Phenolic Compounds and Biological Activity of Kitaibelia vitifolia

Abstract

This study was aimed at evaluating the antioxidant activity and efficacy of the ethanolic extract of the endemic plant species Kitaibelia vitifolia in inhibiting the growth of selected fungi and bacteria. Antimicrobial activity was tested using the broth dilution procedure for determination of minimum inhibitory concentration (MIC). MICs were determined for eight selected indicator strains. The highest susceptibility to K. vitifolia ethanolic extract among the bacteria tested was exhibited by Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, and Klebsiella pneumoniae ATCC 13883 (MIC=15.62 μg/mL), followed by Escherichia coli ATCC 25922 and Proteus mirabilis ATCC 14153 (MIC=31.25 μg/mL), and Proteus vulgaris ATCC 13315 (MIC=62.50 μg/mL). Of the fungi, Candida albicans ATCC 10231 (MIC=15.62 μg/mL) showed the highest susceptibility, and Aspergillus niger ATCC 16404 (MIC=31.25 μg/mL) had the lowest. Results showed that K. vitifolia extract possesses antioxidant activity, with total antioxidant capacity of 75.45±0.68 μg of ascorbic acid/g and 50% inhibition concentration values of 47.45±0.55 μg/mL for 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity, 35.35±0.68 μg/mL for inhibitory activity against lipid peroxidation, 95.25±0.52 μg/mL for hydroxyl radical scavenging activity, and 31.50±0.35 μg/mL for metal chelating activity. Total phenolics, flavonoids, condensed tannins, and gallotannins were 85.25±0.69 mg of gallic acid (GA)/g, 45.32±0.55 mg of rutin/g, 54.25±0.75 mg of GA/g, and 41.74±0.55 mg of GA/g, respectively. The phenolic composition of K. vitifolia extract was determined by high-performance liquid chromatography. Rosmarinic acid was found to be the dominant phenolic compound of the extract.

Introduction

T he use of traditional medicinal plants for primary health care and other purposes has progressively increased worldwide in recent years. Plants communicate with their environment by producing a diverse range of chemicals. These secondary metabolites are a common feature of specific plants and plant families. Many plant secondary metabolites have antimicrobial properties that make plant extracts and products successful in the treatment of bacterial, fungal, and viral infections.^1
–3 The different parts of plants (root, leaf, flower, fruit, stem, and bark) are used to effectively treat numerous diseases. Their antioxidant and antimicrobial properties affect a range of physiological processes in the human body, thus providing protection against both free radicals and growth of undesirable microorganisms.

Free radicals as highly reactive intermediaries lead to oxidative tissue damage and, therefore, potential damage of each molecular type. Free radicals occur in a cell as a result of the effect of different external factors such as ultraviolet and X-ray radiation, chemical reactions, and some metabolic processes. The accumulation of these species causes serious diseases, including cardiovascular diseases, premature aging, cancer, inflammatory diseases, etc.^4

–9 However, synthetic antioxidants, such as butylated hydroxytoluene and butylated hydroxyanisole, widely known for their ability to terminate the chain reaction of lipid peroxidation, have been proved to be carcinogenic and cause liver damage.¹⁰

Both bacterial resistance to a large number of antibiotics and the capacity of plants to synthesize biologically active substances are reasons for the increasing importance given to the use of plant-derived products in bacterial control. The use of plants in the food industry to replace synthetic preservatives, antioxidants, or other food additives has increased significantly over recent years.¹¹

Many species of herbs are active antioxidants, mainly because of the content of phenolic compounds.¹² Phenolic compounds are ubiquitous in plants; flavonoids and other plant phenolics, such as phenolic acids, stilbenes, tannins, lignans, and lignins, are important in the plant for normal growth and development and for defense against infection and injury. These compounds are commonly found in both edible and nonedible plants, and they have been reported to have multiple biological effects, including antioxidant activity.¹³ Various investigations have implied that total phenolic compounds are closely related to antioxidant activity,¹⁴ with flavonoids and tannins being major plant compounds with antioxidant activity.¹

