Modern Approaches to Formation and Management ...

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SHEE «Kherson State Agricultural University» Pomeranian University in Slupsk

Proceedings of the Conference

«Modern Approaches to Formation and Management of Anthropogenic and "atural Biocoenosis in the Countries of Eastern Europe» Section 1 Land resources and the efficiency of their use

KHERSO" 2015

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UDK 615.012.1: 582.949.2: 581.3 A"TIMICROBIAL SCREE"I"G OF ETHA"OLIC EXTRACT OF COELOGYIE CRISTATA LI"DL. (ORCHIDACEAE) LEAVES LYUDMYLA BUYUI1, DSCI HALYIA TKACHEIKO2, PHD MARIOLA TRUCHAI2, PHD LYUDMYLA KOVALSKA1, PHD OLEKSAIDR GYREIKO1, PHD student 1

M.M. Gryshko Rational Botanical Garden, Rational Academy of Science of Ukraine, Timiryazevska Str.1, Kyiv, 01014, Ukraine; 2 Institute of Biology and Environmental Protection, Pomeranian University in Slupsk, Arciszewski Str. 22b, 76-200 Slupsk, Poland; Abstract The present study was conducted to determine antibacterial and antifungal potential of ethanolic extract of Coelogyne cristata leaves against Gram-positive (Staphylococcus aureus) and Gram-negative bacterial strains (Pseudomonas aeruginosa and Escherichia coli) as well as against fungus Candida albicans.Our results showed that the ethanolic extract of C. cristata leaves exhibited strong activity against the Gram-positive bacterial strain (27 mm of inhibition zone diameter for Staphylococcus aureus), and moderate activity against the Gram-negative bacteria (13 mm for Escherichia coli) and Candida albicans (14 mm of inhibition zone diameter). Pseudomonas aeruginosa appeared to be less sensitive to the extract(the inhibition zone diameterwas 10 mm).Our results showed that ethanolic leafextract of C. cristatahas potent antimicrobial activity against Staphylococcus aureus and antifungal properties against Candida albicans. These results form a good basis for further pharmacological investigations of medicinal effects of orchids. Keywords:Coelogyne cristata, Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Streptococcus pneumonia, antimicrobial activity Introduction Numerous orchid species are traditionally used as herbal remedy for microbial infections and many other ailments, but the potential of most of the orchid species for therapeutic use is yet to be explored.Recent studies show that orchid extracts have several health-promoting benefits, including diuretic, antirheumatic, antiinflammatory, anticancirogenic, hypoglycemic activities, antimicrobial, anticonvulsive, relaxation, neuroprotective, and antiviral activities (Pérez Gutiérrez, 2010). Orchids have been used as a source of medicine 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, diarrhea, muscle

