First determination of some phenolic compounds and antimicrobial activities of Geranium ibericum subsp. jubatum: A plant endemic to Turkey

This paper includes the results of the first study about the phenolic characteristics and antimicrobial analyses of Geranium ibericum subsp. jubatum species found in Turkey . In the present work, the phenolic contents of different parts of the G. ibericum (flower, root, leaf) were determined with high-performance liquid chromatography (HPLC)–DAD (diode-array detector) and liquid chromatography (LC)–MS/MS (mass spectrometry). The following phenolic compounds were investigated: catechin, protocatechuic acid, gallic acid, ellagic acid, chlorogenic acid, 4-hydroxybenzaldehyde, p -coumaric acid, rutin, naringenin, kaempferol . Based on the results obtained, the roots and flowers of the plant are found to be very rich in ellagic acid (3473.57 µg g-1 dry plant) and catechin (2228.76 µg g-1 dry plant). The amount of chlorogenic acid (54.570 µg g-1 dry plant) is also high in the roots. The amounts of protocatechuic acid (122.5 µg g-1 dry plant) and gallic acid (725.34 µg g-1 dry plant) are high in the leaves. In addition, the total extract of G. ibericum obtained from leaf, flower, and root was tested against 6 gram-negative bacteria and Candida albicans . The G. ibericum extract was nearly as effective as commercial antibiotics at some concentrations (500-750 µg µL-1) for Acinetobacter baumannii , Klebsiella pneumonia , Proteus mirabilis , and Bacillus cereus .


HPLC-DAD analysis
The reverse-phase C18 column (150 mm × 4.6 mm, 5μm; Fortis) was used for the chromatographic separation of compounds. A slight modification was made in the mobile phase reported in the literature, which consisted of A: 2% acetic acid-ultrapure water, and B: 50%-50% acetonitrile-ultrapure water including 0.5% acetic acid. The following mobile phase gradient program was used for the separation of phenolic compounds: 0-8 min (7% B), 8  The injection volume was adjusted to 20 μL, the mobile phase flow rate to 0.7 mL min -1 , and the column temperature to 25 °C. The diode-array detector (DAD) was operated at four different wavelengths (260, 280, 308, 324 nm). The wavelengths determined in DAD were selected according to the maximum absorption wavelengths by examining the spectra of phenolic standards.

LC-MS/MS analysis
The ODS Hypersil (4.6 × 250 mm, 5 µm) column was used in liquid chromatography (LC)-MS/MS (mass spectrometry) analyses. For mobile phases, water containing 0.1% formic acid, and B methanol was used. The mobile phase solvent program was adjusted to 0 and 0-1 min 100% A, 1-22 min 5% A, 22-25 min 5% A, and 25-30 min 100% B. The flow rate was set to 0.7 mL min -1 , the column temperature to 30 °C, and the injection volume was 20 µL. The total extracts were studied at the second and third tests as there had been no differences between root, leaf, and flower extracts in terms of antimicrobial activity. The antimicrobial activity and minimal inhibitory concentrations (MIC) were determined by the disc diffusion and minimum dilution methods, respectively. The experiments were performed according to the method described by [18]. The extract was diluted to 300-1000 µg µL -1 with DMSO (20%). Ofloxacin (OFX) (10 μg disc -1 ), netilmicin (NET30) (30 μg disc -1 ), and sulbactam (SCF) (30 kg disc -1 ) were used as positive references for the disc diffusion method. Maxipime (Bristol-Myers Squibb, New York, NY, USA) in concentrations of 7.81-500 µg µL -1 was used as a positive reference for minimal inhibitory concentration determination; 10 μL of DMSO (20%) was the negative reference. Each experiment was performed three times.

Results and discussion
The total phenolic content analysis and antioxidant capacity (using ABTS and DPPH methods) of G. ibericum were determined spectrophotometrically as reported by Ay et al. [19]. As in that study, the FRAP method used to determine the antioxidant capacity was applied in paper and the results were evaluated. In addition, in the continuation of these studies, we aimed to determine the phenolic characteristics of these endemic species extracts with chromatographic studies and antimicrobial activities. Since it dissolves low-molecular-weight polyphenols, methanol was used as the extraction solvent. It should be noted that the compounds detected by the chromatographic method and the antimicrobial capacity assessment belong only to the methanol extract, taking into account that the components to be dissolved will change as the solvent changes [20].

Iron (III) reduction/antioxidant force (FRAP) method results
A calibration curve based on absorbance measurements was constructed by using different concentrations of Trolox. Using this curve, antioxidant activity was expressed as Trolox equivalent antioxidant capacity (TEAC, μM); the graph of the column is given in Figure 1. All samples used in the FRAP method were analyzed by diluting 1:100 from the stock solution.
The iron (III) reduction forces of the samples are 207, 410, and 221 µM TEAC for flower, root, and leaf, respectively and are shown in Figure 1. When the iron (III) reduction/antioxidant strengths of the extracts were compared, the root extract had the highest iron extracting potential and the highest antioxidant activity. The lowest iron (III) reduction/antioxidant strength was found in the flower extract. These results are similar to those of the previous study involving ABTS and DPPH methods [19].

