Słupskie Prace Biologiczne

Nr 13 ss. 37-54 2016

ISSN 1734-0926 Przyjęto: 7.11.2016 © Instytut Biologii i Ochrony Środowiska Akademii Pomorskiej w Słupsku Zaakceptowano: 16.01.2017

ANTIMICROBIAL SCREENING OF THE VARIOUS EXTRACTS DERIVED FROM THE LEAVES AND PSEUDOBULBS OF COELOGYNE SPECIOSA (BLUME) LINDL. ()

Lyudmyla Buyun1 Halyna Tkachenko2 Zbigniew Osadowski2 Anna Góralczyk2 Lyudmyla Kovalska1 Oleksandr Gyrenko1

1 M. Gryshko National Botanical Garden National Academy of Science of Ukraine Timiryazevska St. 1, Kiev 01014, Ukraine e-mail: [email protected] 2 Pomeranian University in Słupsk Institute of Biology and Environmental Protection Department of Zoology and Animal Physiology Arciszewski St. 22b, 76-200 Słupsk, Poland

ABSTRACT

The present study was conducted to investigate in vitro antimicrobial activity of various extracts obtained from vegetative parts of Coelogyne speciosa against Gram- positive (Staphylococcus aureus ATCC 25923) and Gram-negative bacteria (Esche- richia coli ATCC 25922). The leaves and pseudobulbs of C. speciosa , culti- vated under glasshouse conditions, were sampled at M.M. Gryshko National Botani- cal Garden (NBG), National Academy of Science of Ukraine. Freshly collected leaves and pseudobulbs were washed, weighted, crushed, and homogenized in 96% ethanol, methanol, ethyl acetate, hexane, and dichloromethane (in proportion 1:19) at room temperature. Antimicrobial activity was determined using the agar disk dif- fusion assay (Bauer et al. 1966). The present study has shown that ethanolic extracts from leaves and pseudobulbs of C. speciosa exhibited strong activity against S. au- reus (inhibition zone diameter were 21.5 mm and 19 mm, respectively), while meth- anolic extract from leaves and pseudobulbs revealed mild activity (8.1 and 8 mm). Moreover, it has been observed that ethyl acetate, hexane and dichloromethane ex- tracts obtained from leaves and pseudobulbs of C. speciosa revealed no antibacterial

37 activity against S. aureus. Our results also showed that ethanolic extract from leaves of C. speciosa exhibited strong activity against E. coli (inhibition zone diameter was 21 mm), while other extracts from pseudobulbs revealed minimum activity (inhibition zone diameter was 12 mm). Those extracts in ethyl acetate, hexane and dichloro- methane both from the leaves and pseudobulbs revealed no antibacterial activity against S. aureus. Thus, the research showed that ethanolic extracts of C. speciosa pos- sess antibacterial potency against S. aureus and E. coli and may be used as natural an- tiseptics and antimicrobial agents in medicine and veterinary practice. Considering the medicinal importance of the tested microorganisms, the findings of this research are considered to be very promising in the perspective of new drug development from sources.

Key words: Coelogyne speciosa Lindl., Staphylococcus aureus, Escherichia coli, antimicrobial activity, orchids, agar disk diffusion assay

INTRODUCTION

Living plant collections in the botanic gardens are underutilized worldwide re- sources for plant conservation (Cibrian-Jaramillo et al. 2013). Beside, many groups of plants are valuable sources of diverse compounds, possessing broad spectrum of biological activity. In particular, orchids have been used as a source of medicines for millennia to treat different diseases and ailments including tuberculosis, paralysis, stomach disorders, chest pain, arthritis, syphilis, jaundice, cholera, acidity, eczema, tumour, piles, boils, inflammations, menstrual disorder, spermatorrhea, leucoderma, diarrhoea, muscular pain, blood dysentery, hepatitis, dyspepsia, bone fractures, rheu- matism, asthma, malaria, earache, sexually transmitted diseases, wounds and sores. In addition, many orchidaceous preparations are used as emetic, purgative, aphrodis- iac, vermifuge, bronchodilator, sex stimulator, contraceptive, cooling agent and rem- edies in scorpion sting and snake bite (Hossain 2011). Pharmacological studies have revealed antimicrobial, antioxidant, hepatoprotective, anti-inflammatory, anti-arthritic and wound healing properties of some orchids in pre-clinical studies (Bulpitt 2005, Panda and Mandal 2013). Orchids belong to the largest family of angiosperms, with approximately 880 genera and 27,800 species (Givnish et al. 2016). Today, nearly 50 orchid species are widely used in different systems of medicine (Panda and Mandal 2013). Some of the species like tessellata (Roxb.) Hook. ex G. Don, Dactylorhiza incarnata subsp. incarnata, nobile Lindl. have been already documented for their proven medicinal values (Kong et al. 2003). Pseudobulbs are most commonly used to cure ailments, followed by leaves, roots, tubers/rhizomes and flowers. Major local uses include aphrodisiacs, energizers, and treatments of skin burns, fractured or dis- located bones (both of humans and cattle), headaches, fever, and wounds. Other uses include insect repellent, blood purifier, skin fungi, antidote against snake bites and scorpion stings, inducement of abortions and recovery from child birth. Orchids are mainly used as paste, powder or juice, solely or mixed with milk, honey or wheat flour. Orchid extracts are either consumed orally or applied externally. Fresh orchid

