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Abstract

Introduction

Cladonia rangiformis, also known as reindeer lichen, has been used for various remedies in folk medicine. The rich nutritional value of this lichen is reflected in the fact that it contains atranorin, which has been proven to have numerous biological activities.

Objective

The chemical composition, antioxidant, anticholinesterase, and antigenotoxic activities of the C. rangiformis acetone extract were investigated. The novelty of this work is that anticholinesterase activity, protective effect on human lymphocytes, and antioxidant activity by the cupric-reducing antioxidant capacity (CUPRAC) method of C. rangiformis extract were determined for the first time, as well as gas chromatography - mass spectrometry (GC-MS) profile.

Methods

Chemical composition was carried out by high-performance liquid chromatography (HPLC)-diode array detector and GC-MS. Antigenotoxic effect was evaluated in human lymphocytes by cytokinesis-block micronucleus assay. Total phenolic content, DPPH and ABTS radical scavenging capacities, CUPRAC, and total reducing power were determined spectrophotometrically. Determination of acetylcholinesterase activity was performed by Ellman's colorimetry assay. The statistical analysis of variance (One-way ANOVA) was performed using Origin software package version 7.0.

Results

HPLC method was used to identified 3 compounds with atranorin as predominant constituent (97.2%). Fumarprotocetraric and atraric acid were represented in a much smaller mutually approximate amount, 0.3 and 0.5%, respectively. Rangiformic acid was the most abundant volatile compound. Acetone extract of C. rangiformis (2 μg/mL) decreased the frequency of micronuclei by 15.5% while the radioprotectant amifostine reduced the frequency of occurrence of micronuclei by 11.4%. On the contrary, at a concentration of 1.0 mg/mL the extract exhibited an activating effect on cholinesterase (2.0%), while at a concentration of 10.0 mg/mL it showed a weak inhibitory effect (7.3%). The total phenol content was 132.71 μg gallic acid equivalents per mg of dry extract. The IC₅₀ value for the DPPH experiment was 16.19 mg/mL and for ABTS was 11.80 mg/mL. Cupric reducing capacity and total reducing power were 17.81 μg Trolox equivalents per mg of dry extract and 0.45 μg ascorbic acid equivalents per mg of dry extract, respectively.

Conclusion

In addition to previously identified compounds in the acetone extract, atraric acid was identified for the first time by using HPLC method, and orcinol, β-orcinol, lauric alcohol, atranol, and rangiformic acid by using GC-MS method. The results of biological activity proved that acetone extract had antioxidant capacity and weak anticholinesterase activity. Reduction of the number of micronuclei in human lymphocytes, classified C. rangiformis as promising substances source with beneficial effects on DNA damage.

Keywords

anticholinesterase activity antigenotoxic effect antioxidant activity bioactivity chemical composition

Introduction

Lichens are complex organisms that arise from representatives of 2 different empires: algae or cyanobacteria (the photobiont) which live among filaments of multiple fungal species (the mycobiont). Photobionts and mycobionts live in unique symbiotic relationship and unequally depend on each other. Thus, mycobionts give lichens their shape and appearance (they often contribute 80%-90% of lichen biomass), providing protection against desiccation by supplying water to the algae, protect them from sunlight, and absorb mineral nutrients from the substrate (soil, trees, rock) on which lichens were found.¹ The photobionts in turn, synthesize organic nutrients as products of photosynthesis, in such a way that reduces atmospheric carbon dioxide into organic carbon sugars to feed both symbionts.²

Lichens produce a number of secondary metabolites, known as “lichen substances,” mainly from fungal metabolism.³ These metabolites include aliphatic, cycloaliphatic, aromatic, and terpene components (depsides, depsidones, dibenzofurans, xanthones, and terpene derivatives). Depsides, depsidones, tridepsides, and tetradepsides are the most numerous class of metabolites in lichens.⁴ Determining the chemical composition of lichen substances was the subject of interest of many research teams, which also had focus their research on examining the biological activities that lichens possess, including: antibacterial,⁵ antiviral,⁶ antioxidant,⁷ analgesic, antipyretic,⁸ cytotoxic, fungicidal, herbicidal, insecticidal,⁹ and many others. Previous studies have shown that lichens of the genus Cladonia have biological activities such as antibacterial, anticancer, antioxidant, hepatoprotective, antidiabetic, insecticidal, anthelminthic, expectorant, etc¹⁰

