A comparative study of the antimicrobial properties and antioxidant enzyme activities of field-grown and in vitro-propagated plants of endemic Digitalis trojana Ivanina

The antimicrobial properties and antioxidant enzyme activities of field-grown and in vitro-propagated plants of Digitalis trojana Ivanina (Helen of Troy foxglove), a perennial endemic plant species of Turkey, were compared. The field work was carried out in May and July 2014, and plant samples of D. trojana were collected from Kazdağı (Turkey). Propagation of D. trojana was achieved by culturing leaf explants on MS medium supplemented with 13.3 μM 6-benzylaminopurine (BA) and 0.53 μM α-naphthaleneacetic acid (NAA). The antimicrobial activity, plant lipid peroxidation levels and antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR)) activities were analyzed in 12and 17-week-old in vitro-grown Digitalis plants collected in May and July from two different localities at 430 and 1173 m above sea level. Although the in vitro-propagated plants had very low antagonistic activities compared to field-grown plants, they exhibited remarkably similar antibacterial activities against Escherichia coli ATCC 11230, Pseudomonas aeruginosa ATCC 27853 and Bacillus subtilis ATCC 6633. There were no important differences between plants collected from the two localities (430 and 1173 m a.s.l.). Biochemical analysis showed that the antioxidant enzyme (SOD, APX, GR) activities of field-grown plants were higher than in vitro-grown plants. Also, the difference in altitude at which the plants were grown was apparently linked to decreases in antioxidant enzyme activities, except for POX in field-grown plants collected in July.


INTRODUCTION
Digitalis trojana Ivanina is a member of the Plantaginaceae family, known by its common name Helen of Troy foxglove.The plant is a perennial endemic to Çanakkale and Balıkesir, northwestern Turkey [1].According to the Red Data Book of Turkish Plants, the conservation status of this species is classified as vulnerable (VU) [2].Digitalis species are a medicinally important group of plants because of their content of cardenolides or cardiac glycosides (CGs), which are used in human medicine.The cardenolides are effective drugs for treating several cardiac defects [3].They also possess a broad spectrum of biological activities, including anticarcinogenic, acaricidal and antibacterial properties.Recent studies have focused on the anticarcinogenic effects of digoxin and digitoxin [4,5].
During the last two decades, there has been a tremendous rise in infectious diseases (hepatitis, malaria, tuberculosis, etc.) with fatal outcomes.This has been attributed to increased resistance of pathogenic microbes to antimicrobial agents/drugs and the indiscriminate use of synthetic antibiotics.Thus, there is a continual search for new antimicrobials, especially of natural origin [6].In recent years, despite the progress of new technologies and medicine, it has become essential to develop and refine the multipurpose use of natural products.Antimicrobial activity studies with Digitalis species are very limited, as revealed by a literature survey [7,8].Therefore, it is important to assess the antimicrobial activity of in vitro-propagated and field-grown D. trojana.
Plants are sessile organisms with complex protection mechanisms.During stress, reactive oxygen (O 2 ) species (ROS) such as 1 O 2 , H 2 O 2 , O 2 -and HO .increase oxidative stress.During normal metabolism, nonenzymic components and antioxidant enzymes such as ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX), peroxiredoxin (PrxR) and superoxide dismutase (SOD) are essential for ROS detoxification [9].Although ROS are toxic molecules, plants utilize these molecules to respond to changes in the environment and developmental dynamics [10].The higher antioxidant levels in plants growing at higher altitudes enables them to deal with stressors [11,12].
There is no information about the potential link between antioxidant enzymes and the antimicrobial properties of D. trojana and their distribution in Kazdağı.The aims of this work were (i) to determine the antimicrobial and antioxidant enzyme activities of D. trojana, and (ii) to compare the antimicrobial and antioxidant enzyme activities of field-grown plants that were collected from different altitudes in May and July, with those of in vitro-propagated D. trojana.
Antimicrobial properties, lipid peroxidation level, total protein content and antioxidant enzyme activi-ties were analyzed in 12-and 17-week-old in vitropropagated plants and in field-grown plants in May and July, from two different localities in Kazdağı.The plant samples were harvested in three replicates and placed in liquid nitrogen.The samples were stored at -30°C until analysis.

