Antibacterial Properties and Flavonoids Content of Some Mosses Common in Armenia

Iranian Journal of Pharmaceutical Sciences 2020: 16 (4): 31-42 www.ijps.ir Original Article

Inesa Semerjyan, Gayane Semerjyan, Henrikh Semerjyan, Armen Trchounian*

Department of Biochemistry, Microbiology and Biotechnology, Yerevan State University, Yerevan, Armenia.

Abstract The present study was aimed to evaluate the antibacterial potential of ethanol, methanol, acetone, and aqueous extracts of some bryophytes common in Armenia (Mnium spinosum (Voit) Schwaegr, Brachythecium salebrosum (Web. et Mohr) B.S.G., Thuidiumrecognitum (Hedw) Lindb and Dicranum scoparium (Hedw). Antibacterial activity was determined using agar-well diffusion method against five bacterial species (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Salmonella enterica, and S. typhimirium). The results showed that the bryophytes D. scoparium and B. salebrosum possessed high antibacterial activity in methanol extracts, whereas M. spinosum has high antibacterial activity in acetone extract. Antibacterial activity against S. aureus was comparably weaker. Ethanol extract of T. recognitum had greater antibacterial activity than the extract of M. spinosum. Of all those tested for antibacterial activity bryophytes in vitro extracts of D. scoparium have yielded the most promising results. Antibacterial activity might be caused by a high content of flavonoids in bryophytes determined. Thus, the studied bryophytes might be used to develop new antibacterial agents.

Keywords: agar-well diffusion method; antibacterial activity; bryophytes; flavonoids; plant extracts; solvents.

1. Introduction synthesized drugs. Safe, effective and

Corresponding Authors: Armen Trchounian, Department affordable indigenous remedies have become of Biochemistry, Microbiology and Biotechnology, more popular among people living in urban Yerevan State University, Yerevan, Armenia. and rural areas. Therefore, medicinal plants Tel: (+374)60-710520 Email: [email protected] have become an integral part of health care Cite this article as: Semerjyan I, Semerjyan G, Semerjyan system. Ethnobotanical studies are important H, Trchounian A, Antibacterial Properties and Flavonoids Content of Some Mosses Common in to identify the ancient and modern cultures of Armenia, 2020, 16 (4): 31-42. plant use in the world and to preserve the

Medicinal plants are important components original knowledge of medicinal plants. of both traditional and conventional medical Moreover, more attention is paid to medicinal products since ancient times. Most people plants due to their effectiveness, the cost of prefer herbal-based medicine over chemically existing pharmaceutical products and cultural

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preferences. It has been reported that a large They have an interesting feature – they are not number of plants have antimicrobial and attacked by bacteria, fungi or pests. They are a antioxidant potential (1-3). In addition, potential source for medicine because they medicinal plants are used as food, human contain secondary metabolites. Some active medicinal drugs, veterinary medicine and are biomolecules, such as terpenoids and phenolic important for the economy (4). bibenzyls, have been studied for cytotoxicity Bryophytes or “mosses” are the oldest against various human cancer cell lines, their group of terrestrial plants, which include antibiotic, antioxidant, antithrombin, liverworts, hornworts and mosses. This group antiplatelet and neuroprotective activity, and of non-vascular plants includes between their ability to inhibit a number of 25,000 and 28,000 species, and they grow in biochemically important enzymes (7). Crude shady places. Unlike vascular plants, they are extracts or various bioactive compounds were herbaceous and absorb water and minerals isolated from mosses for anticancer efficacy mainly through leaves (5). Bryophytes are on cancer cell lines. used as indicator species for erosion control, The cytotoxic efficacy of bryophytes was heavy metal pollution, detection and reflected in several biochemical markers of the monitoring of radioactivity, as aquatic induction of apoptosis and necrosis, such as bioindicators, radioactivity indicators, as DNA fragmentation, nuclear condensation, material for beds of seed, fuel, drugs and food proteolysis of poly (ADP-ribose) polymerase sources, pesticides, for nitrogen fixation, (PARP), activation of caspases (a family of landscaping with mosses, waste treatment, cysteine aspartic proteases), inhibition of construction, clothing, furnishing, packaging, antiapoptotic nuclear transcriptional factor B, genetic engineering and for tillage, etc. (6). activation of p38 (mitogen-activated protein The active components of bryophytes, found kinase), etc. Some of these mechanisms in most of them are used as antibacterial, actually play a decisive role in the induction of antifungal, cytotoxic, antitumor and apoptosis (9). Despite numerous reports on the insecticidal agents in medical and agricultural biological activity of bryophytes, including fields. The phytochemical agents of their antibacterial activity, it is necessary to bryophytes include a wide range of clarify the effects of various extracts and to biologically active compounds, such as determine their chemical compositions. In carbohydrates, lipids, proteins, steroids, addition, various species growing in different polyphenols, terpenoids, organic acids, regions may have a special chemical alcohols, fatty acids, aliphatic compounds, composition and specific properties, that acetogenins, phenylquinones, aromatic and should be studied. phenolic substances (7, 8). Bryophytes are common in Armenia, but A number of bryophytes has been used in they have been little studied. About 350 traditional medicine to treat various diseases. species of mosses were identified in Armenia

