Determination of total phenol content.

The total content of phenolic compounds was determined using the Folin–Ciocalteau method [21]. In each well on microtitration plate, 100 μL of 10% Folin–Ciocalteu reagent solution was added to 20 μL of each extract (1 mg mL^-1). After incubation (six minutes at room temperature), 80 μL of 7.5% sodium carbonate solution was added. The control contained 100% ethanol instead of the sample. Gallic acid dissolved in distilled water at concentrations of 0.005–0.200 mg mL^-1 was used to construct the calibration curve. After incubation (120 minutes in the dark at room temperature), the absorbance was read at 740 nm using a microtiter plate reader (Multiskan Sky Thermo Scientific, Finland). The total phenol content of the samples was calculated from the calibration curve equation and is presented as gallic acid equivalents, as mg GAE per g dry extract.

Determination of total phenolic acid content.

Total phenolic acid content was determined according to the modified procedure [22]. In each well on microtiter plate, 20 μL of Arnow reagent (10% w/v sodium molybdate and 10% w/v sodium nitrite, 20 μL 0.1 mol L^-1 hydrochloric acid and 20 μL 1 mol L^-1sodium hydroxide were added to 10 μL of each extract (1 mg mL^-1). Afterwards, another 100 μL of distilled water was added. The control contained 100% ethanol instead of the sample. To construct the calibration curve, caffeic acid dissolved in 50% ethanol at concentrations of 0.0078–1 mg mL^-1 was used. Immediately after all components were added, the absorbance was read at 490 nm using a microtiter plate reader (Multiskan Sky Thermo Scientific, Finland). The total phenolic acid content of the samples was calculated from the calibration curve equation and represented ascaffeic acid equivalents, as mg CAE per g dry extract.

Determination of total flavonoid content.

The procedure [23] was used to determine the total flavonoid content. In each well on microtitern plate, 205 μL of 80% ethanol, 5 μL of 10% aluminum nitrate nonahydrate, and 5 μL of 1 mol L^-1potassium acetate solution were added to 50 μL of each extract (1 mg mL^-1). The control contained 100% ethanol instead of the sample. Quercetin, dissolved in 96% ethanol at concentrations of 0.005–0.200 mg mL^-1 was used to construct the calibration curve. The absorbance was read after incubation (40 minutes at room temperature) at 415 nm using a microtiter plate reader (Multiscan Sky Thermo Scientific, Finland). The flavonoid content is calculated from the equation of the calibration curve and is expressed as quercetin equivalents, as mg QE per g of dry extract.

Determination of total flavonol content.

The total flavonol content was determined according to the procedure [22]. In each well on microtiter plate, 40 μL of methanolic solution of aluminium chloride (AlCl₃) (20 mg mL^-1) and 120 μL of methanolic sodium acetate solution (50 mg mL^-1) were added to 40 μL of each extract (1 mg mL^-1). The control contained 100% methanol instead of the sample. Quercetin dissolved in 100% methanol at concentrations of 0.0078–1 mg mL^-1 was used to construct the calibration curve instead of the extract. The absorbance was read at 440 nm after incubation (150 minutes) using a microtiter plate reader (Multiskan Sky Thermo Scientific, Finland). The total flavonol content of the samples was calculated from the calibration curve equation and represented as quercetin equivalents, as mg QE per g of dry extract.

Determination of total triterpenoid content.

The total triterpenoid content was determined according to the following procedure [24]. In each well on microtiter plate, 10 μL of extract (10 mg mL^-1) was added, followed by the addition of 15 μL of vanillin-glacial acid solution (5% v/v) and 50 μL of perchloric acid solution. The reaction mixture was incubated for 45 minutes at 60°C. After cooling to room temperature, 225 μL of glacial acetic acid was added. The control contained 100% methanol (0.005–1 mg mL^-1) instead of the sample. Absorbance was measured at 548 nm, using a Multiskan Sky Thermo Scientific microtiter plate reader, Finland. Ursolic acid dissolved in 100% methanol was used to construct the calibration curve. All measurements were repeated three times. The total triterpenoid content of the samples was calculated from the calibration curve and represented as equivalents of ursolic acids, as mg UAE per g of the extract.

