Chemical composition and antimicrobial activities of essential oil and ethanol extract of Cyperus fuscus L burs from Turkey

Purpose: To investigate the chemical composition of the essential oil of Cyperus fuscus burs as well as the antimicrobial activities of the ethanol extracts and essential oil. Methods: Fresh burs roots of C. fuscus were collected at the flowering stage in an open area in Güdül (Ankara, Turkey). Preparation of the ethanol extract, hydrodistillation of the essential oil, GC-FID-MS analysis, and agar diffusion and MIC agar dilution assays were performed to determine various parameters for the oil and extracts. Results: The major compounds of the essential oil were dehydroaromadendrene (10.7 %), azulenone (8.5 %), α-selinene (7.5 %), α-ylangene (6.0 %) and β-caryophyllene (5.6 %). The essential oil of Cyperus fuscus exhibited activity against Gram-negative bacteria with minimum ınhibitory concentration (MIC) values ranging from 1000 to 31.25 μl/mL. Similarly, the ethanol extract of the burs showed good antimicrobial activity with the MIC of the ethanol extracts on ranging from 1000 μg/mL (Escherichia coli) to 250 μg/mL (Pseudomonas. Aeruginosa, p < 0.05). However, the ethanol extract was inactive against yeast strains. Conclusion: Thus, the essential oil and ethanol extract of the studied plant can potentially be used as antimicrobial agent.


INTRODUCTION
For years, the plants have been utilized by many cultures as flavoring agents and as natural preservatives in food and medical therapeutic agents [1].Some Cyperus species has health benefits and nutritional value.In recent years, they are used as functional foods since they possess high amount of oleic acid, glucose, phosphorus, potassium, vitamins C and E. These burs are also used as a food additive in the preparation of oil for cooking, salad preparation, production of caramel, milk and a refreshing drink [2,3].However, scientific interest on these natural plants have increased especially for the protection of food against antimicrobials.For this reason, the people demand natural foods long shelf-life.These prolong both the long shelf-life of food and prevent new foodborne disease outbreaks caused by pathogenic microorganisms resistant to antibiotics.Essential oil obtained from several plants has antimicrobial [4] as well as antifungal activity [5,6].
The genus Cyperus members has been used in pain, fever, diarrhoea, dysentery and other intestinal problems [7].Characterized by the presence of quinines [8], flavonoids [9,10] and sesquiterpenes [11].The chemical composition of essential oils of different Cyperus species have been reported, the major compounds identified in oil samples are mostly monoterpenes and sesquiterpenes [12][13][14].
There are some studies on the chemical composition of the essential oil of Cyperus species.Essential oils of some Syperus species including C. rotundus [13,15], C. difformis L. and C. arenarius [12] were studied regarding their chemical composition.In addition, chemical compositions of the three Egyptian Cyperus species; C. articulatus, C. esculentus and C. papyrus were showed by comparison [16].In this study we showed the chemical composition of essential oil of C. fuscus and antimicrobial activity of ethanol extracts in the first time.
The chemical composition of the essential oil derived from the burs of Cyperus fuscus in Turkey as well as antimicrobial activities of this oil and ethanolic extract of this plant against human pathogenic bacteria were studied in this work.

EXPERIMENTAL Collection of plant material
The fresh burs of C. fuscus were collected at the flowering stage from, 2009-2010 in July by Erdem, ( 1025) in open area on sandy loam, mudflats found in Yeşilöz Village-Kirmir valley, stream side, 750 m in Güdül (Ankara).The current study area is within the A4 square in the grid-square system adopted for the Flora of Turkey [17,18].The research area lies between latitude N 40°14ʹ 11.09ʺ and longitude E 32° 15ʹ 44.4ʺ.
Plant tissues were pulverized into fine powder.The plant materials were collected and positioned in plastic bags and transported to the laboratory.Voucher specimen was deposited in the Herbarium of Ahi Evran University, Kırşehir-Turkey (Ahi Herb.No 1025).

