This article has Open Peer Review reports available.
Antimicrobial, antimycobacterial and antibiofilm properties of Couroupita guianensis Aubl. fruit extract
© Al-Dhabi et al.; licensee BioMed Central Ltd. 2012
Received: 21 July 2012
Accepted: 22 November 2012
Published: 4 December 2012
Couroupita guianensis Aubl. (Lecythidaceae) is commonly called Ayahuma and the Cannonball tree. It is distributed in the tropical regions of northern South America and Southern Caribbean. It has several medicinal properties. It is used to treat hypertension, tumours, pain, inflammatory processes, cold, stomach ache, skin diseases, malaria, wounds and toothache.
The fruits of Couroupita guianensis were extracted with chloroform. Antimicrobial, antimycobacterial and antibiofilm forming activities of the chloroform extract were investigated. Quantitative estimation of Indirubin, one of the major constituent, was identified by HPLC.
Chloroform extract showed good antimicrobial and antibiofilm forming activities; however it showed low antimycobacterial activity. The zones of inhibition by chloroform extract ranged from 0 to 26 mm. Chloroform extract showed effective antibiofilm activity against Pseudomonas aeruginosa starting from 2 mg/mL BIC, with 52% inhibition of biofilm formation. When the chloroform extract was subjected to HPLC-DAD analysis, along with Indirubin standard, in the same chromatographic conditions, it was found that Indirubin was one of the major compounds in this plant (0.0918% dry weight basis).
The chloroform extract showed good antimicrobial and antibiofilm properties. Chloroform extract can be evaluated further in drug development programmes.
KeywordsAntimicrobial Antimycobacterial Antibiofilm Couroupita guianensis Indirubin
Couroupita guianensis Aubl. (Lecythidaceae) is commonly called Ayahuma and the Cannonball tree. It is an evergreen tree allied to the Brazil Nut (Bertholletia excelsa) and is native to tropical northern South America and the Southern Caribbean . The trees are grown extensively in Shiva temples in India. Hindus revere it as a sacred tree because the petals of the flower resemble the hood of the Naga, a sacred snake, protecting a Shiva Lingam, the stigma. The tree also produces globular brown woody, indehiscent, amphisarcun (double fleshy) fruits of an astonishing size, almost the size of a human head . It is widely planted in tropical and subtropical botanical gardens as an ornamental; it does well under cultivations and it is used to feed animals. Native Amazonian people from Amazonian region and other states of the north region of Brazil use infusions or teas obtained from the leaves, flowers, and barks of Couroupita guianensis to treat hypertension, tumours, pain, and inflammatory processes . The Cannonball tree possesses antibiotic, antifungal, antiseptic and analgesic qualities. The trees are used to cure cold and stomach ache. Juice made from the leaves is used to cure skin diseases, and shamans of South America have even used tree parts for treating malaria. The inside of the fruit can disinfect wounds and young leaves cure toothache . Chemical studies of this species showed the presence of α-amirin, β-amirin, β-sitosterol, nerol, tryptanthrine, indigo, indirubin, isatin, linoleic acid, carotenoids and sterols [5–10]. In the flowers, it was possible to identify eugenol, linalool and (E,E)-farnesol where as triterpenoid esters of fatty acids as β-amirin palmitate were characterized in the leaves . Indirubin is a purple 3,2′bisindole, and is a constituent of indigo natural. Indigo natural is a dark blue powder prepared from the leaves of a number of medicinal plants including Baphicacanthus cusia (Acanthaceae), Polygonum tinctorium (Polygonaceae), Isatis indigotica (Brassicaceae), Indigofera suffrutticosa (Fabaceae) and Indigofera tinctoria (Fabaceae) . Indigo, naturally is used in traditional Chinese medicine as a hemostatic, antipyretic, antiinflammatory, and sedative agent in the treatment of bacterial and viral infections . In the present communication we report the antimicrobial, antimycobacterial and antibiofilm forming activities of the chloroform extract of the fruit of C. guianensis. The HPLC finger print of the chloroform extract together with quantification of Indirubin as marker is also given.
Materials and methods
Fresh fruits of Couroupita guianensis were collected during June 2011 from Loyola College Jesuit garden, Chennai, India. The species was identified by a plant taxonomist at Entomology Research Institute, Loyola College, Chennai, India. A voucher specimen (No. ERI/ETHPH/CQ/225) was deposited at the herbarium of the institute.
Preparation of plant extract
The collected fruit was shade dried at room temperature and powdered. 1 kg of fruit powder was extracted with chloroform at room temperature for 48 hrs. The extract was evaporated to dryness at 40°C under reduced pressure.