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pain, blood dysentery, hepatitis, dyspepsia, bone fractures, rheumatism, asthma, malaria, earache, sexually transmitted diseases, wounds and sores. Besides, many orchidaceous preparations are used as emetic, purgative, aphrodisiac, vermifuge, bronchodilator, sex stimulator, contraceptive, cooling agent and remedies in scorpion sting and snake bite (Hossain, 2011). The medicinal orchids belong mainly to the genera: Calanthe, Coelogyne, Cymbidium, Cypipedium, Dendrobium, Ephemerantha, Eria, Galeola, Gastrodia, Gymnadenia, Habenaria, Ludisia, Luisia, Revilia and Thunia (Szlachetko, 2001). Coelogyne comprises over 200 species distributed throughout southeast Asia with main centers of diversity in Borneo, Sumatra and the Himalayas. Most species areepiphytes and occur in primaryforests (Gravendeel, 2000). Coelogyne cristata is the type species of the genus Coelogyne. It has high ornamental value as a cut flower. It produces its graceful racemes of white flowers with yellow splotches on the throat, and has a long lasting and fragrant scent (Naing et al., 2011). C. cristata is small to moderately sized sympodial epiphyte or lithophyte grows to 15-30 cm tall with two dark green leaves at the top of each pseudobulb. Pseudobulbs are oblong-ovoid, on a stout rhizome, closely spaced to 5-6 cm apart, smooth.The 15-30 cm arched inflorescence is pendent to suberect and emerges from the base of mature pseudobulbs, usually before new growth starts. Five to eight longlasting, showy flowers open simultaneously on each inflorescence. Flowers very large, about 8cm across; sepals and petals white, lip white with yellow keels between side-lobes and 2 golden-yellow blotches on mid-lobe, column white. They are sometimes fragrant and last four to five weeks if kept dry and cool. The specific epithet refers to the crested form of the keels on the lip of the flowers.For a time in the past, these plants were knows as Cymbidium speciosissimum D. Don. (www.aos.org). C. cristataplants grow epiphytically on trees, sometimes as lithophytes on rocks in shaded, lower and upper montane forest (up to 2500 m) of the northeast of India, Tibet, Nepal and into China where they may experience wet summers and cold winters. Some areas may be quite bright in winter but many areas a covered in fog or cloud and sunlight is much lower (http://coelogynes.com/). C. cristata is used for the treatment of fractured bones in folk-tradition of Kumaon region, Uttarakhand, India (Sharma et al., 2014). In Myanmar, the pseudobulb of C. cristata is used in making traditional medicine for dysentery and diarrhea (Naing et al., 2011). Sharma et al. (2014) proposed that ethanolic extract of C. cristata and its pure compound coelogin have potential in the management of postmenopausal osteoporosis. Pseudobulb of C. cristata infusion is used in constipation and also acts as aphrodisiac (Dhiman Anil, 2004). C. cristata contains the phenanthrenes coeloginanthridin (3,5,7-trihydroxy-1,2-dimethoxy-9,10dihydrophenanthrene), a 9,10-dihydrophenanthrene derivative, and coeloginanthrin, the corresponding phenanthrene analogue, coelogin and coeloginin (Majumder et al., 2001).In light of earlier reports on structurally similar compounds, coeloginanthridin and coeloginanthrin may have biological activities of phytoalexins and endogenous plant growth regulators (Majumder et al., 2001).

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Fig. 1. Coelogyne cristataLindl.specimenin M.M. Gryshko National Botanical Garden (Kyiv, Ukraine).

Fig. 2. Flower of Coelogyne cristataLindl.

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The present study was conducted to determine 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 strainsas well as againstfungus Candida albicans. Materials and methods The leaves of orchids, cultivated under glasshouse conditions, were sampled at M.M. Gryshko National Botanical Garden (NBG), National Academy of Science of Ukraine in September, 2015.Since 1999 the whole collection of tropical and subtropical plants (including orchids) has the status of a National Heritage Collection of Ukraine.Beside that, NBG collection of tropical orchids was registered at the Administrative Organ of CITES in Ukraine (Ministry of Environment, registration No. 6939/19/1-10 of 23 June 2004). The collected leaves were brought into the laboratory for antimicrobial studies. Freshly crushed leaves were washed, weighted, and homogenized in 96% ethanol (in proportion 1:10) at room temperature. Coelogyne cristata leaf extracts were screened for antimicrobial activity using disc diffusion methods(Bauer et al., 1966).Gram-negative bacteria Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922), as well as Grampositive bacteriumStaphylococcus aureus (ATCC 25923) were used as test organisms.Antimicrobial activity of crude extract of the plant sample was evaluated by the paper disc diffusion method. Cultures of Gram-positive and Gram-negative bacteria were suspended in sterile solution of 0.9% normal saline and the turbidity adjusted equivalent to that of a 0.5 McFarland standard. All the cultures were inoculated 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 370C for 24 h. A negative control disc was impregnated with 50 µL of sterile ethanol used in each experiment. The antimicrobial activities of the extracts tested were evaluated at the end of the inoculated period by measuring the inhibition zone diameter around each paper disc in millimeters. The presence of inhibition zones around each of well after the period of incubation was regarded as the presence of antimicrobial action whereas the absence of any measurable zone of inhibition was interpreted as absence of antimicrobial effect. Zone diameters were determined. The fungal organism used for the present study was Candida albicans. The plates were incubated at 27oC. The plates were then observed for the zone of inhibition produced by the antifungal activity of C. cristata. All statistical calculation was performed on separate data from each bacterial strains. Results and Discussion The results of antimicrobial activity of ethanolic extract of C. cristata leaves are presented in Figs 3-5.