38 flowers are used to induce vomiting by exposure to a dominant foul smell. Local communities in and also commonly eat freshly cut species of Coelogyne in the forest when they feel thirsty (Subedi et al. 2011). A wide range of chemical compounds has been identified from medicinal orchids (Subedi et al. 2011). Studies have reported the isolation of huge number of important phytochemicals from different genera of orchids such as alkaloids, flavonoids, stil- benoids, anthocyanins, triterpenoids, orchinol, hircinol, cypripedin, bibenzyl deriva- tives, phenanthrenes, jibantine, nidemin and loroglossin which are present in leaves, pseudobulb, roots, flowers or in the entire plant (Pant 2013). Coelogyne speciosa (Blume) Lindl. (Photos 1 and 2) belongs to the section Spe- ciosae, distributed from (), all over Malesia to the islands in the Pacific Ocean (Gravendeel 2000). The epithet speciosa (which is Latin for beautiful) refers to the showy flowers. Pseudobuls are ovoid and clustered, obtusely 4-angled when young. Leaves are one or two per pseudobulbs. Leaf blade is obovate-lanceolate to linear-lanceolate to lanceo- late; apex acuminate or cuspidate; main nerves 3-5 (Gravendeel 2000). Synanthous in- florescences, characteristic feature for C. speciosa, are predominant in the section Speciosae: the inflorescence-bearing shoot has an immature pseudobulb hidden in the basal scales and the young leaf or leaves on top of this pseudobulb start swelling and leaf or leaves fully develop (Gravendeel and de Vogel 1999) (Figs 1 and 2). The flowers open in succession (starting with the basal one) (www.orchidspecies. com/coelogynespeciosa.htm). Artificial pollination treatments, which we carried out under glasshouse condi- tions, have shown that Coelogyne speciosa is largely self-sterile. Barbara Graven- deel in Holland has initiated detailed research into Coelogyne speciosa, backed by extensive DNA analysis (Gravendeel and de Vogel 1999, Gravendeel 2000).

Photo 1. A specimen of Coelogyne speciosa Lindl., cultivated at M.M. Gryshko National Bo- tanical Garden (Kyiv, Ukraine)

Source: own research

39

Photo 2. The general view of Coelogyne speciosa flower

Source: own research

Moreover, many species of Orchidaceae family also possess antimicrobial activi- ty (Pérez Gutiérrez 2010).

Fig. 1. The branching pattern of shoot system Fig. 2. Scheme of the structure of C. speci- of Coelogyne speciosa osa elementary shoot: a) leaf; b) pseudo-

Source: own research bulb; c) buds; d) rhizome; e) node; f) inter- node

Source: own research

40 Therefore, the aim of this study was to evaluate the antimicrobial activity of the crude extracts obtained from leaves and pseudobulbs of C. speciosa prepared in dif- ferent solvent systems against Staphylococcus aureus, a clinically important micro- organism responsible for many infections, as well as against Escherichia coli to sup- port the use of these extracts as novel natural products for compounded bacterial- treatment modalities.

MATERIALS AND METHODS

Collection of Plant Material. The leaves and pseudobulbs of C. speciosa plants, cultivated under glasshouse conditions, were sampled at M.M. Gryshko National Botanical Garden (NBG), National Academy of Science of Ukraine. Since 1999, the whole collection of tropical and subtropical plants (including orchids) has the status of a National Heritage Collection of Ukraine. Besides that, NBG collection of tropi- cal orchids was registered at the Administrative Organ of CITES in Ukraine (Minis- try of Environmental Protection, registration No. 6939/19/1-10 of 23 June 2004). Preparation of Plant Extracts. Freshly collected leaves and pseudobulbs were washed, weighted, crushed, and homogenized in 96% ethanol, methanol, ethyl ace- tate, hexane, and dichloromethane (in proportion 1:19) at room temperature. Conse- quently five kinds of solvent systems were used to extract the active ingredients from the leaves and pseudobulbs of Coelogyne speciosa. Bacterial Growth Inhibition Test of Plant Extracts by the Disk Diffusion Method. Antimicrobial activity was determined using the agar disk diffusion assay (Bauer et al. 1966). Cultures of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were suspended in sterile solution of 0.9% normal saline and the tur- bidity adjusted equivalent to that of a 0.5 McFarland standard. Culture was inoculat- ed onto Mueller-Hinton (MH) agar plates. Sterile filter paper discs impregnated with 50 μL of extract dilutions were applied over each of the culture plates. Isolates of bacteria were then incubated at 37oC for 24 h. The plates were then observed for the zone of inhibition produced by the antifungal activity of C. speciosa. As negative control, disc impregnated with 50 μl of sterile ethanol, methanol, ethyl acetate, hex- ane, and dichloromethane was used in each experiment. The antimicrobial activities of the extracts tested were evaluated at the end of the inoculation period by measur- ing the inhibition zone diameter around each paper disc in millimeters. The plates were observed and photographs were taken. For each extract six replicate trials were conducted against each organism. Zone diameters were determined and averaged. Statistical analysis. All statistical calculations were performed on separate data from each bacterial strains (Zar 1999). All statistical calculation was performed on separate data from each individual with STATISTICA 8.0. The following zone di- ameter criteria were used to assign susceptibility or resistance of bacteria to the phy- tochemicals tested: Susceptible (S) ≥ 15 mm, Intermediate (I) = 11-14 mm, and Re- sistant (R) ≤ 10 mm (Okoth et al. 2013).

