Fatty acids, bioactive substances, antioxidant and antimicrobial activity of Ankyropetalum spp., a novel source of nervonic acid;Ácidos grasos, bioactivos y actividades antioxidante y antimicrobiana de Ankyropetalum spp., una nueva fuente de ácido nervónico

Ácidos grasos, bioactivos y actividades antioxidante y antimicrobiana de Ankyropetalum spp., una nueva fuente de ácido nervónico. Los extractos de Ankyropetalum se obtuvieron usando dos medios de extracción diferentes (Soxhlet y baño ultrasónico). A estos extractos se les determinó el contenido fenólico, flavonoides, DPPH, FRAP, y la actividad antimicrobiana. Además, se determinó la composición de ácidos grasos mediante GC-MS. Se encontraron valores altos de contenido fenólico y flavonoide total en A. reuteri y A. gypsophiloides, respectivamente. Los valores de DPPH y FRAP fueron altos en A. arsusianum y A. gypsophiloides, respectivamente. Se obtuvieron mejores resultados utilizando metanol como disolvente y Soxhlet como extractor. Los resultados mostraron que los extractos parecen ser razonablemente efectivos contra los organismos ensayados, incluidos los aislados clínicos. Los resultados más prometedores se obtuvieron con todos los extractos USB de especies contra la Cándida parapsilosis. Es notable que los niveles de ácido nervónico en A. arsusianum y A. reuteri alcanzaron el 40%. A diferencia de otras fuentes de ácido nervónico en el mundo, la ausencia de ácido erúcico en el aceite vegetal aumenta el valor de estas plantas.


INTRODUCTION
Ankyropetalum Fenzl is a small genus which includes only three species in the world. It belongs to the Caryophyllaceae family with 32 genera and 494 species in Turkey (Davis, 1982). The species are A. arsusianum Kotschy ex Boiss., A. reuteri Boiss. and Hausskn. and A. gypsophiloides Fenzl (Barkoudah, 1962). The gene center of the genus is Turkey, and the taxa of this genus are spread mainly throughout South-Eastern Anatolia and partially in the Mediterranean region (Ozcelik and Muca, 2010). While A. reuteri is endemic and belongs to the EN category, the other species are rare and found in South-east Anatolia and within the borders of neighboring countries (Ekim et al., 2000;Korkmaz and Ozcelik, 2011). The Ankyropetalum, Gypsophila and Saponaria members are known as "Çöven Otu" in Turkey. It is generally difficult to distinguish the Ankyropetalum from the perennial Gypsophila species. Ankyropetalum and Gypsophyla are used for similar purposes. A. gypsophiloides has been used in the preparation of a local food and A. reuteri has been mixed with straw and used as animal feed (Ozcelik and Muca, 2010). They are also used for the production of tahini halvah, foam halvah, Turkish delight, herbed cheese, çöven bread, and the manufacturing of chemical cleaner, fire extinguisher manufacturing, liquor making, and soap making (Korkmaz and Ozcelik, 2011).
Until the development of synthetic medicines from prehistoric times, plants were the basis of almost all medical treatments (Djeridane et al., 2006). Because of the less harmful effect than its synthetic counterparts, there is still interest in traditional herbal products today (Mocan et al., 2018). Herbal products contain biologically highly active compounds such as phenolic compounds, flavonoids, flavonols, and tocopherols, which play an important role in human nutrition and health (Mocan et al., 2018). It has been reported that there is an inverse relationship between the formation of human diseases and the consumption of various antioxidant plants (Dudonne et al., 2009). Therefore, research on the identification of antioxidative compounds is an important issue. Due to potential health risks and toxicity, there has also been an increased interest in the use of natural antioxidants in foodstuffs or medical materials in recent times (Djeridane et al., 2006). Fatty acids play an essential role in many body functions (Elias, 1983). Nervonic acid, one of the critical fatty acids, has been suggested to help maintain brain health, increase brain function, reduce fatigue, and accumulate less fat in the blood (Mohanty et al., 2013). There is a tendency for the use of nervonic acid to be added to foods for the treatment of neurological diseases such as Alzheimer's, multiple sclerosis, adrenoleukodystrophy and people with Zellweger's syndrome (Tang et al., 2013). It is also used as a medication in the symptomatic treatment of patients with schizophrenia, psychosis and attention deficit (Krishnan, 2009).
Since these species are endemic or rare and furthermore, they are not cultured, they are almost at the point of extinction. There are a limited number of studies on the taxonomy, ecology and economic importance of Ankyropetalum. Ankyropetalum is an economic plant with its commercial potential due to its addition to food. Despite its medicinal and economic value, there is no published biochemical or bioactivity study on this plant. Therefore, in this study, the total phenolic and flavonoid contents, antioxidant and antimicrobial activity of A. arsusianum, A. reuteri and A. gypsophiloides were investigated to find new potential sources of natural antioxidants. On the other hand, according to a GC-MS analysis, plant oils were found to be a novel source of nervonic acid.