The following Gram positive, Gram negative bacteria, clinical isolates and fungi were used for the experiment.
Gram positive bacteria
Bacillus subtilis MTCC 441, Micrococcus luteus MTCC 106, Enterobacter aerogenes MTCC 111 and Staphylococcus aureus MTCC 96.
Gram negative bacteria
Shigella flexneri MTCC 1457, Salmonella paratyphi-B, Klebsiella pneumoniae MTCC 109, Pseudomonas aeruginosa MTCC 741, Proteus vulgaris MTCC 1771 and Salmonella typhimurium MTCC 1251.
Escherichia coli (ESBL-3984,), Escherichia coli (ESBL-3904), Klebsiella pneumoniae (ESBL-3971), Klebsiella pneumoniae (ESBL-75799), Klebsiella pneumoniae (ESBL-3894), Klebsiella pneumoniae (ESBL-3967) and Staphylococcus aureus (MRSA).
Candida albicans MTCC 227 and Malassesia pachydermatis; The reference cultures were obtained from Institute of Microbial Technology (IMTECH), Chandigarh, India-160 036 and Department of Microbiology, Christian Medical College, Vellore, Tamil Nadu, India.
Preparation of inoculum
Bacterial inoculums were prepared by growing cells in Mueller Hinton Broth (MHB) (Himedia, Mumbai) for 24 hrs at 37°C. Yeast was grown on Sabouraud Dextrose Broth (SDB) (Himedia, Mumbai) at 28°C for 48 hrs.
Antibacterial activity was carried out using disc diffusion method . Petri plates were prepared with 20 mL of sterile Mueller Hinton Agar (MHA). The test cultures were swabbed on the top of the solidified media and allowed to dry for 10 min. A specific concentration (5 mg/disc) of the chloroform extract was loaded to each disc. The loaded discs were placed on the surface of the medium. Negative control was prepared using respective solvents. Streptomycin was used as positive control. The plates were incubated for 24 hrs at 37°C for bacteria and for 48 hrs at 28°C for fungi. Zones of inhibition were recorded in millimetres and the experiment was repeated twice.
The anti-TB activity of chloroform extract was evaluated against standard sensitive strain M. tuberculosis H37Rv and rifampicin isolate M. tuberculosis XRD-1. The mycobacterial cultures were obtained from Clinical Microbiology Division, Indian Institute of Integrative Medicine, Jammu 180 001, India. The minimum inhibitory concentration (MIC) was determined using broth micro-dilution assay [15, 16]. The experiment was performed in sterile Middlebrook 7H9 broth supplemented with 10% ADC (BD Biosciences, USA). The above-mentioned test bacteria were grown to mid-log phase (10–12 days) at 37°C with shaking in the test media (Middlebrook 7H9 broth supplemented with 10% ADC). Stock solution (1 mg/mL) of chloroform extract was prepared in DMSO and 6.4 μl volume of these stock solutions were added to the wells of a 96 well U bottom microtitre plates (Tarson, Mumbai, India) and nine 2 fold serial dilutions of the compound were prepared in 100 μl of test media. The turbidity of the cultures was adjusted to be equivalent to 1 McFarland turbidity standard (~1 x 107 CFU/mL), which was further diluted to 1:10 in test media and a 100 μl volume of this diluted inoculum was added to each well of the plate, resulting in a final inoculum of 5 x 105 CFU/mL. The final concentrations of the chloroform extract after the addition of inoculums ranged from 0.12 to 32 μg/mL. Rifampicin in the concentration range from 0.12 to 32 μg/mL was used as control drug in the experiment. Periphery wells of the plate were filled with sterile distilled water to prevent evaporation of media in the wells. The plates were incubated at 37°C under 5% CO2 for 3 weeks. Inhibition of growth was determined both by visual examination and with a spectrophotometer at an OD600 (Multiskan spectrum; Thermo Scientific, USA). The lowest concentration of the compound showing no turbidity was recorded as MIC.
Effect of chloroform extract of C. guianensis on biofilm formation
The effect of the Chloroform extract on biofilm forming activity of P. aueroginosa was tested on 24-well polystyrene plates. Chloroform extract at concentrations of 1–5 mg/mL were added in LB containing the bacterial suspension at 106 CFUmL/1. The plates were incubated for 24 h at 37°C. After incubation, biofilm was stained with 0.4% crystal violet. The biofilm inhibitory concentration (BIC) was determined as the lowest concentration that produced visible disruption in biofilm formation and significant reduction in the readings when compared with the control wells at OD570nm. Thus, the BIC was determined by both spectrophotometric quantification and also by microscopic visualization. For visualization of biofilms by light microscopy, the biofilms were allowed to grow on glass pieces (1/1 cm) placed in 24-well polystyrene plates supplemented with the extracts (1–5 mg/mL) and incubated for 24 h at 37°C. Crystal violet staining was performed as described above. Stained glass pieces were placed on slides with the biofilm pointing up and were inspected by light microscopy at magnifications of X40. Visible biofilms were documented with an attached digital camera (Nikon Eclipse Ti 100) .
