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Infectio

Print version ISSN 0123-9392

Infect. vol.21 no.3 Bogotá July/Sept. 2017

https://doi.org/10.22354/in.v21i3.675 

Artículos originales

Composición química y actividades antimicrobianas y antioxidantes del aceite esencial de Echinophora platyloba

Chemical composition, antimicrobial and antioxidant activities of Echinophora platyloba essential oil

Fourough Mahdian1 

Mohaddese Mahboubi2 

Ebrahim Rahimi3 

Maryam Moslehi Shad1 

1 Department of Food Science and Technology, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, IR Iran

2 Department of Microbiology, Medicinal Plant, Research Center of Barij, Kashan, IR Iran

3 Department of Food Hygiene and Public Health, Faculty of Veterinary Medicine, Shahrekord Branch, Islamic Azad University, Shahrekord, IR Iran


Resumen

Objetivo:

Echinophora platyloba se utiliza tradicionalmente como agente antimicrobiano con el fin de preservar los productos caseros de deterioro. El objetivo de este estudio fue evaluar la composición química del aceite esencial, actividad antimicrobiana y antioxidante del aceite esencial de E. platyloba de la provincia de Shahr-e-Kord.

Materiales y métodos:

la composición química del aceite esencial de E. platyloba mediante técnicas de GC-MS y GC y evaluar su efecto antibacteriano frente a Staphylococcus aureus, Salmonella enterica y Helicobacter pylori por ensayos de difusión en disco y de micro dilución en caldo. La actividad antioxidante del aceite esencial de E. platyloba se evaluó por los radicales ABST. β-ocimeno y α-cariofileno eran los componentes principales del aceite esencial de E. platyloba. El aceite esencial mostró la actividad antibacteriana prometido contra S. aureus, seguido de S. enterica y H. pylori. El aceite esencial presenta actividad antioxidante del aceite esencial mostró la IC50 de 0. 32 mg/ml que fue mayor que la IC50 de ácido ascórbico (0.20 mg/ml).

En conclusión

: Aceite esencial de E. platyloba ha se recomienda ß-ocimeno y quimiotipo α-cariofileno y su uso como agentes antioxidantes y antibacterianas como sus usos tradicionales.

Palabras clave: Aceite esencial de E. platyloba; β-ocimeno; α-cariofileno; antimicrobianos; antioxidantes

Abstract

Objective:

Echinophora platyloba is traditionally used as antimicrobial agent in order to preserve the home-made products from deterioration. The aim of this study was to evaluate chemical composition of essential oil, antimicrobial and antioxidant activity of E. platyloba essential oil from Shahr-E-Kord city, Chaharmahal and Bakhtiari Province.

Materials and methods:

chemical composition of E. platyloba essential oil by GC and GC-MS techniques and evaluate its antibacterial effect against Staphylococcus aureus, Salmonella enterica and Helicobacter pylori by disc diffusion and micro broth dilution assays. The antioxidant activity of E. platyloba essential oil was evaluated by ABST radicals. β-ocimene and α-caryophyllene were the main components of E. platyloba essential oil. The essential oil showed the promised antibacterial activity against S. aureus, followed by S. enterica and H. pylori. The antioxidant activity evaluation of essential oil showed the IC50 of 0.32 mg/ml that was higher than the IC50 of ascorbic acid (0.20 mg/ml).

In conclusion

, E. platyloba essential oil has β-ocimene and α-caryophyllene chemotype and its use as antioxidant and antibacterial agents is recommended as its traditional uses.

Keywords: E. platyloba essential oil; β-ocimene; α-caryophyllene; antimicrobial; antioxidant

Introduction

Echinophora platyloba, a plant from Umbelliferae family is used in Iranian Traditional medicine as antimicrobial agent for protection of home made products from spoilage1. According to its traditional uses, there are some studies that evaluate the antimicrobial activities of E. platyloba extracts against dermatophytes (Trichophyton sp, Microsporum sp and Epidermophyton flucosum)2,3, Candida albicans1,2,4,5, Listeria monocytogenes, Alcaligenes faecalis, Serratia marscescenes, Providencia rettgeri6, Staphylococcus aureus, and Pseudomonas aeruginosa7. The antimicrobial activity of E. platyloba essential oil against L. monocytogenes8-10, Bacillus cereus9, B. subtilis8,9, S. aureus 8,9, E. coli O157H78,9, P. aeruginosa, Candida sp (C. albicans, C. tropicalis), Rhodotorula sp (R. rubra and R. mucilaginosa)9, and Aspergillus niger8 were confirmed. Other biological activities of E. platyloba extracts such as antioxidant11, anti-parasitic12, and estrogenic activities13,14were confirmed. Although, there are some studies that evaluate the antimicrobial activity of E. platyloba essential oil8-10,15, but for the first time, we evaluate the antibacterial activity of E. platyloba essential oil against Helicobacter pylori, Salmonella enterica and Staphylococcus aureus as the most important pathogenic bacteria. Furthermore, because geographical locations affect on chemical compositions of essential oils16, analyses of chemical compositions of E. platyloba essential oil is most important for any biological evaluation.

