Biological activities of Umbilicaria crustulosa ( Ach . ) Frey acetone extract

This paper reports for the first time the effect of an acetone extract of Umbilicaria crustulosa on the micronucleus distribution of human lymphocytes, and on the cholinesterase activity and antioxidant activity by the cupric ion reducing antioxidant capacity (CUPRAC) method. Additionally, the total phenolic compounds (TPC) and the antioxidant properties were estimated via DPPH, ABTS and TRP assays. Moreover, the antibacterial activity against two Gram-positive and three Gram-negative bacteria were determined. Acetone extract of U. crustulosa at concentration of 1 and 2 μg mL-1 decreased a frequency of micronuclei (MN) by 10.8 and 16.8 %, respectively, acting more or slightly less than the synthetic protector amifostine (AMF, WR-2721, 11.4 %, at concentration of 1 μg mL-1). The tested extract did not inhibit cholinesterase activity nor did it exhibit activity toward the examined bacteria. The extract reduced the concentration of DPPH and ABTS radicals by 88.7 and 96.2 %, respectively. Values for total reducing power (TRP) and cupric reducing capacity (CUPRAC) were 0.6197±0.0166 μg ascorbic acid equivalents (AAE) per mg of dry extract, and 19.7641±1.6546 μg trolox equivalents (TE) per mg of dry extract, respectively. The total phenol content was 350.4188 ±14.587 μg gallic acid equivalents (GAE) per mg of dry extract. The results of the present study showed that U. crustulosa acetone extract is a promising candidate for in vivo experiments considering its antioxidant activity and protective effect on human lymphocytes.


INTRODUCTION
Lichens of the genus Umbilicaria are generally known as rock tripe.These species grow on siliceous rocks usually in the Alpine vegetation zone and the Northern hemisphere. 1 Some of these species are used as food and represent a part of folk medicine. 2,3Umbilicaria crustulosa (Ach.)Frey is known as a crusty novel lichen.Serina and Arroyo found two U. crustulosa chemotypes in samples growing in Spain: gyrophoric acid and crustinic acid chemotype.Beside crustinic and gyrophoric acids, Umbilicaria species are characterized by the presence of lecanoric, hiascic, ovoic, umbilicaric, salazinic and norstictic acids. 3,4he presence of phenolic groups in the structure of the constituents of U. crustulosa extracts results in their antioxidant activity. 3,5Antioxidants could prevent oxidation of biomolecules in food as well as in human cells.Industrial antioxidants are mostly synthetic components which could have negative impacts on human health. 6Natural antioxidants are very important source of protection against free radicals, which could be related to many disease such as Alzheimer's disease, atherosclerosis, emphysema and many forms of cancer. 7,8A decrease in the amount of acetylcholine in the brain is the hallmark of Alzheimer's disease.Some synthetic cholinesterase inhibitors are used for the treatment of disease but an alternative approach could be the utilization of natural anticholinesterases. 9 According to published data, Hypogimnia physodes extracts and lichens secondary metabolites have an impact on micronucleus (MN) expression in peripheral blood lymphocytes. 10,11Micronuclei result from lesions/adducts at the level of DNA or chromosomes, or at the level of proteins directly or indirectly involved in chromosome segregation.The cytokinesis-block micronucleus technique enables micronuclei to be specifically scored in cells that have completed nuclear division and is, therefore, not influenced by variations in cell division kinetics, and it has been shown to be a sensitive and reliable index of chromosome damage. 12aking into account the above-mentioned biological activities of the lichens, the aim of present study was to evaluate for the first time effect of U. crustulosa acetone extract on the micronucleus distribution in human lymphocytes, and the effect on cholinesterase and antioxidant activity by the CUPRAC method.
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Extraction
Extracts were prepared in triplicate according to the following procedure: powdered lichen material (10 g) was first subjected to ultrasound-assisted extraction with acetone (50 mL; 30 min in an ultrasound bath, type UZK 8; manufactured by Maget, Bela Palanka, Serbia.Subsequently, the extract was left in the dark (at room temperature) for an additional five days.Dry residues of the extracts were obtained using a rotary evaporator with the water bath set at 40 °C.The extract yield was 7.4±0.5 %.

HPLC analysis
HPLC analysis was performed on an Agilent, Zorbax Eclipse XDB-C18, 5 μm, 4.6 mm×150 mm column using an Agilent 1200 series liquid chromatograph (equipped with a diode array detector (DAD), Chemstation Software (Agilent Technologies), a quaternary pump, an online vacuum degasser, auto sampler and a thermostated column compartment.The mobile phase, methanol-water-formic acid (80:20:0.2volume ratios), was pumped at a flow rate of 0.5 mL min -1 , the injection volume was 5 μL (concentration 10 mg of the dry extract per 1 mL of acetone), at 25 °C.The spectra were acquired in the range 190-400 nm and the chromatogram plotted at 254 nm.Identification was conducted using retention times and UV spectra.
The statistical analysis was performed using Origin software package version 7.0.The statistical significance of difference between the data pairs was evaluated by analysis of variance (one-way ANOVA) followed by the Tukey test.Statistical difference was considered significant at p < 0.01 and p < 0.05.