Plant materials
Three species of Ankyropetalum Fenzl were used in this study. The samples were collected from the natural habitats around Kahramanmaraş, Hatay and Gaziantep in the summer of 2015 (Table 1). The plants were identified according to the Flora of Turkey (Davis, 1982

Sample preparation and extraction
The plants were dried in the shade for about a week and then pulverized by grinding in a laboratory blender. Methanol and ethanol were used in two different extraction methods. Total phenolics, total flavonoids, antioxidant activity and antimicrobial activity were determined in these obtained extracts.

Extraction method (SOX method)
The extraction was carried out using a Soxhlet apparatus at 60 °C for 6 hours with the addition of solvent (100 ml) to 10 g of plant material. After removal of the solvent in a vacuum rotary evaporator at 40 °C, the extract was kept at -20 °C for further analysis.

Extraction method (USB method)
Solvent (100 ml) was added to 10 g of plant and extraction was carried out in the Ultrasonic Water Bath for 1 hour at room temperature. The samples were centrifuged for 15 min at 3500 rpm. After centrifuging, the eluted liquid fraction was collected in another tube and the plant sample was extracted again as described above. The extracts were combined and the solvent was removed in a vacuum rotary evaporator at 40 °C and the extract was kept at -20 °C for further analysis.

Determination of total phenolic and flavonoid content
Folin-Ciocalteau colorimetric method was used to determine the total phenolic contents of the fractions (Blainski et al., 2013). Flavonoid compounds were extracted in the ultrasonic bath using 50 ml of 80% methanol:water (v/v) with 0.5 g of powdered plant samples for 20 min. Samples were centrifuged at 14000 rpm for 5 min. The total flavonoid content of the extracts was evaluated using spectrophotometry (Chang et al., 2002). All extracts were tested in triplicate to confirm the reproducibility of the results.

DPPH and FRAP analysis
Scavenging free radical potentials were analyzed using 1,1-Diphenyl-2-picrylhydrazyl (DPPH) (Brand-Williams et al., 1995). Ascorbic acid was used as positive control. The antioxidant activity was expressed as IC 50, which denotes the concentration of sample required to scavenge 50% of the DPPH free radicals. The FRAP (The Ferric reducing antioxidant power) analysis was made according to Benzie and Strain (1996). Plant extracts (50 µl) were transferred to 2 ml eppendorf tubes and 600 µl FRAP agent were added. Absorbance was measured at 593 nm. The results were calculated as µmol ascorbic acid equivalent/g dry plant weight using the ascorbic acid (10-1000 µmol⋅l -1 ) calibration chart. Results were given in µmol/g dry plant weight. All extracts were tested in triplicate to confirm the reproducibility of the results.