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A B Fig. 3. Antimicrobial activity of ethanolic extract of C. cristata leaves against Gram-positive (A, Staphylococcus aureus) and Gram-negative (B, Escherichia coli) bacterial strainsmeasured as inhibition zone diameter. All microorganisms tested were susceptible to the leaf extract of C.cristata.Extract of C. cristata displayed least inhibitory activity against test fungus (mean diameter of growth of inhibition zones was 14.0 mm) (Fig. 3).

Fig. 4. Antifungal activity of ethanolic extract of C. cristata leaves against Candida albicansmeasured as inhibition zone diameter. Our results showed that the ethanolic extract of C. cristata leaves exhibited strong activity against the Gram-positive bacterial strain (27 mm of inhibition zone diameter for Staphylococcus aureus) (Fig. 3A), and moderate activity against the Gram-negative bacteria (13 mm for Escherichia coli) (Fig. 3B) and Candida albicans (14 mm of inhibition zone diameter) (Figs 4, 5). Pseudomonas aeruginosa appeared to be less sensitive to the extract(the inhibition zone was 10 mm) (Fig. 5).

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Fig. 5. The antimicrobial activity of ethanolic extract of C. cristata leaves against bacterial strains and Candida albicans measured as inhibition zone diameter. Many plant species widely explored for antimicrobial compounds fall into the family Orchidaceae. Subedi (2011) revealed that Aerides multiflora, Calanthe puberula, Coelogyne flaccida, Coelogyne nitida, Coelogyne punctulata, Coelogyne stricta, Cymbidium iridioides, Dendrobium eriiflorum, Dendrobiumfugax, Luisia trichorrhiza and Pholidota imbricatawere possessed a broad spectrum ofantibacterial activity. Sensitivity decreased from Escherichia coli >Bacillus subtilis>Pseudomonas aeruginosa>Salmonella typhi>Klebsiella pneumonia (Subedi, 2011). Two antifungal compounds, lusianthrin and chrysin, were isolated from the seedlings of Cypripedium macranthos var. rebunense that had developed shoots (Shimura et al., 2007). The former had a slightly stronger antifungal activity than the latter, and the antifungal spectra of these compounds were relatively specific to the nonpathogenic Rhizoctonia spp. orchid plants equip multiple antifungal compounds and use them at specific developmental stages; lusianthrin maintains the perilous symbiotic association for germination and chrysin helps to protect adult plants (Shimura et al., 2007). Matu and van Staden (2003) screened the aqueous, hexane and methanol extracts of 12 plant species, traditionally used in Kenya for treatment of ailments of infectious and/or inflammatory nature for in vitro antibacterial and anti-inflammatory activities. Antibacterial activity was tested using the agar diffusion method while anti-inflammatory activity was tested using the cyclooxygenase (COX-1) assay. The highest activity was found in the hexane extracts of Spiranthes mauritianum (Matu and van Staden, 2003). Vaz et al. (2009) examined antimicrobial activity of endophytic fungi isolated from the leaves, stems and roots of 54 species of tropical orchid species present in Brazilian ecosystems. For this purpose 382 filamentous fungi and 13 yeast isolates were obtained and cultured to examine the production of crude extracts. The multivariate statistical analyses conducted, indicate that the extracts of endophytic fungi, isolated from leaves of terrestrial orchids in semideciduous forest were more