Therefore, we analyze the chemical compositions of E. platyloba essential oil, and then its antimicrobial activities against three important pathogens and the antioxidant activity were evaluated.

Materials and methods

Plant Materials, extraction of essential oil by hydrodistillation method

Aerial parts of Echinophora platyloba at full flowering stage were collected from Shahr-E-Kord city, Chaharmahal- Va-Bakhtiari province, South-West of Iran in June 2015 and were authenticated and deposited in Agricultural Research Center of Chaharmahal-Va-Bakhtiari province, Iran (Sample no:1376). The samples were grinded and subjected to hydrodistillation by Clevenger type apparatus for 3 h. The separated essential oil was dried and kept in a dark vial at a cold place until the analysis.

Analysis of E. platyloba essential oil by Gas Chromatography (GC) and Gas Chromatography-Mass spectrum (GC-MS)

The chemical composition of E. platyloba essential oil was analyzed using GC and GC-MS. The GC apparatus was equipped with agilent technology (HP) 6890 system, capillary column of HP-1MS (30 m × 0.25 mm, film thickness 0.25 μm). The oven temperature program was initiated at 40°C, held for 1 min then raised up to 230 °C at a rate of 3°C/min held for 10 min. Helium was used as a carrier gas at a flow rate 1.0 ml/min. The detector and injector temperatures were 250 and 230°C, respectively. The GC/MS analysis was conducted on a HP 6890 GC system coupled with 5973 network mass selective detector with a capillary column the same as above, carrier gas helium with flow rate 1 ml/min with a split ratio equal to 1/50. The programmed injector and oven temperature was identical to GC. The compounds of the oil were identified by comparison of their retention indices (RI), mass spectra fragmentation with those on the stored Wiley 275.L, Wiley 7n.1 mass computer library, and NIST (National Institute of Standards and Technology), as well as comparison of the fragmentation pattern of the mass spectra with data published in the literature17.

Microbial strains

The antibacterial activity evaluation was conducted against Helicobacter pylori ATCC 26695, Salmonella enterica BAA-708 and Staphylococcus aureus ATCC 25923. H. pylori, S. enteric and S. aureus were cultured on sheep blood (5%) supplemented brucella agar and Xylose lysine deoxycholate agar and Manitol salt agar, respectively. The microbial strains were incubated at 30-35 ºC in suitable conditions. One or two colonies of each strain were suspended in saline solution and their turbidities were adjusted to 0.5 McFarland by Spectrophotometer instruments (1×108 CFU/ml).

Evaluation the Antibacterial activity of E. platyloba essential oil

The antibacterial activity evaluation of essential oil was evaluated by disc diffusion assay and micro-broth dilution assay. In disc diffusion assay, the inhibition zone diameters (in millimeter) of E. platyloba essential oil were determined by inoculating the above suspended microbial strains on Muller Hinton Agar (Merck) and sheep blood (5%) supplemented Muller hinton Agar by sterile cotton swab. Sterile disks containing 10 μl of essential oil were placed on the inoculated plates. The plates were incubated and then the inhibition zone diameters (mm) were measured and were recorded as means ± standard deviation (SD)18.

For determining the MIC (minimal inhibitory concentration) and MBC (minimal Bactericidal concentration) values of essential oil in micro-broth dilution assay, the essential oil were dissolved in DMSO and then it was diluted in distilled water. 50 μl of each dilution was added into the wells of 96 micro titer plates. The microbial suspensions were diluted to 106 CFU/ml. Then, 50 μl of diluted microbial suspensions were added to each well and were incubated at 37ºC for 24 h.

The first wells with no turbidity and the first well without any growth on solid media were determined as MIC and MBC values, respectively19.