It is known that the biological activities of lichens have a therapeutic effect on various diseases and conditions and that they have been used in the traditional medicine of many countries for treatment from ancient times.¹¹ For example, Cladonia pyxidata is a valued folk remedy used for the treatment of whooping cough and as an antipyretic.¹² Interestingly, in the Great Lakes region of North America, newborn babies were bathed in water that had previously been boiled with the lichen C. rangiferina.¹³

The micronucleus (MN) test enables the determination of the genotoxicity and chromosomal instability, which is believed to be a major contributor to cancer progression. This test is based on the detection of genetic damage caused by chemicals through the formation of MN in treated cells. The number of micronuclei (MN) serves as an indicator of DNA damage.¹⁴

The cholinesterase inhibitors (anticholinesterase) block the normal breakdown of acetylcholine, which increases its concentration level and prolongs the action of acetylcholine as a neurotransmitter in the central and peripheral nervous system. These inhibitors are considered to delay the progression of some diseases or disorders, such as Alzheimer's disease, Parkinson's disease, and dementia of Lewy bodies.¹⁵

Antioxidants of natural origin have attracted special interest due to their effectiveness in preventing destructive processes caused by oxidative stress. They have the ability to stabilize or deactivate free radicals, often before they can attack a biological cell. Lichens could be a good source of natural antioxidants because they are rich in secondary metabolites in the form of phenolic compounds, well known for their antioxidant properties.⁷

Previous phytochemical investigation on Cladonia rangiformis acetone extract showed the presence of atranorin, fumarprotocetraric and protocetraric acid¹⁶; rangiformic and norrangiformic acid.¹⁷ Fatty acids and their esters, atraric acid, phytol and orsellinalaldehyde were identified after transesterification of the lichen sample.¹⁶

Kotan et al.¹⁸ proved a significant protective effect of the C. rangiformis methanol extract against oxidative stress caused by the carcinogenic substance aflatoxin B1. Also, the cytotoxic effects of the C. rangiformis chloroform extract on human breast cancer MCF-7 cells were previously studied.¹⁹ The studies of the radical scavenging ability and reducing power capacity on the Tunisian lichen C. rangiformis acetone extract showed that it possesses antioxidant activity.²⁰ Kocakaya et al.²¹ found ABTS radical removal effect of the C. rangiformis methanolic extract.

Despite the new research presented in this paper and everything already known about the lichens, the potential of lichens as a source of drugs has not been fully explored. Therefore, and bearing in mind published paper dealing with both high-performance liquid chromatography (HPLC) and gas chromatography - mass spectrometry (GC-MS) C. rangiformis composition,¹⁶ the aim of this investigation was designated as determination of the chemical composition of C. rangiformis acetone extract using HPLC and GC-MS methods. Additionally, total phenolic content (TPC), DPPH and ABTS scavenging radical capacity, cupric-reducing antioxidant capacity (CUPRAC) and total reducing power (TRP) as well as the effect on the formation of MN in human blood plasma lymphocytes and on serum cholinesterase activity were examined. As far as we know, the effect of acetone C. rangiformis extract on serum cholinesterase activity, antigenotoxic effect, antioxidant activity using the CUPRAC method and GC-MS profile were investigated for the first time.

Materials and Methods

Lichen Material

The lichen sample of C. rangiformis Hoffm. was collected in the Quercus cerris forest, in the village of Duga poljana on the slopes of Suva planina (492 m above sea level; coordinates 43°11′ N, 22°04′ E). Herbarium voucher has been deposited in the Herbarium of the Department of Biology and Ecology of the Faculty of Sciences and Mathematics, University of Niš, under the acquisition number 10 889. The identification of lichen was performed by Dr Bojan Zlatković from the Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš.

The collected lichen thallus was air dried in a place protected from light, for 10 days. The dry lichen material was stored in a dark place and protected from moisture at room temperature.

Preparation of Lichen Extract

The dried and powdered lichen material (10 g) was extracted with acetone (50 mL; Sigma Aldrich) in triplicate and left in the ultrasonic bath (30 min; Maget, Serbia),²² and then in a dark place and at room temperature, for 5 days. The dry extract was obtained using a rotary vacuum evaporator (KNF Laboxact, Germany) at 40 °C. The extract yield was 7.6 ± 0.5%. Dry acetone extract was used to investigate biological activities.