Kitaibelia vitifolia is a member of the Malvaceae family. No literature data are available on the traditional use of K. vitifolia. However, the extract of the plant was found to contain a high amount of rosmarinic acid. Rosmarinic acid has several interesting medicinal and biological activities, including antiviral, antibacterial, anti-inflammatory, and antioxidant activities. The presence of rosmarinic acid in medicinal plants, herbs, and spices has beneficial and health-promoting effects.^16,17 Rosmarinic acid is versatile, being used in food preservatives, cosmetics, and medical applications. It has been reported to occur in several taxonomically non-related families of the plant kingdom.¹⁸ The biological activity of extracts of different plants, such as rosemary (Rosmarinus officinalis L.), sage (Salvia officinalis L.), thyme (Thymus vulgaris L.), and lavender (Lavendula angustifolia Mill.), has been associated with phenolic compounds, including rosmarinic acid. Rosmarinic acid is found in various species of Lamiaceae used commonly as culinary herbs.^18,19 In view of the above, the present study was designed to evaluate the potential use of K. vitifolia in both nutrition and disease treatment.

Materials and Methods

Chemicals used

All standards for high-performance liquid chromatography (HPLC) analysis were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and Alfa Aesar (Karlsruhe, Germany). Acetonitrile and phosphoric acid were of HPLC grade (Tedia Co., Fairfield, OH, USA). Ethanol was of analytical grade (Aldrich Chemical Co., Steinheim, Germany).

Plant material

The test plant was collected at Ilijak Hill (Central Serbia) in May/June 2009. The plant was collected at the flowering stage. The species was identified, and a voucher specimen (16350 BEOU, Lakušić Dmitar) was deposited at the Department of Botany, Faculty of Biology, University of Belgrade, Belgrade, Serbia.

Extract preparation

Plant samples (10.0 g) were extracted by 96% ethanol or ethanol (100.0 mL) as a solvent. The extraction process was carried out using an ultrasonic bath (model B-220, Branson Instruments, Smith-Kline Co., Danbury, CT, USA) at room temperature for 1 hour. After filtration, 5 mL of the liquid extract was used for extraction yield determination. The solvent was removed by a rotary evaporator (Devarot, Elektromedicina, Ljubljana, Slovenia) under vacuum and was dried at 60°C to constant weight. The dried extracts were stored in glass bottles at 4°C to prevent oxidative damage until analysis. The above-ground part of plants was collected.

Spectrophotometric measurements

Spectrophotometric measurements were performed using an ultraviolet-visible spectrophotometer (model MA9523-SPEKOL 211, ISKRA, Horjul, Slovenia).

Test microorganisms

The antimicrobial activity of the plant extract was tested in vitro against the bacteria Staphylococcus aureus ATCC 25923, Klebsiella pneumoniae ATCC 13883, Escherichia coli ATCC 25922, Proteus vulgaris ATCC 13315, Proteus mirabilis ATCC 14153, and Bacillus subtilis ATCC 6633 and the fungi Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404. The fungi were cultured on potato-glucose agar for 7 days at room temperature of 20°C under alternating light/dark conditions; then they were cultured on a new potato-glucose substrate for another 7 days. The culturing procedure was performed four times, after which pure cultures required for determination were obtained. The identification of the test microorganisms was confirmed by the Laboratory of Mycology, Department of Microbiology, Institute Torlak, Belgrade, Serbia.

Minimum inhibitory concentration

Minimum inhibitory concentration (MIC) values of the extract and cirsimarin against the test bacteria were determined using a microdilution method in 96-well microtiter plates.²⁰ All tests were performed in Muller–Hinton broth with the exception of yeast, for which cSabouraud dextrose broth was used. A total of 100 μL stock solution of oil (in methanol, 200 μL/mL) and cirsimarin (in 10% dimethyl sulfoxide, 2 mg/mL) was pipetted into the first row of the plate. Fifty microliters of Mueller–Hinton or Sabouraud dextrose broth (supplemented with Tween 80 at a final concentration of 0.5% [vol/vol] for analysis of oil) was added to the other wells. Fifty microliters from the first test wells was pipetted into the second well of each microtiter line, and then 50 μL of scalar dilution was transferred from the second to the 12th well. Ten microliters of resazurin indicator solution (prepared by dissolving a 270-mg tablet in 40 mL of sterile distilled water) and 30 μL of nutrient broth were added to each well. Finally, 10 μL of bacterial suspension (10⁶ colony-forming units/mL) and yeast spore suspension (3×10⁴ colony-forming units/mL) was added to each well. For each strain, the growth conditions and the sterility of the medium were checked. The standard antibiotic amracin was used to control the sensitivity of the tested bacteria, whereas ketoconazole was used as the control against the tested yeast. Plates were wrapped loosely with cling film to ensure that bacteria did not become dehydrated and prepared in triplicate, and then they were placed in an incubator at 37°C for 24 hours for the bacteria and at 28°C for 48 hours for the yeast. Color change was then assessed visually. Any color change from purple to pink or colorless was recorded as positive. The lowest concentration at which color change occurred was taken as the MIC value. The average of three values was calculated and represented the MIC for the tested compounds and standard drug.