41

RESULTS

The present study has shown that ethanolic extracts from the leaves and pseudo- bulbs of C. speciosa exhibited strong activity against S. aureus (inhibition zone di- ameter were 21.5 mm and 19 mm, respectively), while methanolic extract from leaves and pseudobulbs revealed mild activity (8.1 and 8 mm) (Figs 3 and 4). More- over, it has been observed that ethyl acetate, hexane and dichloromethane extracts obtained from leaves and pseudobulbs of C. speciosa revealed no antibacterial activ- ity against S. aureus.

Fig. 3. Antimicrobial activity of various extracts obtained from the leaves of C. speciosa against Staphylococcus aureus measured as inhibition zone diameter (n = 6)

Source: own research

Fig. 4. Antimicrobial activity of various extracts obtained from pseudobulbs of C. speciosa against Staphylococcus aureus measured as inhibition zone diameter (n = 6)

Source: own research

42 The results of antimicrobial activity of various extracts obtained from the leaves and pseudobulbs of C. speciosa against E. coli are presented in Figs 5 and 6. Our re- sults also showed that ethanolic extract from leaves of C. speciosa exhibited strong activity against E. coli (inhibition zone diameter was 21 mm) (Fig. 5), while others extracts from pseudobulbs revealed minimum activity (inhibition zone diameter was 12 mm) (Fig. 6). Those extracts in ethyl acetate, hexane and dichloromethane both from the leaves and pseudobulbs revealed no antibacterial activity against S. aureus (Figs 5 and 6).

Fig. 5. Antimicrobial activity of various extracts obtained from leaves of C. speciosa against Escherichia coli measured as inhibition zone diameter (n = 6)

Source: own research

Fig. 6. Antimicrobial activity of various extracts obtained from pseudobulbs of C. speciosa against Escherichia coli measured as inhibition zone diameter (n = 6)

Source: own research

43 Detailed data regarding the zones of inhibition by the various plant extracts were recorded and presented in Figs 7 and 8.

A B Fig. 7. Antibacterial activity of ethanolic extracts obtained from leaves (A) and pseudobulbs (B) of C. speciosa against S. aureus measured as inhibition zone diameter

Source: own research

A B Fig. 8. Antibacterial activity of ethanolic extracts obtained from leaves (A) and pseudobulbs (B) of C. speciosa against E. coli measured as inhibition zone diameter

Source: own research

DISCUSSION

In the present study, the effect of extracts obtained from leaves and pseudobulbs of C. speciosa prepared in various solvents on the growth of S .aureus and E. coli was investigated in vitro. The results revealed the antimicrobial potential of ethanol- ic extracts. All the test organisms were susceptible to ethanolic extracts obtained from leaves and pseudobulbs of C. speciosa with inhibition zone diameter between 12-21.5 mm. E. coli was more resistant to ethanolic extracts obtained from leaves and pseudobulbs of C. speciosa and the diameter of inhibition zone around the rest ranged from 11-13.5 mm. The results obtained from the present study are consistent with earlier reports, which found that other species of Coelogyne have noticeable phytochemical, antimi-