32 Antibacterial Properties and Flavonoids Content of Some Mosses Common in Armenia

(10), most of them in the forest zone (10, 11), identified by Dr. A. Poghosyan (Department of and some of them are highly specialized and Botany and Mycology, Yerevan State have a specific ultrastructure (11). For some University (YSU), Armenia) and deposited in species grown in Armenia, especially the Takhtadjyan Herbarium of the Department Brachythecium campestre, Pelliaepiphilla, of Botany and Mycology, YSU (Vouchers no. Tortularuralis and others, the chemical 13450, 13451, 13452 and 13453, respectively). composition and activity of some enzymes involved in metabolism of purine bases have 2.2. Plant Extraction been reported (12). This activity depends on Plant material was carefully collected from heavy metal ions (Cu2+, Cd2+), polluting the the soil and washed thoroughly with distilled environment, therefore it is assumed that these water to remove the adhering soil or mosses serve as bioindicators for monitoring extraneous particles of dust. the state of the environment. In addition, there For microbiological studies, green parts of are some species with a high content of mosses (without rhizoids) were used, which secondary metabolites that may have were washed with liquid soap and running antimicrobial activity (7), therefore, mosses distilled water. Mosses were dried at room with high antibacterial activity can be used to temperature. After that, the mosses were develop antimicrobial drugs. Such a study is of placed in a flask and extracted with methanol, considerable interest due to the problem of ethanol, acetone or water (1 g moss per 20 mL bacterial resistance to antibiotics (3, 13, 14) solvent). The extraction was carried out on a and may be a useful tool to combat this magnetic stirrer at 18-20 0C for 48 h. After problem. Plant derived antimicrobials are also that, the samples were centrifuged at 1500 rpm considered safer than their synthetic for 10 min. The supernatants obtained were counterparts due to their natural origin (12). dried at 37 0C. The resulting powder was In this study, the species of bryophytes dissolved in 580 μL of dimethyl sulfoxide common in Armenia were screened for their (DMSO). Samples were sterilized, as antibacterial activity and main phytochemical described (14). It should be noted that the components. This study will enable the use of solvents used are usually applied with various bryophytes to develop new antibacterial drugs. mosses (7, 16-18) and other plant extracts (2, 3, 13), and they are also used in folk medicine. 2. Materials and Methods 2.1. Plant Materials 2.3. Test Microorganisms The bryophytes Mnium spinosum Antibacterial activity was tested in vitro Schwaegr, Brachythecium salebrosum B.S.G., against Escherichia coli, Bacillus subtilis, Thuidium recognitum Lindb and Dicranum Staphylococcus aureus, Salmonella enterica scoparium were collected in Armenia (at an and S. typhimirium. The strains E. coli VKPM- altitude of ∼1450 m). The plants were M17 (Russian National Collection of