Determination of total coumarin content.

Determination of the total content of coumarin was performed according to the procedure [25] with modifications. In each well on microtiter plate, 2 μL of extract (10 mg mL^-1), 8 μL of distilled water, and 2 μL of lead acetate trihydrate solution (5% w/v) was added. Another 28 μL of distilled water was added to each well, followed by 160 μL of 0.1 mol L^-1 hydrochloric acid. The reaction mixture was incubated for 30 minutes at room temperature. The control contained 100% methanol instead of the sample. Absorbance was measured at 320 nm using Multiskan Sky Thermo Scientific microtiter plate reader, Finland. Coumarin dissolved in methanol (0.005–1 mg mL^-1) was used to construct the calibration curve. All measurements were repeated three times. The total coumarin content of the samples was calculated from the calibration curve equation and is presented as coumarin equivalents, as mg CE per g of the dry extract.

Liquid chromatography-mass spectrometry.

Separation of compounds of interest was performed using a Dionex Ultimate 3000 UHPLC system equipped with a diode array detector (DAD) that was connected to TSQ Quantum Access Max triple-quadrupole mass spectrometer equipped with heated electrospray ionization probe (HESI-II, Thermo Fisher Scientific, Bremen, Germany) in negative ionization mode.

A Syncronis C18 column (100 × 2.1 mm, 1.7 μm particle size) at 40°C was used for compounds separation: Flow rate was set of 0.3 mL/min and the mobile phase was consisted of (A) water + 0.1% formic acid and (B) acetonitrile. Linear gradient program was used as follows: 0.0–1.0 min 5% B, 1.0–14.0 min from 5% to 95% (B), 14.0–14.1 min from 95% to 5% (B), and 5% (B) for six minutes.

Parameters of the ion source and the other MS data necessary for quantification were as previously described in the literature [26]. The MS data were acquired in negative mode in m/z range from 100 to 1000. Full scanning (FS), product ion scanning (PIS) and neutral loss scanning (NLS) were conducted for the qualitative analysis. Collision-induced fragmentation experiments were performed with collision energy set to 30 eV. A selected reaction monitoring (SRM) experiment for quantitative analysis was performed using two MS² fragments for each compound, which were previously defined as dominant in PIS experiments.

A 10 g/L stock solution of a mixture of all standards was prepared in methanol. Dilution of the stock solution with mobile phase (acetonitrile:H₂O = 1:1) yielded the working solution at concentrations of 0.10, 0.25, 0.50, 0.75, and 1.00 mg/L. Quantification of all compounds was performed with pure commercial standards. Calibration curves were obtained by plotting the peak areas of the compounds to the peak area of the compounds in the standard solution. Calibration curves revealed good linearity, with R² values exceeding 0.99. Phenolic acids were identified by direct comparison with commercial standards and expessed as mg per 100 g. Xcalibur software (version 2.1) was used for instrument control, data acquisition, and data analysis.

DPPH assay.

The DPPH free radical scavenging activities of the extracts were analyzed according to the following procedure [27] with slight modifications. DPPH solution (40 μg mL^-1) was prepared in methanol. To each well on a microtiter plate was added 20 μL of sample (in triplicate) of appropriate concentration (1000, 500, 100, 50, and 10 μg mL^-1) and 180 μL of DPPH solution. As a negative control, 20 μL of methanol was used instead of a sample. BHA, BHT, and AA were used as positive controls. The reaction plates were kept in the dark at a room temperature for 30 minutes, after which the absorbance was measured at 517 nm using Multiskan Sky Thermo Scientific Microtiter plate reader, Finland. The inhibition of DPPH radicals in the test sample was calculated using the following formula and expressed as a percentage (%): (1) where Ab is the absorbance of negative control (without sample) and As is the absorbance of the sample at different concentration and the positive controls, as well. The results are presented as the mean of percentage of DPPH radicals inhibition ± standard error.

Determination of total reducing potential.