The extract yield was calculated based on the following formula:²³

total yield (%) = \frac{total extract mass}{mass of lichen} \times 100

For the purpose of HPLC and GC-MS analysis, dry lichen material (2 g ) were extracted with 10 mL acetone (Sigma Aldrich), using an ultrasonic bath (Maget) for 30 min, and left in a dark place and at room temperature overnight. The extracts were filtered using a microfilter (0.45 μm; Whatman, Sigma Aldrich) into vials for HPLC and GC-MS analysis.

HPLC Analysis

HPLC analysis was performed as previously described with some changes.²⁴ Analysis was carried out on an Agilent, Zorbax Eclipse XDB-C18, 5 μm, 4.6 × 150 mm column, by using a liquid chromatograph (Agilent 1200 series), equipped with a diode array detector, Chemstation Software (Agilent Technologies), a quaternary pump, an online vacuum degasser, an autosampler and a thermostatted column compartment. The mobile phase, methanol/water/formic acid = 80/20/0.2 (v/v/v) (Sigma Aldrich), was pumped at a flow rate of 0.5 mL/min, the injection volume was 5 μl (concentration 10 mg of the dry extract per 1 mL of solvent, filtered through 0.45 μm filter), at 25 °C. The spectra were acquired in the range 190 to 400 nm and chromatograms plotted at 254 nm. Identification of components was achieved by comparing retention times and UV spectra of constituents from the literature^25,26 and preisolated component (atranorin) under identical experimental conditions.²⁷

GC-MS Analyses

The chemical composition of the acetone extract of C. rangiformis is investigated by GC-MS (in triplicate), which was carried out using a 7890/7000B GC-MS/MS triple quadrupole system (Agilent Technologies, equipped with a Combi PAL auto sampler). GC-MS analysis was performed as previously described with some changes.²⁸ The fused silica capillary column HP-5MS (5% phenylmethylsiloxane, 30 m × 0.25 mm, film thickness 0.25 μm) was used. The injector and interface operated at 250 and 300 °C, respectively. The temperature program: 70 °C for 2.25 min, 5 °C/min to 300 °C, then isothermally held for 10 min. The carrier gas was helium (Messer) with a flow of 1.0 mL/min. 2 μl of samples were injected split ratio 5:1. Post run: Back flash for 1.89 min, at 280 °C, with helium at 50 psi. MS conditions were as follows: Ionization voltage of 70 eV, acquisition mass range 50 to 650, scan time 0.32 s. The percentage composition was computed from the TIC peak areas. Constituents were identified by comparison of their linear retention indices (relative to C₈—C₄₀ alkanes on the HP-5MS column) with literature values,^29-31 and their MS with those from Wiley 6, NIST02 and Mass Finder 2.3, by the application of the AMDIS software (The Automated Mass Spectral Deconvolution and Identification System, Ver. 2.1, DTRA/NIST, 2011).

Cytokinesis-Block MN Assay

Cytokinesis-block MN (CBMN) assay was performed as previously described.^32,33 The blood samples were obtained at the Medical Unit of Nuclear Facilities of Serbia in accordance with the current health and ethical regulations in Serbia (Parliament of the Republic of Serbia. Law on Health Care. Official Gazette of the Republic of Serbia 2005; 107: 112-161).

The cell culture lymphocytes were treated with 1.0, 2.0 and 3.0 μg/mL of the examined acetone extracts. Previously, studies showed that lichen extracts and isolated secondary metabolites at the applied concentration of 2.0 μg/mL being the most effective. Higher concentrations of extracts and compounds under the study causes smaller decrease in the frequency of MN.^34-38 Therefore, the concentrations of 1.0, 2.0, and 3.0 μg/mL were chosen for this study.

Amifostine WR-2721 (98% S-2 [3-aminopropylamino]-ethylphosphothioic acid; Marligen-Biosciences) at concentration of 1 μg/mL was used as a negative control. The alkylating agent mitomycin C (MMC) (Bristol-Myers Squibb), prepared by diluting the drug in phosphate buffer (Phosphate Buffered Saline, pH 7.4, liquid, sterile-filtered, suitable for cell culture; Merck; prod. num. 806 552), and added at a final concentration of 0.1 μg/mL in lymphocytes cultures was used as a positive control. Three experiments were performed for each sample. The results are expressed as the means ± standard deviation (SD).

All cultures were incubated in a thermostat at 37 °C. Treatment with the investigated extract lasted 19 h, after which all cultures were washed with pure medium, transferred to 5 mL of fresh RPMI 1640 medium (RPMI 1640 Medium + GlutaMAX + 25 mM HEPES; Invitrogen-Gibco-BRL) and incubated an additional 72 h for MN.