44 crobial, antioxidant and anticancer activity (Sahaya Shibu et al. 2013). According to Sahaya Shibu and co-workers (2013), C. nervosa exhibits potential antimicrobial, antioxidant and anticancer properties. The previous investigations on pharmacologi- cal properties of orchid species, belonging to Coelogyne genus, have found that these plants are sources of phytochemicals which are able to initiate different biolog- ical effects including antimicrobial activity (Pérez Gutiérrez 2010, Sarmad Moin et al. 2012, Sahaya Shibu et al. 2013, Pramanick 2016). Preliminary phytochemical screening for the presence of alkaloids, carbohy- drates, glycosides, saponins, terpenoids, steroids, flavonoids, phenolic compounds, protein, gum and mucilages, phytosterol, tannins and phlobatannins were carried out. The screening of antimicrobial, antioxidant and anticancer activities was also performed. Preliminary phytochemical analysis revealed that the ethanol and aque- ous extracts shown the maximum phytochemical constituents. This analysis shows the presence of alkaloids, carbohydrates, glycosides, saponins, terpenoids, steroids, flavonoids, phenolic compounds, protein, phytosterol, tannins and phlobatannins in ethanol extract. The result obtained is similar to the results of Sarmad and co- workers (2012) and Mazumder and co-workers (2010) in Coelogyne stricta and Pa- pilionanthe teres. All the extracts of C. nervosa were tested for antimicrobial and an- tioxidant activities. The ethanolic extract of C. nervosa showed the maximum zone of inhibition against the bacteria Pseudomonas aeruginosa (15 mm), Enterococcus faecalis (14.3 mm), Salmonella enterica (12 mm), Bacillus subtilis (11 mm) and Corynebacteria spp. (9 mm). The ethanolic extract showed the maximum antibacte- rial activity against all the microorganisms tested, which shows the minimum inhibi- tory concentration (MIC) value in the range between 400 to 600 (μg/ml). The water extract possessed strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 126 μg/ml) and it shows cytotoxic activity towards the MCF-7 cancer cell line (IC50, 292.8 μg/ml). Several compounds like 9,10-Dihydro-5H-phenanthro- (4,5 bcd)-pyrans and pyrones were isolated from a number of species like Coelo- gyne, Pholidota, and Otochilus (Veerraju et al. 1989) whereas, trans-3,4,30,50-tetra- hydroxystilbene is reported to possess a chemopreventative agent with anti-leukemic activity and is being extensively studied for various cancers including colorectal and lung cancer as well as in cardiovascular diseases (Wolter et al. 2002). The anticancer activity results states that the IC50 value of water extract on MCF-7 cell line was 292.8 μg/ml which shows the plant have anticancer activity (Sahaya Shibu et al. 2013). In a study conducted by Paulomi and co-workers (2013), the bioactivity of few unexplored orchids (Aerides odorata, Acampe papilosa and Acampe ochracea that are native to North-East India) against kanamycin and ampicillin resistant E. coli was found out. All the three plants showed good antibacterial property against the selected strains of E. coli. Among the three strains, the ampicillin resistant variety showed strong inhibition (average zone diameter of 7.0 mm), the kanamycin re- sistant variety showed comparatively less inhibition (average zone diameter of 5.3 mm) and the (MTCC) variety showed the least (average zone diameter of 5.0 mm). The treatment in the antibiotic suspended medium showed less inhibition for acetone extract (average zone diameter of 5.3 mm) against ampicillin resistant variety than water extract (average zone diameter of 6.3 mm) against kanamycin resistant variety (Paulomi et al. 2013).

45 The antimicrobial effect showed by C. speciosa is in agreement with previous an- timicrobial effects produced by numerous orchids. To screen for bioactive chemicals, Subedi and co-workers (2011) have tested the extracts of traditionally used plant parts (either stems or pseudobulbs) of a subset of the orchids used in traditional medicines or involved in trade for their antibacterial activity against common intestinal and harmful bacteria (Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus) us- ing both a disc diffusion method and Minimum Inhibitory Concentration Assay (MIC). Results of Subedi and co-workers (2011) suggest that traditional uses of wild orchids against infectious diseases by local communities are based on antibacterial properties which could be used for the development of alternative drugs. All the extracts showed activity against one or more test bacteria. Aerides multiflora, Calanthe puberula, Coe- logyne flaccida, Coelogyne nitida, Coelogyne punctulata, Coelogyne stricta, Cymbidi- um iridioides, Dendrobium eriiflorum, Dendrobium fugax (syn. Flickingeria fugax), Luisia trichorrhiza and Pholidota imbricata showed a broad spectrum antibacterial ac- tivity. In contrast, Bulbophyllum umbellatum, Coelogyne flaccida and Malaxis acu- minata reduced growth of only a single strain. The minimal inhibitory concentration ranged from 0.9375 ± 0.44194 for Dactylorhiza hatagirea and Epigeneium amplum up to 2.0833 ± 0.72169 mg/ml for Cymbidium iridioides and Pholidota imbricata, mak- ing these the most and least potent extracts, respectively. Sensitivity decreased from E. coli > B. subtilis > S. aeruginosa > P. aeruginosa > S. typhi > K. pneumonia (Sub- edi et al. 2011). Antibacterial activities of different extracts of epiphytic orchids (Eria spicata, Bulbophyllum affine, Vanda cristata, Rhynchostylis retusa, Dendrobium nobile, Dendrobium amoenum, Coelogyne cristata, Pholidota imbricata) were tested against 5 species of bacteria (Gram-positive bacteria S. aureus and Bacillus subtilis and Gram-negative bacteria Vibrio cholerae, E. coli and Klebsiella pneumonia) and anti- fungal activities were tested against 3 species of fungi (Candida albicans, Rhyzopus stolonifer and Mucor spp.) (Marasini and Joshi 2012). Only Coelogyne stricta (leaf) and Dendrobium amoenum were shown good activity against Klebsiella pneumonae but Pholidota articulata and P. imbricata have showed good activity against E. coli. Eria spicata, Bulbophyllum affine, Vanda cristata and Rhynchostylis retusa have showed weak activity against all bacteria. Dendrobium nobile displayed good inhibi- tion against S. aureus but weak one against all other bacteria. All plants extract ex- hibited very good inhibition against S. aureus. Pholidota imbricata and C. cristata have showed the highest inhibition against V. cholera and S. aureus respectively. Af- ter evaluating the zone of inhibition values of various extracts, two extracts with highest zone of inhibition value were taken for MIC test by two fold serial dilution method. MIC of extract of C. cristata was tested against S. aureus and that of P. im- bricata against V. cholerae. The MIC of C. cristata against S. aureus was observed at 31.25 mg/ml and that of P. imbricata against V. cholerae was observed at 62.5 mg/ml. The minimum bactericidal concentration (MBC) values of the extracts of P. imbricata against V. cholerae and C. cristata against S. aureus were observed at 125 mg/ml and 250 mg/ml respectively. Coelogyne stricta (pseudobulb), C. stricta (leaf) and Dendrobium amoenum exhibited no activity while P. imbricata and P. ar- ticulata extracts revealed good activity against fungal organisms. Rest of the extracts

