Chemical composition, antimicrobial, antioxidant and anthocyanin activities of mosses (Cinclidotus fontinaloides (Hedw.) P.Beauv. and Palustriella commutata (Hedw.) Ochyra) gathered from Turkey

Abstract Cinclidotus fontinaloides (Hedw.) P. Beauv. and Palustriella commutata (Hedw.) Ochyra were used in this study. The chemical composition of mosses in the ethanol and water extract was determined using GC-MS and FT-IR. Antioxidant activities of moss extracts were tested by total phenol and ABTS methods. Trolox equivalent value (TEAC) of mosses was calculated as 26 ± 0.32 and 10 ± 0.22 mg/g. The amounts of the flavonoid compounds were calculated as mg gallic acid. Gallic acid equivalent value of mosses was calculated as 587 ± 0.55 and 496 ± 0.22 mg/g. The extracts antimicrobial activity was tested against Escherichia coli (ATCC 8739), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 10145) and Candida albicans (ATCC 10231). Significant antibacterial effect was observed for C. fontinaloides and P. commutata in ethanol extract. Total anthocyanin activities of C. fontinaloides and P. commutata were determined as 50 ± 0.17 and 144 ± 0.33 mg/L.


Introduction
Mosses and liverworts are small, low-growing plants and constitute the phylum Bryophyta, which is phylogenetically placed between vascular plants and algae. Bryophyta has more than 22,000 members all over the world, and nearly 3000 bryophytes are reported to have medicinal value and therefore, the members of this unique division in the plant kingdom are now increasingly used as new sources of pharmaceuticals (Friederich et al. 1999;Saroya 2011;Gokbulut et al. 2012). Bryophytes are considered as a 'remarkable reservoir' of new, natural products or secondary compounds, many of which have shown interesting biologic activity. These activities can be presented as: antimicrobial, antifungal, cytotoxic, antitumour, vasopressin (VP) antagonist, cardiotonic, allergy causing, irritancy and tumour effecting, insect anti-feedant, insecticidal, molluscicidal, piscicidal, plant growth regulatory, superoxide anion radical release inhibition and 5-lipoxygenase, calmodulin, hyaluronidase and cyclooxygenase inhibition features of bryophytes. Some of the latest results also predicted the beneficial influence of bryophytes in AIDS therapy (some bibenzyles of liverworts) (Asakawa 2007, Sabovljevic et al. 2001Pejin et al. 2012Pejin et al. , 2013. The biological characteristics of the terpenoids and aromatic compounds isolated from bryophytes show antibacterial and anti-fungal activity (Miller & Rice-Evans 1999;Barros et al. 2007;Veljıć et al. 2010).
The aim of this work was to identify chemical composition and to test the antimicrobial, antioxidant and anthocyanin activities of ethanol and water extracts of the moss species of Cinclidotus fontinaloides (Hedw.) P.Beauv. and Palustriella commutata (Hedw.) Ochyra (Nigde, Turkey).
The bacterial model that was chosen for this study is the most commonly used bacteria strains and methods. The moss species used in this research are specific to the region (Nigde) in Turkey. The mosses obtained from Nigde region were tested for the antimicrobial and antioxidant properties for potential active agent or drug use.

GC-MS and FT-IR identification of extracts
Data obtained from GC-MS from the sample of water and ethanol extracts of C. fontinaloides and P. commutata were given, respectively, in Table S1-S3. A total of 160 compounds were determined from water extract of C. fontinaloides by GC-MS as shown in Figure S2a. Among these compounds with higher antioxidant, antimicrobial and anthocyanin activities were methyl-d3 1-dideuterio-2-propenyl ether, hexamethylcyclotrisiloxane and 1-hexanol and are given in Table S1. A total of 152 compounds were determined from ethanol extract of C. fontinaloidesas illustrated in Figure S2b. From these compounds with higher antioxidant, antimicrobial and anthocyanin activities were phenacylthiocyanate, butanenitrile, benzoic acid, nonanoic acid and tetradecane. The lists of these compounds were given in Table S1. A total of 160 compounds were determined from water extract of P. commutata and the corresponding GC-MS spectra were given Figure S2c. From these compounds with higher antioxidant, antimicrobial and anthocyanin activities were benzaldehyde, benzene, 1,3-dichloro-, 3-hexene, 1-(1-methoxyethoxy)-(E)-are exist tetradecane and given in Table  S2. A total of 152 compounds were determined from ethanol extract of P. commutata as the corresponding GC-MS spectra are given in Figure S2(d). Phenacylthiocyanate, p-xylene, oxime-, methoxy-phenyl-and butyrolactone were found to have higher antioxidant, anti-bacterial and anthocyanin activities as given in Table S3. FT-IR spectrum of the ethanol extracts of C. fontinaloides and P. commutata is given in Figure S3. In the spectrum of C. fontinaloides, the most notable peaks observed at 3500-3100 cm −1 belong to the -OH, 2915 for -CH 3 andCH 2 stretching frequencies. And the following stretching frequencies can be assigned as 2848 cm −1 for -CH, 2238 cm −1 for C≡C, 1979 cm −1 for C-H, 1709 cm −1 for RCH=CH 2 , 1462 cm −1 for NH 3 , and 1032 cm −1 for -CH 2 and -OH. In the FT-IR spectrum of P. commutata, the peaks observed between 3500 and 3000 cm −1 belong to the -OH , and the peaks at 2915 cm −1 for -CH 3 vibrations. Also, the following vibrations are assigned as: 2848 cm −1 for ≡CH, 2927 cm −1 for -CH 3 , 1589 cm −1 for RCH = CH 2 , 1436 cm −1 for NH 3 , 1249 cm −1 for -CH and 1032 cm −1 for -CH 2 , -OH stretching frequencies.

