Investigation of Some Biological Activities of Extracts Centranthus longiflorus subsp. longiflorus

: In this study, the effects of above and below-ground extracts of Centranthus longiflorus subsp. longiflorus plant, commonly found in Turkey, on antioxidant, antimicrobial and DNA damage were evaluated. Plant extracts were prepared by applying three different solvents (hexane, methanol and ethanol). The antimicrobial activity tests of the extracts were performed using four different standard strains and one yeast. DPPH, total phenolic content calculation and CUPRAC methods were applied for antioxidant activity studies. Additionally, the effects of plant extracts on DNA damage were investigated using pBR322 plasmid DNA. According to the data obtained, especially the below-ground hexane (MIC value:375µg/mL) extract showed more antimicrobial activity than other plant extracts, and it was found to be more effective Gram negative bacteria. The highest antioxidant activity was determined in extracts prepared with above (IC 50 value of methanol extract:4.5mg/mL) and below-ground (IC 50 value of methanol extract:5.7mg/mL) methanol. The above (93,9 µg GAE/mL) and below-ground (96.9 µg GAE/mL) methanol extracts were seen to have high total phenolic content. It has also been observed that above-ground hexane and methanol extracts have no effect on pBR322 plasmid DNA, but other extracts affect pBR322 plasmid DNA in the direction of degradation or deformation. Especially, the extracts of the above and below-ground ethanol had the effect of completely eliminating the open ring form. Therefore, it was concluded that this taxon could be widely used in the treatment and prevention of oxidative stress-related diseases in the future.

extract solution (1 mg/mL) were completed with methanol to 2.3 mL and 50 µl of Folin-Ciocalteu reagent was added. After 3 min, 150 µl of 2% (w/v) Na2CO3 solution was added and incubated for two hours at room temperature. The absorbance of the samples was read at 760 nm (Thermo Scientific Genesys 10S UV-VIS Spectrophotometer) against the blank, which did not contain a test sample. The results were expressed as µg(GAE)/mL (extract).

DPPH Free Radical Scavenging Activity
The antioxidant activities of the plant samples were evaluated by testing the DPPH free radical scavenging activity. Butylated hydroxy anisole (BHA) was used as a standard antioxidant. For this purpose, 50 µl of different concentrations (3-10 mg/mL) of plant extracts were incubated with 2850 µl of DPPH solution (6x10 -5 M) in dark and at room temperature for 30 minutes. At the end of this period, the absorbance was measured 517 nm against the blank sample [21]. The DPPH % was calculated according to formula (I) and the results were expressed as IC50 value. IC50 refers to the concentration of plant at the moment when half of the DPPH amount was scavenging.
Inhibition %= (ADPPH·-Asample) / ADPPH·x100 (I) Respectively, ADPPH refers to· the DPPH radical in the absence of plant extract and Asample refers to the DPPH radical in the presence of plant extract absorbance (at 517 nm).

Cu (II) Reducing Activity (CUPRAC)
In order to determine the antioxidant capacity of Trolox equivalent (TEAC), Cu (II) reduction activity test was performed by CUPRAC (Cupric Ion Reducing Antioxidant Capacity) method. 1 mL of 10 -2 M CuCl2, 1 mL of 7.5x10 -3 M neocuproine and 1 mL of 1 M NH4Ac were placed in a test tube, respectively. These plant extracts were put into tubes at certain concentrations (10-100 µg/mL) and diluted with dH2O to 4.1 mL. After the tubes were kept closed for 30 minutes at room temperature, the absorbance values at 450 nm were measured. Results were expressed as trolox equivalent (µmol trolox/mg extract) antioxidant capacity (TEAC CUPRAC) utilizing formula (II) [22].

TEACCUPRAC = A(plant extract) / A(Trolox) (II)
A(plant extract) and A(trolox) in Formula (II) refer to the molar absorption coefficient for plant extract and trolox, respectively.

Antimicrobial Activity
Antimicrobial activity was performed using the MIC (Minimum Inhibitory Concentration) method [23]. Microorganisms were obtained from Ondokuz Mayıs University. Gram positive (Staphylococcus aureus ATCC 6538P, Bacillus cereus ATCC 7064), Gram negative (Escherichia coli W3110, Pseudomonas aeruginosa ATCC 27853) and a yeast (Candida albicans ATCC 10231) were used. Stock solutions of the used extracts were prepared at a concentration of 40 mg/mL. Extracts were dissolved in DMSO. The last tube without bacterial growth was determined as MIC value. MIC values obtained in the study were shown as µg/mL.

