In Vitro Antimicrobial Activityof Cynodon dactylon (L.) Pers. (bermuda) Against Selected Pathogens S. Abdullah, J. Gobilik and K. P. Chong Abstract Cynodon dactylon (L.) Pers. is a type of perennial grass that possesses great medicinal values. In this study, the antimicrobial activity of the plant crude extract from seven different solvents (acetone, chloroform, diethyl ether, ethanol, ethyl acetate, methanol, and n-pentane) was investigated against some pathogens (Bacillus cereus, Bacillus subtilis, Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumonia) using disc diffusion method and thin-layer chromatographic (TLC) bioassay for plant-SPE extracts against Aspergillus niger. Crude extraction showed that ethanolic extraction produced highest yield (7.065 %) followed by methanolic (5.420 %) and chloroform (3.550 %) extraction. The lowest yield was obtained from n-pentane extraction (0.500 %). The antimicrobial study revealed broad spectrum of antimicrobial activity from ethanol (7.0–10.0 ± 0.0–1.0 mm) and ethyl acetate (7.0–12.0 ± 0.0–1.0 mm) extracts against all of the bacterial pathogens. Both methanol and acetone extracts showed activity to B. cereus (8.0 ± 0.0 mm) and B. subtilis (7.0 ± 0.0 mm), while chloroform extract showed activity to B. subtilis (7.0 ± 0.0 mm) and S.pyogenes (8.3 ± 0.6 mm), respectively. Diethyl ether extraction showed activity only to S. pyogenes (7.3 ± 0.6 mm), while no activity was observed from n-pentane extraction. Great antimicrobial activity were observed for both ethyl acetate and ethanol SPE-based extracts (SBE) with size of inhibition ranging from 8.0 ± 0.0 mm to 15.7 ± 0.6 mm for ethyl acetate SBE and 8.0 ± 0.0 mm to 13.0 ± 0.0 mm for ethanol SBE. No significant antimicrobial activity was observed from thin-layer chromatographic bioassay against A. niger. S. Abdullah K. P. Chong (&) Sustainable Palm Oil Research Unit (SPOR), School of Science and Technology, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia e-mail:
[email protected] J. Gobilik School of Sustainable Agriculture, Universiti Malaysia Sabah, Sandakan Campus, Mile 10, Sg. Batang 90000 Sandakan, Sabah, Malaysia e-mail:
[email protected] R. Pogaku et al. (eds.), Developments in Sustainable Chemical and Bioprocess Technology, DOI: 10.1007/978-1-4614-6208-8_29, Springer Science+Business Media New York 2013 227 bactericidal. glycosides. 2011). it also created the problem in controlling the growth of infectious diseases caused by the pathogens. Plants produce large number of organic compounds such as alkaloids. Keywords Cynodon dactylon Crude extract SPE-based extract TLC bioassay Antimicrobial Introduction Medicinal plants play a very important role in pharmaceuticals industry in developing alternative drugs to overcome the pitfalls possessed by the synthetic drugs (Yadav et al. and even in semi-arid climates (Watson and Dallwitz 1992). Besides used in remedy. It is found almost everywhere in tropical. Pongsak and Parichat 2010. Staphylococcus aureus. Albert-Baskar and Ignacimuthu 2010). dactylon is used as a rejuvenator and for wound healing (Mangathayaru et al. and allergic effect (Singh et al. Chong et al. diuretic activity.228 S. chemotherapeutic. and phenolics as secondary metabolites which are used as defensive mechanism against respective pathogens (Francisco and Pinotti 2000. Scientifically. However. 2011). 2010). flavonoids. C. . subtropical. 2011). which are highly associated with nosocomial infection. terpenoids. This study will report the antimicrobial activity of C. antiulcer activity. it is tested to have antidiabetic effect. there is a constant need for new and effective therapeutic agents. Traditionally. These compounds possess medicinal values and become an attractive subject for researchers to develop new antibiotics (Joy et al. but also widely used by human. pan-tropical species of grass which belongs to the family Poaceae. there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases from medicinal plants. Cynodon dactylon is a perennial. It is not only widespread. Besides the tougher jobs to search for more effective drugs against the pathogens. antioxidant. 2012). and Pseudomonas aeruginosa. Syahriel et al. 2009). Previous studies show that many plant secondary metabolites act as bioactive compounds. right heart failure protective activity. Given the alarming incidence of antibiotic resistance in pathogens raises concern among the medical practitioners. and bacteriostatic agents (Singh and Gupta 2008. 