46 showed moderate or even very weak activity against selected fungal pathogens. An- tibacterial activities shown by C. cristata, P. articulata and P. imbricata extracts were more active than other extracts. P. imbricata and P. articulata extracts have found intermediate activity but all others extracts have showed very less activity or even failed to show activity against all different five fungal organisms (Marasini and Joshi 2012). Rashmi and co-workers (2015) have determined antibacterial and radical scav- enging activity of extracts of four epiphytic orchids, namely Luisia zeylanica, Pholidota pallida, Dendrobium nutantiflorum and Coelogyne breviscapa collected at different places of Western Ghats of Karnataka, India. Antibacterial and radical scavenging activity of orchid extracts was determined by Agar well diffusion and DPPH free radical scavenging activity. Two Gram-positive bacteria viz., B. subtilis and B. coagulans and two Gram-negative bacteria viz., E. coli and Salmonella typhi were used. In study of Rashmi and co-workers (2015), it was observed that the ex- tracts of all orchids exhibited inhibitory effect against all tested bacteria, but to a varied extent. Among orchids, marked antibacterial activity was displayed by C. breviscapa as indicated by wider zones of inhibition. Other orchids exhibited more or less similar inhibitory activity against test bacteria. Among Gram-positive and Gram-negative bacteria, B. subtilis and E. coli were inhibited to higher extent by extract of orchids respectively. Extracts were shown to inhibit B. coagulans and S. typhi to more or less similar extent. Moreover, all orchid extracts displayed dose dependent scavenging of radicals i.e., on increasing the concentration of extract, scavenging efficacy was also increased. Highest and least scavenging potential was exhibited by extract of L. zeylanica and P. pallida respectively as revealed by IC50 values. The scavenging effect of orchid extracts is in the order – L. zeylanica (62.62 µg/ml) > C. breviscapa (71.91 µg/ml) > D. nutantiflorum (91.46 µg/ml) > P. pallida (128.31 µg/ml). Reference antioxidant i.e., ascorbic acid displayed higher scaveng- ing activity (1.12 µg/ml) when compared to orchid extracts. The observed bioactivi- ties of orchid extracts could be ascribed to the presence of phytochemicals, in par- ticular phenolic compounds. In study of Rashmi and co-workers (2015), a positive correlation between the phenolic content and radical scavenging activity of orchid extracts was observed, i.e., extracts possessing high phenolic content exhibited marked scavenging of DPPH free radicals. Aqueous, ethanolic and chloroform soluble extracts of leaf and pseudobulb of Bulbophyllum neilgherrense (5.50 w/v) were screened for their antibacterial poten- tial against five species of bacteria, viz., E. coli, S. aureus, B. pumilus, P. aeruginosa and P. putida (Priya and Krishnaveni 2005). The zone of inhibition was the largest when alcoholic extract was used. Results of Priya and Krishnaveni (2005) revealed that the extracts, though less effective than streptomycin, proved inhibitory to bacte- rial growth in vitro. The growth inhibitory effect was more pronounced in ethanolic extracts of leaf and pseudobulb followed by chloroform and aqueous extracts. The ethanolic leaf extract was found more effective against E. coli, S. aureus and P. aeruginosa than B. pumilus and P. putida. The pseudobulb extract was found more effective against P. aeruginosa and P. putida (Priya and Krishnaveni 2005). In our previous study (Buyun et al. 2016, Tkachenko et al. 2015, 2016, Góralczyk et al. 2016), we have reinforced the assumption that Coelogyne species could be poten-