DNA Interaction
Determination of the plant extracts effect on plasmid DNA (pBR322) was made according to agarose gel electrophoresis method [24]. For this purpose, 1% Agarose gel was prepared in TBE (1X) buffer. 120 µg/mL plant extracts were interacted with 0,5 µg/mL pBR322 plasmid DNA at 37 0 C for 2 hours. After incubation samples were mixed with 6X loading dye and loaded on 1 % agarose gel. Electrophoresis was carried out 100 v for 80 min. The gel, then, was stained with EtBr (Ethidium Bromide) and the bands were imaged with the aid of the UV transilluminator (Cleaver Clear View).

RESULTS and DISCUSSION
Saponins, flavonoids, phenols and tannins are among the secondary metabolites in plants and play an important role in anticancer and antioxidant activities [25][26][27]. The iridoids, fatty acids and 16 phenolic compounds are isolated from the above and below ground parts of C. longiflorus subsp. longiflorus [6][7][8]11]. For this, the above-ground and root parts of this taxon are used in the prevention and treatment of diseases such as soothing, antispasmodic, anticholytic, familial hypercholesterolemia, coronary artery disease and colon cancer in traditional Turkish medicine [10,28]. On the other hand, scientists have stated that methanol is widely used and it is an effective solvent for the extraction of antioxidants [29,30].

Total Phenolic Compounds
The standard graph was prepared using gallic acid in order to calculate the total phenolic content and shown in Figure 1. The phenolic contents of above and below-ground extracts were given in Table 1. When total phenolic results of above and below-ground extracts of C. longiflorus subsp. longiflorus were examined, above and below-ground methanol extracts were seen to have high total phenolic content. Especially above and below-ground hexane extracts had the lowest total phenolic content compared to other extracts of this subspecies. For this, above and below-ground methanol extracts of studied subspecies may be preferred as natural antioxidant sources in the future.

Free Radical Scavenging Analysis
The free radical scavenging activity results of the above and below-ground extracts were given in Figure 2 and in Table 2. According to these results, IC50 values of above and belowground hexane extracts could not be calculated since they do not have radical extinguishing capabilities. In particular, IC50 value of the above and below-ground methanol extracts had the highest antioxidant capacity. This situation is related to the high concentration of phenolic compounds in the above and below-ground methanol extracts. Antioxidant activity occurs due to the natural polyphenolic compounds present in plant extracts [31]. The amount of polyphenolic compounds in plants is important, since plants containing high polyphenolic compounds will be important antioxidant sources. We think that above and below-ground methanol extracts of C. longiflorus subsp. longiflorus may be used as an antioxidant source.     Inhibition % Plant extract (mg/mL) AG hexane BG hexane AG ethanol BG ethanol AG methanol BG methanol development of new drug formulations and the preparation of new food supplements. In a study on C. longiflorus growing in Lebanon, three different tests (DPPH, H2O2 and iron chelating) were applied to plant extracts prepared with aqueous and methanol. The tests showed that antioxidant activity in C. longiflorus extracts were 80%, 70% and 50%, respectively [4]. In our study, it was determined that above and below-ground extracts especially prepared with methanol had significant antioxidant activity. Briefly, it is possible to think that the above and below-ground extracts prepared with methanol of this subspecies contain more polyphenols compounds than other extracts. Also, Aliyazıoğlu et al. [11] put forward that methanol extracts of C. longiflorus showed strong antioxidant activity. In short, our antioxidant results are consistent with antioxidant results of Rammal et al. [4], Aliyazıcıoğlu et al. [11], Zengin et al. [32]. This is due to the use of the same organic solvent and similar antioxidant tests in four studies. Similar antioxidant results were reported in Gagea fibrosa (Desf.) Schulte & Schultes fil.-leaves methanol extract and Romulea ramiflora Ten subsp. ramiflora-bulb methanol extract by Mammadov et al. [19]. On the other hand, Turan and Mammadov [33] reported the highest antioxidant activity in acetone extracts of Cyclamen alpinum Dammann ex. Springer. Molyneux [34] put forward that phenolic compounds can act as free radical scavengers based on their hydrogen-donating property. The above findings indicate that the antioxidant activity of methanol extract is associated with a high phenolic compound level. On the other hand, it can potentially be used to prevent oxidative stress-related and aging-associated diseases, since this taxon has numerous secondary metabolites and neutralizes free radicals in the body [35]. Aliyazıcıoğlu et al. [11] emphasized that C. longiflorus had rich phenolic compositions, antioxidant activities and potential for using as raw materials in the pharmaceutical and food industries in the prevention and treatment of various diseases due to oxidative stress.
In Turkey, antioxidant activities of some plants used in traditional medicine (including Centranthus longiflorus) were investigated by Çoban et al. [36]. Plant samples were collected from Ankara-Kızılcahamam. The above-ground extracts of plant samples were prepared using aqueous and ethanol. The above-ground ethanol extract of C. longiflorus was found to have the most potential antioxidant activity. Hence, Çoban et al. [36] reported that the above-ground ethanol extract can be considered as the best antioxidant source. Nevertheless, it is plausible to suggest that the ethanol extracts of this species may contain more polyphenolic compounds, because the polyphenolic compounds were determined to exhibit potent antioxidant activities [37,38]. The above and below-ground parts of Heliotropium samolifolium Bunge subsp. erzurumicum Dönmez were extracted in the presence of different organic solvents (hexane, chloroform, ethyl acetate, ethanol, ethanol+aqueous and aqueous). Especially above ground ethanol+aqueous, chloroform and below-ground ethanol extracts of the H. samolifolium subsp. erzurumicum were reported to demonstrate the highest antioxidant activity [39]. In the biological activity studies of the endemic Iris kirkwoodiae Chaudhary, especially aqueous and methanol extracts exhibited very high antioxidant activity [40]. The antioxidant findings of Çoban et al. [36], Sağlam and Kandemir [39], Emaduldeen [40] are compatible with the antioxidant findings of our study. This may be due to the use of similar organic solvents and similar antioxidant methods. Similar organic solvents cause to appear of similar secondary metabolites, although plant species are different.