2001). Zwenger and Basu 2008. Abdullah et al. dactylon against some common pathogens such as Escherichia coli. The development of drug-resistant pathogens that are mostly involved in nosocomial infection has raised concern among medicinal practitioners (Adalet et al. considerable amount of attention of antimicrobial substances derived from higher plants is immerging in recent years. the plant is also used as forage for animals and as turfgrass (Wu et al. most of the synthetic drugs possess side effect to the consumers (Chaudhari et al. Meanwhile. Hence. As a result. 2007). anticancer potentials. tannins. the assessments on plant-derived antibiotics are still under investigation. It was believed that the intense used of a number of synthetic antimicrobial drugs which contributed to the development. 2007. chloroform. . dactylon was collected near the Universiti Malaysia Sabah’s (UMS) area in Kota Kinabalu. Voucher (jgobilik 1090/2011) was kept at School of Sustainable Agriculture.In Vitro Antimicrobial Activity 229 Methodology Plant Collection (Cynodon Dactylon) Wild ecotype of C. n-pentane. Dried aliquots were stored in -20 C for further bioassays. The aliquots were taken to dryness using purified nitrogen gas. Methanol absolute (1 ml) was used to activate the sorbent and further equilibrated with sterile deionised distilled water (1 ml). Institute of Tropical Biology and Conservation. Approximately. v/v). Plant Extraction The whole part of C. The soaking process was repeated three times for each extraction to obtain a complete extraction. UMS. In this study. The extracts obtained were then evaporated and concentrated under reduced pressure (768–7 mmhg) using RotaVaporTM (BUCHI) to obtain 1 ml of extract per 10 g of plant sample. Samples were collected in the evening during the daylight time. and a duplicate was submitted to BORH Herbarium. the plant samples were cleaned thoroughly with distilled water to remove soil and dirt. only ethyl acetate and ethanol extracts were used for SPE-based extract bioassay. Samples were then loaded into the cartridges and washed with 1 % methanol (1 ml) to remove any impurities from the samples and finally eluted with 2 ml of methanol:acetonitrile (1:1. Prior to extraction. dactylon which was cleaned by distilled water was shadedried for 24 h in a drying chamber at 40–50 C and powdered using a mechanical blender (Waring Commercial Blender). ethyl acetate. Solid-Phase Extraction StrataTM-X 33um Polymeric Sorbent reverse-phase (200 mg/6 mL) (Phenomenex) cartridges with 12-cartridge manifold system was used. Malaysia. and methanol–Merck) and shaken on a platform shaker (LabCompanionTM) at 150 rpm with temperature of 25 C to obtain various plant extracts. and maintained in a mini-nursery in campus. ethanol. diethyl ether. 100 g of plant powder was later soaked into 200 mL of different solvents (acetone. Aliquots were then kept in -20 C temperature for further use. UMS. sowed. coli. Chloramphenicol antibiotic discs (1 mg/ml. before it is suspended in potato dextrose broth (PDB) prior to TLC bioassay.. which helps to diffuse the extract into the medium. Approximately. Malaysia. aureus. TLC plates were incubated in a moist chamber for 48 h at 25–27 C to spot any antimicrobial property of the separated compounds from the plant. 0. incubated at 37 C for 24 h before grown in nutrient broth (NB) and adjusted according to McFarland standard to achieve approximately 1 9 108 CFU/ml before introducing to the test media.3 paper (6 mm diameter) were prepared for the disc diffusion bioassay. Sterile discs of Whatman No.230 S. The discs loaded with test extracts. Approximately. The plates were kept at room temperature for 30 min. All extract concentrations were standardised to 100 mg/mL. their corresponding solvents. Kota Kinabalu. Aspergillus niger was obtained from stock culture of UMS. Mueller-Hinton Agar (MHA) medium was prepared and sterilised in an autoclave at 121 C for 15 min at 15 psi before transferring it to sterilised petri dish. Streptococcus pyogenes. niger was cultured on potato dextrose agar (PDA) for approximately 7–10 days. S. the test microorganisms were subcultured on nutrient agar (NA). The microbial cultures were preserved in 30 % glycerol stock solution at -85 C temperature. For the negative control discs. Abdullah et al.niger which was suspended in PDB. 60 lL of the respective solvents was loaded to separate discs. and the antibiotic discs were placed on the media with the help of a sterile forceps carefully with adequate spacing between each other. Klebsiella spp.1 ml culture of bacterial pathogens adjusted according to McFarland standard was placed on the MHA media and spread throughout the plate using spread plate technique. Test Microorganisms Pure cultures of eight different pathogens (Bacillus cereus. E. The A. Bacillus subtilis. 