47 tial antibacterial or antifungal resource. We have determined antifungal potential of eleven species of orchids namely Coelogyne viscosa Lindl., C. cristata Lindl., C. law- renceana Rolfe, C. pandurata Lindl., C. assamica Linden & Rchb.f., C. fimbriata Lindl., C. ovalis Lindl., C. asperata Lindl., C. speciosa (Blume) Lindl., C. tomentosa Lindl., and C. brachyptera Rchb.f. against Candida albicans. Ethanolic orchid extracts resulted in considerable suppression of growth of C. albicans. The orchid extracts from various species of Coelogyne genus displayed varied antifungal potency. Among or- chids selected, marked antifungal efficacy was observed in case of C. speciosa (mean diameter of growth of inhibition zones was 19.7 mm), C. ovalis (18.2 mm), C. brachyp- tera (17.2 mm), and C. assamica (17.1 mm). Extract of C. cristata displayed least in- hibitory activity against test fungus (mean diameter of growth of inhibition zones was 14.0 mm). Orchids were shown to exhibit antifungal activity against a variety of mold species. Our results showed that different extracts of epiphytic orchids from Coelogyne genus have potent antifungal properties against Candida albicans. Antifungal activities shown by C. speciosa, C. ovalis, C. brachyptera, and C. assamica extracts were most active then other extracts (Tkachenko et al. 2015). Moreover, ethanolic orchid leaf ex- tracts resulted in considerable suppression of growth of S. aureus. The orchid extracts from various species of Coelogyne genus displayed varied antimicrobial potency. Among orchids selected, marked antimicrobial efficacy was observed for C. cristata (mean diameter of growth of inhibition zones was 27.5 mm), C. tomentosa (26 mm), C. lawrenceana (26 mm), C. brachyptera (26 mm), C. viscosa (25.5 mm), C. pandura- ta (24.5 mm), and C. fimbriata (24 mm). Thus, our results showed that different ex- tracts of epiphytic orchids from Coelogyne genus have potent antimicrobial properties against S. aureus (Tkachenko et al. 2016). Previously, we have investigated the antibacterial effects of ethanolic extract of Coelogyne brachyptera leaves against specific Gram-positive (Staphylococcus aure- us ATCC 25923 and methicillin-resistant S. aureus locally isolated) and Gram- negative bacteria (Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922) and fungal strain (Candida albicans) (Buyun et al. 2016a). Our results showed that the ethanolic extract of C. brachyptera leaves showed strong activity against the Gram-positive bacterial strains (20 mm diameter of inhibition zone for S. aureus and 26 mm for meticillin-resistant S. aureus), and moderate activity against the Gram-negative bacteria (14 mm for E. coli and 11 mm for P. aerugino- sa). Extract of C. brachyptera has displayed strong inhibitory activity against test fungus Candida albicans (mean diameter of inhibition zone was 17.2 mm). Our study revealed the good antimicrobial and antifungal activities of ethanolic leaf ex- tracts of C. brachyptera. Yet, this research illustrates that a Gram-positive bacterium was more susceptible to the ethanolic leaf extracts of C. brachyptera as compared to Gram-negative bacteria species. Beside, extract of C. brachyptera leaves has dis- played strong inhibitory effect against test fungus C. albicans (Buyun et al. 2016a). We also determined antibacterial and antifungal potential of ethanolic extract of C. cristata leaves against Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa and Escherichia coli) bacterial strains (Góralczyk et al. 2016). All microorganisms tested were susceptible to the leaf extract of C. cristata. Extract of C. cristata displayed the least inhibitory activity against test fungus (mean diameter of growth of inhibition zones was 14 mm). Our results showed that the etha-

48 nolic extract of C. cristata leaves exhibited strong activity against the Gram-positive bacterial strain (27 mm of inhibition zone diameter for S. aureus), and moderate activi- ty against the Gram-negative bacteria (13 mm for E. coli). P. aeruginosa appeared to be less sensitive to the extract (the inhibition zone was 10 mm). In our present study we conclude that ethanolic extract of C. cristata leaves has potent antimicrobial activi- ty against S. aureus (Góralczyk et al. 2016). The ethanolic extract of C. ovalis leaves showed strong activity against S. aureus (27 mm of inhibition zone diameter), while ethanolic extract from pseudobulbs revealed less activity (22 mm). Methanolic and ethyl acetate extracts obtained from C. ovalis leaves also showed appreciable antimicrobial activity (32 mm and 35 mm, respectively), whereas those extracts from pseudobulbs revealed no antibacterial activity against S. au- reus (Buyun et al. 2016c). Our study has shown that ethanolic extracts from leaves and pseudobulbs of C. tomentosa exhibited strong activity against S. aureus (inhibition zone diameter were 29 mm and 30 mm, respectively), while methanolic extract from leaves and pseudobulbs revealed less profound activity (18 mm and 10 mm, respectively). Moreover, it has been observed that ethyl acetate extract obtained from C. tomentosa pseudobulbs also showed appreciable antimicrobial activity (25 mm), while those ex- tracts from the leaves, as well as hexane and dichloromethane extracts both from the leaves and pseudobulbs revealed no antibacterial activity against S. aureus. Hence, the overall results of the present investigation provide evidence that the crude extracts ob- tained from leaves and pseudobulbs of C. tomentosa could be considered as promising natural antimicrobial products (Buyun et al. 2016b). The ethanolic extract from leaves and pseudobulbs of C. huettneriana exhibited strong activity against E. coli (inhibition zone diameter were 28 mm and 13 mm, respectively), while others extracts from leaves and pseudobulbs revealed minimum activity. Similarly, it has been observed that etha- nolic extract obtained from leaves and pseudobulbs also showed appreciable antimicro- bial activity against S. aureus (19.5 mm and 21 mm, respectively), while those extracts in ethyl acetate, hexane and dichloromethane both from the leaves and pseudobulbs re- vealed no antibacterial activity against S. aureus (Buyun et al. 2016d). Also, the results obtained from the present research showed antimicrobial potential of C. speciosa extracts against S. aureus. So these plants extracts can be used as anti- septics and antimicrobial agents in medicine and veterinary. The antibacterial activity in Coelogyne may be due to presence of alkaloids, bibenzyl derivatives, flavonoids, phenanthrenes and terpenoids. Clinical studies confirmed diuretic, anti-rheumatic, an- ti-inflammatory, anti-carciogenic, antimicrobial, anticonvulsive, relaxation, neuro- protective and antivirus activities (Pérez Gutiérrez 2010). Kovács and co-workers (2008) have furnishes an overview of the hydroxy or/and methoxy-substituted 9,10-dihydro/phenanthrenes, methylated, prenylated and other monomeric derivatives, dimeric and trimeric phenanthrenes and their biological ac- tivities. A fairly large number of phenanthrenes have been reported from higher plants, mainly in the Orchidaceae family, in the species of Dendrobium, Bulbophyl- lum, Eria, Maxillaria, Bletilla, Coelogyna, Cymbidium, Ephemerantha and Epiden- drum. A few phenanthrenes have been found in the Hepaticae class and Dioscore- aceae, Combretaceae and Betulaceae families as well. These plants have often been used in traditional medicine, and phenanthrenes have therefore been studied for their cytotoxicity, antimicrobial, spasmolytic, anti-inflammatory, antiplatelet aggregation,