CUPRAC Analysis
The standard graph of Trolox was shown in Figure 3, the trolox equivalent of plant extracts are given in Figure 4 and in Table 3. The εT value of the trolox was calculated as 1.66 x 10 4 L.mol -1 .cm -1 . In antioxidant studies, Cu 2 + reduction is used to determine electron donation activity. According to trolox equivalents, the above and below-ground methanol extracts were found to have high antioxidant activity. Aliyazıcıoğlu et al. [11] used ferric reducing/antioxidant power (FRAP) assay in methanol sample of C. longiflorus. Trolox equivalent was found to be high in blossom (114 μmol Trolox/100 g DW) and trunk (156 μmol Trolox/100 g DW). In our study, the highest torolox equivalent was seen in above (126 µmol trolox/mg extract) and below-ground (132 µmol trolox/mg extract) methanol extracts. These data were showed parallelism with the results found in other antioxidant methods in our study. Similar Cu 2+ reduction (CUPRAC) results were found in gall and leaves ethanol extracts of Andricus quercustozae by Azmaz et al. [41].

Antimicrobial Analysis
MIC values of above and below-ground of plant extracts were given in Table 4. The below-ground hexane extract was found to have strong antimicrobial activity on E. coli and P. aeruginosa. However, the below-ground hexane extract was observed to show intermediate antimicrobial activity on B. cereus and S. aureus. The above-ground hexane extract had low antimicrobial activity on E. coli, P. aeruginosa and C. albicans. Also, the below-ground ethanol extract of this subspecies demonstrated antimicrobial activity on only B. cereus bacterium (  Aliyazıoğlu et al. [11] reported that aqueous and methanol extracts of C. longiflorus showed strong antimicrobial effect against Mycobacterium smegmatis ATCC607 bacterium. In our study, the strongest antimicrobial activity was seen in below-ground hexane extracts. Especially, below-ground hexane extracts have an activity on the Gram-negative, positive bacteria and yeast used in this study. This effect was seen higher in Gram negative bacteria than Gram positive bacteria. Therefore, it would be more appropriate to prepare an antimicrobial drug on Gram negative bacteria rather than Gram positive bacteria from extracts of this taxon. On the other hand, there are no antimicrobial effects on bacteria of above and below-ground ethanol and methanol extracts except below-ground ethanol. Our antimicrobial results do not match the antimicrobial results of Aliyazıoğlu et al. [11]. Such a case can be attributed to the collection of plant samples from different localities at different times and the use of different antimicrobial methods and different microorganism. Also, Aliyazıcıoğlu et al. [11] have made extraction process without separating above and below ground the plant materials. The aqueous and ethanol extracts of Eryngium creticum Lam. and C. longiflorus distributing in Lebanon were tested with five different bacteria (Staphylococcus epidermidis CIP 444, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853) by Makki et al. [10]. Aqueous extracts were reported to exhibit strong antibacterial activity in both plants. The reason why Makki et al. [10] and our antimicrobial results are incompatible may be due to the use of different bacterial strains and different organic solvents in the study. Also, this case may be due to the different cell wall structures of Gram-positive and Gram-negative bacteria.
In another antimicrobial research [39], the above and below-ground extracts of H. samolifolium subsp. erzurumicum, which distribute around Erzurum, were applied to Gram positive (Staphylococcus aureus ATCC 25923, Micrococcus luteus NRLLB 1018), Gram negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) standard bacterial strains and a yeast (Candida albicans ATCC 10231). All above and below-ground extracts of the subsp. erzurumicum except above-ground hexane and above-ground ethanol+aqueous were obtained to show intermediate antifungal activity on C. albicans. The below-ground ethanol extract was found to exhibit moderate activity on S. aureus, while the below-ground ethanol extract to exhibit stronger antimicrobial activity on M. luteus and P. aeruginosa. The below-ground ethanol extract of the H. samolifolium subsp. erzurumicum did not have any antimicrobial activity on only E. coli. Our antimicrobial findings do not match with the antimicrobial findings of Sağlam and Kandemir [39]. It is thought that this is due to the use of different plant extracts in the studies. Since different plant extracts have different secondary metabolites, the effects of these metabolites on microorganisms are different.