Total phenolic content and antioxidant activity
Determination of the total phenolic content (TPC) and four antioxidant assays, i.e., DPPH and ABTS scavenging radical capacity, CUPRAC (cupric reducing antioxidant capacity) and TRP (total reducing power), were performed as previously described. 13,14All spectrophotometric assays were conducted on a Perkin Elmer lambda 15 double beam UV/Vis spectrophotometer.For all the above-mentioned experiments, the concentration of the sample solution was 15 mg of extract per mL of methanol.All analyses were performed in triplicate.The results are presented as mean ± standard deviation (SD).

Cholinesterase activity
Assessment of the effect of the extract on cholinesterase activity was performed as previously described. 15The activity was measured spectrophotometrically using a Konelab 20 analyzer (Thermo Fisher Scientific, Helsinki, Finland) with thermostated flow cells, length 7 mm (at a wavelength 405 nm).The sample concentration was 10 mg of dry extract per 1 mL of DMSO.A solution of neostigmine bromide at a concentration of 200 µg mL -1 was used as the reference standard.

Antimicrobial activity
Antimicrobial activity was evaluated against two Gram-positive (Bacillus subtilis subsp.spizizenii ATCC 6633 and Staphylococcus aureus ATCC 6538) and three Gram-negative (Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027 and Salmonella abony NCTC 6017) bacteria.Analysis was performed according to the National Committee for Clinical Laboratory Standards (NCCLS). 16Each test was performed in triplicate.The sample concentration was 1 mg per disk (diameter 12 mm).Streptomycin and chloramphenicol were used as positive controls, at a concentration of 10 mg and 30 mg per disk, respectively.

HPLC analysis
The HPLC chromatogram of the acetone extract of U. crustulosa and the UV spectra of the identified constituents are presented in Fig. 1.
Chemical composition and structures of the constituents are given in Table I.The most abundant component of the acetone extract of U. crustulosa is the tridepside gyrophoric acid and this sample could be classified as a gyrophoric acid chemotype. 4he major components of the extract were the didepside lecanoric acid and the tridepside gyrophoric acid, comprised of two and three, respectively, orsellinic acid units joined with ester bonds at the para-positions.Crustinic acid is an exception, as it has the ester bonded at the meta-position of the third ring.No direct evidence is available on the biosynthesis of meta-depsides.The co-occurrence and interconversion of the depsides meta-and para-scrobiculin, which exist in dynamic equilibrium, in Lobaria scrobiculata led to the hypothesis that meta-depsides are formed from para-depsides via an intramolecular rearrangement after hydroxylation of the carbon at the meta-position of the B-ring. 17

Cytokinesis-block micronucleus assay (CBMN)
The extract was tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using the cytochalasin-B blocked MN assay at concentrations of 1.0, 2.0 and 3.0 μg mL -1 .The frequencies and distribution of MN in human lymphocytes were scored.The statistical significance of difference between the data pairs was evaluated by analysis of variance (Oneway ANOVA) followed by the Tukey test.The results are presented in Table II.The cell cultures treated with amifostine (radioprotectant, previously known as WR-2721) at a concentration of 1 μg mL -1 resulted in a decrease of the MN frequency of 11.4 % compared to the control cell cultures (statistically significant, p < 0.05).The U. crustulosa extract at a concentration of 2 μg mL -1 gave a _________________________________________________________________________________________________________________________ (CC) 2017 SCS.
Available on line at www.shd.org.rs/JSCS/BIOLOGICAL ACTIVITIES OF U. crustulosa 147 decrease in the MN frequency of 16.38 %, which was higher than that with amifostine, while at a concentration of 1 μg mL -1 , the effect was 10.8 %, which is slightly less than the effect of amifostine.The U. crustulosa extract at a concentration of 3 µg mL -1 was less effective (6.2 %) than amifostine.
Since the number of micronuclei serves as an indicator of DNA damage, these results indicate that the examined extract at a concentration of 2 μg mL -1 protects DNA.Previous results of H. physodes extracts suggested a similar conclusion.A diethyl ether extract of the lichen H. physodes at a concentration of 1 μg mL -1 caused a significant decrease in the frequency of micronuclei, 17.4 %, while it was less effective at a concentration of 2 μg mL -1 . 11he effect of examined extracts on cell proliferation was investigated by determining the cytokinesis-block proliferation index (CBPI), calculated as [(M I + 2M II + 3(M III + M IV )]/N, where M I-IV represent the number of cells with 1 to 4 nuclei, respectively, and N is the number of cells scored. 18The comparable CBPI values of extracts, amifostine WR-2721 untreated cells have confirmed absence of the impact on the cell proliferation.This fact is important for validity of the results because MN expression is dependent on cell division.