Antioxidant evaluation of E. platyloba essential oil

For evaluating the potency of E. platyloba essential oil against ABTS free radicals: A solution containing ABTS (7 mM) in per-sulfate (2.45 mM) (1:1) was prepared. This solution was kept in dark place for 12-16 h and then was diluted to 1:25. Three ml of diluted solution was added to 40 μl of different concentrations of essential oil. After 15 min, the absorbance of solutions was read at 734 nm, the inhibition percent of essential oil were estimated in this way: I% = [Ablank_ Asample/Ablank]×100, where Ablank was the absorbance of control and Asample is the absorbance of different concentrations of essential oil. Ascorbic acid was used as control20.

Results and discussion

Chemical composition of E. platyloba essential oil

Twenty one components were identified in E. platyloba essential oil that represented 90.6% of total oil composition. β-Ocimene (46.4%), α-phellandrene (12.1%), β-myrcene (8.5%), limonene (6.1%) were the main components of E. platyloba essential oil from ChaharMahal-E-Bakhtiari (South- West of Iran), followed by linalool (3.2%), α-pinene (3.0%), terpinolene (2.5%), p-cymene (1.8%) and δ-3-carene (1.4%). Thymol and carvacrol were not found in this essential oil (Table 1).

Table 1 Chemical composition of E. platyloba essential oil by GC and GC-MS 

Some studies have evaluated the chemical composition of E. platyloba essential oils from different parts of Iran. β-ocimene (28%-68%) were reported as the first main component of E. platyloba essential oils from four studies21-24, while the second main component has been different in these studies, and furanone (6.2%), α-decalactone (8.4%), γ-phellandrene (24.2%), and δ-3- carene (16.2%) were reported as the second main components of essential oil from Golpayegan21, Tehran23, northwest (Maragheh istrict)22, Shalamzar24, Iran; respectively. Asarone (10.2%), anethole (7.4 %), eugenol (6.7%) and dimethyl styrene (6.6 %) were the main components of E. platyloba essential oil from Torbat-Heydariye (Khorasan Razavi Province, Iran)8.

Thymol (27.2%), trans-ocimene (20.9%) and carvacrol (7.2%) were reported as the main components of E. platyloba essential oil from ChaharMahal VA Bakhtiari province, Iran9, while thymol and carvacrol was not found in our essential oil from ChaharMahal VA Bakhtiari province. The precise location of sampling from ChaharMahal VA Bakhtiari province has not en determined in Saei-Dehkordi et al study. Geographical location, harvesting time and many other unknown factors can affect on chemical composition of E. platyloba essential oil.

The first main component of E. platyloba essential oil from our study was according to the essential oils from Golpayegan21, Tehran23, northwest (Maragheh district)22, and Shalamzar24, different geographical regions of Iran, while the second main component of our essential oil was α-phellandrene, and its isomer (γ-phellandrene) were found only in the E. platyloba essential oil from Maragheh district, the northwest of Iran22.

Therefore, four different chemotypes were reported for E. platyloba essential oil until now including 1):β-ocimene, δ-3-carene 2):thymol, trans-ocimene, carvacrol 3):Asarone, anethole, eugenol; 4) β-ocimene, α-caryophyllene.

Antimicrobial activity of E. platyloba essential oil

The antimicrobial activity of E. platyloba essential oil against three bacteria including Gram negative (Salmonella enterica, Helicobacter pylori), Gram positive (Staphylococcus aureus) ones by disc diffusion assay showed the least inhibition zone diameter was for Helicobacter pylori (17 mm), followed by Salmonella enterica (20 mm). The large inhibition zone diameter was for S. aureus (23 mm). 10 μl of E. platyloba essential oil had higher inhibition zone diameter than tetracycline as antibiotic.

The antimicrobial evaluation by micro-broth dilution assay against above bacteria had the MIC values between 0.5-1.1 μl/ml and MFC values between 1.25-2.5 μl/ml, respectively. The sensitive bacteria to E. platyloba essential oil was S. aureus (MIC and MBC values of 0.5 and 1.25 μl/ml), followed by S. enterica (MIC and MBC values of 1 and 2.5 μl/ml). The less sensitive bacteria to E. platyloba essential oil were H. pylori (MIC and MBC values of 1.1 and 5 μl/ml) (Table 2).