Preparation of extracts for antimicrobial activity
The overhead parts of the each located D. trojana plant were dried at room temperature (26±3ºC) in the dark and pulverized.Each dried, pulverized sample (15 g) was extracted with 150 mL of different solvents (ethanol, methanol, ethyl acetate and acetone) (Merck, Darmstadt, Germany) by Soxhlet equipment for 24 h [15].For in vitro plant extraction, dried (at 50°C for 5 days) tissue powders (1 g) of each plant (12-17 weeks-old) were dissolved separately in 20 mL of 80% (v/v) ethanol overnight on a rotary shaker at room temperature.The extract was filtered using Whatman filter paper no. 1 and the extracts were stored in sterile screw-capped bottles at 4°C.

Antimicrobial activity screening
The antagonistic effects of each plant against bacteria and yeast samples were detected by measuring the diameter zones of inhibition (mm).Empty sterilized antibiotic disks were each imbrued with extract (50 µL).All the bacterial and yeast cultures were incubated at 35±0.1°C for 24 h and 25±0.1°C for 48 h by inoculation into nutrient broth and malt extract broth, respectively.An inoculum containing 10 6 bacterial cells or 10 8 yeast cells/mL was spread on Mueller-Hinton agar (Oxoid Lt., Hampshire, UK) plates (1 mL inoculum/plate) [16].Inhibition zones formed on agar plates were evaluated in mm.Studies were performed in triplicate.Treatments with penicillin (P10), ampicillin (AMP20) and nystatin (NYS30) served as positive controls, and treatments with ethanol, methanol, ethyl acetate and acetone without plant materials served as negative controls.For quantitative antimicrobial analyses, minimum inhibitory concentration (MIC) values of all samples were determined [17].Streptomycin was used as a reference antibiotic for bacteria MIC values.

Lipid peroxidation level
The leaf lipid peroxidation level (500 mg) of 12-and 17-week-old in vitro plants and collected field-grown plants in Kazdağı was assayed by determining the level of malondialdehyde (MDA).The content of thiobarbituric acid reactive substance (TBARS) was assayed by MDA [18].

Antioxidant enzyme assay
A frozen plant sample (500 mg) was placed in liquid nitrogen and homogenized in 50 mM sodium phosphate buffer (pH 7.8) containing 1 mM EDTA Na 2 for SOD, CAT and GR analyses.For the determination of APX, the extraction buffer contained 5 mM ascorbate.The analysis of POX required grinding a 500-mg sample with 50 mM of sodium acetate buffer (pH 6.5).All homogenates were centrifuged at 21000xg for 30 min at 4°C and the supernatants were used for determination of the enzyme activities and total protein content.The total protein content of the enzyme extract was determined according to Bradford [19].SOD (EC 1.15.1.1)activity was measured as the inhibition of photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm [20][21].CAT (EC 1.11.1.6)activity was measured by measuring the initial rate of disappearance of H 2 O 2 in the reaction mixture at 240 nm [22].The reaction mixture contained H 2 O 2 and 0.1 mM EDTA in 0.05 M Na-phosphate buffer (pH 7) and the enzyme extract.GR (EC 1.6.4.2) activity was measured according to Foyer and Halliwell [23].The reaction mixture contained Na-phosphate buffer (pH 7.8), glutathione disulfide (GSSG), NADPH.Na 4 and the extract.NADPH oxidation was determined at 340 nm.One unit of GR was defined as 1 mmol mL -1 GSSG reduced per min.APX (EC 1.11.1.11)activity was measured according to Nakano and Asada [24].One unit of APX was defined as 1 mmol mL -1 ascorbate oxidized in the reac-tion mixture containing 0.05 M Na-phosphate buffer (pH 7), 0.5 mM ascorbate, 0.1 mM EDTA.Na 2 , 1.2 mM H 2 O 2 , and the enzyme extract at 290 nm per min.POX (EC 1.11.1.7)activity was measured according to Kanner and Kinsella [25].The reaction mixture contained 50 mM Na-acetate buffer (pH 6.5), 90 mM H 2 O 2 , 100 mM pyrogallol and the enzyme extract.The activity was spectrophotometrically followed at 300 nm.A Mecasys Optizen Pop UV/Vis spectrophotometer was used for all spectrophotometric analyses.