Determination of total phenolic content

Total phenols were estimated according to the Folin–Ciocalteu method.²¹ The extract was diluted to the concentration of 1 mg/mL, and aliquots of 0.5 mL were mixed with 2.5 mL of Folin–Ciocalteu reagent (previously diluted 10-fold with distilled water) and 2 mL of NaHCO₃ (7.5%). Aliquots were left for 15 minutes at 45°C, and then the absorbance was measured at 765 nm with a spectrophotometer against a blank sample. Total phenols were determined as gallic acid (GA) equivalents (mg of GA/g of extract), and the values are presented in Table 1 as means of triplicate analyses.

Table 1.

Total Phenolics, Flavonoids, Condensed Tannins, and Gallotannins and Total Antioxidant Capacity of the K. vitifolia Extract Tested

Component	Value
Total phenolics (mg of GA/g)	85.25±0.69
Flavonoids (mg of RU/g)	45.32±0.55
Condensed tannins (mg of GA/g)	54.25±0.75
Gallotanins (mg of GA/g)	41.74±0.55
Total antioxidant capacity (μg of AA/g)	75.45±0.68

AA, ascorbic acid; GA, gallic acid; RU, rutin.

Determination of flavonoid content

Total flavonoids were determined according to the method of Brighente et al.²² Two percent aluminum chloride (0.5 mL) in methanol was mixed with the same volume of methanol solution of plant extract. After the mixture was left for 1 hour at room temperature, the absorbance was measured at 415 nm with a spectrophotometer against a blank sample. Total flavonoids were determined as rutin (RU) equivalents (mg of RU/g of dry extract), and the values are presented in Table 1 as means of triplicate analyses.

Determination of condensed tannins

The method for determination of condensed tannins relies on the precipitation of proanthocyanidins with formaldehyde.²³ Total phenolic content was measured using the Folin–Ciocalteu reagent as described before. A 0.5 mol equivalent of phloroglucinol was added for every GA equivalent in the extract. The calculated amount of phloroglucinol, followed by 1 mL of a 2:5 (vol/vol) HCl/H₂O solution and 1 mL of formaldehyde solution (13 mL of 37% formaldehyde diluted to 100 mL in water), was added to an aliquot of 2 mL of the extract soluted in methanol. After overnight incubation at room temperature, the levels of unprecipitated phenols were estimated in the supernatant by the Folin–Ciocalteu method. The precipitate contains proanthocyanidins and the known amount of phloroglucinol, which is always quantitatively precipitated. The concentration of condensed tannins was calculated as the residuum of the total phenolic and unprecipitated phenolic concentrations and expressed as GA equivalents. The results are given in Table 1 and presented as means of triplicate analyses.

Determination of gallotannins

Gallotannins are hydrosoluble tannins containing a GA residue esterified to a polyol. Gallotannins can be detected quantitatively using the potassium iodate assay. This assay is based on the reaction of potassium iodate with galloyl esters,²³ which will form a red intermediate and ultimately a yellow compound. The concentration of the red intermediate can be measured spectrophotometrically at 550 nm. The reaction was performed by adding 1.5 mL of a saturated potassium iodate solution to 3.5 mL of extract at a temperature over 40°C until maximum absorbance was reached (regardless of time). Gallotannins were determined using GA as the standard. The results are given in Table 1 and presented as means of triplicate analyses.

