Antiproliferative and Antimicrobial Activities of Selected Bryophytes

One-hundred and sixty-eight aqueous and organic extracts of 42 selected bryophyte species were screened in vitro for antiproliferative activity on a panel of human gynecological cancer cell lines containing HeLa (cervix epithelial adenocarcinoma), A2780 (ovarian carcinoma), and T47D (invasive ductal breast carcinoma) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and for antibacterial activity on 11 strains using the disc-diffusion method. A total of 99 extracts derived from 41 species exerted ≥25% inhibition of proliferation of at least one of the cancer cell lines at 10 μg/mL. In the cases of Brachythecium rutabulum, Encalypta streptocarpa, Climacium dendroides, Neckera besseri, Pleurozium schreberi, and Pseudoleskeella nervosa, more than one extract was active in the antiproliferative assay, whereas the highest activity was observed in the case of Paraleucobryum longifolium. From the tested families, Brachytheciaceae and Amblystegiaceae provided the highest number of antiproliferative extracts. Only 19 samples of 15 taxa showed moderate antibacterial activity, including the most active Plagiomnium cuspidatum, being active on 8 tested strains. Methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus were the most susceptible to the assayed species. This is the first report on the bioactivities of these 14 species.


Introduction
In the era of drug development based on high-throughput pharmacological screening, there is an increasing demand for molecules to be tested. Besides large synthetic libraries, natural products are of primary importance because of their pharmacological activities and high structural diversity. In the last decades, 22% of all the newly approved drug molecules have been natural product derivatives (semisynthetic), 4% have been genuine natural products, and 13% have been made by total synthesis bearing the pharmacophore of a natural product [1]. Two of the most important research topics in drug development are anticancer agents and antibiotics. According to the World Cancer Report apoptosis [29,30] and inhibit p-glycoprotein-mediated multidrug resistance [31]. Some ent-kauranes induced apoptosis through a caspase-8-dependent pathway [32,33]. In the case of diterpene jungermanenones, a caspase-independent pathway, together with the inhibition of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), is suggested [34]. Marchantin C, a compound with a different structure from all the previously known microtubule inhibitors, decreased the quantity of microtubules at the G2/M phase in human tumor cells and decreased the polymerisation rate of tubulin, similarly to in [30,35].
The aim of our work was to carry out an extensive in vitro bioactivity assay on 42 bryophyte species native to Hungary to identify taxa with remarkable antiproliferative and antimicrobial activities. Further, the phytochemical and bioactivity data of the assayed species were reviewed.