30 lg/disc) were used as positive control to compare the antimicrobial activity. Antimicrobial Activity Study Thin-layer chromatographic bioassay: Thin-layer chromatography (TLC) (Silica Gel 60 F254—Merck) bioassay coupled with A. P. aeruginosa. Dactylon ethanol and ethyl acetate extracts were applied on TLC plate which developed in ethyl acetate:hexane (1:9) solvent system and then examined under UV light (265 and 325 nm) to spot the presence of compounds of interest. Disc diffusion bioassay: Antimicrobial activity of the plant extracts was evaluated using disc diffusion method according to Kirby-Bauer method as described by Lalitha (2011). For thin-layer chromatographic (TLC) bioassay. and Streptococcus pneumonia) were obtained from Queen Elizabeth Hospital. . and 60 ll (6 mg) of the extracts were loaded to each disc. Prior to antimicrobial activity study. niger culture was used as preliminary screening for antimicrobial activity. The plates later were sprayed with 7–10 day-old A. 2 mg of C. 0 mm) for ethanol extract while B.In Vitro Antimicrobial Activity 231 The plates were then incubated at 37 C for 24 h in an incubator to determine the antibacterial activity of the respective solvent extraction of C. zone of inhibition in diameter was measured and recorded. Results Extraction Yield Different solvents have different resolving strength towards the plant constituents which resulted in different yield as shown in Table 1. dactylon with different solvents Solvent Acetone Chloroform Diethyl ether Ethanol Ethyl acetate Methanol n-pentane Yield (%) 2.420 %) and chloroform extraction (3.065 1. The lowest yield was obtained from n-pentane extraction with only 0. The extracts were unable to inhibit the growth of A.0 mm and 10.0 mm) for ethanol extract while S. From Table 2.9605 3. both ethanol and ethyl acetate extracts showed significant effect to almost all the tested pathogens with the size of inhibition between 7.923 7. Disc diffusion bioassay (crude extract): Ethanol and ethyl acetate extracts of the plant exhibited broad spectrum of antimicrobial activity.0 mm) for ethyl acetate extract.500 % of yield.0 and 12.0 mm for ethyl acetate extract. The greatest activity observed was against S.0 mm) for ethyl acetate extract. From all of the extractions. niger after incubated for 48 h. 2). 1.0 mm).218 5. However.065 %) followed by methanolic extraction (5. Antimicrobial Activity Thin-layer chromatographic bioassay: Separation of bioactive compounds from C.0 mm) and Table 1 Extraction yield of C.550 0. as shown in Fig. Most of the extractions using non-polar solvents produced lower yield.0 ± 0. ethanolic extraction produced the highest yield (7. Methanol extract showed very weak activity against Streptococcus pneumoniae (7.0 ± 0. After incubation. cereus (12. pneumonia (7.0 mm for ethanol extract and 7. The other extraction yield results are as shown in the Table 1.500 . The least activity observed was against B.0 ± 0.0 ± 0.0 ± 1.0 ± 1.0 ± 1. cereus (8. dactylon.0 ± 0.0 ± 0. dacylon for ethanol and ethyl acetate extracts was the best in ethyl acetate:hexane (1:9) solvent system which produces higher resolution and greater separation.420 0. while both methanol and acetone showed weak activity against B. the antimicrobial properties from both ethanol and ethyl acetate extracts were not detected (Fig. pyogenes (10.550 %).subtilis (7.0 ± 1. dactylon by TLC. Meanwhile. observed under UV light (i = 254 nm. .0 ± 0. E. pyogenes (7. P.A) and ethanol (EtOH) extracts of C. coli. a E.A:Hexane (2:18) solvent system. aeruginosa. subtilis (7.232 S. Abdullah et al. b EtOH extract B. a Ethyl acetate extract.3 ± 0. niger was able to grow on the plate after incubated for 48 h. Chloroform extract showed inhibition to two pathogens tested. 