicrobial agents include the resistance of Enterococcus to vancomycin,2 Staphylococcus to methicillin,3,4 Escherichia coli to third-generation cephalosporins,5 Pseudomonas aeruginosa to quinoline and imipenem,6,7 and Candida albicans to the azole antimicrobial agents.8
Demand for the use of synthetic compounds as sources of medicine is considerably reduced due to the certainty of undesirable effects. Producers and consumers have gained understandings about the need to seek safe and natural resources to compete with infectious diseases. Thus, a novel of natural resource obtained from certain plant species could be of interest.9 Plants are known as bioreactors and are economically safe for production of antimicrobial agents. Essential oil compositions
Medicinal Plants Processing Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Department of Pharmacognosy, School of Pharmacy, Shiraz University of Medical Sciences, P.O. BOX: 71345-1583, Shiraz, Iran Department of Medical Mycology and Parasitology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Department of Phytopharmaceuticals (Traditional Pharmacy), School of Pharmacy and Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Corresponding Author: Mohammad M. Zarshenas, PhD, Department of Phytopharmaceuticals (Traditional Pharmacy), School of Pharmacy, Shiraz University of Medical Sciences, PO Box 71345-1583, Shiraz, Islamic Republic of Iran. Email: [email protected]
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Journal of Evidence-Based Complementary & Alternative Medicine
from respective herbs are known as appropriate agents for antimicrobial properties. The quality of oil is simply dependent on the climatic conditions in which the plants are cultivated.10,11 With reference to the findings and experiences of traditional medicine, many medicinal plants have been introduced for the management of human ailments. There are numerous citations of medicinal herbs and spices in medical and pharmaceutical manuscripts of traditional Persian medicine.12,13 One of the most famous natural preparations reported by early Persian practitioners was herbal medicinal oil. With regard to its active components, this form has being prepared by various methods.14 One of these methods was the hydrodistillation of spicy and aromatic plant parts. The product of this preparation method is currently known as essential oil.14 Trachyspermum ammi (L.) Sprague (TA) from the family Apiaceae is an old herb with various medical properties. There are medieval and traditional reports on the effects of Trachyspermum ammi oil and hydrosol on neural disorders such paralysis, tremor, and palsy as well as chronic pains. Trachyspermum ammi extract has been previously applied for the management of ophthalmic and otic infections. Seeds were known to be effective against respiratory and gastrointestinal diseases. Trachyspermum ammi was also reported as an aphrodisiac, galactagogue, and diuretic agent. Persian practitioners also reported the use of the fumigation form of Trachyspermum ammi seeds in female genital disorders. Toxicologically, topical decoction of Trachyspermum ammi seeds was reported as an analgesic agent for pain caused by scorpion’s bite.15 The oil extracted from seeds of Trachyspermum ammi from the family Apiaceae has shown considerable antibacterial, antiviral,16 antifungal,17 antitussive,18 anti-inflammatory,19 and analgesic20 effects as well as antioxidant, bronchodilatory,21 and antitumor activities.22 Since the total essential oil of Trachyspermum ammi has shown antimicrobial effects, the current work aimed to evaluate the antimicrobial activities of the herb’s essential oil and respective fractions against hospital-acquired pathogens.
Materials and Methods Extraction and Fractionation of the Essential Oil Seeds of Trachyspermum ammi were collected from Firozabad district in Fars province and identified by Ms S. Khademian, botanist in the Department of Traditional Pharmacy, Shiraz University of Medical Sciences. A sample of seeds was deposited in the Shiraz School of Pharmacy Herbarium with a specified number. Using a Clevenger apparatus, essential oil of Trachyspermum ammi was extracted using the hydrodistillation procedure.11 The essential oil obtained was subsequently fractionated using column chromatography packed with silica gel as stationary phase and eluted with petroleum ether–diethyl ether by increasing polarity (1:0; 1:1; 0:1). Three fractions (petroleum ether as F1: 400 cc; petroleum ether–diethyl ether as F2: 400 cc; and diethyl ether as F3: 100 cc) were collected and concentrated for the next step. The yields for F1, F2, and F3 fractions were 53.39%, 44.75%, and 0.84%, respectively.
