Antimicrobial and Antiproliferative Activities of Homalothecium Sericeum (Hedw.) Schimp

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Antimicrobial and antiproliferative activities of homalothecium sericeum (hedw.) schimp. extracts

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ANTIMICROBIAL AND ANTIPROLIFERATIVE ACTIVITIES OF Homalothecium sericeum (Hedw.) Schimp. EXTRACTS

Pinar Oztopcu-Vatan1,*, Filiz Savaroglu1, Cansu Filik-Iscen2, Selda Kabadere3, Semra Ilhan1 and Ruhi Uyar3

1 Department of Biology, Faculty of Art and Sciences, Eskisehir Osmangazi University, 26480 Eskisehir, Turkey 2 Department of Elementary Education, Faculty of Education, Eskisehir Osmangazi University, 26480 Eskisehir, Turkey 3 Department of Physiology, Faculty of Medicine, Eskisehir Osmangazi University, 26480 Eskisehir, Turkey

ABSTRACT of plant species [2]. Even though a few reviews concerning the biologically active chemical constituents of bryophytes The purpose of the current study was to investigate the have been published [2-4], the chemistry of bryophytes has possible antimicrobial and antiproliferative properties of been neglected for a long time for the following reasons: Homalothecium sericeum (Hedw.) Schimp. (Brachythe- it is very small morphologically, difficult to collect in large ciaceae). In our study, we first obtained 7 different extracts amounts as pure samples, difficult to identify, and consid- (methanol, chloroform, acetone, A, B, C and D) by two ered to be nutritionally useless to humans. However, the different extraction processes. The antimicrobial activity of Chinese, Europeans and North Americans have been using these extracts was then assessed by using the well diffusion bryophytes since time immemorial as a traditional medi- method against 6 bacterial and 7 fungal strains. cine to cure cuts, burns, external wounds, bacteriosis, pul- monary tuberculosis, neurasthenia, fractures, convulsions, None of the extracts influenced the growth of the fungi. scalds, uropathy, pneumonia etc. [4-7]. One of the features Although all of these extracts carried a variety of inhibitory that have helped bryophytes survive and maintain their potentials against just Pseudomonas aeruginosa, the ex- place in today’s flora is their content of biologically active tracts of acetone, A and C showed much better antibacte- compounds [1]. There is evidence confirming the antibiotic rial activities with minimum inhibitory concentrations of activity of bryophytes against prokaryotic cells and fungi 23.4, 93.8 and 375 µg/mL, respectively. Therefore, the [7, 8]. Dulger et al. [9] investigated the antimicrobial activ- possible cytotoxic activities of acetone extracts A and C of ity of methanol extracts from the aerial parts of eight dif- H. sericeum were determined by applying 3-(4,5-dimethyl- ferent mosses species with the disc diffusion method, and thiazol-2yl)- 2,5-diphenyltetrazolium bromide assay (MTT) concluded that methanol extracts possess mainly a moder- on rat glioma cell line (C6). Extract C was the most effec- ate activity against Gram positive and negative bacteria tive on C6 cell viability and, 85 and 170 µg/mL reduced and a weak antiyeast activity. Ilhan et al. [10] studied the the cell survival ratio by 39 and 86% in 24 h, respectively. antimicrobial activity of methanol and acetone extracts This study demonstrates for the first time that acetone and from Palustriella commutata and indicated that methanol C extracts from Homalothecium sericeum may be useful extract weakly affected some bacteria, but that it was as antibacterial and antiproliferative agents in the future; inactive against yeast and mould strains. In another study, hence, further purification of these extracts is anticipated. the antimicrobial activity of ethanol extracts of 15 Indian

mosses was evaluated and it was found that only 7 of them were active against all the organisms tested [7]. Bodade et KEYWORDS: Bryophyta, Homalothecium sericeum, antibacterial, al. [1] investigated the antimicrobial activity of different antifungal, antiproliferative. extracts and indicated that all extracts showed varying effects against all the test fungi and bacteria. Further evidence was supplied by Spjut et al. [11], who tested 184 species of mosses and 23 species of liverworts for antitu- 1. INTRODUCTION mor activity and found that while the extracts of 43 species were active, those of 75 species were toxic to the test Bryophytes belong to a group of the oldest known land mice. The most activity was found in Brachytheciaceae, plants, which includes liverworts, hornworts and mosses [1]. Dicranaceae, Grimmiaceae, Hypnaceae, Mniaceae, Neck- More than 22.000 members of the mosses exist, spread out eraceae, Polytrichaceae, and Thuidiaceae. Since Homa- throughout the world, accounting for approximately 5.5% lothecium sericeum belongs to the family of Brachythe- ciaceae and for the aforementioned reasons, we chose to * Corresponding author investigate the properties of this moss.