Approximately 2 × 10⁶ blood lymphocytes were set up in 5 mL RPMI-1640 medium supplemented with 15% of calf serum and 2.4 μg/mL of phytohemaglutinin (Invitrogen-Gibco-BRL). One hour after initiating the cell stimulation, lichen extract dissolved in water was added to the samples. The incidence of spontaneously occurring MN in control samples was scored. Cytochalasin B (Invitrogen-Gibco-BRL, Vienna, Austria) at a final concentration of 6 µg/mL was added to the samples after 44 h of culture, and the lymphocyte cultures were incubated for a further 24 h. After 72 h of culture, the cells were washed with 0.9% NaCl (Merck, Sharp and Dohme GMBH.), collected by centrifugation and treated with hypotonic solution at 37 °C. The hypotonic solution consisted of 0.56% KCl + 0.9% NaCl (mixed in equal volumes). The cell suspension was prefixed in methanol/acetic acid (3:1), washed 3 times with fixative, and dropped onto a clean slide. The slides were air dried and stained with alkaline Giemsa 2% (Sigma-Aldrich, Vienna, Austria). At least 1000 binucleated (BN) cells per sample were scored, registering MN.

The effects of investigated compounds on cell proliferation were estimated by the cytokinesis-block proliferation index. For the analysis of MN, only BN cells with well-preserved cytoplasm were scored (under a light microscope with a 40 × 10 magnification). The number of BN cells with 1, 2, 3, or more MN was then tabulated. The data for each treatment were expressed as the frequency of MN per 1000 BN cells.

The statistical analysis was performed using Origin software package version 7.0. The statistical significance of the 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 < .01 and P < .05.

TPC and Antioxidant Activity

TPC and 4 antioxidant assays: DPPH and ABTS scavenging radical capacity, CUPRAC and TRP were performed as previously described.^39,40 All spectrophotometric assays were conducted on a double beam UV/VIS spectrophotometer Perkin Elmer lambda 15. For all above-mentioned experiments concentration of sample solution was 15 mg of extract per mL of methanol (Sigma Aldrich), while the concentration of all standards was 0.1 mg/mL. All analyses were performed in triplicate. Results are presented as mean ± SD.

Cholinesterase Activity

Assessment of extract effect on cholinesterase activity was performed as previously described.⁴¹ Activity was measured spectrophotometrically using a Konelab 20 analyzer (Thermofisher Scientific) with flow thermostatted cells, length 7 mm (at wavelength 405 nm). Sample concentration was 10 mg of dry extract per 1 mL of DMSO (Sigma Aldrich). Solution of neostigmine bromide (Sigma Co. St. Louis, SAD) at a concentration of 200 μg/mL was used as a reference standard.

Results and Discussion

HPLC Analysis

HPLC was used to analyze the phenolic composition of acetone extract of C. rangiformis. The results are presented in Table 1. The HPLC chromatograms of extract and atranorin are shown in Figures 1 and 2.

Figure 1.

High-performance liquid chromatography (HPLC) chromatogram of acetone extract of Cladonia rangiformis.

Figure 2.

High-performance liquid chromatography (HPLC) chromatogram of atranorin.

Table 1.

Relative Presence of Components of Cladonia rangiformis Acetone Extract (Expressed as a Percentage of the Total Chromatogram Area at 254 nm), Chemical Structures, Class of the Compounds and Their Absorption maxima of the UV spectrum.

Component	Retention time (Rt) (min)	%	Absorption maxima (nm) of the UV spectrum	Compound class
Atraric acid	4.6	0.5	220, 270, 305	Monocyclic aromatic compound
Fumarprotocetraric acid	5.0	0.3	212, 238, 314	Depsidone
Atranorin	22.0	97.2	210, 252, 312	Depside
Total identified (%)		98.0

According to the HPLC chromatogram, a 3 phenolic compounds were identified, representing 98.0% of total examined extract. A depside atranorin was the main component, with a presence of 97.2% in the tested extract. Monoaromatic atraric acid and depsidone fumarprotocetraric acid were considerably less represented (with 0.5% and 0.3%, respectively).

Atranorin is one of the most common lichen secondary metabolite, characteristics for numerous lichen families. Studies on the bioactive properties of atranorin have revealed antibacterial, antifungal, antioxidant and cytotoxic activity, anti-inflammatory properties, and photoprotective capacity.⁴²

It was found in previous research that atranorin, fumarprotocetraric acid, and protocetraric acid were present in the acetone extract of C. rangiformis.¹⁶ Atranorin was the main component, which is in agreement with our study. Atraric acid has not been identified in C. rangiformis extract by HPLC, up to now.