Gas Chromatography/Mass Spectrometry Analysis of the Total Essential Oil and Respective Fractions Gas chromatography/mass spectrometry analysis was carried out using Agilent Technologies gas chromatograph model 5975C, which was equipped with a HP-5 capillary column (phenyl methyl siloxan, 30 m 0.25 mm internal diameter) and was connected to a mass spectrometer operating at 70 eV of ionization energy and a mass range of 30 to 600 m/z. Helium was selected as the carrier gas with a flow rate of 1 mL/min, and the split ratio was 1:30. The injector and detector temperatures were set at 250 C and 280 C, respectively. Column temperature was programmed linearly from 60 C to 250 C (rate of 5 C/ min) and then held for 10 minutes at 250 C. Retention indices were determined using retention times of injected n-alkanes before injection of samples under the same chromatographic conditions. Identification and quantification of constituents via the gas chromatography/mass spectrometry procedure was based on the comparison of those mass spectra with Willey (nl7) and Adams library spectra as well as with those reported in a previous investigation.23
Evaluation of the Antimicrobial Activities Antibacterial activities of essential oil and respective fractions were evaluated against standard species of Staphylococcus aureus (ATCC 25923 and ATCC 700698), Escherichia coli (ATCC 25922 and ATCC o157), Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 11700), Streptococcus pyogenes (ATCC 8668), as well as clinical isolates of Escherichia coli and Pseudomonas aeruginosa collected from the Dr Faghihi Hospital (Shiraz, Iran). Antifungal activities of essential oil and respective fractions were evaluated against 9 American Type of Culture Collection (ATCC) strains of fungi including Candida albicans (ATCC 10261), Candida krusei (ATCC 6258), Candida glabrata (ATCC 90030), Candida parapsilosis (ATCC 4344), Aspergillus flavus (ATCC 64025), and Aspergillus fumigates (ATCC 14110), candida dubliniensis (ATCC 7987), Aspergillus oryzae (CBS 818.72), Aspergillus clavatus (CBS 14.65). In addition, the antimicrobial activities of the essential oil and related fractions were also examined against 6 clinical isolates of Candida albicans identified by polymerase chain reaction–restriction fragment length polymorphism.24 The susceptibility of all clinical isolates of bacteria and fungi against selected antibiotics were examined by broth microdilution methods.25 Values of each minimum inhibitory concentration were determined using the broth microdilution method recommended by the CLSI with some modifications. To determine the antimicrobial activities against yeast species, serial dilutions of the essential oil (0.0625-32 mL/mL) were prepared in 96-well microtiter plates using RPMI-1640 media (Sigma, St Louis, MO) buffered with MOPS (Sigma). To determine the antibacterial activities, serial dilutions of essential oil (0.0625-32 mL/mL) were prepared in Mueller-Hinton media (Merck, Darmstadt, Germany). Test yeasts or bacteria strains were suspended in the medium. Using a spectrophotometric method, cell densities were adjusted to 0.5 McFarland standards at a wavelength of 530 nm (stock suspension of 1-5 106 cells/mL for yeasts and 1-1.5 108 cells/mL for bacteria). Approximately 0.1 mL of the working inoculums was added to the microtiter plates, which were incubated in a humid atmosphere at 30 C for 24 to 48 hours (for yeast) or at 37 C for 24 hours (for bacteria). A total of 200 mL of the uninoculated medium was included as a sterility control (blank). In addition, growth controls (medium with inoculums without essential oil) were also included. Growth in each well was compared to that of the control well. Minimum inhibitory concentrations were visually determined and defined
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Table 1. Chemical Compositions of Trachyspermum ammi Essential Oil and Respective Fractionsa. No.