Determination of total antioxidant capacity

The total antioxidant activity of the K. vitifolia extract was evaluated by the phosphomolybdenum method.²⁴ The assay is based on the reduction of Mo(VI) to Mo(V) by antioxidant compounds and subsequent formation of a green phosphate/Mo(V) complex at acid pH. A total of 0.3 mL of sample extract was combined with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95°C for 90 minutes. Then the absorbance of the solution was measured at 695 nm with a spectrophotometer against the blank after cooling to room temperature. Methanol (0.3 mL) in the place of extract was used as the blank. Ascorbic acid (AA) was used as the standard, and total antioxidant capacity was expressed as milligrams of AA per gram of dry extract. The results are given in Table 1.

Determination of 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity

The method used by Takao et al.²⁵ was adopted with suitable modifications from Kumarasamy et al.²⁶ 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (8 mg) was dissolved in methanol (100 mL) to obtain a concentration of 80 μg/mL. Serial dilutions were carried out with the stock solution (1 mg/mL) of the extract. Solutions (2 mL each) were then mixed with DPPH (2 mL) and allowed to stand for 30 minutes for any reaction to occur, and the absorbance was measured at 517 nm. AA, GA, and butylated hydroxytoluene were used as reference standards and dissolved in methanol to make the stock solution with the same concentration (1 mg/mL). The control sample was prepared containing the same volume without test compounds or reference antioxidants. Ninety-five percent methanol was used as a blank.

The 50% inhibition concentration (IC₅₀) value, defined as the concentration of the test material that leads to 50% reduction of the free radical concentration, was calculated as micrograms per milliliter through a sigmoidal dose–response curve.

Determination of inhibitory activity against lipid peroxidation

Antioxidant activity was determined by the thiocyanate method.²⁷ Serial dilutions were carried out with the stock solution (1 mg/mL) of the extracts, and 0.5 mL of each solution was added to linoleic acid emulsion (2.5 mL, 40 mM, pH 7.0). The linoleic acid emulsion was prepared by mixing 0.2804 g of linoleic acid and 0.2804 g of Tween-20 as emulsifier in 50 mL of 40 mM phosphate buffer, and the mixture was then homogenized. The final volume was adjusted to 5 mL with 40 mM phosphate buffer, pH 7.0. After incubation at 37°C in the dark for 72 hours, a 0.1-mL aliquot of the reaction solution was mixed with 4.7 mL of ethanol (75%), 0.1 mL of FeCl₂ (20 mM), and 0.1 mL of ammonium thiocyanate (30%). The absorbance of this mixture was measured at 500 nm, and the mixture was stirred for 3 minutes. AA, GA, α-tocopherol, and butylated hydroxytoluene were used as reference compounds. To eliminate the solvent effect, the control sample, which contained the same amount of the solvent added to the linoleic acid emulsion in the test sample and reference compound, was used.

Measurement of ferrous ion chelating ability

The ferrous ion chelating ability was measured by the decrease in absorbance at 562 nm of the iron(II)–ferrozine complex.^28,29 One milliliter of 0.125 mM FeSO₄ was added to 1.0 mL of sample (with different dilutions), followed by 1.0 mL of 0.3125 mM ferrozine. The mixture was allowed to equilibrate for 10 minutes before measurement of the absorbance.

Determination of hydroxyl radical scavenging activity

The ability of K. vitifolia to inhibit non–site-specific hydroxyl radical-mediated peroxidation was carried out according to the method described by Hinneburg et al.³⁰ The reaction mixture contained 100 μL of extract dissolved in water, 500 μL of 5.6 mM 2-deoxy-d-ribose in KH₂PO₄–NaOH buffer (50 mM, pH 7.4), 200 μL of premixed 100 μM FeCl₃ and 10⁴ mM EDTA (1:1 vol/vol) solution, 100 μL of 1.0 mM H₂O₂, and 100 μL of 1.0 mM aqueous AA. Tubes were vortex-mixed and incubated at 50°C for 30 minutes. Then, 1 mL of 2.8% trichloroacetic acid and 1 mL of 1.0% thiobarbituric acid were added to each tube. The samples were vortex-mixed and heated in a water bath at 50°C for 30 minutes. The extent of oxidation of 2-deoxyribose was estimated from the absorbance of the solution at 532 nm. The percentage inhibition values were calculated from the absorbance of the control and of the sample, where the controls contained all the reaction reagents excep the extract or positive control substance. The values are presented as means of triplicate analyses.