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active against Escherichia coli, whereas extracts of endophytic fungi from roots of rupicolous orchids collected in rock fields were more active against Candida krusei and Candida albicans. One isolate of Alternaria sp. and one isolate of Fusarium oxysporum presented the strongest antibacterial activity. Three Fusarium isolates, Epicoccum nigrum, and Sclerostagonospora opuntiae showed the greatest minimum inhibitory concentration values against the pathogenic yeasts (Vaz et al., 2009). Sharma et al. (2014) revealed that ethanolic extract of C. cristata and its compound coelogin promote osteoprotective activity in ovariectomized estrogen deficient mice. Feeding of ethanolic extract to ovariectomized estrogen deficient mice led to significant restoration of trabecular microarchitecture in both femoral and tibial bones, better bone quality and also devoid of any uterine estrogenicity. Subsequently, coelogin, a pure compound was isolated from ethyl acetate fraction of C. cristata and evaluatedin vitro osteoblast cell cultures. Treatment of coelogin to osteoblasts led to enhanced alkaline phosphatase activity (a marker of osteoblast differentiation), mineral nodule formation and mRNA levels of osteogenic markers (Sharma et al., 2014). Moreover, other species of Orchidaceaefamilyalso possess antimicrobial activity. The antimicrobial effects of vanillin and vanillic acid were verified against several species and strains of Listeria monocytogenes, Listeria innocua, Listeria grayi, and Listeria seeligeri. Vanillin and vanillic acid, isolated from Vanilla planifolia, could prove useful either alone or in mixtures for the control of Listeria spp. in food products (Delaquis et al., 2005). Moreover, vanillin has the potential to preserve fruit juices and soft drinks that are low in both lipid and protein content against Saccharomyces cerevisiae and Candida parapsilosis (Fitzgerald et al., 2004). Antimicrobial activity of C. cristata can be explained by a fairly large number of phenanthrenes.Preliminary phytochemical analysis, conducted on Coelogyne nervosa(Sahaya Shibu, 2013) has revealed that the ethanolic extracts showed the maximum phytochemical constituents. In addition, the ethanolic extract showed the maximum antibacterial activity against all the microorganisms tested. The phytochemical analysis of C. nervosa (Sahaya Shibu, 2013) showed the presence of alkaloids, carbohydrates, glycosides, saponins, terpenoids, steroids, flavonoids, phenolic compounds, protein, phytosterol, tannins and phlobatannins in ethanolic extract. This result is similar to the results of Sarmad Moin et al.(2012) for Coelogyne stricta. Majumder et al. (2001) isolated from the orchid C. cristatacoeloginanthridin, a 9,10-dihydrophenanthrene derivative, and coeloginanthrin, the corresponding phenanthrene analogue, which earlier afforded coelogin (1a) and coeloginin (1b). The structures of coeloginanthridin and coeloginanthrin were established as 3,5,7trihydroxy-1,2-dimethoxy-9,10-dihydrophenanthrene (2a) and 3,5,7-trihydroxy-1,2dimethoxyphenanthrene (2c), respectively, from spectral and chemical evidence including the conversion of coeloginanthridin triacetate (2b) to coeloginanthrin triacetate (2d) (Majumder et al., 2001). They have been reported from higher plants, mainly in the Orchidaceae family, in the species Dendrobium, Bulbophyllum, Eria, Maxillaria, Bletilla, Coelogyna, Cymbidium, Ephemerantha and Epidendrum. These plants have often been used in traditional medicine, and phenanthrenes have therefore