The reducing potential of extracts was analyzed by the method [28] and following the procedure [29] with modifications. The reduction capacity is based on the transformation of Fe³⁺ to Fe²⁺ ferric ions, which results in the development of a blue-green color which intensity can be measured spectrophotometrically. Sample (20 μL, concentrations of 1000, 500, 100, 50, and 10 μg mL^-1), 40 μL of phosphate buffer (0.2 mol L^-1, pH 6.6) and 40 μL of 1% potassium ferricyanide were added to the reaction mixture. The blank was prepared in the same manner as the reaction mixture, with 20 μL of appropriate solvent instead of sample. After an incubation of 20 minutes at 42°C, 40 μL of 10% trichloroacetic acid, 40 μL of distilled water and 8 μL of 0.1% iron trichloride were added. The absorbance was measured at 700 nm after incubation at room temperature for 10 minutes using Multiskan Sky Thermo Scientific, Finland microtiter plate reader. BHA, BHT, and ascorbic acid were used as positive controls and data are presented only against ascorbic acid as the same results were obtained. The reducing potential of the samples is expressed as μmol of ascorbic acid equivalents per gram of dry extract (μmol AAE per g of dry extract).

β-carotene/linoleic acid assay.

The β-carotene/linoleic acid assay was performed according to a described procedure [30] with modifications. In solution of β-carotene in chloroform (125 μL, 4 mg mL^-1), linoleic acid (6.25 μL), and Tween 40 (50 mg) were dissolved. In the prepared emulsion, 125 μL of chloroform was added. After dissolution, the chloroform was evaporated using a rotary evaporator at 40°C (Buchirotavapor R-114) and the dry residue was dissolved with 25 mL of distilled water. Extracts (concentrations of 1000, 500, 100, 50, and 10 μg mL^-1) and standards (BHA, BHT, and AA) were prepared in triplicate. 200 μL of emulsion and 28 μL of test substance (extracts, standards, 100% methanol–as negative control) were added to each well on the microtiter plate. Absorbance was measured at 490 nm immediately after sample and reagent addition (t₀ = 0) and two hours (t₁₂₀ = 120) after incubation using Multiskan Sky Thermo Scientific Microtiter plate reader, Finland. The antioxidant activity of the samples was determined by monitoring the inhibition of β-carotene dye loss, using the following equation: (2)

WhereA₁₂₀ and C₁₂₀ were the absorbances measured at t₁₂₀ minutes for samples and negative control, respectively, while C₀ represent the absorbance of the negative control at t₀. The values obtained are presented as the mean of three replicates ± standard error.

α-amylase inhibition assay.

The Caraway-Somogy iodine/potassium iodide method, according to Zengin et al [31] was used with modifications. In the test well (S), 25 μL of extract (concentration of 1000, 500, 100, 50, and 10 μg mL^-1), 50 μL of α-amylase solution (0.5 mgmL^-1) in sodium phosphate buffer (0.1 mol L^-1, pH 6.8, with 6 mM NaCl) was added. After 10 minutes at 37°C, 50 μL of 0.2% starch was added and the plate was incubated for another 10 minutes at 37°C. The reaction was terminated by the addition of 25 μL of 1mol L^-1 hydrochloric acid, followed by the addition of 100 μL of Lugol’s solution. Acarbose was used as a standard. The absorbance was measured at 630 nm using Multiskan Sky Thermo Scientific, Finland microtiter plate reader. The percentage of inhibition (% I) of α-amylase was calculated using the following equation: (3)

Where enzyme control (C₁) contained buffer instead of sample, substrate control (C₂) contained buffer instead of enzyme, while the sample color control (B) contained buffer instead of enzyme and starch. All measurements were done in triplicates. The results are presented as mean ± standard error.

α-glucosidase inhibition test.