GC-MS Analyses

The chemical composition of volatile components of C. rangiformis acetone extract is shown in Table 2. The TIC chromatogram is shown in Figure 3.

Figure 3.

Gas chromatography and mass spectrometry (GC-MS) chromatogram of acetone extract of Cladonia rangiformis.

Table 2.

Relative Abundance of Identified Volatile Components (%), Experimental (RI) and Literature (RL) Retention Indices, Chemical Formula, MW and Chemical Structures; tr - Content Less Than 0.1%.

Components	RI	RL	Chemical formula	MW, g/mol	%
1,3-Dihydroxy-5-methylbenzene (syn. orcinol)	1370	1369⁴⁴	C₇H₈O₂	124.052	tr
1,3-Dihydroxy-4,5-dimethylbenzene (syn. β-orcinol)	1418	1419²⁸	C₈H₁₀O₂	138.068	0.4
n-Dodecanol (syn. lauric alcohol)	1472	1474⁴⁵	C₁₂H₂₆O	186.198	tr
2,6-Dihydroxy-4-methylbenzaldehyde (syn. atranol)	1550	1549²⁸	C₈H₈O₃	152.147	10.6
Methyl 2,4-dihydroxy-3,6-dimethylbenzoate (syn. atraric acid)	1709	1706²⁸	C₁₀H₁₂O₄	196.073	19.4
(2S)-2-[(2S)-1-methoxy-1-oxohexadecan-2-yl]butanedioic acid (syn. rangiformic acid)	2647	−	C₂₁H₃₈O₆	369.263	66.6
Total amount (%)					97.0

The most intense peak on the chromatogram belongs to rangiformic acid, aliphatic acid, which accounts 66.6% of the total chemical composition of C. rangiformis volatile components. This aliphatic acid does not absorb UV and therefore it could not be detected by HPLC.⁴³

The second most abundant compound in tested extract was atraric acid (19.4%), which was also identified by the HPLC method in this study. It can be assumed that the atraric acid identified by GC-MS has a dual origin. One part was formed by degradation under GC-MS⁴⁴ conditions and the other part was present in the initial extract. Significant amount of atranol (10.6%) was detected in lichen sample, followed by β-orcinol with a very low percentage (0.4%). Orcinol and lauric alcohol were identified in trace amounts in the tested extract.

The HPLC analysis of C. rangiformis acetone extract showed the presence of atranorin with 97.2% of total examined extract. After the degradation process (during GC-MS analysis) of the depside atranorin in a first step mainly generates atraric acid and haematommic acid. After haematommic acid decarboxylation, atranol is formed.⁴⁴

The previous phytochemical investigation by HPLC-MS on C. rangiformis acetone extract showed the presence of rangiformic and norrangiformic acid.¹⁷ Fatty acids and their esters, atraric acid, phytol and orsellinalaldehyde were identified by GC-MS after transesterification of the dried and ground lichen sample.¹⁶ To the best of our knowledge, our results of the GC-MS profile of the acetone extract of C. rangiformis presented here would be the first.

Cytokinesis-Block MN Assay

The acetone extract of C. rangiformis was tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using CBMN assay at concentrations of 1.0, 2.0 and 3.0 µg/mL. The frequency and distribution of MN in human lymphocytes were scored. Formation of MN due to an alkylating agent, MMC, and prevention of MN formation by a DNA repair system agent, amifostine WR-2721, was determined. The possible clastogenic, anticlastogenic or modulating effects of investigated extract was determined on the basis of the action of these 2 agents. The results are presented in Table 3.

Table 3.

Frequency of Micronuclei, Proliferation index of Cytokinesis Block, Distribution and Frequency of Micronuclei in Cells Treated with Different Concentrations of Acetone Extract of Selected Lichen species.