1 n-Decane 2 r-Cymene 3 g-Terpinene 4 Thymol 5 Carvacrol 6 Nonadecane Identification Unknown compounds
— 33.73% 48.07% 17.41% — — 99.21% 0.79%
— 15.45% 14.84% 69.71% — — 100 —
— — — 95.32% 1.04% 1.07% 97.43 2.57%
10.02% 10.1% 6.35% 63.02% — — 89.48% 10.52%
999.6 1025 1064 1199 1290 1301
MS-KI MS-KI MS-KI MS-KI MS-KI MS-KI
Abbreviations: MS, mass spectrometry; KI, retention index. a Compounds have been identified via combination of both mass spectra and retention indices. KI values represent the retention indices calculated against C8-C24 n-alkanes on the mentioned column. Components have been sorted according to the retention indices on DB-5 MS capillary column.
as the lowest concentration of the essential oil that produced 95% growth reduction compared to the growth in the control well. Each experiment was performed in triplicate. Moreover, media from the wells with fungi showing no visible growth on Sabouraud dextrose agar (Merck, Darmstadt, Germany) as well as media from wells with bacteria showing no visible growth on Mueller-Hinton agar (Merck, Darmstadt, Germany) were further cultured to determine the minimum fungicidal concentration and minimum bactericidal concentration. Minimum bactericidal concentrations and minimum fungicidal concentrations were determined as the lowest concentration yielding no more than 4 colonies. This concentration corresponds to a mortality of 98% of the organisms in the initial inoculums.
Results and Discussion Gas chromatography/mass spectrometry analysis of the total essential oil of Trachyspermum ammi revealed that g-terpinene (48.07%), r-cymene (33.73%), and thymol (17.41%) were the major constituents. Table 1 presents the chemical compositions related to the Trachyspermum ammi essential oil and the isolated fractions. In all fractions thymol was found to be the main component at a much higher amount compared to that of the total oil. The effectiveness of Trachyspermum ammi essential oil and its respective fractions on the studied microorganisms is presented in Tables 2 and 3. According to the results, antibacterial activities of total essential oil on either gram positive or gram negative bacteria were similar. Thus, the total oil could cease the growth of both bacteria at the concentration ranging from 0.5 to 4 mL/mL. It was observed that F1 possessed bactericidal effects on both gram negative and positive groups at the different concentrations employed. F2 exhibited more impact on gram positive bacteria as compared to the total oil and F1. The effect of F2 on isolates of Escherichia coli and Pseudomonas aeruginosa was similar to that of the total oil, but was higher than total oil and F1 in regard to Salmonella isolate. Effects of pure thymol on both gram negative and positive bacteria were found to be similar. The bacteriostatic and also bactericidal effects of thymol was found at the range of 1 to 8 mg/mL. Application of 1 to 2 mL/ mL from the total oil possessed both bacteriostatic and bactericidal effects on resistant Escherichia coli. The bacteriostatic and bactericidal effects of F1 on Escherichia coli were found at 2 to 4 mL/mL. F2 showed the strongest activity with bacteriostatic and
bactericidal effects at concentration ranges of 0.5 to 1 mL/mL and 0.5 to 2 mL/mL, respectively. With regard of the antifungal activities, results revealed that F2 exhibited the best fungistatic and fungicidal activities on the Aspergillus isolates. Numerous studies have been performed on the analysis of Trachyspermum ammi essential oil. Several investigations reported that thymol, g-terpinene, and r-cymene were major constituents of respective seed samples,26-28 whereas others reported carvacrol as main component as well.29-31 Similar to some previous investigations, in the current work, g-terpinene (48.07%), r-cymene (33.73%), and thymol (17.41%) were found as the main constituents.11,32 Investigations have shown that differences in the rate and type of chemical composition within extracted essential oil of Trachyspermum ammi may due to the location of cultivation and geographical conditions as well as collection time and the extraction procedure.11,28 Till now, there have been some studies on the pharmacological effects of Trachyspermum ammi essential oil. Trachyspermum ammi volatile oil and respective fractions were assessed for muscle relaxant, anticholinergic, and xanthinelike activities. This in vivo study showed that a carvacrolrich fraction was highly effective and possessed the relaxant activity.33 An investigation on the fumigant toxicity of Trachyspermum ammi essential oil revealed the potential role of the oil as a fumigant for stored-product insects.26 In an in vitro study, the antioxidant and antimutagenic activities of Trachyspermum ammi were evaluated. Results indicated that the related effects were due to the presence of phenolic terpenoids.22 The analgesic effects of Trachyspermum ammi essential oil were confirmed in an in vivo study.34 Apart from these investigations, the antimicrobial effects of Trachyspermum ammi essential oil have been evaluated more frequently.10,16,17,29 Previous researches have indicated that the antibacterial activity of Trachyspermum ammi essential oil is attributed to the presence and amount of thymol, carvacrol, g-terpinene, and r-cymene.32 In spite of the numerous experimental and animal studies on Trachyspermum ammi essential oil, only one human study has been performed, to study the analgesic effects of the essential oil.35 More recently, since the medicinal herb species were selected for drug discovery, many investigators focused their 3