Anti-proliferative properties of C. longiflorus extracts collected from Lebanon were also examined. It was suggested that the leaf and stem parts of this plant had significant antioxidant and anti-proliferative activities, due to the presence of some secondary metabolites (alkaloids, coumarins, saponin, polyphenols, volatile oils, flavonoids) in the leaves and stem of C. longiflorus [42]. In a study, sedative, anticonvulsant and behavior modification activities of subsp. longiflorus extracts were investigated. In the effects of aqueous extract (100 mg/kg) compared with diazepam, aqueous was found to have sedative and anticonvulsant effects similar to those produced by diazepam (5 mg/kg) [43].

DNA Interaction
The DNA interaction results were shown in Figure 5. 1 and 2 lanes belong to pBR322 DNA+H2O and pBR322 DNA+DMSO control groups, respectively. The extracts of aboveground hexane (Lane 3) and above-ground methanol (Lane 7) had no effect on pBR322 plasmid DNA, while the extracts of the above and below-ground ethanol (Lanes 5 and 6) had the effect of completely eliminating the open ring form. The below-ground methanol extract (Lane 8) had a disintegrating effect on the open ring structure. However, the below-ground hexane extract (Lane 4) was found to have an effect on increasing the concentration of open ring form. In a DNA interaction study, the below and above-ground chloroform and aqueous, aboveground hexane, below-ground ethyl acetate, ethanol extracts of endemic H. samolifolium subsp. erzurumicum showed a destructive effect to the structure of pBR322 plasmid DNA [39]. In the biological activity studies of the endemic Iris kirkwoodiae, all extracts except root dichloromethane and aqueous extracts showed protection for plasmid DNA against UV and H2O2 [40]. In another DNA interaction study, samples of endemic Iris galatica Siehe. were extracted with hexane, methanol dichloromethane and aqueous, all of the plant extracts were found to protect DNA against the harmful effects of UV and H2O2 [44]. Also, below-ground ethanol extracts of Leucojum aestivum L. were displayed highly effect on pBR322 plasmid DNA [45]. In a similar study with endemic Linaria corifolia Desf., the above and below-ground ethanol, ethyl acetate and dichloromethane extracts were determined to show protective activity on plasmid DNA [46]. The plasmid DNA results of the mentioned-above studies are compatible with plasmid DNA results in our study.

CONCLUSION
Some biological and antioxidant activities of extracts of C. longiflorus subsp. longiflorus collected from the vicinity of Tortum (Erzurum) were determined. According to our antioxidant results, both above and below-ground methanol extracts can be used as antioxidant source in many areas. Our antimicrobial data showed that the below-ground hexane extracts of this plant may be added in the content of drugs used in the treatment of diseases Gram-positive and negative bacteria, yeast borne. In addition, above-ground hexane extracts can be included into the structure of drugs for the treatment of Gram negative and yeast diseases. Especially, the above and below-ground ethanol extracts of investigated subspecies were seen to have significant effect on pBR322 plasmid DNA. Briefly, above and below-ground hexane, methanol and ethanol extracts of this plant may be used in many areas of the pharmaceutical industry. Although there are some studies related to this subspecies, this study will contribute to other studies in this field as a new literature source. The study on DNA damage was done by us for the first time.