Standardization of chloroform extract by HPLC-DAD analysis
Sample (chloroform extract) (10 mg in 100 mL) and standard Indirubin (5 mg in 100 mL) were dissolved in methanol. The solutions were filtered through a membrane filter (pore size 0.20 μm) prior to HPLC analysis.
HPLC analysis was carried out on a Waters Alliance 2695 separations Module with photodiode array detector (Waters, 2996). The LC column was an YMC pack ODS A (150 mm × 4.6 mm, 5 μm) column. Two mobile phases A and B were used at flow rate of 1.0 mL/min. The mobile phase was filtered through a 0.45 μm filter, and degassed by vacuum, followed by sonication. Mobile phase A consisted of water with 0.1% orthophosphoric acid and mobile phase was B acetonitrile. Separation was carried out at room temperature. A gradient was used, starting at 95% A, changing to 10% A linearly in 15 min. After elution the column was re-equilibrated for 3 min under the initial conditions. The HPLC profile of C. guianensis chloroform extract was compared with that of standard compound, Indirubin which was best detected at 254 nm.
Statistical analysis was performed using SPSS. Values were expressed as mean ± SD. A Duncan–ANOVA test was used to compare parameters between groups and a Dunnett–ANOVA test to compare between tests and control.
Results and discussion
Antimicrobial activity of the chloroform extract of C. guianensis fruits (5 mg/mL)
E. coli (ESBL-3984)
E. coli (ESBL-3904)
K. pneumoniae (ESBL-3971)
K. pneumoniae (ESBL-75799)
K. pneumoniae (ESBL-3894)
K. pneumoniae (ESBL-3967)
S. aureus (MRSA)
Minimum inhibitory concentration of the chloroform extract of C. guianensis against M. tuberculosis
Chloroform extract MIC (μg/mL)
M. tuberculosis H37Rv (HR-Sen) ATCC 27294
M. tuberculosis XRD-1
BIC of chloroform extract against the biofilm forming activity of Pseudomonas aeruginosa strain at 24 h
0.546 ± 0.00145
0.581 ± 0.00176
0.637 ± 0.00644
0.712 ± 0.00318
0.941 ± 0.00260
1.706 ± 0.00240
The bioactive compounds present in the extracts might have interfered with the adherence of P. aueroginosa by releasing the adhesion compound lipoteichoic acid from the streptococcal cell surface . P. aeruginosa has emerged as one of the most problematic Gram-negative pathogen, with an alarmingly high antibiotic resistance rate [22, 23]. The activity of C. guianensis might be due to its ability to complex with cell wall  and thus inhibiting the microbial growth . An important step in biofilm development is the formation of the characteristic biofilm architecture . Cell surface charge and CSH play a crucial role in bacterium–host cell interactions . There are several reports regarding plant extracts interfering in the biofilm formation of Gram-negative bacteria  and Gram-positive bacteria [28–30]. Biofilm-associated diseases caused by Gram-positive bacteria include caries, gingivitis, periodontitis, endocarditis and prostatitis . Many forms of streptococcal infections, especially recurrent and chronic infections, are associated with the formation of bacterial biofilms . This phenomenon is also observed in antibiotics where the sub-inhibitory concentrations (sub-MICs) of antibiotics, although not able to kill bacteria, can modify their physicochemical characteristics and the architecture of their outermost surface and may interfere with some bacterial functions.
The chloroform extract of the fruit of C. guianensis showed good antimicrobial activities but low antimycobacterial activity against tested strains. The antibiofilm forming activity of the chloroform extract showed a pronounced effect on inhibition of biofilm formation at low concentrations with 52% inhibition.
We are grateful to Addiriyah Chair for Environmental Studies, Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, P.O. Box 2455, Saudi Arabia for financial assistance.