HPLC analysis

Individual phenolic compounds were quantified by reversed-phase HPLC analysis. Samples were injected in the Waters (Milford, MA, USA) HPLC system consisting of model 1525 binary pumps, a thermostat, and a model 717+ autosampler connected to a Waters model 2996 diode array detector. Chromatograms were gathered in three-dimensional mode with extracted signals at specific wavelengths for different compounds (370, 326, and 254 nm, respectively). Separation of phenolics was performed in a Symmetry C-18 RP column (125×4 mm, 5-μm-diameter particles) (Waters) connected to the appropriate guard column. Two mobile phases, A (0.1% phosphoric acid) and B (acetonitrile), were used at a flow rate of 1 mL/minute with the following gradient profile: the first 20 minutes from 10% to 22% B, the next 20 minutes of linear rise up to 40% B, followed by 5 minutes of reverse to 10% B and an additional 5 minutes of equilibration time. Data acquisition and spectral evaluation for peak confirmation were carried out by Waters Empower 2 software. All standards for HPLC analysis were of analytical grade and were purchased from Sigma and Alfa Aesar.

Statistical analysis

The results are presented as mean±SD values of three determinations. Statistical analyses were performed using Student's t test and one-way analysis of variance. Multiple comparisons of means were performed by Least Significant Difference test. A probability value of P<.05 was considered significant. All computations were made by using SPSS version 11.0 statistical software (SPSS Inc., Chicago, IL, USA). IC₅₀ values were calculated by nonlinear regression analysis from the sigmoidal dose–response inhibition curve.

Results And Discussion

Phenolic compounds and flavonoids have been reported to be associated with antioxidant action in biological systems, mainly due to their reduction–oxidation properties, which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides.³¹ One of the more prominent properties of flavonoids is their excellent radical scavenging ability. It is also a valuable aspect for therapeutic and prophylactic applications of flavonoids (e.g., after infection, inflammation, burns, or radiation injury).³² The activity of crude methanol extracts is due to the presence of flavonoid monomers and polymers (condensed tannins), hydrolyzable tannins, and phenolics. Polyphenolic compounds from plants such as condensed and hydrolyzable tannins have been shown to be powerful antioxidants.³³ Furthermore, it was reported that tannins are 15–30 times more effective in quenching peroxyl radicals than simple phenolics. Therefore, tannins should be considered as important biological antioxidants.³⁴ The results on total phenolic, flavonoid, condensed tannin, and gallotannin content, and total antioxidant capacity are given in Table 1. Total phenolics, flavonoids, condensed tannins, and gallotannins were 85.25±0.69 mg of GA/g, 45.32±0.55 mg of RU/g, 54.25±0.75 mg of GA/g, and 41.74±0.55 mg of GA/g, respectively. The results showed that the ethanolic extract of K. vitifolia possesses antioxidant activity, with total antioxidant capacity being 75.45±0.68 μg of AA/g.

IC₅₀ values were determined for each measurement: 47.45±0.55 μg/mL for DPPH free radical scavenging activity, 35.35±0.68 μg/mL for inhibitory activity against lipid peroxidation, 95.25±0.52 μg/mL for hydroxyl radical scavenging activity, and 31.50±0.35 μg/mL for chelating ability (Table 2).

Table 2.

Antioxidant Activity of the K. vitifolia Extract Tested and Standards

	IC₅₀ (μg/mL)
Plant species	DPPH scavenging activity	Inhibitory activity against lipid peroxidation	Metal chelating activity	Hydroxyl radical scavenging activity
K. vitifolia	47.45±0.55	35.35±0.68	31.50±0.35	95.25±0.52
GA	3.79±0.69	255.43±11.68	—	59.14±1.10
AA	6.05±0.34	>1,000	—	160.55±2.31
BHT	15.61±1.26	1.00±0.23	—	33.92±0.79
α-Tocopherol	—	0.48±0.05	—	—

Results are mean±SD values from three experiments. The 50% inhibitory concentration (IC₅₀) values were determined by nonlinear regression analysis.