49 antiallergic activities and phytotoxicity (Kovács et al. 2008). Finally, it should be noted, that occurrence of phenanthrenes and dihydrophenanthrenes in orchids is considered as accumulation of orchid phytoalexins in response to infection of orchid plant by mycorrhizal fungi (Fisch et al. 1973). It has been reported that many natural products including pigments, enzymes and bioactive components are soluble in water, which explains the highest yield of ex- tract, while some of the solvents especially acetone are selective for tannins (Rao 2004). Different solvents have various degrees of solubility for different phytocon- stituents (Manilal and Sathishkumar 1986). Therefore, we suggest that different an- timicrobial activity of extracts obtained from C. speciosa diluted with various sol- vents is determined by different agents presented in extracts. A number of phytochemicals such as alkaloids, bibenzyl derivatives, flavonoids, phenanthrenes etc. have been reported from orchids. Presence of these phytochemi- cals provides antimicrobial, antitumor, anti-inflammatory, antiviral activities, etc. A number of members of orchid family are used as potent inhibitor against bacteria and also proved to be a potent antimicrobial agents. The methanolic extract from dif- ferent parts of orchids has shown antimicrobial activity. A broad spectrum antibacte- rial activity (24 bacteria and protozoan) was exhibited by Pseudovanilla foliata (syn. Galeola foliata) leaves and stem bark extracted with petrol, dichloromethane, ethyl acetate, butanol and methanol. A very good level of activity was demonstrated by the dichloromethane and ethyl acetate fractions (Khan and Omoloso 2004). Moreover, endophytic fungi isolated from different orchids could be of potential antibacterial or antifungal resource. Antimicrobial activity of ethanolic extract of fermentation broth of 53 endophytes (30 isolates from Dendrobium devonianum and 23 endophytic fungi from D. thyrsiflorum) was explored by Xing and co-workers (2011) using agar diffusion test. 10 endophytic fungi in D. devonianum and 11 in D. thyrsiflorum exhibited antimicrobial activity against at least one pathogenic bac- terium or fungus among 6 pathogenic microbes (E. coli, B. subtilis, S. aureus, Can- dida albicans, Cryptococcus neoformans, and Aspergillus fumigatus). Out of the fungal endophytes isolated from D. devonianum and D. thyrsiflorum, Phoma dis- played strong inhibitory activity (inhibition zones in diameter > 20 mm) against pathogens. Epicoccum nigrum from D. thyrsiflorum exhibited antibacterial activity even stronger than ampicillin sodium. Fusarium isolated from the two Dendrobium species was effective against the pathogenic bacterial as well as fungal pathogens (Xing et al. 2011). Vaz and co-workers (2009) have examine antimicrobial activity of endophytic fungi isolated from the leaves, stems, and roots of 54 species of Or- chidaceae collected in a Brazilian tropical ecosystem. In total, 382 filamentous fungi and 13 yeast isolates were obtained and cultured to examine the production of crude extracts. Thirty-three percent of the isolates displayed antimicrobial activity against at least one target microorganism. The extracts of endophytic fungi isolated from leaves of terrestrial orchids in semideciduous forest were more active against E. coli, whereas extracts of endophytic fungi from roots of rupicolous orchids collected in rock fields were more active against Candida krusei and Candida albicans. Among the fungi that were screened in the study, 22 isolates held their antimicrobial activi- ties after replication and were therefore selected for assessment of the MIC, which ranged from 62.5 to 250 µg/ml and 7.8 to 250 µg/ml against bacteria and fungi, re-