The total phenolic content and antioxidant activity
TPC analysis showed a high content of phenolic components in the acetone extract of U. crustulosa, 350.4188±14.587µg GAE mg -1 due to the presence of the tridepside gyrophoric acid and the didepside lecanoric acid (total 92.0 % of the sample).It was not possible to compare the present results expressed as galic acid equivalents (GAE) with those of Kosanic et al. because they expressed the amount of total phenolic compounds as the pyrocatechol equivalent. 19he assessment of the DPPH and ABTS scavenging activity showed that extract reduced the concentration of DPPH and ABTS radicals by 88.7 and 96.2 %, respectively, which is consistent with previously published results.Namely, Kosanic et al. found that the scavenging activity of a methanol extract of U. crustulosa was 79.85 %, while Buçukoglu et al. found that the values of the DPPH scavenging activity of gyrophoric and leconaric acids were 50.96 and 32.48 %, respectively. 19,20he value of the total reducing power ability (TRP, ability of antioxidants to reduce hexacyanidoferrate (III) to hexacyanidoferrate (II), which leads to an increase in the absorbance of the reaction mixtures) was 0.6197±0.0166mg ascorbic acid equivalents (AAE) per mg dry extract weight.Again, it was not possible to compare the obtained results with those previously published due to the different expression of the results (absorbance vs. AAE). 19he CUPRAC assay has some advantages compared to the other experiments since the reagent is stable, accessible, low-cost, and the reaction (reduction of a cupric neocuproine complex (Cu(II)-Nc) by antioxidants to the cuprous form (Cu(I)-Nc)) is realized at nearly physiological pH.The result obtained for the U. crustulosa acetone extracts (19.7641±0.0166mg trolox equivalents (TE) per mg dry extract) is close to the highest CUPRAC capacity of mushrooms (21.738-4.164mg TE per mg dw). 13

Cholinesterase activity
Results obtained from the screening of the interaction of the extract with cholinesterase from pooled human serum showed that U. crustulosa possess a weak activation effect on the cholinesterase activity.In the conducted experiment, neostigmin bromide (commercial cholinesterase inhibitor) inhibited cholinesterase to extent of 96.6 % while U. crustulosa extract manifested weak activation effect on cholinesterase to an extent of 1.6 %.

Antibacterial
The results of the antibacterial assays against two Gram-positive (B.subtilis subsp.spizizenii ATCC 6633 and S. aureus ATCC 6538) and three Gram--negative bacteria (E. coli ATCC 8739, P. aeruginosa ATCC 9027 and S. abony NCTC 6017) showed that the acetone extract of U. crustulosa had no activity against the tested bacteria at a concentration of 1 mg per disk.These results were not entirely in accordance with those previously published.Namely, Rankovic   20,21 Since gyrophoric acid participated 68.5 % to the extract composition, the results obtained in the present study could be explained by its inactivity.

TABLE I .
Composition (as percentages of the total peak area of the HPLC chromatograms recorded at 254 nm) and structures of the constituents of the U. crustulosa extract TABLE II.Incidence of MN, cytokinesis-block proliferation index, distribution of MN per cells (mean ± SD) and frequency of MN in cell cultures of human lymphocytes treated with different concentration of Umbilicaria crustulosa acetone extract (AE); MN/1000 Bn cells - incidence of micronuclei in 1000 binucleated cells; % Bn cells with micronuclei; MN/Bn cellsincidence of micronuclei in binucleated cells; CBPI -cytokinesis-block proliferation index; Frequency of MN -incidence of MN present as % from control groups in cell cultures of human lymphocytes treated with different concentration of extracts; a* -statistically significant difference from control at p < 0.05 et al. and Kosanic et al. reported no activity toward Escherichia coli, Botrytis cinerea and Candida albicans but moderate antimicrobial activity of methanol and acetone extracts of U. crustulosa to several bacteria and fungi, such as: Bacillus mycoides, Bacillus subtilis, Enterobacter cloaceae, Klebsiella pneumoniae, Staphylococcus aureus, Aspergillus flavus, Aspergillus fumigatus, Fusarium oxysporum, Mucor mucedo, Paecilomyces variotii , Penicillium purpurescens, Penicillium verrucosum, Saccharomyces cerevisiae, Trichoderma harzianum. 2,19Bucukoglu et al. and Candan et al. found that gyrophoric acid was not effective against B. subtilis, E. coli, K. pneumoniae, P. aeruginosa and Salmonela typhimurium.