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Cancer cells and pathogens that have gained resis- separately concentrated by rotary evaporation to dryness in tance to drug therapy are an increasing public health prob- vacuum (extract D, yield 2.44%). lem. Yet, there are a few really effective antifungal prepa- These yields from different extracts were then weighed, rations currently available for the treatment of fungal attack dissolved in dimethyl sulphoxide (DMSO) to final concen- in agriculture, an occurrence which can be economically tration of 200 mg/mL and stored at 4°C for further use. devastating. The logical progression of this knowledge would be to screen plants for any constituents that could 2.3. Test microorganisms show activity against pathogenic fungi, bacteria and/or cancer cells. To achieve this aim, the most investigated taxa The bacterial strains were recovered from long term at present are from the angiosperms group whereas very storage at –85°C in the cryobank. The bacteria were re- little data is available about other groups of plants, in par- freshed in Nutrient Broth (Merck, Germany) at 35–37°C, ticular bryophytes [5, 9, 10, 12, 13]. Therefore, we investi- and then inoculated on Nutrient Agar (Merck) plates to gated, for the first time, the possible antimicrobial poten- check the microbial purity. The moulds were refreshed in tial of Homalothecium sericeum (Hedw.) Schimp. against Malt Extract Agar (Merck) at 27°C. The strain numbers some bacterial and fungal species. In addition, only the most and sources of the acquired microorganisms are listed in effective antimicrobial extracts were tested to determine Table 1. their possible anticancer activity on a glioma cell line (C6). TABLE 1 - Bacterial and fungal strains used for antimicrobial activity test.

2. MATERIALS AND METHODS Bacterial strains Fungal strains aBacillus subtilis cAspergillus flavus NRRL B-209 ATCC 9807 2.1. Plant material bEnterococcus faecalis aAspergillus fumigatus ATCC 29212 NRRL 163 Plant material was collected from Hekimdag village on b c the Sundiken Mountains (Eskisehir, Turkey), at a height of Escherichia coli Aspergillus niger ATCC 25922 ATCC 10949 1240 m, in May 2006, and identified by Dr. F. Savaroglu, bPseudomonas aeruginosa aAspergillus parasiticus Eskisehir Osmangazi University. A voucher specimen (Sa- ATCC 27853 NRRL 465 varoglu 29) was deposited at the Herbarium of our depart- cSalmonella typhimurium dFusarium graminearum ment. ATCC 14028 (wild type) bStaphylococcus aureus dFusarium solani ATCC 25923 (wild type) d 2.2. Preparation of the extracts Geotrichum candidum