Preparation of ethanol extract
The harvested plant bruses were dried in shadow, pulverized and mixed with 50 % ethanol in solvents using a Soxhlet apparatus and extracted for 72 hours.The solution then filtered through a Whatman no. 1 filter paper and the solvent were evaporated.Finally, the crude forms of the crystals were obtained and stored in a refrigerator until further use.The ethanol extract was diluted to concentrations ranging from 250 to 1000 μg/ml, with DMSO [19].

Extraction of essential oil
Hydro-distillation method was used to obtain the essential oil using a modified Clevenger apparatus.A total of 100 g burs parts of fresh plant material were used.The resulting essential oil (EO) was dried over anhydrous sodium sulphate prior to filtration, and kept at 4°C until the analysis.

Gas chromatography-mass spectometry (GC-FID/GC-MS) analysis
GC-FID / MS analysis of the essential oils were done in Plant Products and Biotechnology Research Laboratory (BUBAL), Firat University, by means of Hewlett Packard-Agilent 5973N GC-MS system with 6890 GC equipped with a flame ionization detector (FID).The study was performed by simultaneous injection in the same instrument.HP-5 MS column (30 m×0.25 mm i.d., film thickness (0.25 μm)) was used in both analysis with helium as the carrier gas in GC-MS.The injection volume was 1.0 μL of diluted solution (1/100) of oil in n-hexane.The temperature of the injector was 250°C, and the flow rate was 1.3 mL/min.(splitless mode).The GC oven temperature was preserved at 70°C for 2 min and automated to 150°C at a rate of 10 °C/min and then kept constant at 150°C for 15 min to 240°C at a rate of 5°C/min.A series of nalkanes were used as reference points in the calculation of retention indices (RI).MS were occupied at 70 eV and a mass range of 35-425.The determination of the compounds was based on comparison of their retention indices (RI), and mass spectra with those acquired from authentic Wiley (7 th version) and Nist 98 libraries.

Test microorganisms
The ethanol extract was tested against 11 microorganisms; L. monocytogenes (

Agar diffusion assay
The agar diffusion test method applied by Aneja and Joshi [20] was used to observe the antimicrobial activity of both oil and ethanolic extract.The microbial suspension were swabbed and spread on nutrient agar.The plates were cultured in 0.1 mL (10 5 -10 6 cells/mL) and subsequently 8 mm wide wells were bored within these agar plates using a sterile cork borer.The each wells were aseptically filled with 100 μL of ethanolic extract (250, 500 and 1000 μg/mL) and 20 μL of essential oil was placed on the inoculated agar.The plates were incubated overnight at 37 o C for bacteria and at 20-22 o C for 5 days for yeast cultures.Microbial growth was determined by measuring the diameter of zone of inhibition.For each strain, a negative control was maintained where DMSO was used in place of extract.In order to determine the sensitivity of gram negative and gram positive bacteria, Gentamycin (20 g/disc) was chosen as standard, while Nystatin (50 g/disc) was selected as Standard for yeasts (Table 2).Each experiment was carried out three times and mean values are presented.

Minimum ınhibitory concentration (MIC) assay
Tube dilution method was used to determine MIC of the extracts.The essential oil and extract of Cyperus fuscus were dissolved in 10 % dimethylsulfoxide (DMSO).The highest concentration (1000 μg/mL) was prepared at the beginning.Then serial two-fold dilutions were prepared resulting the concentrations ranging from 31.25 to 1000 μg/mL in 15 mL sterile test tubes containing nutrient broth and 100 µL of the bacterial suspension containing 10 8 CFU/mL of respective test organisms.The tubes were incubated at 37 ºC in an incubator for 24 h for bacteria and 22 o C for 48 h for yeast.A tube containing nutrient broth without extract was used as control.The least extract concentration inhibiting the growth of the test organisms was considered as MIC (Table 2).

Statistical analysis
A completely randomized experimental design was used with three replications in factorial arrangements.One-way analysis of variance was also used.Tukey HSD multiple comparison tests were used to find out which group originated the difference between the groups.The normality assumption in the analyses was tested by Kolmogorov-Smirnov test.Statistical analyses were performed using SPSS (Version 20.0, SPSS Inc, USA) statistical package program.In the analyses, significance level was set at p < 0.05.