The potential of extracts in α-glucosidase inhibition was analyzed according to the procedure [32]. pNPG was used as a substrate. In the test well (S), 120 μL of extract (concentration of 1000, 500, 100, 50, and 10 μg mL^-1) and 20 μL of enzyme solution (0.5 U mL^-1) in potassium phosphate buffer (0.1 mol L^-1, pH 6.8) were added. After five minutes at 37°C, 20 μL of 5 mM pNPG was added and the plate was incubated for 20 minutes at 37°C. The reaction was stopped by adding 80 μL of 0.2 mol L^-1 sodium carbonate in buffer. Acarbose was used as positive control. The absorbance was measured at 405 nm using Multiskan Sky Thermo Scientific microtiter plate reader, Finland. The α-glucosidase inhibition percentage (% I) was calculated using the following equation: (4) where negative control (C) contained buffer instead of sample, while sample color control (B) contained all components except enzyme, instead of which buffer was added. All measurements were repeated three times. The results are presented as mean ± standard error.

Acetylcholinesterase inhibition test.

The activity of the extracts in acetylcholinesterase inhibition was analyzed based on the procedure [33] with modifications. Briefly, the reaction mixture (S) was prepared by adding 140 μL of sodium phosphate buffer (0.1 mol L^-1, pH 7), 20 μL of DTNB solution, 20 μL of extract solution (in buffer containing 5% DMSO, concentrations of 1000, 500, 100, 50, and 10 μg mL^-1) and 20 μL of acetylcholinesterase solution (5 U mL^-1) in Tris-HCl buffer (20 mmol L^-1, pH 7.5). Sample-free mixture was used as a negative control (C) and galantamine as a positive control, while the sample color control contained buffer instead of enzyme. Each treatment wasdone in triplicate. The absorbance was measured using Multiskan Sky Thermo Scientific, Finland, microtiter plate reader at a wavelength of 412 nm. The inhibition percentage (I) was calculated using the following equation and the results are presented as mean ± standard error: (5)

Tyrosinase inhibition assay.

The activity of the samples in tyrosinase inhibition was analyzed according to the slightly modified procedure [34]. The reaction mixture (S) was prepared by adding 80 μL of sodium phosphate buffer (0.1 mol L^-1, pH 7), 40 μL of tyrosinase solution (46 U L^-1) and 40 μL of sample (concentrations of 1000, 500, 100, 50, and 10 μg mL^-1). The sample-free mixture was used as a negative control (C) and kojic acid as a positive control, while the sample color control contained buffer instead of enzyme. After the addition of 40 μL of L-DOPA buffer solution and incubation for 30 minutes at 25°C, the absorbance was measured using Multiskan Sky Thermo Scientific, Finland, microtiter plate reader at a wavelength of 475 nm. Each treatment was done in triplicate. The inhibition percentage (I) was calculated using the following equation and the results are presented as mean ± standard error: (6)

Cell line cultivation.

To examine the biocompatibility and immunomodulatory effects of moss extracts, four cell lines were cultured: MRC-5 (fibroblast cell line), BV2 (microglia), HCT-116 (colon cancer) and MDA-MB-231 (breast cancer). All cells were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA). Cell lines were maintained in culture as monolayers at 37°C in a humidified atmosphere with 5% CO₂, in a complete nutrient medium, with 10% FCS, glucose and 10 μL mL^-1 of antibiotics (penicillin/streptomycin). All cell lines were cultured in culture medium for 24 h and then treated with the corresponding extracts for another 24 h.

Cell line treatment.

Depending on the number of cells, appropriate dilutions were made for application to a 96-well microtiter plate. Cells were evenly seeded in the appropriate density. The nutrient medium was used as a negative control. The test extracts of H. ciliata were first dissolved in DMSO, however they were afterwards diluted in DMEM so that their final concentration in the culture was 10 μg mL^-1.

MTT assay.