Table 2 Antimicrobial activity of E. platyloba essential oil 

Although, there are three studies that evaluate the anti-staphylococcal

activity of E. platyloba essential oil against S. aureus8-10but their chemical compositions were different with our study. The essential oil with thymol, trans-ocimene and carvacrol had the MIC value of 448 μg/ml9 and the essential oil with asarone, anethole and eugenol as main components had the MIC of 500 μg/ml against S. aureus8. MIC value of E. platyloba essential oil with ocimene (26.5%), 2,3-Dim thylcyclohexa-1,3-diene (9.9%), α-pinene (7.7%) against S. aureus was 12500 ppm10. The results of two different studies by others showed the chemical composition of E. platyloba essential oil had no effect on the antibacterial activity of this oil against S. aureus (MIC=500 μg/ml)8,9.

The antibacterial activity of E. platyloba essential oil is related to its main components. Our essential oil was containing α-caryophyllene or α- humulene and β-ocimene as main components showed promised antibacterial activity especially against Gram positive bacteria, S. aureus. The antibacterial activity of α-caryophyllene has been confirmed against S. aureus25. Furthermore, α-caryophyllene showed anti-inflammatory effects26. Therefore, the presence of α-caryophyllene in E. platyloba may donate the anti-inflammatory effect to this essential oil that make it as suitable candidate for inflammatory- infectious diseases or other inflammatory diseases.

Antioxidant activity of E. platyloba essential oil

The antioxidant evaluation showed the IC50 for E. platyloba essential oil and ascorbic acid were 32 and 20 ppm, respectively. In fact, the antioxidant activity of E. platyloba essential oil was a little lower than ascorbic acid as control (Fig 1).

Figura 1 The antioxidant evaluation of E. platyloba essential oil by ABTS free radicals 

Today, the free radicals threaten the health, and many scientists persuade the people to use the natural antioxidants27,28. Furthermore, the use of chemical synthetic antioxidants in different industries has been limited due to their adverse effects on human health29. E. platyloba essential oil with (Z)-β-Ocimene (26.7%), δ -3-carene (16.2%) and limonene (6.6%) as the main components showed the IC20 of 1.1 mg/ml in DPPH system11. The oil with thymol (27.2%), trans-ocimene (20.9%) and carvacrol (7.2%) had the IC50 of 50 μg/ml9.

According to the results, the geographic region seems to be important in the composition of essential oils. As our results and other studies have been shown, the IC50 of this essential oil can change and the lower IC50 or high antioxidant activity is related to the E. platyloba essential oil with higher phenolic compound such as thymol, carvacrol (thymol, transocimene and carvacrol chemotype), followed by β-ocimene and α-caryophyllene chemotype (0.3 mg/ml) and then E. platyloba essential oil with (Z)-β-Ocimene, δ-3-carene and limonene (IC20 1.1 mg/ml) (Table 3).

Table 3 The comparison between the results of this study with other study on of E. platyloba essential oil 

Therefore, E. platyloba essential oil can be used as natural antioxidant in preserving the products and humans from deterioration of free radicals.

Conclusion

E. platyloba essential oil of our study had β-ocimene and α-caryophyllene as the main components; furthermore, three chemotypes were reported for E. platyloba essential oil including 1):β-ocimene, δ -3-carene 2):thymol, trans-ocimene, carvacrol 3):Asarone, anethole, eugenol; 4) β-ocimene, α-caryophyllene. In β-ocimene chemotype, the second main component was different in many studies but chemical composition of our study was according to the report of Hassanpouraghdam and his colleagues with different eographical origin22. E. platyloba essential oil with Z- β-ocimene and α-caryophyllene showed promised antibacterial activity against Gram positive bacteria (S. aureus), followed by Salmonella enterica. The less sensitive bacteria were anaerobe Gram negative bacteria, Helicobacter pylori. Our hemotype showed the low antioxidant activity than thymol, trans-ocimene, and carvacrol chemotype but had higher antioxidant activity than that of E. platyloba essential oil with (Z)-β-Ocimene, δ-3-carene and limonene.

Acknowledgements

This study was supported by Shahr-E-kord Branch, Islamic Azad University, Shahrekord, Iran.

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Recibido: 08 de Octubre de 2016; Aprobado: 03 de Febrero de 2017

Correo electrónico: mahboubi@barijessence.com mahboubi1357@yahoo.com * Autor para correspondencia. Department of Microbiology, Medicinal Plant, Research Center of Barij, Kashan, Iran. TeleFax: 098 8644465112 Correo electrónico: mahboubi@barijessence.com mahboubi1357@yahoo.com

Conflict of interests The authors have no conflict of interests to declare

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