Antimicrobial activity properties
The ethanol extracts of the moss are tested against five common bacteria and the data of antimicrobial activity for C. fontinaloides and P. commutata are given in Table S4. MIC (Minimal Inhibitory Concentration) value of mosses (C. fontinaloides and P. commutata) was determined as 100 mg/10 mL and over 100 mg/10 mL against Escherichia coli and Bacillus cereus. MIC value of the extract of C. fontinaloides and Palustriella commutate was determined as 100 mg/10 mL using Staphylococcus aureus and Candida albicans. MIC value of moss extract was determined over 100 mg/10 mL for Pseudomonas aeruginosa as reported in Table S4. MIB (Minimal Bactericidal Effect) values of ethanol extracts of C. fontinaloides and P. commutata were determined as 200 mg/10 mL, and over 200 mg/10 mL against Escherichia coli and Bacillus cereus. MIB value of the extract of mosses (C. fontinaloides and P. commutata) was estimated as 200 mg/10 mL using Staphylococcus aureus and Candida albicans. MIB values of mosses (C. fontinaloides and P. commutata) were determined over 200 mg/10 mL against Pseudomonas aeruginosa bacteria as given in Table S4. It is clear that the ethanol extract of the used mosses possesses some antimicrobial activity that may be useful for medicinal applications.

Antioxidant activity
Total antioxidant activities of C. fontinaloides and P. commutata were measured by the ABTS method and calculated according to the following equation: Trolox equivalent value (TEAC) of mosses was calculated as 26 ± 0.32 and 10 ± 0.22 mg/g from the equation where a is the slop of measurement, 3.4396 is the slope of standard and f is dilution factor.
Folin-Ciocalteu; amounts of the extracted compounds were calculated as mg gallic acid (μg GAE/mL −1 extract). Total phenol amount of mosses calculated using Folin-Ciocalteu method at 760 nm wavelength and inhibition % from the following equation: Absorbance (760 nm) = 0.009 [Gallic Acid] + 0.1034 Gallic acid equivalent value of extract of mosses was calculated as 99 ± 0.94 and 84 ± 0.35 μg/ mL. The mosses investigated in this study had potential use for antioxidant materials where that could be applicable in medicine, food or agriculture.

Anthocyanin activities
Anthocyanins are water-soluble naturally coloured substances that provide a variety of colours such as pink, red, violet, blue and purple to many fruits, vegetables and flowers. The change can occur with change in pH, is one of the main properties of these compounds. This property is due to the presence of various pigments changing structures at different pHs. Anthocyanin activity of extracts of mosses was calculated from the following equation: where MW is 449.2 g, ε (molar absorptivity coefficient) is 13,000 and least 1 cm. Total anthocyanin activity (as cyanidin glucoside) of C. fontinaloides and P. commutata were determined as 50 ± 0.17 and 144 ± 0.33 mg/L. In the present study, total anthocyanin activities (as cyanidin glucoside) of C. fontinaloides and P. commutata were determined as 50 mg/L (0.05 mg/ mL) and 144 mg/L (0.14 mg/mL).

Experimental
Experimental details relating to this article are provided as supplementary material, alongside Tables S1-S4 and Figures S1-S2.

Conclusion
The MIC and MIB values of the ethanol extracts of mosses were determined and significant antibacterial effect was detected for both moss species about > 100 and > 200 mg/10 mL, respectively. Total antioxidant activities of C. fontinaloides and P. commutata were determined by ABTS method, and Trolox equivalent value (TEAC) of mosses was calculated as 26 ± 0.32 and 10 ± 0.22 mg/g. The amounts of the flavonoid compounds were calculated as mg gallic acid (mg GAE /g extract). Gallic acid equivalent value of mosses was calculated as 587 ± 0.55 and 496 ± 0.22 mg/g. These results are the proof that C. fontinaloides and P. commutata extract possesses potent antioxidant activity. The significant proportion of the antioxidant activity is caused by phenolic substances in mosses structure. Total antosiyanid activities (as cyanidin glucoside) of C. fontinaloides and P. commutata were determined as 50 ± 0.17 and 144 ± 0.33 mg/L. To sum up, it was demonstrated that the extract of the mosses investigated here has great potential to be used in medicine, cosmetics and pharmaceutical applications as well as food and agricultural use.