Fresh gametophytic samples of H. sericeum were (wild type) a USDA, Agricultural Research Service, Peoria, IL, US; b Faculty of treated with 0.8% Tween 80 aqueous solution to remove Medicine, Department of Microbiology, Eskisehir Osmangazi Univer- epiphytic hosts normally found on the surface, extensively sity, Turkey; c Faculty of Science, Department of Biology, Anadolu washed in tap and distilled water, and dried on filter paper University, Turkey; d Faculty of Art and Science, Department of Biol- at room temperature. Extraction procedures were applied as ogy, Eskişehir Osmangazi University, Turkey ATCC: American Type Culture Collection; NRRL: Northern Regional described elsewhere [14, 15]. Extraction was carried out Research Laboratory through two different processes. At first, 10 g of the sample in powder was extracted 2.4. Determination of antimicrobial activity with 250 mL of 80% methanol, chloroform and acetone for The moss extracts were studied for their antibacterial 8 h using soxhlet equipment. After filtering with Whatman and antifungal activities through the well diffusion method, filter paper (#1), all extracts were concentrated by rotary eva- according to the National Committee for Clinical Labora- poration to dryness in vacuum (yield= 2.10, 1.07 and 2.60 %, tory Standards [16, 17]. Even though there are various respectively) and stored in desiccators for future use. methods for the screening of antimicrobial effects, the well diffusion method was preferred since it is a modification of The second extraction process was completed in four the disc diffusion method and based on those described for steps. First, 30 g of gametophytic plant sample in powder standardized testing of antibiotics. This method can be used was extracted with 250 mL of petroleum ether for 8 h using to simply determine whether or not antibacterial activity is soxhlet equipment (extract A, yield 0.45%). At the second present [18]. step, fat-free air-dried material (15 g) was extracted four times with methanol: water (70:30, v/v) at 40°C, 30 min. The bacterial test cultures were incubated in Mueller- The extract was then concentrated to dryness in vacuum Hinton Broth (MHB) at 35-37ºC until they were visibly (extract B, yield 2.83%). The third and fourth extracts turbid. The density of these cultures was adjusted to a tur- were prepared as follows: fat-free air-dried material (15 g) bidity equivalent to that of the 0.5 McFarland standard used was extracted four times with methanol:water (70:30 v/v) to standardize the inoculum’s density (at 625 nm, 0.08- at 40°C, 30 min, and this was concentrated in vacuum, and 0.1 absorbance) with sterile saline. The bacterial cul- the aqueous phase was extracted with ethyl acetate at room tures adjusted to this standard contained approximately temperature. This was then concentrated to dryness in 1x108 CFU/mL [16, 17]. Alternatively, to induce spore vacuum (extract C, yield 0.95%). The aqueous solution was formation, the molds were grown on Potato Dextrose Agar