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Industrial Microorganisms, Institute of absorbance of the solutions was measured at Genetics and Selection of Industrial 415 nm wavelength using spectrophotometer Microorganisms, Moscow, Russia), B. subtilis GENESYS 10S UV-VIS (Thermo Scientific, WT-A1 (isolated from a soil sample), St. Germany). The total flavonoid content was aureus MDC 5233 (Microbial Depository calculated from the catechin calibration curve Center, National Academy of Sciences (NAS), and expressed as mg catechin equivalent per g Yerevan, Armenia), S. typhimirium MDC 1759 of dried plant material. (wild type), S. enterica K3, isolated from infected pigs and provided by Dr. M. 2.6. Data Processing Mkrtchyan (Institute of Molecular Biology, Data Processing was done using Excel NAS, Yerevan, Armenia) were used. 2013 Microsoft program. Statistical analysis was done using one-way analysis of variance 2.4. Determination of Antibacterial Activity (ANOVA). The validity of differences Antibacterial activity was determined by between different series of experiments (n=5) the agar well diffusion method (19). Plant was evaluated by Student P-test: the value extracts were tested for antibacterial activity P<0.05 was considered, as valid. through an agar platе previously infected with microorganisms, 5µL of the studied extracts of 3. Results and Discussion moss were instilled in the wells. 3.1. Antibacterial Activity of Moss Extracts Dimethylsulfoxide (DMSO) was used as a The results on the antimicrobial activity of control. All plates were kept at low plant extracts (Figures 1-4) showed that temperature for 1-1.5 h for sample diffusion, antibacterial activity of the bryophyte M. and then incubated at 37 0C for 24 h. After spinosum (Figure 1) against B. subtilis and E. incubation, bactericidal and bacteriostatic coli was greater in acetone extract, forming an zones were visible on the agar plate. inhibition zone of 2.2 cm, while the aqueous and methanol extracts had a zone of 2.0 cm. 2.5. Determination of Total Flavonoids Antibacterial activity against St. aureus was Content comparatively weaker, and ethanol extract had Total flavonoid content in ethanol extract almost no antimicrobial effect. Then, the of mosses was evaluated by the AlCl3 method, bryophytes B. salebrosum (Figure 2) and D. as described by Andreeva and Kalinkina (20). scoparium (Figure 4) have high antibacterial 500 µL of plant extract was taken into a test activity in methanol extracts. Moreover, the tube, 100 µL of 10% AlCl3 was introduced, bryophyte T. recognitum had a higher then 100 µL of 1 M CH3COONa was added, antibacterial activity in ethanol extract (Figure then 2800 µL of distilled H2O was also added. 3) than M. spinosum. Particulalry, methanol The tubes were incubated at room temperature extracts of D. scoparium possessed high for 30 min to complete the reaction. The antimicrobial activity against E. coli, forming