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Table 2. Minimum Inhibitory and Bactericidal Concentrations of Trachyspermum ammi Essential Oil and Related Fractions. Essential Oil Bacteria
MIC MBC MIC MBC MIC MBC MIC MBC (mL/mL) (mL/mL) (mL/mL) (mL/mL) (mL/mL) (mL/mL) (mg/mL) (mg/mL)
Gram positive Bacillus cereus (ATCC 11778) Streptococcus sobrinus (ATCC 27607) Enterococcus faecalis (ATCC 11700) Streptococcus salivarius (ATCC 9222) Streptococcus mutans (ATCC 35668) Streptococcus pyogenes (ATCC 8668) Streptococcus sanguinis (ATCC 10556) Staphylococcus aureus (ATCC 29213) Staphylococcus aureus (ATCC 25923) Staphylococcus aureus (ATCC 700698) Gram negative Multidrug-resistant Pseudomonas aeruginosa (1) Sensitive strain Pseudomonas aeruginosa (3) Salmonella paratyphi B Salmonella paratyphi A Pseudomonas aeruginosa (ATCC 27853) Escherichia coli (ATCC 25912) Escherichia coli (ATCC o157)
2 4 2 1 1 0.5 2 0.5 1 2 4 2.66 1 2 4 0.5 1
2 4 4 1 2 0.5 4 1 2 2 32 16 2 2 4 0.5 1
4 4 2 1 1 0.5 4 1 1 2 8 9.33 1 2 16 1 1
4 8 4 2 2 0.5 8 1 2 2 32 32 2 2 16 2 1
1 2 1 0.5 0.5 0.25 1 0.5 0.5 1 2 4.66 0.5 1 4 0.5 1
1 4 2 0.5 1 0.25 2 0.5 0.5 1 16 16 1 1 4 0.5 1
4 4 2 1 0.5 1 8 2 4 2 8 8 1 2 8 4 4
4 8 4 1 1 1 8 2 4 2 32 32 1 2 8 8 8
Abbreviations: MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; ATCC, American Type of Culture Collection.
attention toward the production of natural and herbal chemical compositions that may contain antibacterial and antifungal properties. It has been observed that aqueous or alcoholic extraction as well as essential oil compounds yielded from various types of plants may be capable of having antimicrobial effects under laboratory conditions. Previously, it has been reported that Trachyspermum ammi essential oil exhibits antibacterial activity against several resistant bacteria as well as antifungal effects.36 Similar to a previous study,12 our research showed that application of 0.5 to 4 mg/mL of the Trachyspermum ammi essential oil can simply prohibit the hospital-acquired infections generated usually from Staphylococcus aureus, Escherichia coli, and Enterococcus, Pseudomona aeroginosa. On the contrary, a recent investigation showed that a lower amount of Trachyspermum ammi essential oil (0.3 mg/mL) could inhibit the growth of Staphylococcus aureus strains.37 This finding may be due to the high concentration of thymol in the employed sample. In our assessment, a low concentration range of Trachyspermum ammi essential oil (0.0625 to 1 mL/mL) showed fungistatic activities against Candida and Aspergillus species. These findings were also similar to those reported previously.38 An investigation indicated that the effectiveness of Trachyspermum ammi essential oil on gram positive is more than gram negative bacteria.39 Taken as a whole, no certain assessments have been performed on the antimicrobial activities of fractions isolated from Trachyspermum ammi essential oil. Thus, the current work focused on the evaluation of the mentioned fractions. According to the data of gas chromatography/mass spectrometry analysis, total oil involved g-terpinene and r-cymene as the main components, while thymol encompassed over 60% of the
constituents in F1 and F3 and also over 90% of those in F2 (Table 1). The evaluation of antibacterial and antifungal activities of Trachyspermum ammi essential oil has exhibited that these properties were predominantly seen more in F2 rather than F1 and F3, and further increases of thymol concentration in F2 was the cause to generate such efficacy. Therefore, potent antimicrobial activity of F2 compared to the standard sample of thymol could be due to the synergism combination effect of thymol, nonadecane, and carvacrol in this fraction. A previous investigation has shown that the combination of carvacrol and thymol will have considerable synergism affects.40 The result of our study confirmed that the mechanism of activity of the essential oil and its respective fractions can be varied from those in synthetic antibiotics. Previous studies have reported that the composition of phenolic compounds in the essential oil is able to dissolve microorganism membrane and generate pores to produce the antibacterial effects.41 Application of these types of medicaments not only offers great merits over synthetic drugs but also simply prohibits any side effects and has no harm to the environment. Accordingly, such herbal medicaments, either the total essential oil or isolated fractions, can be introduced as good candidates to be applied in combination with conventional antibiotics against nosocomial infections. As the toxicity assessment of Trachyspermum ammi essential oil has not yet been examined, it is remarkable that this investigation should be performed in large and intensive human studies.