Results
In the course of the in vitro screening for antiproliferative and antimicrobial activities, 168 extracts belonging to 42 bryophyte species, 35 genera, and 20 families were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the disc-diffusion method, respectively. The antiproliferative activities of the extracts were assessed on human cervical (HeLa), ovarian (A2780), and breast (T47D) cancer cell lines using two final concentrations (10 and 30 µg/mL). The results of the antiproliferative assays are presented in Table 1. Extracts prepared with n-hexane (A), CHCl 3 (B), aqueous MeOH (C), and H 2 O (D) were investigated for their cytostatic effects. A total of 98 extracts representing 41 species exerted ≥25% inhibition of proliferation of at least one of the cell lines at 10 µg/mL. The numbers of active A/B/C/D fractions were 24/38/20/16, respectively. In the case of 25 extracts (from 17 species), the inhibition was ≥50% on at least one of the cell lines at this concentration. This higher inhibition was most characteristic to B extracts (13), followed by A (7), C (4), and D (1). More than one extract was active in the case of six species, namely, Brachythecium rutabulum, Climacium dendroides, Encalypta streptocarpa, Pleurozium schreberi (A and B), Neckera besseri, and Pseudoleskeella nervosa (A, B, and C). At 30 µg/mL, 36 samples belonging to 26 species were inactive (25 of these were D extracts); further analysis of these extracts did not seem to be prospective. Compounds responsible for bioactivity were less polar in the analysed samples. The highest activity (78.54% inhibition on HeLa at 10 µg/mL) was observed in the case of the B extract of Paraleucobryum longifolium. Moreover, this extract was active on all the cell lines, and activities at 10 µg/mL were not much lower than those at 30 µg/mL (46.84-78.54% vs 56.87-83.93%). Interestingly, in the case of this species, only the CHCl 3 extract had remarkable activities. Concerning the sensitivity of the cell lines, the measure of inhibition was more pronounced in the cases of HeLa and T47D than A2780. On HeLa, 16 extracts; on T47D, 10 extracts; and on A2780, only 3 extracts exerted >50% inhibition at 10 µg/mL. From the tested families, Brachytheciaceae (with Brachythecium rutabulum, Homalothecium philippeanum, and Pseudoscleropodium purum) and Amblystegiaceae (with Amblystegium serpens and Hygroamblystegium tenax) provided the highest numbers of active extracts.
The antimicrobial activity of the tested bryophytes, determined by the disc-diffusion method on 11 standard strains, seemed to be sporadic and of low intensity. From the 42 tested species, only 19 samples of 15 taxa showed moderate antibacterial activity ( Table 2). None of the extracts were active on Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 35218, and Klebsiella pneumoniae ATCC 700603. Methicillin-resistant Staphylococcus aureus ATCC 43300 and Staphylococcus aureus ATCC 29213 proved to be the strains most susceptible to the examined species. Among the fractions with different polarities, the relatively apolar n-hexane and chloroform extracts demonstrated antibacterial activities. The aqueous and remnant MeOH fractions were inactive. From the tested species, Plagiomnium cuspidatum seemed to be the most prospective for further analysis of its antibacterial effects and constituents responsible for this activity, being active on eight tested strains.
There was a notable correlation between the antiproliferative and antimicrobial activities: 7 of the 14 species with antimicrobial activities and 7 of the 15 species with >50% inhibitory activity at 10 µg/mL on any of the cancer cell lines also possessed the other activity. Amblystegium serpens, Brachythecium rutabulum, Cirriphyllum piliferum, Climacium dendroides, Paraleucobryum longifolium, Plagiomnium affine, and Pseudoscleropodium purum were active in both assays.  Amblystegium serpens  Table 3). Ethnopharmacological data are scarce. Barbula unguiculata has been used as an analgesic and to reduce fever, and Bryum argenteum has been used as an antipyretic and as an antifungal agent in folk medicine [36]. The available data on biologically active species generally do not confirm or support our observations. In Anomodon viticulosus, previously only fatty acids were detected [37,38]; there are no data in literature on its antiproliferative and antimicrobial effects. In the case of Atrichum undulatum, the presence of the reported sterols [38], carotenoids [38], and fatty acids [39] may not be related to the antiproliferative activity; however, coumarin glycosides [40] may have some role in this effect. The DMSO (dimethyl sulfoxide) and aqueous extracts of this species were active against certain bacteria [41,42]; however the extracts analysed by us did not have such an effect. For Brachytheciastrum velutinum, only the presence of phenolic acids and flavonoids has been reported [43]. Brachythecium rutabulum possessed remarkable antibacterial activity [44], including against Staphylococcus aureus, as also demonstrated in our experiments. Bryum argenteum, with a confirmed flavonoid content [45,46], was reported to be active against different bacterial and fungal strains [8,47,48]. However, in our antimicrobial assay, it was not active. For Calliergonella cuspidata, only the presence of fatty acids [49] and antioxidant activity was reported [50], with no reference to the antimicrobial activity first observed by us. In the case of Climacium dendroides, both antiproliferative and antimicrobial effects have been reported [51]. From the secondary metabolites of this species, flavonoids and chromenones [51,52] might be related with these activities; however, the role of these compounds has not been investigated. Homalothecium philippeanum was antiproliferative in our assay but, contrary to previous results [53], exerted no antibacterial activity against Staphylococcus aureus. From Plagiomnium affine, which was active in both of our assays, only the presence of flavonoids has been reported previously [54]. Two other Plagiomnium species (P. cuspidatum and P. undulatum) had antimicrobial activities in our tests, with literature references only to their flavonoid contents [55][56][57][58]. In contrast to previous reports [53,59], Pleurozium schreberi did not exert antimicrobial activity. The sesquiterpene and flavonoid content of Porella platyphylla [60][61][62][63] may be related to its antiproliferative activity, but there are no reports on the anticancer effect of this species. From Pseudoscleropodium purum, only sterols, triterpenes, and essential oil have been reported [64][65][66]. The antimicrobial activity of Rhytidium rugosum was reported previously [53]. As part of our experiments, we carried out an LC-MS-based characterisation of the most biologically active extracts. Altogether, 58 compounds were identified from 9 different extracts (Table S4), the majority of which were acids and terpenoids. The limitation of this approach was that it allowed the identification of already known compounds. Further experiments will aim at the isolation and identification of bioactive constituents. Table 3. Literature data on the chemistry (C) and bioactivities (B) of the studied species.

Discussion
The present investigation aimed at screening for antiproliferative and antimicrobial activities of selected bryophytes collected in Hungary. Our results and the lack of extensive scientific data on biologically active species suggest the necessity of further phytochemical and biological investigations. For the 14 species having antiproliferative or antimicrobial effects, this is the first report on their bioactivities.

Plant Material
Bryophytes were collected in the Northern Medium Mountains (Hungary) in September and October of 2014 and were identified by Péter Szűcs. Voucher specimens for each plant were deposited at the herbarium of the Institute of Pharmacognosy, University of Szeged. Extracts were prepared according to the method described previously [91]. Briefly, air-dried, powdered plant material was extracted using MeOH with the use of an ultrasonic bath. After filtration and evaporation, the residues were dissolved in 50% aqueous MeOH and subjected to solvent-solvent partition between n-hexane (extracts A) and CHCl 3 (extracts B), and the remnant gave extracts C. The residual plant materials were dried and extracted with boiling H 2 O. The filtered extracts were freeze-dried, affording extracts D.