Microorganisms and culturing
The bacteria and yeast were obtained from the culture collection of the

Antimicrobial activity assay
The antimicrobial activity of the ethanol and methanol extracts of A. arsusianum, A. gypsophiloides and A. reuteri were determined by the well diffusion method (Collins et al., 1989). Mueller Hinton Agar (Difco) and Sabouraud Dextrose Agar (SDA) (Oxoid) plates were inoculated with a standardized inoculum, giving 1.5x10 8 bacteria and 2.1 x 10 3 cfu ml -1 yeast (Collins et al., 1989). The wells (6 mm) were prepared with a cork borer and filled with 50 µl of extract (20 mg·ml) dissolved in dimethyl Fatty acids, bioactive substances, antioxidant and antimicrobial activity of Ankyropetalum spp., a novel source of nervonic acid • 3 sulfoxide (DMSO). The plates inoculated with bacteria and yeast were then incubated at 37 and 30 °C for 24 and 48 hours, respectively. The inhibition zones produced were measured and the presence of antimicrobial substances was evaluated after an incubation period.

MIC determination
Minimum inhibition concentrations (MIC) of the extracts were determined according to the micro dilution method (Collins et al., 1989) in culture broth media. The extracts presented an inhibition zone in the well-diffusion method dissolved in DMSO and mixed with the Mueller Hinton and Sabouraud dextrose broth in a designed volume. Later, a dilution series was accomplished in micro well plates. As a control, culture medium and DMSO were set as growth control as well as test dilution for sterility control. After inoculation of the test well, 5 µl of organisms were changed, and the plates were incubated for 24/48 hours. The results were stated as mg·ml -1 .

Determination of fatty acid content
The fatty acid content was analysed in a Shimadzu 2025 gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector (FID) and a column TR-CN100, 60 m × 0.25 mm × 0.20 mm. He was used as carrier gas at a flow rate of 1.5 ml·min -1 . Initial column oven temperature was 80 °C for 2 min, then elevated to 140 °C at a rate of 5 °C⋅min -1 (maintained for 2 min at 140 °C), and then elevated to 240 °C at 3 °C·min -1 (maintained for 5 min at 240 °C). The detector and injection temperatures were programmed to 250 and 240 °C, respectively. Peak areas were used to calculate the relative percentage of the fatty acids as total fatty acid.

Statistical analysis
The statistical analysis appropriate for the entirely randomized (3x2x2) x3 factorial design with three replicates was performed. The hypotheses for the means of the primary and interaction effects were tested using the analysis of variance (ANOVA). Since factors of "solvent" and "extractor" have two levels, the direct use of F test is adequate for the related comparisons. If the results of the F test were significant, the means were determined to be statistically different. In addition, for the factors with more than two levels, comparisons of means were made by Tukey's test at the 0.05 significance level (Efe et al., 2000).