2 Thin-layer chromatographic bioassay of ethyl acetate (E. b: Ethanol extract Fig. A.0 mm) and S. subtilis (except for the diethyl ether and n-pentane extracts) and S. aureus. subtilis (7. B. pyogenes (except for acetone and n-pentane extracts) were more sensitive towards most of the extracts. dactylon. E. n-pentane extract did not inhibit any of the tested pathogens. In the present study.0 mm). S.A extract. ii = 365 nm).0 ± 0.6 mm). 1 Separation of bioactive compounds from C.6 mm). Meanwhile. Fig. Diethyl ether extract exhibited the least activity only to S. pyogenes (8.3 ± 0. B. 0 In Vitro Antimicrobial Activity 233 .3 24. 30 lg/disc) Bacillus cereus Bacillus subtilis Escherichia coli Klebsiella spp.0 ± 0.0 ± 0.0 8.d n.0 n.d n.d 8.d n-pentane *Values presented are means of three replicates.3 ± 0.0 7.0 ± 0.d n..0 8.0 21.0 7.d n.d n.0 ± 0.d n.d n.3 23.0 7.d n.d n.0 22.6 Ethanol 12.d n.6 0.3 ± 0.3 ± 0.d n.0 1.d n.8.0 8. dev.0 ± 0.0 9.d 7.0 8.3 ± 0. ± stand.d n.0 ± 0.0 ± 0.0 Ethyl acetate 8.d n. Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pyogenes Streptococcus pneumoniae Acetone Table 2 Antimicrobial activity of C.3 ± 0.0 ± 1.6 n.d n.d Chloroform n.6 10.d n.0 24.3 ± 0.6 0.0 CHL ± ± ± ± ± ± ± ± 1.0 9. each disc loaded with approx.dactyloncrude extracts with the respective solvents against pathogens Tested microbial pathogens Size of inhibition zone (mm) of the plant extract with the respective solvents* 24.0 10.0 8.0 ± 1.d n.0 n. 60 ul or 12 mg/disc of plant extract n.0 7.3 20.0 0.6 1.0 ± 0.0 7.d n.0 ± 1.d 7.0 ± 0.0 ± 0.0 n.0 n.0 ± 0.0 22.0 ± 0.d Diethyl ether 9.0 ± 0.d n.d Methanol n.0 ± 0.d n.d n.d n.6 n.0 0.d 7.d n.6 8.0 ± 0.d n.d = not detected C CHL = Chloramphenicol (1 mg/ml.6 8.0 1. The weakest activity was observed against Klebsiella spp. the greatest activity for ethyl acetate SBE was observed against B.0 n. As shown in Table 3.0 ± 0. higher than the crude extract (12. In accordance to the bioassay of the ethyl acetate crude extract. D EtOH SPE-based extract (flush portion).7 ± 0. SBE): Flush portion from both ethyl acetate and ethanol SPE-based extracts exhibited greater antimicrobial properties in contrast to the elute portion of the extracts.0 18.0 ± 0.d = not detected.7 19. each disc loaded with approx.0 ± 0. . and S.0 ± 0.0 ± 0. A EA SPE-based extract (elute portion).0 0.0 ± 0. C EA SPE-based extract (flush portion).0 ± 0. pneumoniae were only sensitive to ethanol and ethyl acetate extracts.6 11. and some activity against S. B EtOH SPE-based extract (elute portion).0 8.0 7. and P.0 7. 60 ul or 12 mg/disc of plant extract.0 mm of inhibition diameter.0 mm of inhibition diameter.0 mm of inhibition diameter.0 ± 0.0 9. P. Table 3 Antimicrobial activity of C.0 ± 0.0 ± 0.0 n. dactylon EtOH. SPE = Solid-Phase Extraction CHL = Chloramphenicol (1 mg/ml.0 ± 1.0 ± 0.0 mm).0 8.0 *Values presented are means of three replicates.d 8. cereus with 15. Strong inhibition to all the pathogens’ growth observed on standard chloramphenicol disc proved none of the microorganisms tested were chloramphenicol resistant.0 7.0 mm of inhibition diameter. respectively. aureus (9.0 10.0 7.7 ± 0.7 ± 0.0 7.3 25.0 13.0 ± 0.0 ± 0.0 7.0 ± 0.0 ± 0.0 24. EA = Ethyl acetate.0 ± 0. respectively.234 S.0 to 15.6 0. Ethanol SBE also showed activity against S.0 ± ± ± ± ± ± ± ± 0. and S.0 8. Abdullah et al.0 mm). pyogenes with 8.0 ± 0.0 ± 1.0 ± 0. The weakest activity for ethanol SBE was observed against Klebsiella spp.0 19.0 10.7 20.0 n. in contrast to ethanol crude extract in which the greatest activity was observed against S.0 0. both ethyl acetate and ethanol SBEs showed significant activity to almost all the tested pathogens with size of inhibition ranging from 8. ± stand. n.and EA SPE-based extraction against pathogens after 17-h incubation Tested microbial pathogens Size of inhibition zone (mm)* A Bacillus cereus Bacillus subtilis Escherichia coli Klebsiella spp.0 10. Disc diffusion bioassay (for solid-phase extraction-based extract.0 9. The greatest activity for ethanol SBE was against B.0 ± 0.0 ± 0.0 mm of inhibition diameter.0 C D CHL 15. No growth or activity was observed on negative discs.0 ± 1.0 ± 0. S.0 ± 1.d 7. cereus with 13.0 mm to 8.0 ± 0.6 0. pyogenes (10. aeruginosa. 