Determination of phenolic compounds by chromatographic methods
The phenolic contents of Geranium are given in Table 3. Chromatograms of the standard mixture and extracts are given in Figures 2 and 3a,b,c.
The results show that the amounts of ellagic acid, catechin, gallic acid, protocatechuic acid, and chlorogenic acid are remarkably high. The roots and flowers of the plant are very rich in ellagic acid and catechin. The amount of chlorogenic acid in the root is high, and the amounts of protocatechuic acid and gallic acid in the leaves are high. There is also a high amount of gallic acid in the flowers. p-Coumaric acid and kaempferol amounts were higher than those of other phenolics. According to the results of the phenolic contents, the antioxidant strength of the root is significantly higher than that of the leaves and flowers. According to the results given in Table 2, the highest phenolic content can be seen in the root and flower.
A simple extraction was carried out with the samples, and the quantity of many phenolic compounds was determined by HPLC-DAD and LC-MS/MS. This study was the first to investigate the phenolic content of an endemic Geranium species. Although the essential oil content for similar Geranium species has been examined in the literature, the phenolic component content has not frequently been studied. In one of the studies, 17 phenolic compounds were searched for, and 8 of them could be determined. The amounts of phenolic compounds found in the sample study in mg g -1 as follows: caftaric acid 1.30, caffeic acid 2.41, hyperoside 1.64, isoquercitrin 2.58, rutin 1.71, quercitrin 0.42, quercetin 0.82, kaempferol 0.19 [21]. In another study with G. carolinianum, 5 bioactive components were investigated by performing water extraction; only gallic acid and elagic acid were analyzed. The gallic acid results found in that study are about 10 times higher than what we found. The elagic acid results were approximately the same [22].
In a similar study, some of the main phenolics of the G. sibiricum plant were determined. In this study, the amount of gallic acid was higher than the amount we detected [23]. Apart from this, there was no commonality in the determined compounds. In our study, 18 phenolic compounds were searched for, 11 of which could be quantified. The components with the highest amounts were ellagic acid, catechin, gallic acid, and protocatechuic acid. Total ellagic acid, catechin, gallic acid, and protocatechuic acid amounts were 3.99, 3.19, 1.23, and 0.23 mg g -1 , respectively, in root + leaf + flower.
Ellagic acid has been reported to play a protective role against the negative effects of various stress sources. It has also been shown to activate apoptotic pathways, leading to the reduction of cancer and other chronic diseases [24].  Catechin, an important flavonoid, has been demonstrated to effectively inhibit metastases, which may result from inhibition of metalloproteinases [25]. It has also been shown that catechin inhibits lipid oxidation by delaying the consumption of lipid-soluble antioxidants and is an effective antioxidant in human blood plasma [26].
Gallic acid, a polyphenol, has been shown to inhibit carcinogenesis in animal experiments and in vitro cancer cell lines. In addition to drug development, it reduces the risk of developing cancer when taken as a food supplement [27].
When studies on Geranium spp. are examined, it is seen that some subspecies belonging to the Geraniaceae family contain a substance called geraniin that has many important biological activities such as anticancer, antimicrobial, antiviral, and antihyperglycemic activities [28]. When geraniin is hydrolyzed, it produces gallic acid, ellagic acid, corilagin, and brevifolin carboxylic acid [29]. It has been found that these metabolites released by the hydrolysis of geraniin have a range of bioactive properties including antioxidant and free radical scavenging activity [30,31]. However, studies have not shown that all Geranium spp. contain these four components as the main components [1].

Antimicrobial activity
The antimicrobial effects and MIC values of the G. ibericum subsp. jubatum extract (GIJE) were determined by the disc diffusion method and minimum dilution methods at concentrations ranging from 300 to 1000 µg mL -1 . (Table 4). Since no differences have been observed between different parts of the plant, it was decided to study the total G. ibericum extract (GE). Contrary to expectations, the highest dose (1000 µg mL -1 ) showed no antimicrobial activity and the second highest dose showed effectiveness against only 2 bacteria (Acinetobacter baumannii and Proteus mirabilis). GIJE is thought to be completely insoluble and precipitated at high concentration, and consequently showed no antimicrobial activity. The MIC values were as follows: 300 µg mL -1 for P. mirabilis, B. cereus, and S. epidermidis; 400 µg mL -1 for A. baumannii, K. pneumonia, E. aeruginosa, P. aeruginosa, S. aureus, B. subtilis, and C. albicans; 500 µg mL -1 for C. freundii, S. pneumonia, and MRSA ATCC 67101. Significant differences were not observed in terms of being gram-negative or gram-positive. GE was nearly as effective as commercial antibiotics in some concentrations against A. baumannii, K. pneumonia, P. mirabilis, and B. cereus. Lis-Balchin and Deans [32] reported that the extracts obtained from 8 different members of the Geraniaceae    family showed significant antibacterial activity against at least 18 out of 25 bacteria and very slight activity against fungi. Radulović et al. [33] studied the volatile oils obtained from G. sanguineum and G. robertianum. The oils showed strong action against E. coli and A. fumigatus. Giongo et al. [34] showed that the antimycobacterial and antimicrobial activities of geranium-oil-loaded nano-capsules were similar to those of free oil. The geranium oil-loaded nano-capsules were not effective in inhibiting the formation of germ tubes of C. albicans [32]. However, GE showed antimicrobial activity against C. albicans in this study. In the literature, there are a few studies about the antimicrobial activity of the Geranium species, but none of them concern G. ibericum. This study is the first study which evaluates the antimicrobial activity of GIJE.

Conclusion
In this study, the phenolic characters and antimicrobial activity of extracts of the different parts of the endemic G. ibericum were explored. Eighteen phenolic compounds were investigated; 11 of them were detected with chromatographic methods. The antioxidant and antimicrobial activities of the plant were determined. Geranium species are used in alternative medicine because of these properties. The main components that are generally isolated in this plant are catechin, ellagic acid, gallic acid, and protocatechuic acid. These components are known to have important functions. According to the results of antimicrobial activity experiments, the use of geranium extract seems to be a promising tool in overcoming pathogen microorganisms. Further studies of other phenolic compounds (such as geraniin) and essential oils of this species should be carried out; results obtained from such a study in Turkey would make a comparison possible with other Geranium species in the world.