Sample (concentration μg/mL)	MN/1000 Bn cells	Bn cells with MN (%)	MN/Bn cells	CBPI	Frequency of MN (%)
Control	26.3 ± 0.55	2.17 ± 0.07	1.22 ± 0.04	1.64 ± 0.03	100.0
Amifostine (1.0)	23.3 ± 0.44^a*	1.91 ± 0.07	1.22 ± 0.07	1.61 ± 0.04	88.6
MMC (0.1)	33.4 ± 1.82^a^{,^b*}	2.99 ± 0.17	1.15 ± 0.04	1.71 ± 0.07	127.0
CR (1.0)	23.1 ± 0.82^a^{,^c}	1.97 ± 0.07	1.17 ± 0.03	1.61 ± 0.04	87.8
CR (2.0)	22.3 ± 0.51^a^{,^c}	1.81 ± 0.09	1.23 ± 0.05	1.66 ± 0.01	84.8
CR (3.0)	24.1 ± 1.31^c*	2.02 ± 0.07	1.19 ± 0.03	1.62 ± 0.05	91.6

Abbreviations: CR, acetone extract of lichen Cladonia rangiformis; MMC, mitomycin C; MN/1000 Bn cells, number of micronuclei per 1000 examined binuclear cells, (mean value ± SD); Bn cells, binuclear cells with a micronucleus, (mean value ± SD); MN/Bn cell, number of micronuclei/binuclear cell, (mean value ± SD); CBPI, cytokinesis block proliferation index, (mean value ± SD); Frequency of MN, frequency of micronuclei presented as % in relation to control groups in culture of human lymphocytes treated with different concentrations of extracts; Statistically significant difference between pairs of data was evaluated by analysis of variance (One-way ANOVA) followed by Tukey's test. Statistical difference was considered significant at P < .01 and P < .05.

Statistically significant difference P < .01 compared to the control group.

^a*

Statistically significant difference P < .05 compared to the control group.

^b*

Statistically significant difference P < .05 compared to amifostine.

^c*

Statistically significant difference P < .05 compared to mitomycin C.

Treatment with the alkylating agent MMC, as a positive control, at a concentration of 0.1 μg/mL resulted in a significant (P < .05) increase in MN frequency of 27.0% compared to control cell cultures. Cell cultures treated with amifostine VR-2721, as a negative control, at a concentration of 1 μg/mL showed a significant reduction (P < .01) in MN frequency of 11.4% compared to control cell cultures.

Cladonia rangiformis acetone extracts (CR) at a concentration of 1.0 and 2.0 μg/mL showed a significant decrease in the frequency of MN by 12.2% and 15.5% (respectively) compared to control cell cultures, while the same extract at a concentration of 3.0 μg/mL caused a decrease in the frequency of MN by 8.4%. The analyzed acetone extract of lichen gave better results in reducing the number of binuclear lymphocytes with MN compared to amifostine at the concentration of 1.0 μg/mL. It seems that the optimal concentration of the extract for reducing the number of MN is 2.0 μg/mL, which is in agreement with the published results of other lichen species.^34-38

In this paper, for the first time, we report the antigenotoxic effect of acetone C. rangiformis extract.

The human in vitro micronucleus test has become a fast and reliable assay for mutagenicity testing. In this study, we found that tested extract exerted a beneficial effect on control lymphocyte cells giving a significant decrease in the frequency of MN in comparison with the control cell cultures. Since the number of MN serves as an indicator of DNA damage, these results indicate that tested extract protects DNA by reducing the number of MN, and decreases a lipid peroxidation of lymphocytes mostly induced by superoxide anion radicals.

The results of previous research pointed out that atranorin (at a concentration of 2.0 µg/mL) reduced the frequency of MN by 11.1%, while in the same experiment amifostine (1.0 µg/mL) diminished micronucleus frequency by 22.5%.⁴⁶

Considering the above, it can be assumed that in addition to atranorin, other constituents affect the activity of the extract.

Kotan et al.¹⁸ determined that the methanolic extract of C. rangiformis (at a concentration of 5 and 10 μg/mL) showed an antigenotoxic effect by reducing the genotoxicity induced by aflatoxin B1, which may reduce the risk of carcinogenesis. The cytotoxic effects of the chloroform extracts of lichens C. rangiformis and C. convulta on human breast cancer MCF-7 cells were previously studied.¹⁹

There are also other reports of lichen secondary metabolites showing inhibitory activity against various cell cancers. Kosanić et al.⁴⁷ proved the significant cytotoxic effect of atranorin and fumarprotocetraric acid isolated from lichens of the genus Cladonia. Benn et al.⁴⁸ showed marginal cytotoxicity of rangiformic acid isolated from C. retipora.

TPC and Antioxidant Activity

The antioxidant potential of acetone extract of C. rangiformis was evaluated by determining its ability for a DPPH and ABTS radical scavenging, TRP, CUPRAC and its TPC. The results of antioxidant activity are expressed in equivalents of the used standards and for DPPH and ABTS also as IC₅₀ values (Table 4 and Table 5).