The effects of tested extracts on cell viability were determined by MTT assay. To each well 100 μL of cells were added, following 1 × 10⁴ cells (MRC-5, BV2, MDA-MB-231) and 5 × 10⁴ (HCT-116). For the treatment of MRC-5, HCT-116 and MDA-MB-231 cell lines, 50 μL of the test substance (E1, E2, E3) and 50 μL of full DMEM were added to each well. Control column contained 100 μL of complete DMEM. For the treatment of the BV2 cell line, 50 μL of stimulator—LPS (lipopolysaccharide) and 50 μL of the test substance (E1, E2, E3) were added to each well. Control column contained 50 μL LPS and 50 μL full DMEM. After 24 h incubation, 100 μL of the medium was removed from each well and 10 μL of MTT was added. After 3 h incubation (at 37°C in a humidified atmosphere with 5% CO₂), 100 μL of 10% SDS with 1N hydrochloric acid was added and after the dissolution of the precipitated crystals, the absorbance was measured at 540 nm (Microplate Reader LKB 5060–006, LKB Instruments, Vienna, Austria). The results are presented as a percentage of viability (for MRC-5, HCT-116 and MDA-MB-231) and as metabolic activity (for BV2), from three independent experiments performed in quadruplicate relative to the control (non-treated cell lines) given a value of 100%.

NBT assay.

In order to determine the effects of tested extracts on the production of ROS and their potential anti-oxidative effects, NBT assay was used. The setup of the experiment was identical to the one used when performing the MTT test. After 24 h incubation, 100 μL of the medium was removed from each well and 10 μL of NBT was added. After 3 h incubation (at 37°C in a humidified atmosphere with 5% CO₂), 100 μL of 10% SDS with 1N hydrochloric acid was added and after the dissolution of the precipitated crystals, the absorbance of the solution was measured at 540 nm (Microplate Reader LKB 5060–006, LKB Instruments, Vienna, Austria). The results are represented as the mean values of the ROS index, calculated based on the absorbance of the samples made in quadruplicate (n = 4) and are expressed as an index relative to the control.

Determination of NO production.

For the determination of nitric oxide (NO) production, the Griess reaction was applied. Each treatment was done in quadruplicate and there was a control (non-treated cells) for each extract. BV2 cells were stimulated with LPS. 50 μL of cells were seeded and 50 μL of Griess reagent was added to each well (A:B = 1:1). After 10 minutes the absorbance of the solution was measured at 520 nm (Microplate Reader LKB 5060–006, LKB Instruments, Vienna, Austria). After incubation with the test extracts, NO production was calculated (the results are presented as nitrite concentration (μmol L^-1) equivalent to NO production).

Level of total phenolic (acid) contents in extracts.

The total phenolic content values of the extracts decrease in the following order: E2>E3>E1. The highest concentration of total phenolics is shown to be in extract E2 and E3, whereas the total phenolic content of the E1 is lower, as shown in Table 2.

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Table 2. Distribution of secondary metabolites in Hedwigia ciliata extracts.

https://doi.org/10.1371/journal.pone.0246810.t002

Based on the obtained results, the best extracting solvent was the mixture of water:ethanol (50:50, vol%). According to the obtained results, extracts E1 and E2 exhibited similar phenolic acid content, while the highest concentration of phenolic acids was shown to be in E3 extract, which indicated a significantly higher level of these compounds than in the other tested extracts.

Level of total flavonol and flavonoid contents in extracts.

The results presented in Table 2 have shown that flavonols were only present in extract E3, while low content was observed in E1 and E2. Experimental data presented in Table 2 also demonstrated that the concentration of total flavonoids varied among different extracts. The values decrease in the following order: E3>E1>E2. The highest concentration of flavonoids was found in extract E3, almost twice the value of the other solvents, E1 and E2.

Level of total triterpenoid and coumarin contents in extracts.

The highest triterpenoid content was obtained in E1, whereas the lowest total triterpenoid content occurred in E3 (Table 2). According to the obtained results (data not shown), no traces of coumarins were found in the tested extracts.

Liquid chromatography–mass spectrometry analyses of extracts.

Using the Liquid Chromatography—Mass Spectrometry (LC/MS) analyses, the presence of different phenolic and flavonoid compounds was determined and it was observed that they differed among the corresponding extracts depending on their polarity. Based on the obtained results, extracts E1 and E2 showed higher concentration of phenolic compounds compared to E3 extract. In the ethanolic (E1) extracts, the most prevalent was shown to be protocatechuic acid, while for the E2 extract measured value was half the previous value. The second most common compound was p-hydroxybenzoicacid (Table 3). Other compounds such as gallic acid, caffeic acid, quercetin 3-O-rutinoside, apigenin, naringenin and kaempferol were also detected in E1 and E2 extract, however at lower concentrations, ranging from 1.00–2.45 g per 100 g. In contrast, the values for the other tested compounds were lower than 1.00 g per 100 g in the less polar, E3 extract.