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(PDA) slants at 27ºC for 5 to 7 days. After being counted dium (control) or medium with extracts at concentrations of with Thoma slide, the spore concentration was adjusted to 0.17, 1.7, 17, 85 or 170 µg/mL for 24 h. Each tested con- 106 CFU/mL with sterile 0.1% Tween 80 for each mold. centration was inoculated in at least eight wells, and all Mueller-Hinton agar (MHA) and Sabouraud Dextrose Agar experiments were performed at least three times. (SDA), sterilized in a flask and cooled to 45-50°C, were distributed to the sterilized Petri dishes (9 cm). The entire 2.7. MTT assay surface of the MHA plates and Sabouraud 4% glucose Drug cytotoxicity screening was determined by using 3- medium (SGM) plates was inoculated with the bacteria (4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide and fungi by spreading through a sterile swab dipped into (MTT) colorimetric assay [21]. MTT assay measures cell the adjusted suspensions [19]. Six wells, each 6 mm in dia- viability, proliferation or cytotoxicity based on the ability meter, were cut out of the agar and 20 µL of the extract of the mitochondrial succinate-tetrazolium reductase sys- solutions were placed into each well. The plates were tem to convert the yellow tetrazolium MTT to a purple for- incubated with bacteria at 37°C for 24 h or with fungal mazan dye. The amount of dye produced is proportional to strains at 30°C for 48 h. Following these incubations, the the number of metabolically alive cells [22]. MTT solution dishes were kept at 4°C for 2 h. The diameters of the in- (25 µL) was added to each well and incubated at 37°C for hibition zones were measured in millimeters. Penicillin 4 h. After all the medium had been removed from the and tetracycline (Bioanalyse) were used as a positive con- wells, the formazan crystals were dissolved in 100 µL trol for bacteria; amphotericin B (Sigma) as a positive con- DMSO for 5 min at room temperature. The absorbance of trol for fungi; and DMSO was used as negative control. formazan dye was read at 550 nm using a microplate reader All assays were done in duplicate. (Bio-Tek Instruments), and cell survival percentages were calculated according to the following formula: absorbance 2.5. Minimum inhibitory concentration (MIC) of treated cells in each well x 100 / mean absorbance of MIC was determined by the micro dilution method us- control cells. The dose response curves were calculated ing a 96 well plate according to NCCLS [17, 20]. Primar- for all extracts at the above-mentioned concentrations and ily, 100 µL of MHB or Sabouraud Dextrose Broth (SDB) expressed as the mean percent fraction of control ± SEM. was placed in each well. The stock solutions of the extracts were diluted and transferred into the first well, and serial All statistical analyses were performed using one-way dilutions were performed so that concentrations in the analysis of variance (ANOVA) and followed up by Tukey’s range of 1.5-1500 µg/mL were obtained. The inoculums multiple comparison tests. were adjusted to contain approximately 105 CFU/mL bacteria and 104 CFU/mL fungi, as described above. One hun- dred µL of the inoculums was added to all the wells and 3. RESULTS the plates were incubated at 37ºC for 24 h for bacteria or at 30ºC for 48 h for fungi. Antimicrobial activity was detected The bacterial and fungal strains used for the antimicro- by adding 20 µL of 0.5% TTC aqueous solution. The MIC bial activity test are listed in Table 1. The results of the value was taken as the lowest concentration of the extract antibacterial activities of H. sericeum extracts in different that inhibited any visible bacterial or fungal growth, as in- solvents on test microorganisms are represented in Table 2. dicated by TTC staining after incubation [16, 20]. Penicil- The growth of fungi was not influenced by any of these lin and tetracycline were again used as the reference anti- extracts. Among the tested bacteria, P. aeruginosa was biotic control. found to be the most sensitive to all extracts. Only extract C showed a moderate inhibitory effect on B. subtilis (9 mm), 2.6. Cell culture and treatments followed by E. faecalis (9 mm). The applied concentra- DMSO dissolved extracts of acetone, A and C of H. tions of all extracts of H. sericeum did not demonstrate sericeum were diluted further in Dulbecco’s Modified Ea- any activity against E. coli, S. typhimurium and S. aureus. gle’s Medium (DMEM) at a ratio of 1:10 and the maximum Upon comparing all the extracts, the acetone extract exhib- concentration of DMSO was adjusted to 0.1%. All ex- ited the highest antimicrobial activity against P. aeruginosa tracts were prepared immediately prior to use and pro- (14 mm). With the exception of B and D (9 mm), all the tected from light. other extracts demonstrated a strong inhibitory effect against 2 P. aeruginosa. Table 3 illustrates the MIC ranges of three The C6 cells in 75 cm flasks were cultured in DMEM extracts against P.aeruginosa. The MIC values of ace- supplemented with 10% fetal bovine serum (FBS), 1% tone, extracts A and C, against bacterial pathogens ranged penicillin-streptomycin solution at 37°C in a humidified from 23.4 - 375 µg/mL. With the exception of P. aerugi- atmosphere of 5% CO2. When confluence was achieved, nosa, the applied concentrations of methanol, chloroform, the C6 cells were trypsinized and viability was accessed by and acetone, extracts A, B and D, did not demonstrate any trypan blue dye exclusion and found to be higher than 98%. inhibitory effect against any of the tested bacteria. After trypsinization, 250 µL samples of medium containing approximately 2x104 cells/well were seeded into 96-well Acetone extract of H. sericeum at 0.17, 1.7, 17 and tissue culture plates and incubated for 24 h. Following this 85 µg/mL concentrations did not change C6 growth, but at incubation period, the medium was replaced with only me- 170 µg/mL inhibited about 16 % in 24 h (p<0.001). At con-

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centrations of 1.7, 17 (p<0.01), 85 and 170 (p<0.001) TABLE 3 - Minimum inhibitory concentrations (MIC, µg/mL) of µg/mL extract A decreased C6 cell viability by 11, 10, 14 three extracts of H. sericeum against P. aeruginosa. and 13 % in 24 h, respectively. However, extract C at 85 Extracts MIC (µg/mL) and 170 µg/mL concentrations reduced C6 viability by 39 Acetone 23.4 and 86 % in 24 h, respectively (p<0.001, Fig. 1). Extract A 93.8 Extract C 375.0 Penicillin (µg/mL) <1.5 Tetracycline (µg/mL) <1.5

TABLE 2 - Antibacterial activities of Homalothecium sericeum extracts as inhibition zones (mm).