30 lg/disc) Klebsiella spp.0 1. aeruginosa with 7.0 ± 0.0 ± 0.0 ± 0.0 0.0 ± 0. EtOH = Ethanol. pyogenes.0 ± 0.0 7. pneumonia. respectively.0 11. with 11. subtilis (11.0 mm for ethanol SBE. Pseudomonas aeruginosa Streptococcus pyogenes Streptococcus pneumonia Staphylococcus Aureus 8. aureus with 10.0 8.0 mm) followed by B.0 ± 0.0 mm of inhibition diameter.0 mm).6 mm for ethyl acetate SBE and from 13.0 8.0 8.0 7.0 18.0 ± 0.0 n.0 ± 0.0 ± 0.6 1.. dev.d n. and S.0 23.d B ± 0.0 ± 0.d 8.0 ± 0. other solvents such as ethyl acetate were able to resolve other trace bioactive constituents which are not being able to be resolved by ethanol in greater amount. polar solvents were able to produce higher yield. the age of the plant. Najafi et al. such as alkaloids. Singh and Gupta 2008). percentage humidity of the harvested material. Cardiac glycosides from C. 2007). Previously. 2009). To a large extent. dactylon were previously studied to possess antiarrhythmic activity against ischaemia or reperfusion-induced arrhythmias and cardioprotective properties in tested rat (AlbertBaskar and Ignacimuthu 2010. the outer membrane acts as a great barrier to many environmental substances including antibiotics. 2009). terpenoids. the plant has been studied to contain many bioactive constituents which contributed to its antimicrobial activity (Syahriel et al. gram-negative bacteria were more resistant to antibiotics than gram-positive bacteria. flavonoids. Due to the great extent of pharmacological effects exert by the plant glycoside. explaining the significant antimicrobial activity which levelled to the ethanolic extraction. Carbohydrate and fatty acid derivatives from natural source have been proven to possess broad-spectrum antimicrobial activity (Nobmann et al. and bioactivity of the extracts (Mandal et al. 2012). 2012). The antibacterial activity of ethanol extract was believed to be due to the presence of active principle in the extracts such as saponins. etc. and terpenoids which might be responsible for the broad spectrum of antibacterial activity compared to the other extracts (Kafaru 1994. situation and time of harvest. In gram-negative bacteria. Meanwhile. The resistance is due to the differences in their cell wall composition. From the extraction yield in Table 1. tannins. A large number of constitutive plant compounds have been reported to have antimicrobial activity. phenolics.In Vitro Antimicrobial Activity 235 Discussion Higher plants consist of wide range of bioactive compounds. 2008). This implies that most of the plant constituents are polar compounds such as saponins and the phenolics. toxicity. the antimicrobial activity exhibited by the plant in the present study could be induced by the derivatives from this carbohydrate constituent. the abundance of glycoside and its derivatives were identified from the plant (Syahriel et al. Presence of thick murine layer in the cell wall prevents the entry of the inhibitors (Madigan et al. saponins. From the previous study. which were utilised by the plants themselves as a defensive mechanism and to maintain the plant biological activities (Zwenger and Basu 2008). and the method of extraction are possible sources of variation for the chemical composition. Biological actions are primarily due to these components in a very complicated concert of synergistic or antagonistic activities. Mixtures of such chemicals show a broad spectrum of biological effects and pharmacological properties. Generally. The present study revealed that there is no significance difference between gram-negative and gram-positive bacteria in terms of susceptibility to the crude extracts although for the extracts other than ethanol . Higher resolving strength of ethanol in regards to its yield percentage consequently enables it to resolve comparatively more bioactive compounds which might explain the considerable antimicrobial activity compared to the other solvents. M. A. 108. Queen Elizabeth Hospital for their support in supplying the biological specimens. G. 181–184. V. A. and ethyl acetate extracts. P. P. Bose.. Kabir. Azap. & Ignacimuthu. J. Brazilian Archives of Biology & Technology. Chaudhari. PHCOG REV: Review article. Mathew. & Garjani. B. (2011). M. Journal of Ethnopharmacology. P. Joy (eds.. K. caffeic acid and 4-hydroxybenzoic acid against Ganoderma boninense. 4. 123. Dunlap... Evaluation of the immunomodulatory and DNA protective activities of the shoots of Cynodon dactylon. V. 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