50 spectively. One isolate of Alternaria sp. and one isolate of Fusarium oxysporum pre- sented the strongest antibacterial activity. Three Fusarium isolates, Epicoccum nigrum, and Sclerostagonospora opuntiae showed the greatest MIC values against the pathogenic yeasts (Vaz et al. 2009). The work also signifies the earlier findings on various antimicrobial activity of orchid (Pérez Gutiérrez 2010) and validates the findings that the presence of phytochemicals in orchid may be the reason for this ef- ficacy.

CONCLUSIONS

The examined ethanolic extracts obtained from leaves and pseudobulbs of C. spe- ciosa showed different antibacterial activities against S. aureus and E. coli, while methanol, ethyl acetate, hexane, and dichloromethane extracts had no antibacterial ac- tivities. The ethanolic extracts from leaves exhibited notable antimicrobial activity against S. aureus and E. coli (inhibition zone diameter were 21.5 and 20 mm). The ob- served activity may contribute to the reasons why the leaves are used for infectious and inflammatory conditions in ethnomedicine. The research showed that ethanolic extracts of C. speciosa possess antibacterial potency against S. aureus and E. coli. These findings led the authors to suggest that these extracts may be used as a natural antiseptics and antimicrobial agents in medicine and veterinary practice. Nevertheless, despite the promising results, more research should be carried out to further evaluate the roles of particular compounds, isolated from all parts of orchid plants, attributable to antimicrobial activity.

REFERENCES

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SUMMARY

Orchids have been used all over the world in traditional healing and treatment system of a large number of diverse diseases. Extracts and metabolites of these

53 plants demonstrate useful pharmacological activities, i.e. diuretic, antirheumatic, an- ti-inflammatory, anticarcinogenic, hypoglycemic, antimicrobial, anticonvulsive, re- laxation, neuroprotective, and antivirus, activities (Pérez Gutiérrez 2010). Phyto- chemical studies in the genus Coelogyne revealed that these plants have a wide array of secondary compounds resulting in profound biological activity, antimicrobial, in particular (Pant 2013, Sahaya et al. 2013). In this regard, it is important to investi- gate scientifically those plants which have been used in traditional medicines as po- tential sources of novel antimicrobial compounds. So the present study was conduct- ed to investigate in vitro antimicrobial activity of various extracts obtained from vegetative parts of Coelogyne speciosa against Gram-positive (Staphylococcus aure- us ATCC 25923) and Gram-negative bacteria (Escherichia coli ATCC 25922). The leaves and pseudobulbs of C. speciosa plants, cultivated under glasshouse condi- tions, were sampled at M.M. Gryshko National Botanical Garden (NBG), National Academy of Science of Ukraine. Freshly leaves and pseudobulbs were washed, weighted, crushed, and homogenized in 96% ethanol, methanol, ethyl acetate, hex- ane, and dichloromethane (in proportion 1 : 19) at room temperature. Antimicrobial activity was determined using the agar disk diffusion assay (Bauer et al. 1966). The present study has shown that ethanolic extracts from leaves and pseudobulbs of C. speciosa exhibited strong activity against S. aureus (inhibition zone diameter were 21.5 mm and 19 mm, respectively), while methanolic extract from leaves and pseu- dobulbs revealed mild activity (8.1 and 8 mm). Moreover, it has been observed that ethyl acetate, hexane and dichloromethane extracts obtained from leaves and pseu- dobulbs of C. speciosa revealed no antibacterial activity against S. aureus. Our re- sults also showed that ethanolic extract from leaves of C. speciosa exhibited strong activity against E. coli (inhibition zone diameter was 21 mm), while others extracts from pseudobulbs revealed minimum activity (inhibition zone diameter was 12 mm). Those extracts in ethyl acetate, hexane and dichloromethane both from the leaves and pseudobulbs revealed no antibacterial activity against S. aureus. The an- tibacterial activity in Coelogyne may be due to presence of alkaloids, bibenzyl de- rivatives, flavonoids, phenanthrenes and terpenoids. Clinical studies confirmed diuret- ic, anti-rheumatic, anti-inflammatory, anti-carcinogenic, antimicrobial, anticonvulsive, relaxation, neuro-protective and antivirus activities (Pérez Gutiérrez 2010). The ob- served activity may contribute to the reasons why the leaves are used for infectious and inflammatory conditions in ethnomedicine. Thus, the research showed that etha- nolic extracts of C. speciosa possess antibacterial potency against S. aureus and E. coli and may be used as a natural antiseptics and antimicrobial agents in medicine and veterinary practice. Considering the medicinal importance of the tested microor- ganisms, the findings of this research are considered to be very promising in the per- spective of new drug development from plant sources.

54