Control bacterial strains Methanol Chloroform Acetone Extract A Extract B Extract C Extract D Control 1* 2** B. subtilis 209 NS NS NS NS NS 9 NS 13±0,2 24±0,1 E. faecalis 29212 NS NS NS NS NS 9 NS 27±0,2 15±0,1 E. coli 25922 NS NS NS NS NS NS NS 30±0,2 25±0,1 P. aeruginosa 27853 11 11 14 13 9 12 9 30±0,2 20±0,1 S. typhimurium 14028 NS NS NS NS NS NS NS 21±0,2 18±0,1 S. aureus 25923 NS NS NS NS NS NS NS 35±0,2 27±0,1 NS: not sensitive; * Penicillin (10 µg/disc); ** Tetracycline (30 µg/disc)

extract of acethone 120 extract A 100 extract C *** ** ** 80 *** *** *** 60

C6 survival (%)C6 survival 40

20 ***

0 C 0,17 1,7 17 85 170 Doses (µg/mL)

FIGURE 1 - The effects of acetone, extracts A and C of H. sericeum on C6 cell survival for 24 h (C: control, **: p<0.01, ***: p<0.001).

4. DISCUSSION Although just extract C had some weak inhibitory activity on both B. subtilis and E. faecalis, all the extracts of In this study, we assayed the antimicrobial and an- H. sericeum demonstrated an inhibitory effect against P. timitotic activity of some extracts of H. sericeum against aeruginosa (acetone>extract A>extract C>methanol= chlo- 6 bacterial, 7 fungal species, and a glioma cell line. Aside roform> extract B=extract D). The acetone extract showed from their effectiveness against bacteria, our results indi- the highest antibacterial action on P. aeruginosa, a Gram cated that none of the extracts had any antifungal activity. negative bacterium. Similarly, Basile et al. [5, 6] reported In contrast, Veljic et al. [23] studied the antimicrobial activ- that the sensitivity of Gram-negative bacteria against ace- ity of methanol extracts of different mosses and indicated tone extracts of Rhynchostegium riparioides and Lunu- that all the tested extracts showed a strong antifungal activ- laria cruciata (Bryophyta) is generally higher than that of ity. Singh et al. [7] also found that 7 moss species were Gram-positive ones. While their MIC result of acetone ex- active on 8 fungi. However, there are some studies on tracts against P. aeruginosa was 16 and 128 µg/mL, respec- selected bryophytes that support our results of no antifun- tively, our best MIC result for acetone extract against P. gal activity [10, 24]. aeruginosa was 23.4 µg/mL. Acetone extract of R. ri-

parioides had a similar antibacterial activity with H. against P. aeruginosa, but extract C has the strongest cyto- sericeum [6]. Conversely, Basile et al. [5] found that P. toxic activity against C6 glioma cells. Since the acetone aeruginosa was not sensitive to the acetone extract of and C extracts of H. sericeum may prove to be a good and Pleurochaete squarrosa, but that E .coli was most sensi- new source of antibacterial and antiproliferative agents, tive. Since antibiotics are generally more active against further studies are needed to clarify the content of these Gram positive than Gram negative bacteria, it is more extracts. difficult to treat the infections of Gram negative bacteria. The antibacterial activity obtained in this study against a Gram negative bacterium may indicate that H. sericeum ACKNOWLEDGEMENT extracts possess more antimicrobial action against Gram negative microorganisms than Gram positive microorgan- This study was supported by Eskisehir Osmangazi isms. P. aeruginosa is frequently associated with infections University Scientific Research Projects Committee (Pro- of the urinary and respiratory tract in humans. P. aerugi- ject No: 2007/19012). nosa infections are also common in patients receiving treatment for severe burns or other traumatic skin damage and in people suffering from cystic fibrosis [25]. REFERENCES Since the acetone extracts A and C of H. sericeum [1] Bodade, R.G., Borkar, P.S., Md Saiful, A. and Khobragade, showed greater antibacterial action, we preferred to study C.N. (2008) In vitro screening of Bryophytes for antimicro- only these extracts on the rat glioma cell line. Only the bial activity. Journal of Medicinal Plants 7(4), 23-28. highest concentration of acetone extract had a partial effect [2] Zinsmeister, H.D. and Mues, R. (1987) Moose als Reservoir for 24 h. 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[27] Spjut, R.W., Cassady, J.M, Mc Cloud, T., Suffness, M., Nor- Fax: +90 222 2393578 ris, D.H., Cragg, G.M. and Edson, C.F. (1988) Variation in cytotoxicity and antitumor activity among samples of the E-mail: [email protected] Moss Claopodium crispifolium (Thuidiaceae). Economic Bo- tany 42(1), 62-72. FEB/ Vol 20/ No 2a/ 2011 – pages 461 - 466

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