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Table 3. LC/MS analyses of the extracts.

https://doi.org/10.1371/journal.pone.0246810.t003

Antineurodegenerative activity.

The results obtained regarding the acetylcholinesterase inhibitory activity are given in the Fig 4A. The data are similar for the all tested extracts, showing moderate to low inhibitory effect in comparison with the positive control, galantamine. The highest acetylcholinesterase inhibition among analyzed samples was noted for the E3 extract. As presented in Fig 4A, all of the tested concentrations of this extract exerted moderate inhibitory activity, in a concentration-dependent manner. Regarding E1 and E2 extracts, notable inhibitory effects were also measured. Interestingly, the obtained inhibition values increased proportionally with the decreasing of sample concentration (Fig 4A). As presented in Fig 4B, all of the extracts exhibited notable effects against tyrosinase, with higher inhibition rate in comparison with the positive control, kojic acid. The most potent inhibitory effects were observed for E1 extract, with a maximum of inhibition at a concentration of 1000 μg mL^-1, which was higher than inhibition value of the kojic acid. The extracts E2 and E3 also showed noticeable inhibitory effects for the highest sample concentration used (Fig 4B), although statistically insignificant for the E2 extract. The values obtained for the other concentrations displayed significant inhibition, and they are decreasing proportionally with the decrease in sample concentration.

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Fig 4.

Antineurodegenerative and anti-neuroinflammatory activity–effects of the corresponding extracts (E1, E2, E3) towards acetylcholinesterase (A) and tyrosinase (B). The results are expressed as the inhibition percentage, as mean value ± SE from an experiment performed in triplicate (* p<0.05; ** p<0.01 different concentrations of extracts vs. corresponding standard, galantamine or kojic acid). (C) Anti-neuroinflammatory activity of the extracts towards BV2 cell line–the effect of investigated extracts on the viability, NO production (measured by nitrite level), and ROS production in vitro after 24 h of exposure. The results are expressed as the mean ± SE from a representative experiment of three independent experiments performed in quadruplicate (** p<0.01 extract vs. LPS-treated cells).

https://doi.org/10.1371/journal.pone.0246810.g004

Anti-neuroinflammatory activity.

Activated microglia release mediators such as inflammatory molecules or neurotrophic factors that display either harmful or beneficial effects on neuronal signaling and survival [36]. The amounts of released factors depend on the intensity of stimulation and on time; therefore we first determined whether microglial cells modulate the metabolic activity and the production of ROS and NO, by following activation by LPS, in a concentration- or a time-dependent manner. For this purpose, microglial cells BV2 were stimulated for 24 h with LPS. Microglial activation was confirmed by an increase in nitrite concentration in the cell culture supernatants (Fig 4C). Regarding the treatment of BV2 cells, as presented in the Fig 4C, the metabolic activity/viability of LPS-stimulated BV2 cells was slowly reduced in comparison with the control non-stimulated cells. After simultaneous treatment with LPS and extracts, a significant normalization in the metabolic activity of BV2 cells was detected, with corresponding extracts showing similar results.

As expected, LPS-stimulated BV2 cells produced large amounts of NO (Fig 4C) compared with the non-treated cells. The results have shown that all of the extracts expressed similar effects in reducing NO production by LPS-stimulated BV2 cells, indicating their potential anti-neuroinflammatory activity.

Furthermore, the ROS production also increased by LPS-stimulated BV2 cells after treatment with the tested extracts in selected concentration (10 mg mL^-1) as presented in Fig 4C. All of the analyzed extracts performed similar effects, suggesting a potential of the selected extracts for increasing of antioxidant mechanisms in investigated cells including ROS production.