Statistical analysis of antioxidant enzyme activities
Each assay was repeated two times independently.The vertical bars in the Fig. 1 indicate the means±standard error of five replicates.The data were subject to one way ANOVA according to randomized plot design in the in vitro-propagated plants and randomized split block design in the field-grown plants (months as the main-plot and altitudes as the sub-plot) using the Statistical Package for Social Sciences (SPSS) ver.20.0.Mean comparison was conducted using Tukey's test at p≤0.05.(Means followed by the same letter are not significantly different).

Antimicrobial activity
The antibacterial and antifungal activities of extracts obtained from field-and in vitro-grown 12-and 17-week-old D. trojana plants against the test microorganisms were assessed by the presence and diameters of the inhibition zones, MIC and MBC (minimal bactericidal concentration) (Tables 1 and 2).There was no inhibition zone in the negative control.All the field-grown and in vitro-propagated plants exhibited antibacterial activity against E. coli, Ps. aeruginosa (except 2J), S. aureus, E. faecalis.Ps. aeruginosa in particular was more susceptible to all extracts of both field-and in vitro-grown plants as compared to all standard antibacterial antibiotics (except 2J).
The extracts exhibited antifungal activity against C. albicans and K. fragilis test fungi, however, the antifungal activity was lower than the activity of the standard antifungal agent NYS.When the activities of extracts of the field-grown plants were compared in terms of different sampling times and locations (May vs. July 2014; 430 vs. 1173 m), we did not observe any significant differences between the two groups of plants.However, the antifungal activity of the fieldgrown plants was higher than that of plants grown in vitro.Further, the in vitro plants produced no significant antagonistic effect against St. typhimurium, S. aureus, E. faecalis, C. albicans and K. fragilis.The microdilution method was used for all plant sample extracts to determine the MICs and MBCs.The extracts had weak effects against the test microorganisms, with MICs and MBCs ranging from 1024 (1000) to 64 (128) µg/mL.These values were considerably lower than for the standard antibiotics.

Lipid peroxidation level, total protein content and antioxidant enzyme activities
A statistically significant change in the total protein content of in vitro-propagated 12-and 17-week-old plants was not observed (Table 3).There were no statistically significant changes in SOD, APX and CAT activities (Fig. 1B, C and E, respectively), although GR and POX activities significantly changed during the growth of plants in vitro (Fig. 1D and F, respec-tively).POX activity in in vitro-propagated 17-weekold plants was significantly increased (52.48%) as compared to in vitro-propagated 12-week-old plants (Fig. 1F), while GR activity decreased (42.94%;Fig. 1D).The TBARS content in field-grown plants was significantly decreased (40.19%) in May and July (11.19%;Fig. 1A) with increased altitude.The total protein content was significantly increased (70.75%) in May and in July (94.28%;Table 3).At higher-altitude SOD activity was significantly decreased (41.48% and 46.96%), as was that of APX (66.54% and 50.87%)  and GR (58.28% and 74.98%).The decreases in CAT and POX activities were nonsignificant (84.32% and 84.32%, respectively) in May and July (Fig. 1B-F).In field-grown plants, the increase in seasonal temperature affected the total protein and TBARS contents and antioxidant enzyme activities.The average TBARS content in July was 136.94% higher than in May (Fig. 1A).In contrast to TBARS, the average total protein content in July was 7.78% lower than in May (Table 3).Compared to the mean enzyme activities in May and July, SOD, APX and GR activities decreased by 8.2%, 11.74%, and 5.75%, respectively (Fig. 1B-D).How-ever, the average CAT and POX activities increased by 40.65% and 6.09%, respectively (Fig. 1E and F).
The average total protein contents of field-grown plants in May and July were, respectively, 17.79% and 26.25% lower than in vitro-propagated plants (Table 3).Also, the average POX activities were, respectively, 46.19% and 42.91% lower (Fig. 1F).The average TBARS contents in May and July were 33.29% and 216.10% higher, respectively, when compared to in vitro-propagated plants (Fig. 1A).Interestingly, the average SOD and APX activities of field-grown plants  The values indicate means±standard error of five replicates.Means followed by the same letter were not significantly different at p≤0.05 as determined by Tukey's multiple range test.Bovine serum albumin (BSA) served as a standard (430 m above sea level; 1173 m above sea level).