Antioxidant assay
Oxidative stress is thought to cause the development and progress of diseases as well as ageing. Many phenolic compounds with biologically essential effects are considered to be the most abundant antioxidants in foods (Mocan et al., 2018;Abeywickrama et al., 2016;Locatelli et al., 2017). Since different radicals and oxidants have different mechanisms of antioxidant response, there is no single way to measure antioxidant capacity (Isık et al., 2015). Therefore, in this study, different solvents and extractors were applied to compare the total phenolic and flavonoid contents of Ankyropetalum species by using Folin-Ciocâlteu and AlCl 3 assays and the results are given in Table 2.
When means of the species and two-way interactions of species x extractor and species x solvent were examined, the highest total of phenolic substances was observed for A. reuteri extract (52.75, 48.04, 48.51 mg GAE⋅g -1 , respectively). Two-way interactions of solvent x indicated the statistical superiority of methanolic extracts over ethanolic extracts in all types. On the other hand, interactions of the x extractor showed that soxhlet was statistically superior to USB in all types (Table 3).
Regarding flavonoid content, A. gypsophiloides shows superiority over other species. As in the total phenolic content, flavonoid content was also found to be poorer than A. arsusianum in comparison to the other species. Soxhlet and methanol were found to be more effective in eliciting the flavonoid contents in the species. Other researchers working with different species of Caryophyllaceae have also reported that methanolic extracts have higher phenolic and flavonoid contents than other solvents (Nikolova et al., 2011;Chima et al., 2014).
The lower the IC 50 in DPPH analysis, the better the free radicals can be scavenged and thus impair the free radical chain reaction (Lim et al., 2007). Antioxidant activities were determined by DPPH and FRAP tests in this study and the results are presented in Table 1. The extracts of A. arsusianum and A. gypsophiloides were more active against DPPH free radical than A. reuteri.
Methanolic extracts were more active than ethanolic extracts; although the extractors did not significantly affect DPPH activity. FRAP results were also found to be similar to DPPH. A. reuteri also showed lower activity than the other two species. Methanolic extracts were more active in all three species. However, unlike DPPH, it was statistically significant that the extracts obtained from the soxhlet were more active than USB extracts.
The phenolic compounds and flavonoids obtained from plants were shown to have abundant antioxidant activity in food products (Van Acker et al., 1996). In general, extracts with high radical scavenging activity had a high phenolic content. However, there was no significant relationship between total phenolic and flavonoid contents and antioxidant activity in this study. Phenolic content was highest in A. reuterii, whereas antioxidant activity was found to be lower than the other two species in both DPPH and FRAP tests. A similar situation was seen in total flavonoid content. According to the results from the DPPH and FRAP tests, A. arsusianum and A. gypsophiloides species had high activity; while A. gypsophiloides alone had superior flavonoid content. This lack of relationship is also present in different studies. For example, Arslan et al., (2013) studied three species of Gypsophila (G. arrostii, G. pilulifera, G. simonii) from the same family and obtained the highest total phenolic content in G. simonii (15.15 mg⋅g -1 ). According to the results from the ABTS and DPPH analyses, G. pilulifera had a stronger antioxidant activity compared to the other two species. A similar result was reported by Stankovic et al., (2015). According to their studies, while the phenolic contents in Hordeum hystrix and Puccinella limosa were found to be low, these plants were shown to have the highest antioxidant activity. On the other hand, the total phenolic content, IC 50 value and FRAP value of G. pilulifera extracts were 6.5 mg⋅g -1 , 4.56 mg·ml -1 and 23.5 µg⋅g -1 , respectively (Yazici and Ozmen, 2017). These values were considerably lower than the values obtained in this study.
There may be several reasons why A. reuterii had lower antioxidant potency than the other two species despite its high phenol content and relatively high flavonoid content. The reaction of DPPH or FRAP may have been reversed with some phenols (Percentage of disappearance in antioxidant activity), or the reaction between DPPH or FRAP and substrate molecules may have been slow (Lim et al., 2007;Huang et al., 2000).

Antimicrobial assay
The inhibitory activity of plant extracts was assayed against seven bacteria and four yeasts. The results showed that both solvent extracts of A. arsusianum, A. gypsophiloides and A. reuteri had substantial inhibitory activity against all the Gram-positive bacteria tested (Table 4). As E. coli, a member of gram-negative bacteria, was inhibited with all extracts, Enterobacter cloaca was the only one inhibited with the A. reuteri extract. However, K. pneumonia was not affected by any of the extracts. According to the general opinion, Gram-negative bacteria are already more resistant than Gram-positive organisms (Stickler and King, 1992). Among the 4 yeast strains, Candida parapsilosis was inhibited by three plant extracts, while C. albicans was inhibited only by A. gypsophiloides with the extract. On the contrary, C. glabrata and Saccharomyces cerevisiae were not affected. It could be an essential property having an inhibitory and noninhibitory activity against pathogenic and nonpathogenic strains, respectively, for food and pharmaceuticals. Concerning extraction method, although neither soxhlet and USB nor methanol and ethanol were found superior to each other, the most promising results were obtained from the activity of A.arsusianum, A.gypsophiloides and A.reuteri USB extracts against C. parapsilosis (MIC: 0.781mg·ml -1 ).
Around the world, numerous plants have been screened by many researchers with different methods against different microorganisms. Here in this study, the extract from A. arsusianum, A. gypsophiloides and A. reuteri obtained with different methods and solvents were tested against common microorganisms. As a result, these extracts seem to be reasonably effective against test organisms, including clinical isolates.