Chemical composition of essential oil
The yield of essential oil belonging to the aerial parts of C. fuscus was obtained as 0.4 % (v/w) yield.The composition of essential oil is presented in Table 1.It is determined that seventy three compounds comprised the 96.4 % of the oil.The major compounds in essential oil studied were dehydroaromadendrene (10.7 %), azulenone (8.5 %), a-selinene (7.5 %), aylangene (6.0 %) and b-caryophyllene (5.6 %) respectively.Many other compounds in minor amounts were also determined.Complex mixtures of monoterpenes and sesquiterpenes were observed in the oil.Sesquiterpenes in C. fuscus oil were found as the main class of terpenoids.The compounds, a-gurjunene, spathulenol, azulene, b-caryophyllene, aselinene, a-ylangene, were comprised the high percentage of sesquiterpenes in the essential oil.Monoterpenes were comprised a little content of the essential oil (Table 1).

Antimicrobial activity
Antimicrobial activities of C. fuscus essential oil are presented in Table 2.This study clearly indicated that C. fuscus essential oil has showed different activities on the bacteria and yeast tested.About the tested bacteria, the oil could not inhibit the growth of some bacteria such as S. typhimurium, E. aerogenes, A. hydrophila, K. pneumoniae, L. monocytogenes and E. feacalis.On the other hand, ethanolic extract the burs of C. fuscus showed antimicrobial activity on 4 bacteria (Table 2).
For E. coli, ethanol extract of C. fuscus exhibited the strongest antibacterial activity (18 mm), followed by A. hydrophila (17 mm).For K. pneumoniae and P. aeruginosa, ethanol extract of C. fuscus exhibited high antibacterial activity (15 mm), while the other bacteria and yeasts showed no activity.The MIC of the ethanol extracts showed that MIC ranged between 1000 and 250 µg/mL.The results showed inhibition of growth of some of the tested microorganisms with various degrees.E. coli (1000 µg/mL), A. hydrophila (500 µg/mL), K. pneumoniae (250 µg/mL) and P. aeruginosa (250 µg/mL) was susceptible whereas other bacteria resistant against ethanol extract.Ethanol extract was not exhibited antifungal activity against C. albicans and C. glabrata strains (Table 2).
Several studies on the antimicrobial activity of essential oils or extracts of other Cyperus species have been reported.It is clear from the previous studies that many species of Cyperus possess high antimicrobial activities [14,23,24].Chemical composition and antimicrobial activity of the Cyperus species were also reported by many researchers.The chemical composition of essential oil and antimicrobial activity of C. leavigatus [14], C. rotundus [24,25], C. esculentus [26] were also studied.It is observed that C. esculentus and C. rotundus have possessed medicinal properties which have been harnessed by traditional medicine practitioners, but only a few of these properties have been proven scientifically [24,26].
The lowest MIC value (250 µg/mL) observed with the ethanol extract on K. pneumoniae and P. aeruginosa.The ethanol extract of C. fuscus has shown the most antibacterial activity against E. coli (Table 2) and this probably supports the reported use of the plant by traditional medicine practitioners in treating diarrhoea and other stomach troubles.
Nowadays, some people are not aware of the use C. fuscus treating stomach or diarrhoea.This plant may be a potential source of a new type of antibiotic with the enlarged studies.Isolating and purifying the bioactive compounds may lead to the development of suitable antibiotic against E. coli, A. hydrophila, K. pneumoniae and P. aeruginosa.As a result it is said that the antimicrobial activity of the ethanolic extract of the C. fuscus were high than the essential oil.In general, it is possible to say that both extracts of the C. fuscus have antimicrobial activities on some bacteria (Table 2).

Table 1 :
Constituents of the essential oil from C. fuscus*

Table 2 :
Antimicrobial activities of ethanol extract and essential oil of C. fuscus burs against microorganisms (MIC and DD (diameter of the inhibition zones))