BHT, butylated hydroxytoluene; DPPH, 2,2-diphenyl-1-picrylhydrazyl.

HPLC analysis showed rosmarinic acid to be the dominant component in the extract, with its proportion being 2.937 mg/g of extract (Fig. 1). A lower content was observed for p-hydroxybenzoic acid and caffeic acid (0.182 mg/g of extract and 0.103 mg/g of extract, respectively) (Table 3). Previous studies showed that rosmarinic acid has many biological activities, including astringent, antioxidant, anti-inflammatory, antimutagen, antibacterial, and antiviral activities.¹⁶ Phenolic compounds such as rosmarinic acid can provide protection against cancer. Moreover, rosmarinic acid contributes to the antioxidant activity of plants used in the cosmetic industry.¹⁶

FIG. 1.

Dominant components of the ethanolic extract of K. vitifolia. See Table 3 for peak assignments. mAU, milli-absorbance units.

Table 3.

Quantitative and Qualitative Contents of Phenolic Components in K. vitifolia Extract

K. vitifolia component number ^a	Component	Retention time (minutes)	Concentration (mg/g of extract)
1	GA	0.889	0.09
4	p-Hydroxybenzoic acid	2.355	0.182
5	Caffeic acid	3.019	0.103
7	Chlorogenic acid	3.696	0.044
8	Syringic acid	4.839	0.042
10	p-Coumaric acid	6.887	0.031
11	Ferulic acid	9.421	0.093
15	Rosmarinic acid	14.071	2.937
17	Quercetin	16.365	0.004

See Figure 1.

The results on antimicrobial activity obtained by the dilution method are given in Table 4; MICs were determined for eight selected indicator strains. The results presented in Table 4 reveal antimicrobial activity of the ethanolic extract of K. vitifolia within the concentration range of 15.62 μg/mL to 62.50 μg/mL. The highest susceptibility to the ethanolic extract of K. vitifolia among the bacteria tested was exhibited by B. subtilis ATCC 6633, S. aureus ATCC 25923, and K. pneumoniae ATCC 13883 (MIC=15.62 μg/mL), followed by E. coli ATCC 25922 and P. mirabilis ATCC 14153 (MIC=31.25 μg/mL) and P. vulgaris ATCC 13315 (MIC=62.50 μg/mL). Among the fungi, C. albicans ATCC 10231 (MIC=15.62 μg/mL) showed the highest susceptibility, and A. niger ATCC 16404 (MIC=31.25 μg/mL) had the lowest.

Table 4.

Minimum Inhibitory Concentrations of the Ethanolic Extract of K. vitifolia and Standard Drugs for Eight Indicator Strains

	MIC (μg/mL)
Microbial strain	K. vitifolia ethanolic extract	Amracin	Ketoconazole
S. aureus ATCC 25923	15.62	0.97	—
K. pneumoniae ATCC 13883	15.62	0.49	—
E. coli ATCC 25922	31.25	0.97	—
P. vulgaris ATCC 13315	62.50	0.49	—
P. mirabilis ATCC 14153	31.25	0.49	—
B. subtilis ATCC 6633	15.62	0.24	—
C. albicans ATCC 10231	15.62	—	1.95
A. niger ATCC 16404	31.25	—	0.97

MIC, minimum inhibitory concentration.

Conclusions

Antioxidant and antimicrobial properties of various extracts of many plants are of great interest in both fundamental science and the food industry since their potential use as natural additives has emerged from a growing tendency to replace synthetic antioxidants by natural ones. The present study confirmed the antimicrobial and antioxidant activities of the ethanolic extract of the Serbian plant K. vitifolia. Determination of polyphenolic components by HPLC analysis revealed the presence of high amounts of rosmarinic acid responsible for the reported antimicrobial activity of K. vitifolia. The results obtained suggest that the extract of the endemic species K. vitifolia shows antimicrobial activity under in vitro conditions against the tested fungi as well as antioxidant activity relative to the control antioxidants.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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