Fatty acid content
The oil content of Ankyropetalum plant extracts was 5.79% for A. arsusianum, 6.86% for A. gypsophiloides and 5.77% for A. reuteri. As a result of the fatty acid analysis of plant extracts, 18, 21 and 26 fatty acids were found in A. arsusianum, A. reuteri and A. gypsophiloides, respectively (Table 5). The major components were nervonic acid (23.66%, 39.76% and 42.88%), butyric acid (10.64%, 19.42% and 21.59%), palmitic, oleic and linoleic acids. Butyric acid was the major SFA in A. arsusianum and A. reuteri (19.42% and 21.59%, respectively), while it was palmitic acid (13.10%) in  A. gypsophiloides. There is a need for saturated fats for energy, hormone production, cellular membranes and organs. Butyric acid reduces virulence (a disease-causing effect) and it is used both in hygiene measures and in protection measures (Van Immerseel et al., 2005). In addition, since butyric acid esters have pleasant odors or flavors, they are often used as food and perfume additives. Some saturated fatty acids are also necessary for important signalling and stabilization processes in the body. Saturated fatty acids that play an important role in these processes are known as palmitic acid, myristic acid and lauric acid (Mohanty et al., 2013).
Ankyropetalum, which contains all three fatty acids, contains palmitic acid predominantly. Parameters associated with significant risk factors for cardiovascular disease have been associated with dietary habits. Olive oil rich in MUFA is one of the main components of the Mediterranean diet (Teres et al., 2008). On the other hand, the literature is increasingly showing the benefits of PUFAs for alleviating cardiovascular, inflammatory, heart diseases, atherosclerosis, autoimmune disorders, diabetes and other diseases (Finley and Shadidi, 2001). It has been observed that MUFAs contribute to the majority of the unsaturated fatty acid content in Ankyropetalum species. In all three species, the major MUFA is nervonic acid and the major PUFA is linoleic acid. It is known that some unsaturated fatty acids, such as nervonic acid, linoleic acid and cis-11 eicosenoic acid are suitable for human nutrition (Carvalho et al., 2006). The studied species contain all three fatty acids, two of which are dominant.
Interest in dietary therapy with nervonic acidcontaining oils and fats has increased with the suggestion of Sargent et al., (1994) that dietary nervonic acid may support the normal synthesis and function of myelin in brain and nerve tissues. This recommendation encouraged the development of refined, nervonic acid-enriched vegetable oil to make experiments on humans and animals. Nervonic acid can be evaluated as a bioactive lipid supplement for the promotion of human and animal health, but it has been reported that nervonic acid-rich vegetable oil with the minimal amount of erucic acid has to be developed to be able to carry out these applications (Guo et al., 2009). In this context, Ankyropetalum oil which does not contain erucic acid at all and which contains nervonic acid at a satisfactory level will be preferred among other sources of nervonic acid.

CONCLUSIONS
Ankyropetalum is consumed locally as food and food additives. However, this plant has not been studied thoroughly enough. For this reason, the bioactive properties of Ankyropetalum have been examined in this study. The results show that Ankyropetalum is rich in phenols and flavonoids, as well as antioxidants and antimicrobials. The effects of extraction methods were evaluated and soxhlet extraction was found to be more effective in total phenolic content and FRAP. Methanol was superior to ethanol as solvent in extraction methods. With high MUFA and PUFA contents, Ankyropetalum oils can be evaluated in various fields as a valuable raw material that can be used in the pharmaceutical, cosmetic, perfume, and food and medicine industry. Ankyropetalum oil is rich in nervonic acid, so it has great potential for the symptomatic treatment of many neurodegenerative diseases such as multiple sclerosis, schizophrenia and Parkinson's disease. Further work on the isolation and identification of bioactive compounds will be beneficial for a better and specifically directed application.

This
work was supported by the Kahramanmaras Sutcu Imam University (Grant no: 2016/3-25YLS). The invaluable help of Prof. Dr. Ashabil Aygan and Dr. Yusuf Ziya Kocabas is also gratefully acknowledged. Fatty acids, bioactive substances, antioxidant and antimicrobial activity of Ankyropetalum spp., a novel source of nervonic acid • 9