Essential oil composition and antibacterial activities of Gypsophila species

Essential oil composition of Gypsophila turcica Hamzaoğlu, Gypsophila pinifolia Boiss. & Hausskn., G. tuberculosa Hub.-Mor., G. eriocalyx Boiss. and G. laricina Schreb. were analyzed by means of gas chromatographymass spectrometry (GC-MS). Thirty six, fourty four, sixty six, forty one and sixty one compounds were identified in the essential oils of G. turcica, G. pinifolia, G. laricina, G. tuberculosa and G. eriocalyx respectively. The major components were determined hentriacontane (12.93 ± 0.4%), 1-octadecanol (8.97 ± 0.1%), hexahydrofarnesyl acetone (6.9 ± 0.09%) and pentacosane (6.63 ± 0.08%) in G. turcica oil, hexadecanoic acid (17.6 ± 0.4%), 1-tetradecanol (7.6 ± 0.1%) and phytol (5.63 ± 0.05%) in G. pinifolia oil, octacosane (6.83%), eicosanal (6.19%), triacontane (6.03%) and heneicosane (5.78%) for G. eriocalyx, hexadecanoic acid (25.3%, 27.0%) and hentriacontane (13.0%, 12.6%) for G. tuberculosa and G. laricina, respectively. Antibacterial activity of G. eriocalyx, G. tuberculosis and G. laricina were investigated against Gram negative (Escherichia coli) and Gram positive (Staphylococcus aureus) bacteria. ARTICLE HISTORY Received: September 09, 2018 Revised: November 30, 2018 Accepted: January 09, 2019


INTRODUCTION
Gypsophila is the 3 th biggest genus in family of Caryophyllaceae to Turkey.Gypsophila species are annual, biennial or perennial herbaceous plants.This genus are distributed mainly in Mediterranean and Iran-Turan areas in Turkey.Gypsophila has 56 species in 10 sections and 33 species are endemic to Turkey [1].By this way, it has made a significant contribution to the biodiversity of Turkey [2].Gypsophila turcica is perennial plant and it was described as a new species in 2012 [3].
Gypsophila species are rich source of triterpene saponin especially in root parts [4,5].Triterpene saponin from this genus are used commercially as medicines, detergent, adjuvants and cosmetics [5,6].Root and barks of the genus used as analgesic, sedative, antipyretic, antiinflammatory, emetic and insecticidal in Turkey [7].Biological activities of the genus seem to be associated with triterpene saponin.Due to the various beneficial biological activities, Gypsophila was the focus of studies that described the phytochemistry of the genus extensively.
As summarized above Gypsophila species have very high medicinal and commercial importance and also contains interesting natural substances.However, according to our literature survey we have not encountered any reports on the essential oil composition of Gypsophila species from Turkey.Additionaly, there is no report on antibacterial activity of essential oils of G. eriocalyx, G. laricina and G. tuberculosa.This prompted us to investigate the essential oil composition and antibacterial activity of Gypsophila genus.To the best of our knowledge this is the first report on the essential oil composition and antibacterial activity of Gypsophila genus.

Plant Materials
Plant materials were collected during the flowering period;

Isolation of the Essential Oils
Aerial parts of the air dried plants subjected to hydrodistillation for 3 h, using a Clevenger-type apparatus to produce essential oils.Condenser of the Clevenger was attached to a microchiller that set to 4°C.Essential oil yields obtained from G. pinifolia, G. turcica, G. laricina, G. eriocalyx and G. tuberculosa 0.03;0.01;0.01;0.01;0.03%(v/w), respectively.The oils were recovered with 1 mL n-hexane and preserved in amber vials under -20°C until the day they were analyzed.

Gas Chromatography/Mass Spectrometry Analysis
The GC-MS analysis was performed with an Agilent 5975C Inert XL EI/CI MSD system operating in EI mode.Essential oil of G. pinifolia and G. turcica were diluted 1/65 and 1/100 (v/v) with n-hexane, respectively.Injector and MS transfer line temperatures were set at 250˚C.Innowax FSC column (60 m (x) 0.25 mm, 0.25 µm film thickness) and helium as carrier gas (1 mL/min) were used in both GC/MS analyses.Splitless injection was employed.Oven temperature was programmed to 60˚C for 10 min.and raised to 220˚C at rate of 4˚C/min.Temperature kept constant at 220˚C for 10 min.and then raised to 240˚C at a rate of 1˚C/min.Mass spectra were recorded at 70 eV with the mass range m/z 35 to 425.