Antiproliferative Assay
The antiproliferative properties of the prepared extracts were determined by means of the MTT assay on a panel of human adherent cancerous cell lines of gynaecological origin containing A2780, HeLa, and T47D cells isolated from ovarian, cervical, and breast carcinomas, respectively. All the cells were purchased from ECACC (European Collection of Authenticated Cell Cultures, Salisbury, U.K.) and were cultivated in minimal essential medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and an antibiotic-antimycotic mixture. All media and supplements were obtained from Lonza Group Ltd. (Basel, Switzerland). The cells were maintained at 37 • C in humidified atmosphere containing 5% CO 2 . Near-confluent cancer cells were seeded onto a 96-well microplate (5000 per well) and attached to the bottom of the well overnight. On the second day, 200 µL of new medium containing the tested substances (at 10 or 30 µg/mL) was added. After incubation for 72 h, the living cells were assayed by the addition of 20 µL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution. MTT was converted by intact mitochondrial reductase and precipitated as blue crystals during a 4 h contact period. The medium was then removed, and the precipitated crystals were dissolved in 100 µL of DMSO during a 60 min period of shaking at 25 • C. Finally, the reduced MTT was assayed at 545 nm using a microplate reader; wells with untreated cells were used as controls [92]. All experiments were carried out on two microplates with at least five parallel wells. Stock solutions of the tested substances (10 mg/mL) were prepared with DMSO. The highest DMSO content of the medium (0.3%) did not have any substantial effect on the cell proliferation. Cisplatin (Ebewe Pharma GmbH, Unterach, Austria), a clinically utilised anticancer drug, was used as a reference agent. The IC 50 values of its antiproliferative action were 12.43, 1.30, and 9.78 µM against HeLa, A2780, and T47D cells, respectively.

Antimicrobial Assay
Antibacterial activities of the extracts against standard bacterial strains were screened for their inhibition zones by the standard disc-diffusion method described previously [93]. The test microorganisms used in this study were 11 international control standard strains. The standard Gram-positive strains were, namely, Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228), Streptococcus agalactiae (ATCC 13813), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (ATCC 19615), and methicillin-resistant Staphylococcus aureus (ATCC 43300). The standard Gram-negative strains were, namely, Escherichia coli (ATCC 35218), Klebsiella pneumoniae (ATCC 700603), and Moraxella catarrhalis (ATCC 43617). Microbial cultures were grown on standard Mueller-Hinton agar plates or Columbia agar +5% sheep blood (COS) plates (bioMérieux, Marcy-l'Étoile, France) at 37 • C under an aerobic or 5% CO 2 environment. The strains were stored in Cryobank vials (MAST Diagnostica, Rheinfeld, Germany) at −70 • C and maintained at 4 • C throughout the study to use as stock cultures. Briefly, bryophyte extracts were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) or water at a concentration of 50 mg mL −1 . The sterile filter paper discs (6 mm in diameter) impregnated with the extracts (10 uL of redissolved extracts) were placed on the plate seeded with the respective bacterial suspensions (inoculums: 0.5 McFarland, 1-2 × 10 8 CFU mL −1 ). The solvent (DMSO) served as the negative control, while ampicillin, erythromycin, imipenem, cefuroxime, and vancomycin antibiotic susceptibility discs were used as the positive control. The plates were incubated at 37 • C for 24 h under aerobic or 5% CO 2 conditions. The diameters of inhibition zones produced by the extracts (including the disc) were measured and recorded. All experiments were carried out in triplicate.

Phytochemical Characterisation of the Extracts
The most active samples were analysed phytochemically by LC-MS. Samples were filtered through 0.45 µm PTFE (polytetrafluoroethylene) syringe filters before analysis. For separation, Kinetex (Phenomenex; XB-C18 and Phenyl-Hexyl, 2.1 × 100 mm, 2.6 um, 100 Å) columns were used. The eluents were the following: A: 0.1% formic acid in MS-grade water; B: 0.1% formic acid in MS-grade acetonitrile. The separation was done with a linear gradient from 5% to 95% B in 35 min with a 0.3 mL/min flow rate. The HPLC instrument was an Agilent 1100 series model consisting of a binary pump, a thermostated autosampler, and a column compartment. The mass spectrometer was a Thermo Q-Exactive Plus Orbitrap equipped with a HESI-II ion source. The mass accuracy was 0.25+/−0.01 ppm in the positive mode and 0.34+/−0.05 in the negative mode at the mass calibration just before the experiment. Acquisition was done in the data-dependent MS 2 scan mode by altering the charge state (positive/negative). The survey scan mass range was set to m/z 80-1000, using the lock masses from the known background ions listed in Table S2. The data-dependent method parameters are shown in Table S3. The acquired MS 2 peak lists were converted to a text file by using the msConvert tool (Proteowizard), and the top 100 MS survey scan peaks were chosen for MS 2 identification against KEGG's small-molecule database, using the MetFrag online search tool. The hits were filtered manually using an 80% matched peak result when the number of MS 2 fragment peaks was at least five.