34 Antibacterial Properties and Flavonoids Content of Some Mosses Common in Armenia a zone of 3.4 cm, and acetone, ethanol and It should also be noted that among aqueous extracts gave zones of 2.1, 2.5 and 2.7 microorganisms, Gram-negative bacteria (E. cm, respectively (Figure 4). In case of S. coli, Erwinia chrysanthemi, Pseudomonas enterica, St. aureus and B. subtilis the activity aeruginosa, S. enterica) were more sensitive to of extracts of D. scoparium was low. The bryophytes; but this activity also depended on methanol extracts of bryophyte B. salebrosum solvents (16, 17). exhibited strong antibacterial activity against Thus, it can be concluded that the E. coli forming a zone of 3.0 cm (Figure 2). bryophytes extracts have high antibacterial No marked inhibition zones were determined activity, and this activity of bryophyte extracts with DMSO, as control (Figures 1-4, wells in depends on solvent used. the centers of plates). Some interesting reports have been 3.2. Phytochemical Screening and Total reviewed to compare data obtained for mosses Flavonoids Content grown in Armenia with the results recorded for The search for potential plant based various bryophytes from other regions, antimicrobials has increased dramatically due including the effectiveness of different to the emergence of multidrug resistance (3, solvents used. Specifically, there is an 13, 14). The identification of plant based interesting report on the high antibacterial antioxidants is another aspect that has gained effects of acetone extract of Polytrichum tremendous importance in protecting cells / juniperinum and methanol extract of Tortella tissues from damage caused by free radicals tortuosa (16). Different antibacterial effects (20). The phenolic compounds present in were obtained with extracts of plants act as powerful antioxidants that can Rhynchostegium vagans A. Jaeger moss: protect cellular structures and mechanisms ethanol extract of R. vagans was the most from free radicals by acting as hydrogen potent with the lowest minimum inhibitory donors and radical scavengers. Antioxidants concentration (MIC) (3.91 to 61.25 µg mL-1) act as free radical scavengers, and thus they and minimum bactericidal/fungicidal help to mitigate the effect of oxidative stress in concentration (MB/FC) (3.91 to 500 µg mL-1) various diseases. Many studies have (17). Importantly, ethanol extracts were found demonstrated the effectiveness of plant to be superior over antibiotics used derived products as a good source of (chloramphenicol and fluconazole) (17). Other antioxidants against various diseases caused results showed that bryophytes Asterella by reactive oxygen species (21). Several angusta had greater antibacterial activity in studies have reported that phenolic methanol extract, and in Targionoa compounds, such as flavonoids and phenolic hyphophylla and Plagochasma articulate the acids present in plants, are responsible for their antibacterial activity was greater in ethanol antioxidant nature (18, 21). extract (18).

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Phytochemical screening indicated the potential candidates for the fight against presence of secondary metabolites, such as multidrug-resistance (22). flavonoids, in mosses (Figure 5). The data The antibacterial activity of flavonoids showed that flavonoids were found in varying depends on the structures, namely on the amounts in the investigated mosses. The substitutions on the aromatic rings. With the largest number of flavonoids was contained in plant extracts possessing antibacterial activity M. spinosum, T. recognitum (Figure 5). increasingly being found, more and more Probably, the antibacterial activity of mosses' flavonoids have proven to be antibacterial extracts was caused by the flavonoids agents, especially with hydrophobic contained. This suggestion seems likely, since substituents, such as prenyl groups. Flavones it is known that flavonoids are synthesized by have been widely investigated for their plants in response to bacterial infection, which antibacterial activity (23); two di-prenylated means that they can be effective antibacterial flavones cuvanon C and moruzin isolated from substances against various microorganisms medicinal plants, were evaluated for their (16). antimicrobial activity using broth Among the various phytochemical microdilution methods. They showed strong compounds of natural origin, flavonoids are activity against Gram-negative bacteria (E. known to play an important role in medicine coli, S. typhimurium) and only limited activity and pharmacology. Flavonoids are biologically against Gram-positive bacteria (S. epidermis, active secondary plant compounds. There are St. aureus). Yin and coworkers (24) also numerous studies suggesting their beneficial isolated a mono-prenylated flavon called effects on human health. Particularly, these corylifol C from Psoralea corylifolia seeds compounds are tested as supportive or and found to be ineffective for inhibiting the alternative therapies for diseases such as growth of St. aureus and S. epidermidis in cancer or type-II diabetes. After oral vitro. These results indicate the importance of administration, they interact with digestive the degree of prenylation for antibacterial enzymes and sugar carriers in the small activity. The substitutive prenyl group intestine. Flavonoid glycosides as well as sometimes reacts with the adjective hydroxyl some aglycones have been reported to reduce group to form a six-member heterocycle, postprandial hyperglycemia in patients with which probably reduces its activity (25). diabetes through interactions with glucose However, further study is required. carriers in the apical membranes of enterocytes (22). It has been also shown that many 4. Conclusion flavonoids interfere with ATP-dependent drug- In this study, all mosses' extracts from efflux transporters which are involved in the bryophytes grown in Armenia demonstrated resistance of cancer cells against various the ability to inhibit the development of cytostatic drugs, and therefore may be bacteria (the diameters of inhibition zone