DISCUSSION
In vitro culture technologies are becoming increasingly important to reveal the antimicrobial and antioxidant enzyme activities of economically important plant species.In plant tissue cultures, the biological activities of natural products do not undergo seasonal variations (limitations) [26,27].In this research, we found that the antimicrobial activity of in vitro-grown plants was lower than in field-grown plants.However, all the plants exhibited antagonistic activity against Ps.aeruginosa.There is no information in the literature about the antimicrobial activity of D. trojana.Benli et al. [8] used extracts from leaves and flowers of D. lamarckii and observed strong growth inhibition against B. subtilis, S. aureus, L. monocytogenes and Shigella sp.bacteria cultures.However, they did not observe any antibacterial effect against Ps.aeruginosa, E. coli and E. faecalis.The difference in the results presented in our study indicates that the two species of Digitalis sp.possess different antimicrobial substances.This suggests that various species of Digitalis sp., including plants that are potentially important sources of medicinal substances, have yet to be identified.Detailed phytochemical investigations are required to determine the types of substances responsible for the antagonistic activities of these medical plants.Ethanol was reported to be the best solvent for extracting antibacterial or antifungal compounds [28].The issues raised in this investigation with ethanol are analogous to those reported in the aforementioned research.It is important that the concentration of extract used in the test can be correlated with high activities of its chemical compounds.We found that the results obtained with the ethanol extract of field-grown plants were higher than the results obtained for the other solvents.
Thus, only ethanol was used for the extraction of in vitro-propagated plants.
Cultivated and in vitro-cultured plants are not as medically potent as wild-grown plants [29,30].Our antimicrobial activity results showed that in vitropropagated plants had lower antimicrobial activity than field-grown plants.It is thought that the production of secondary metabolites in in vitro-propagated plants is lower than that in field-grown plants.Bioactivity testing of in vitro-cultured plants is required to preserve naturally-grown plant populations and to support their use in traditional medicine [31].
Environmental changes such as seasonal variation, light and temperature affect antioxidant enzyme activities in in vitro-propagated plants [32][33][34].Stress conditions inhibit the plant growth rate and antioxidant enzyme activities.We detected a decrease in the TBARS content in both May and July because of higher SOD, CAT, APX and GR activities in field-grown D. trojana plants as compared to in vitro-propagated plants.These results indicate that the antioxidant enzymes of D. trojana were effective at different altitudes.The observed decreases in activities of the antioxidant enzymes (SOD, APX, GR in both months, CAT in July and POX in May) were influenced by the lower atmospheric oxygen concentration at the higher altitude.While there is no research about the antioxidant enzyme activities of D. trojana, many studies on different plants from mountainous regions have shown that the activities of antioxidant enzymes are lower in leaves the higher the altitude.The activities of CAT and Cu/ZnSOD in Pinus mugo leaves collected from 1590 and 1920 m a.s.l. were lower than in samples collected at lower altitudes (1420 m) in the Tatra Mountains, Poland [35].Also, the activities of SOD and POX declined significantly in the leaves of Plantago major with an increase in altitude from 1600 to 3300 m on Mahan Mountain, China [36].On Tianshan Mountain (China), SOD, CAT and GR activities in Polygonum viviparum increased with the increase in altitude (from 2200 to 3900 m) [37].Similarly, in the western part of the French Alps at 2400 m, Soldanella alpina had high activities of SOD, CAT and GR [12].SOD activity in the alpine plant Daphne oleoides was higher than in Teucrium polium from the steppes, in Turkey (Northwest Anatolian Mountains and Central Anatolian steppes) [38].These results are compatible with our results obtained for field-grown plants.This may be one of the reasons why D. trojana is not distributed at altitudes higher than 1173 m.
In conclusion, this study revealed that field-grown D. trojana has higher antimicrobial and antioxidant enzyme activities than in vitro-propagated plants.To our knowledge, this is the first report on the antimicrobial and antioxidant enzyme activities of D. trojana.Our results suggest that field-and in vitro-grown D. trojana can be considered in the development of new antibiotics, and that the antioxidant enzyme activities are affected by increasing altitude and seasonal changes.

Table 2 .
Minimum inhibitory concentration (MIC) of the extracts of field-grown and in vitro (12-and 17-week-old) plants of D. trojana.

Table 1 .
The antibacterial and antifungal activities of extracts obtained from field-grown and in vitro propagated 12-and 17-weekold D. trojana against test microorganisms assessed by measuring the diameter zones of inhibition (mm).

Table 3 .
Total protein content in field-grown and in vitro-propagated Digitalis trojana plants.