Gas Chromatography Analysis
The GC analyses were done with an Agilent 6890N GC system.FID detector temperature was set to 300ºC and same operational conditions applied to a duplicate of the same column used in GC-MS analyses.Simultaneous auto injection was done to obtain the same retention times.Relative percentage amounts of the separated compounds were calculated from integration of the peaks in FID chromatograms.Identification of essential oil components were carried out by comparison of their relative retention indices (RRI) obtained by series of nalkanes (C5 to C30) to the literature and with mass spectra comparison [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27].Mass spectra comparison was done by computer matching with commercial Wiley 8th Ed./NIST 05 Mass Spectra library, Adams Essential Oil Mass Spectral Library and Pallisade 600K Complete Mass Spectra Library.The analysis was carried out in triplicate and the results were given as the mean ± standard deviation.

Antibacterial Assay
Antibacterial activities of the essential oils were tested against two strains; Gram positive Staphylococcus aureus (ATCC 25923) and Gram negative Escherichia coli (ATCC 25922).For the antimicrobial tests, Luria-Bertani broth was used as a growth medium for bacteria.
In order to evaluate antibacterial activity, minimum inhibition concentration (MIC50) values were determined by using broth dilution method.DMSO was used in stock solutions to enhance solubility of the essential oils.Serial dilutions of the stock solutions were prepared on a 96 well plate.After incubation at 37°C for 24 h, bacterial suspension concentrations were standardized to McFarland No: 0.5.Essential oils and bacterial cultures were mixed in the range of 1000-1,95 µg/mL as final concentration.It was paid attention to not exceed 1% final concentration for DMSO.After treatment, the bacteria were incubated at 37°C for 24 h.As negative control, essential oil-free solutions were used.Each test was repeated for three times.Growth analysis was done by using spectrophotometric measurements for MIC determination.Minimum inhibitory concentrations (MIC50) were detected as the minimum concentration at which at least 50% of bacterial growth was missing.
Antibacterial of the oils were evaluated for one Gram (+) and one Gram (-) bacteria by using a broth microdilution assay.G. eriocalyx essential oil showed mild activity on S. aureus (250 µg/mL) but the oil showed very low activity against E. coli (1000 µg/mL).However, G. tuberculosa and G. laricina essential oils did not show any significant activity against tested grains.The results of antibacterial activity of Gypsophila species are given in Table 2.
The essential oil of G. pinifolia, G. tuberculosa and G. laricina had hexadecanoic acid in high amount unlike G. turcica and G. eriocalyx.Essential oils of G. turcica, G. tuberculosa and G. laricina were rich in hentriacontane.But hentriacontane contained at low amount in G. eriocalyx and not detected in G. pinifolia.  RRI Lit.: Relative retention time given in the literature for the compound in similar columns and analysis conditions. 3 The results of the analysis. 4Identification method: RI: identification based on the relative retention times (RRI) of genuine compounds on the HP Innowax column and the literature data; MS: identification based on MS comparison with the database or the literature data, Ac: Identification is done according to RRI and MS values of the authentic compounds.

DISCUSSION and CONCLUSION
Only mild antibacterial activity is observed on G. eriocalyx essential oil against S. aureus.The main compounds of essential oil of G. eriocalyx contained low amount or not detected in other Gypsophila species.Eicosanal is one of the main compound of G. eriocalyx.Antibacterial activity could be correlated with this compound.According to a study from Iran, Gypsophila bicolor was reported to contain germacrene-D, p-cymene, bicyclogermacrene, γdodecadienolactone, terpinolene, cis-β-ocimene and trans-β-ocimene [8] however these compounds were not detected in the G. turcica, G. pinifolia, G. eriocalyx, G. tuberculosa and G. laricina.These differences in the previous literature and present data could be related to different collection times, climatic and soil conditions, ecological factors, methods and instruments employed in analysis or different genotypes.There are very few reports on the essential oil of Gypsophila species therefore it is difficult to produce a comment on the chemosystematic position of this species according to current findings and the existing reports.We believe the results obtained from this research will stimulate further research on the chemistry of Gypsophila species.

Table 1 .
The essential oil composition of five Gypsophila species