36 Antibacterial Properties and Flavonoids Content of Some Mosses Common in Armenia

varied from 0.3 to 3.4 cm)., Antibacterial References activity against E. coli was found in all moss [1] Kumar V, Sharma N, Sourirajan A, Khosla P, extracts. The highest activity was determined Dev K. Comparative evaluation of antimicrobial and antioxidant potential of ethanolic extract and its in extracts of D. scoparium (inhibition zone fractions of bark and leaves of Terminalia arjuna diameter of 3.4 cm), and the lowest activity from north-western Himalayas, India. J. Trad. Compl. was observed in extracts of B. salebrosum Med. (2018) 8: 100-106. (inhibition zone diameter of 0.3 cm). [2] Moghrovyan A, Sahakyan N, Babayan A, Antibacterial activity depended on the solvents Chichoyan N, Petrosyan M, Trchounian A. Essential used. Then, antibacterial activity might be oil and ethanol extract of Oregano (Origanum vulgare associated with flavonoids; among mosses M. L.) from Armenian flora as a natural source of terpenes, flavonoids and other phytochemicals with spinosum and T. recognitum had the highest antiradical, antioxidant, metal chelating, tyrosinase total flavonoid content. inhibitory and antibacterial activity. Curr. Thus, the mosses common in Armenia that Pharmaceut. Design (2019) 25 (16): 1809-1816. we studied have antibacterial activity and can [3] Sahakyan N, Ginovyan M, Petrosyan M, be used as wide range of herbal antibacterial Trchounian A. Antibacterial and anti-phage activity of agents. Especially, extracts of D. scoparium Plantago major l. raw material. Proc. Yerevan State Univ.: Chemistry & Biology (2019) 53 (1): 59-64. can be used in medicine, pharmaceutical and [4] Amalraj A, Gopi S, Medicinal properties of food industry (as natural alternative preventive Terminalia arjuna (Roxb.) Wight & Arn.: A review. agents for combating food poisoning diseases J. Trad. Compl. Med. (2017) 7: 65-78. and preserving food products, avoiding the use [5] Peters K, Treulter H, Doll S, Kidt A.S, of chemical antibacterial agents). Hankemier T, Neumann S. Chemical diversity and classification of secondary metabolites in nine bryophyte species. Metabolites (2019) 9: 222. Acknowledgments [6] Banerjee KS, Srivastava AK. Assessment of The authors would like to acknowledge Dr. diversity and traditional uses of Bryophytes along A. Poghosyan from Department of Botany and some hill roads in a biodiversity hot spot region of Mycology, Yerevan State University India – A case study of mizoram. Int. J. Nat. Resour. (Armenia), for identification of collected plant Ecology Manag. (2019) 4 (3): 73-82. material. Thanks to Dr. L. Hakobyan from [7] Klavina L, Springe G, Nikolajeva V, Research Institute of Biology, Yerevan State Martsinkevich I, Nakurte I, Dzabijeva D, Steinberga I. Chemical composition analysis, antimicrobial activity University (Armenia), for help with improving and cytotoxicity of screening of moss extracts (Moss English. Phytochemistry). Molecules (2015) 20: 17221-17243. The study was done in framework of Basic [8] Chandra S, Chatdra D, Barh A, Pandey K, support from Committee of Science, Ministry Sharma I. Bryophytes: Hoard of remedies, an ethno- of Education, Science, Culture and Sport of medical review. J. Trad. Compl. Med. (2017) 7: 94- Armenia, to Yerevan State University. 98.