Conclusion In current work, the essential oil of Trachyspermum ammi was evaluated for possible antibacterial and antifungal activities
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Fraction 1 MIC (mL/mL)
MIC MBC (mg/mL) (mg/mL)
Abbreviations: MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration.
Candida albicans (A10261) 0.5 0.5 0.5 0.5 0.25 0.25 2 4 16 16 Candida dubliniensis (A7987) 1 1 1 1 0.5 0.5 2 4 32 32 Candida krusei (A6258) 0.5 0.5 0.5 0.5 0.25 0.25 2 2 8 8 Candida parapsilosis (A4344) 0.25 1 0.5 1 0.25 1 2 4 2 8 Candida glabrata (A90030) 0.0625 0.0625 0.125 0.25 0.0625 0.0625 2 2 2 4 Filamentous Aspergillus flavus (A 64025) 0.5 16 1 4 0.5 2 2 16 32 128 Aspergillus fumigatus (A 141 10) 0.0625 4 0.0625 2 0.0625 1 1 8 8 128 Aspergillus oryzae (C818.72) 0.5 1 0.5 1 0.25 0.5 1 2 2 8 Aspergillys clavatus (C514.65) 0.25 0.5 0.25 1 0.25 0.5 1 2 2 16 Clinical isolates Candida albicans (4) 0.44 (0.25-0.5) 0.75 (0.5-1) 0.44 (0.25-0.5) 0.69 (0.25-1) 0.44 (0.25-0.5) 0.5 (0.5) 1.5 (1-2) 3 (2-4) 0.5 (0.25-1) 7.5 (2-16) Candida dubliniensis (2) 0.37 (0.25-0.5) 1 (1) 0.5 (0.5) 1.5 (1-2) 0.25 (0.25) 0.75 (0.5-1) 1.5 (1-2) 3 (2-4) 1.25 (0.5-2) 12 (8-16)
Table 3. Minimum Inhibitory and Fungicidal Activities of Trachyspermum ammi Essential Oil and Respective Fractions.
Journal of Evidence-Based Complementary & Alternative Medicine
against resistant and sensitive strains of microorganisms. Additionally, 3 fractions were isolated from the oil and were assessed for respective activities. F2 possessed considerable activity among the fractions investigated as well as the total oil and pure thymol. Although F2 contained more than 90% of thymol as major constituent, the combination of thymol and other components in this fraction were responsible for the desirable activity. The essentiality of this investigation elucidated that this fraction can be selected for further investigations alone or in combination with current antimicrobial agents. Acknowledgment The authors wish to express their thanks to Shiraz University of Medical Sciences. This paper was derived from Farnoosh Yavari Pharm. D thesis. The authors of this manuscript wish to express their thanks to Shiraz University of Medical Science.
Author Contributions FY MM performed the experimental works wrote the draft and contributed in data gathering and writing the draft version of the article. MMZ completed the draft and contributed in the preparation of the final article. MRM, KZ, and KP contributed in the guidance, revisions, and corrections of the draft and the final version of the article.
Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The authors received no financial support for the research, authorship, and/or publication of this article.
Ethical Approval This work is an experimental and laboratorial study and needs no ethical approval. The study was done at the School of Pharmacy, Shiraz University of Medical Sciences.
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