Table 4.

DPPH and ABTS Scavenging Radical Capacity of Acetone Extract of C. rangiformis. Results are Presented as Mean ± Standard Deviation.

Sample (concentration mg/mL)	DPPH			ABTS
Sample (concentration mg/mL)	μg TE/mg dry extract	RSC (%)	IC₅₀ (mg/mL)	μg TE/mg dry extract	RSC (%)	IC₅₀ (mg/mL)
CR (15.0)	1.8088 ± 0.4545	46.32	16.19	6.8581 ± 0.0453	63.55	11.80
ASK (0.1)	741.6296 ± 62.0585	6.95	0.72	1607.1384 ± 10.7974	97.62	0.05
BHT (0.1)	509.2523 ± 62.9325	0.54	9.17	21.1466 ± 2.0016	4.21	1.18
GAL (0.1)	2467.1546 ± 92.6491	54.50	0.09	1634.8155 ± 49.9388	99.25	0.05

Abbreviations: CR, Cladonia rangiformis; TE, trolox equivalent; standards: ASK, ascorbic acid, BHT, butylated hydroxytoluene, GAL, gallic acid.

Table 5.

TRP, TPC and CUPRAC of Acetone Extract of C. rangiformis. Results are Presented as Mean ± Standard Deviation.

Sample (concentration mg/mL)	TRP	TPC	CUPRAC
Sample (concentration mg/mL)	μg AAE/mg dry extract	µg GAE/mg dry extract	µg TE/mg dry extract
CR (15.0)	0.4490 ± 0.0354	132.7194 ± 2.4443	17.8134 ± 0.2877
ASK (0.1)	52.0464 ± 15.6053	–	1344.7016 ± 51.9135
BHT (0.1)	31.4688 ± 15.6053	–	1110.0840 ± 51.9135
GAL (0.1)	62.0843 ± 15.6053	–	1939.1541 ± 50.0494

Abbreviations: TRP, total reducing power; TPC, total phenolic content; CUPRAC, cupric-reducing antioxidant capacity; CR, Cladonia rangiformis; AAE, equivalent of ascorbic acid; GAE, gallic acid equivalent; TE, trolox equivalent; standards: ASK, ascorbic acid; BHT, butylated hydroxytoluene, GAL, gallic acid. The standards for TPC were not measurable.

As a result of the research, the acetone extract reduced the DPPH radical scavenging activity with a value of 1.80 μg TE/mg dry extract (IC₅₀ = 16.19 mg/mL), while the ability to neutralize ABTS radicals was slightly better (6.85 μg TE/mg dry extract, IC₅₀ = 11.80 mg/mL). Previously, Mendili et al.²⁰ showed that acetone extract of Tunisian lichen C. rangiformis had DPPH antioxidant capacity (IC₅₀ = 23.0 μg/mL). Kocakaya et al.²¹ found ABTS radical removal effect of the methanolic extract of C. rangiformis (2.50 mmol/L/Trolox). According to previous research, there is no data for the analysis of the ability of C. rangiformis acetone extract to neutralize ABTS radicals.

The obtained values for TPC are considerably higher than those previously published for C. rangiformis acetone extract (206.09 μg GAE/g dry extract).²⁰ The study of the reducing power on the Tunisian lichen C. rangiformis acetone extract showed that it possesses antioxidant activity (IC₅₀ = 151 μg/mL).²⁰

For the first time, the antioxidant potential of acetone extract of C. rangiformis lichen was evaluated by determining CUPRAC, the antioxidant ability to reduce Cu (II) to Cu (I). The mentioned method has many advantages over other antioxidant assays, primarily because of applicability to hydrophilic and lipophilic antioxidants and the possibility to perform at physiological pH.⁴⁹ The result obtained by CUPRAC method for examined extract was 17.81 µg Trolox equivalents per mg dry extract. Our previous studies showed that the cupric-reducing capacity of Ramalina capitata, Umbilicaria crustulosa, Peltigera horizontalis, Hypogymnia tubulosa and Evernia prunastri acetone extracts were 6.11, 19.76, 9.94, 46.47 and 20.75 µg TE/mg dry extract, respectively.^34-38 Result obtained in the current study was very near to U. crustulosa and E. prunastri effect but much lower activity than H. tubulosa.