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[9] Dey A, Mukherjee A. Therapeutic potential of (2016) 6: 10-14. bryophytes and derived compounds against cancer. J. [18] Pratima KS. Phytochemical screening and Acute Disease (2015) 4 (3): 236-248. antibacterial activity of Bryophytes. Int. J. Life Sci. [10] Manakyan VA. Results of bryological studies in (2017) 5(3): 405-408. Armenia. Arstoa (1996)5: 15-34 (in Russian). [19] Boyanova L, Gergova G, Nikolov R, Derejian [11] Pogosyan AV, Shahazizyan IV, Nanagulyan S, Lazarova E, Katsarov N, Mitov I, Krastev Z. SG, Zakaryan NA, Eloyan IM. Ultrastructure of Bulgarian propolis against 94 Helicobacter pylori mosses of forest of Arailer mountain (Armenia). strains in vitro by agar-well diffusion, agar dilution Znanie (Knowledge) (2015)12: 73-76 (in Russian). and disc diffusion method. J. Med. Microbiol. (2005) [12] Semerjyan H., Semerjyan G. Use of purine basis 54: 481-483. exchange enzymes for pollution monitoring in [20] Andreeva V, Kalinkina GI. Development of a environment. Biolog. J. Armenia (2017) 69: 63-66 (in method for the quantitative determination of Russian). flavonoids in Artemisia vulgaris. Khim. Rastit. Syr’ya [13] Ginovyan M, Trchounian A. Novel approach to (Chemistry of Plant Raw Materials) (2000)1: 85-88 combat antibiotic resistance: evaluation of some (in Russian). Armenian herb crude extracts for their antibiotic [21] Trchounian A, Petrosyan M, Sahakyan N. Plant modulatory and antiviral properties. J. App.l cell redox homeostasis and reactive oxygen species. Microbiol. (2019)127: 472-480. D. Gupta (Ed.), In: Redox State as a Central [14] Sahakyan N, Petrosyan M, Trchounian, A. The Regulator of Plant-Cell Stress Responses. Springer, activity of Alkanna species in vitro culture and intact Cham. (2016): 25-50. plant extracts against antibiotic resistant [22] Wenzel U. Flavonoids as drugs at the small bacteria. Curr. Pharmaceut. Design (2019) 25(16): intestinal level. Curr. Opin. Pharmacol. (2013) 13 (6): 1861-1865. 864-868. [15] Mehmood N, Zubair M, Rızwan K, Rasool N, [23] Farhadi F, Khameneh B, Iranshahi M, Iranshahy Shahid M, Ahmad V. Antioxidant, antimicrobial and M. Antibacterial activity of flavonoids and their phytochemical analysis of Cichorium intybus seeds structure–activity relationship: An update review. extract and various organic fractions. Iran. J. Pharm. Phytother. Res. (2019) 33 (1): 13-40. Res. (2012)11: 1145-1151. [24] Yin S, Fan C.Q, Wang Y, Dong L, Yue JM. [16] Savaroglu F, Ilhan S, Filik-Iscen C. An Antibacterial prenilflavone deriatives from Prosalea evaluation of the antimicrobial activity of some corylifolia, and their structure-activity relationship Turkish mosses. J. Med. Plants Res. (2011) 5: 3286- study. Bioorg. Med. Chem. (2004) 12 (16): 4387- 3292. 4392. [17] Negi K, Chaturvedi P. In vitro antimicrobial [25] Xie Y, Yang W, Tang F, Chen X., Ren L. efficacy of Rhynchostegium vagans A. Jaerger (moss) Antibacterial activities of flavonoids: structure- against commonly occurring pathogenic microbes of activity relationship and mechanism. Curr. Med. Indian sub-tropics. Asian Pacific J. Trop. Disease Chem. (2015) 22: 132-149.

38 Figures:

Figure 1. Antibacterial activity of acetone, methanol, ethanol and water extracts of M. spinosum. The zones of lack of growth of the tested microorganism under the influence of various extracts of moss M. spinosum are shown. All data are expressed as mean ±SEM of five independent experiments (p< 0.05). For the others, see “Material and methods”.

Figure 2. Antibacterial activity of acetone, methanol, ethanol, water extracts of B. salebrosum. For details, see the legends to Fig. 1.

Figure 3. Antibacterial activity of acetone, methanol, ethanol and water extracts of T. recognitum. For details, see the legends to Fig. 1.

Figure 4. Antibacterial activity of acetone, methanol, ethanol and water extracts of D. scoparium. For details, see the legends to Fig. 1.

40 Antibacterial Properties and Flavonoids Content of Some Mosses Common in Armenia

Figure 5. Total flavonoid content in ethanol extracts of different mosses studied. The total flavonoid content was quantified by the standard curve of catechin (mg g-1). For details, see «Materials and methods».

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