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been studied for their cytotoxicity, antimicrobial, spasmolytic, anti-inflammatory, antiplatelet aggregation, antiallergic activities and phytotoxicity (Kovács et al., 2008). In our present study we concludethat ethanolic extract of C. cristata leaves has potent antimicrobial activity against Staphylococcus aureus and antifungal properties against Candida albicans. These findings form a good basis for further pharmacological investigations of medicinal effects of orchids. Further microbiological studies are to be carried out. References 1. Bauer A.W., Kirby W.M., Sherris J.C., Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol., 1966, 45(4): 493496. 2. Delaquis P., Stanich K., Toivonen P. Effect of pH on the inhibition of Listeria spp. by vanillin and vanillic acid. J. Food Prot., 2005, 68(7): 1472-1476. 3. Dhiman Anil K. Medicinal plants of Uttaranchal state, Chawkhambha Sanskrit Series Office, Varanasi, 2004, 438 p. 4. Fitzgerald D.J., Stratford M., Gasson M.J., Narbad A. The potential application of vanillin in preventing yeast spoilage of soft drinks and fruit juices. J. Food Prot., 2004, 67(2): 391-395. 5. Gravendeel B. 2000. Reorganising the orchid genus Coelogyne: a phylogenetic classification based on morphology and molecules. Leiden (Netherlands), Leiden University. 208 p. 6. Hossain M.M. Therapeutic orchids: traditional uses and recent advances - an overview. Fitoterapia, 2011, 82(2): 102-140. 7. Kovács A., Vasas A., Hohmann J. Natural phenanthrenes and their biological activity. Phytochemistry, 2008, 69(5): 1084-1110. 8. Majumder P.L., Sen S., Majumder S. Phenanthrene derivatives from the orchid Coelogyne cristata. Phytochemistry, 2001, 58(4): 581-586. 9. Matu E.N., van Staden J. Antibacterial and anti-inflammatory activities of some plants used for medicinal purposes in Kenya. J. Ethnopharmacol., 2003, 87(1): 35-41. 10. Naing A.H., Chung J.D., Lim K.B. Plant Regeneration through Indirect Somatic Embryogenesis in Coelogyne cristata Orchid. American Journal of Plant Sciences, 2011, 2, 262-267. 11. Pérez Gutiérrez R.M. Orchids: A review of uses in traditional medicine, its phytochemistry and pharmacology. Journal of Medicinal Plants Research, 2010, 4(8): 592-638. 12. Sahaya Shibu B., Chitra Devi B., Sarmad Moin, Servin Wesley P. Evaluation of bioactive potential of Coelogyne nervosa A. Rich. – an endemic medicinal orchid of Western Ghats, India. Asian Journal of Pharmaceutical and Clinical Research, 2013,6(1): 114-118. 13. Sarmad Moin, Sahaya Shibu B., Servin Wesley P., Chitra Devi B. Bioactive potential of Coelogyne stricta (D. Don) Schltr.: An ornamental and medicinally important orchid. Journal of Pharmacy Research,2012, 5(4): 2191-2196.

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14. SharmaC., MansooriM.N., DixitM., ShuklaP., KumariT., BhandariS.P., NarenderT., SinghD., AryaK.R. EthanolicextractofCoelogynecristataLindley (Orchidaceae) anditscompoundcoeloginpromoteosteoprotectiveactivityinovariectomizedestrogendefi cientmice. Phytomedicine, 2014, 21(12): 1702-1707. 15. Shimura H., Matsuura M., Takada N., Koda Y. An antifungal compound involved in symbiotic germination of Cypripedium macranthos var. rebunense (Orchidaceae). Phytochemistry, 2007, 68(10): 1442-1447. 16. Subedi A. New species, pollinator interactions and pharmaceutical potential of Himalayan orchids. Ph.D. Thesis, LeidenUniversity, The Netherlands, 2011. 17. Szlachetko D. Genera et species Orchidalium. Polish Bot. J., 2001, 46: 11-26. 18. Vaz A.B., Mota R.C., Bomfim M.R., Vieira M.L., Zani C.L., Rosa C.A., Rosa L.H. Antimicrobial activity of endophytic fungi associated with Orchidaceae in Brazil. Can. J. Microbiol., 2009, 55(12): 1381-1391.