The measured antioxidant activity points to the conclusion that the capacity of the extract at a concentration of 15 mg/mL is significantly lower than the antioxidant capacity of the standards used at a concentration of 0.1 mg/mL, but in the range of the antioxidant capacity of extracts of other lichens.^34-38

Anticholinesterase Activity

Lichens can potentially be used for therapeutic purposes as inhibitors of the activity of certain enzymes responsible for the occurrence of some diseases or disorders, such as Alzheimer's disease, Parkinson's disease, and dementia of Lewy bodies.⁵⁰ The cholinesterase activity of C. rangiformis acetone extract and the commercial cholinesterase inhibitor, neostigmine-bromide, was determined. The acetone extract of selected lichen was used for the test at a concentration of 1.0 and 10.0 mg/mL, while the concentration of neostigmine bromide was 0.2 mg/mL.

Acetone extract at a concentration of 1.0 mg/mL exhibited a weak activating effect (2.0%) on cholinesterase activity compared to neostigmine bromide. On the other hand, an inhibitory effect was shown by the C. rangiformis acetone extract at a concentration of 10.0 mg/mL with a contribution of 7.3%. However, the inhibitory effect of the acetone lichen extract is far below the inhibitory effect of the standard cholinesterase inhibitor, neostigmine bromide (96.6%). Based on the results, we conclude that increasing the concentration of acetone extracts also increases the ability to inhibit cholinesterase activity. These are the first reports on the effect of lichen C. rangiformis on cholinesterase inhibitory activity.

Stojanović (2016) examined the effect of atranorin (isolated from Hypogymnia physodes) on cholinesterase activity.²⁷ Compared to neostigmine bromide (as a standard inhibitor), the influence of atranorin on cholinesterase activity was insignificant. At a concentration of 1.0 mg/mL, isolated atranorin showed a weak activating effect (3.6%), while at a concentration of 10.0 mg/mL, it showed a weak inhibiting effect (0.3%). The better inhibiting effect of the C. rangiformis acetone extract compared to atranorin (isolated from H. physodes) is probably due to the presence of other components in the extract that contribute to the inhibitory effect.

In previous research, Luo et al.⁵¹ found that C. macilenta lichen extract showed high cholinesterase inhibitory activity.

Conclusions

The present work provides new insights into the phytochemical composition and biological activity of fruticose lichen C. rangiformis. The HPLC analysis revealed the presence of atranorin as the main component of C. rangiformis extract, while GC-MS analysis enabled the identification of the most dominant volatiles—rangiformic acid, atraric acid, and atranol, for the first time. Determined HPLC composition was similar to previously examined lichen, except of atraric acid which was detected in this study for the first time. It is obvious that the antioxidant capacity of the extract was significantly lower than the capacity of proven antioxidants, ascorbic and gallic acid, but in the range of activity of some lichens, eg R. capitata, U. crustulosa, P. horizontalis, H. tubulosa, and E. prunastri. For the first time, anticholinesterase activity and protective effect on human lymphocytes of C. rangiformis extract were determined, as well as antioxidant activity by the CUPRAC method. The ability of reduction in the number of MN in human lymphocytes, classified C. rangiformis to become a promising source of substances with a beneficial effect on DNA damage.

Footnotes

Authors Contribution

ID contributed to the conceptualization, investigation, and writing-original draft. VM contributed to the formal analysis (antioxidant activity), investigation, resources, and validation. VSJ contributed to the formal analysis (cholinesterase activity), investigation, resources, and validation. MS contributed to the formal analysis (cytokinesis-block micronucleus assay), investigation, resources, and validation. IZ contributed to the investigation, visualization, writing-original draft. GS contributed to the conceptualization, methodology, project administration, supervision, validation, and writing-review and editing.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (grant number 451-03-47/2023-01/200124).

ORCID iDs

Ivana Dimitrijević

Gordana Stojanović

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Cladonia rangiformis Acetone Extract— New Insight into the Chemical Composition and Biological Activity

Abstract

Introduction

Objective

Methods

Results

Conclusion

Keywords

Introduction

Materials and Methods

Lichen Material

Preparation of Lichen Extract

HPLC Analysis

GC-MS Analyses

Cytokinesis-Block MN Assay

TPC and Antioxidant Activity

Cholinesterase Activity

Results and Discussion

HPLC Analysis

GC-MS Analyses

Cytokinesis-Block MN Assay

TPC and Antioxidant Activity

Anticholinesterase Activity

Conclusions

Footnotes

Authors Contribution

Declaration of Conflicting Interests

Funding

ORCID iDs

References