- Lorenzi H: Árvores Brasileiras: Manual de Identificação e Cultivo de Plantas Arbóreas Nativas do Brasil. 1998, Nova Odessa (SP), Brazil: Editora Plantarum, 352 p-Google Scholar
- Mitré M: Couroupita guianensis. IUCN 2012. 1998, IUCN Red List of Threatened SpeciesGoogle Scholar
- Sanz-Biset J, Campos-de-la-Cruz J, Epiquién-Rivera MA, Canigueral S: A first survey on the medicinal plants of the Chazuta valley (Peruvian Amazon). J Ethnopharmacol. 2009, 122: 333-362. 10.1016/j.jep.2008.12.009.View ArticlePubMedGoogle Scholar
- Kumar CS, Naresh G, Sudheer V, Veldi N, Anurag AE: A Short Review On Therapeutic Uses Of Couroupita Guianensis Aubl. Int Res J Pharm App Sci. 2011, 1: 105-108.Google Scholar
- Wong KC, Tie DY: Volatile constituents of Couroupita guianensis Aubl. flowers. J Essent Oil Res. 1995, 7: 225-227. 10.1080/10412905.1995.9698507.View ArticleGoogle Scholar
- Rane JB, Vahanwala SJ, Golatkar SG, Ambaye RY, Khadse BG: Chemical examination of the flowers of Couroupita guianensis Aubl. Indian J Pharm Sci. 2001, 63: 72-73.Google Scholar
- Bergman J, Lindstrom JO, Tilstam U: The structure and properties of some indolic constituents in Couroupita guianensis Aubl. Tetrahedron. 1985, 41: 2879-2881. 10.1016/S0040-4020(01)96609-8.View ArticleGoogle Scholar
- Sen AK, Mahato SB, Dutta NL: Couroupitine A, a new alkaloid from Couroupita guianensis. Tetrahedron Letters. 1974, 7: 609-610.View ArticleGoogle Scholar
- Row LR, Santry CSPP, Suryananayana PP: Chemical examination of Couroupita guianensis. Curr Sci. 1966, 35: 146-147.Google Scholar
- Anjaneyulu ASR, Rao SS: A new ketosteroid from the bark of Couroupita guianensis Aubl. Ind J Chem Sec B: Organic Chemistry. 1998, 37: 382-386.Google Scholar
- Eknat AA, Shivchandraji LK: Amirin palmitate isolation from Couroupita guianensis Aubl. leaves. Indian Drugs. 2002, 39: 213-216.Google Scholar
- Hoessel R, Leclerc S, Endicott JA, Nobel ME, Lawrie A, Tunnah P: Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases. Nat Cell Biol. 1999, 1: 60-67. 10.1038/9035.View ArticlePubMedGoogle Scholar
- Han R: Highlight on the studies of anticancer drugs derived from plants in China. Stem Cells. 1994, 12: 53-63. 10.1002/stem.5530120110.View ArticlePubMedGoogle Scholar
- Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolke RH: Manual of Clinical Microbiology, vol. 6. 1995, Washington DC: ASMGoogle Scholar
- Clinical and Laboratory Standards Institute: Methods for Antimicrobial Susceptibility Testing of Aerobic Bacteria Approved standard M07-A8. 2008, Wayne, PA: National Committee for Clinical Laboratory Standards, 9Google Scholar
- Wallace RJ, Nash DR, Steele L, Steingrube V: Susceptibility testing of slowly growing mycobacteria by a microdilution MIC method with 7H9 broth. J Clinical Microbio. 1986, 24: 976-981.Google Scholar
- Thenmozhi R, Nithyanand P, Rathna J, Karutha Pandian S: Antibioflim activity of coral-associated bacteria against different clinical M serotypes of Streptococcus pyogenes. FEMS Immunol Med Microbiol. 2009, 57: 284-294. 10.1111/j.1574-695X.2009.00613.x.View ArticlePubMedGoogle Scholar
- Regina V, Uma Rajan KM: Phytochemical analysis, antioxidant and antimicrobial studies of fruit rind of Couroupita guianensis (AUBL). Int J Curr Sci. 2012, 221: 262-267.Google Scholar
- Umachigi SP, Jayaveera KN, Ashok Kumar CK, Kumar GS: Antimicrobial, Wound Healing and Antioxidant potential of Couroupita guianensis in rats. Pharmacologyonline. 2007, 3: 269-281.Google Scholar
- Cowan MM: Plant products as antimicrobial agents. Clin Microbiol Rev. 1999, 12: 564-PubMedPubMed CentralGoogle Scholar
- Sun D, Courtney HS, Beachey EH: Berberine sulphate blocks adherence of Streptococcus pyogenes to epithelial cells, fibronectin, and hexadecane. Antimicrob Agents Ch. 1988, 32: 1370-1374. 10.1128/AAC.32.9.1370.View ArticleGoogle Scholar
- Bacq-Calberg CM, Coyotte J, Hoet P, Nguyem-Disteeche M: Microbiologie. 1999, Bruxelles: De Boeck and Larcier, 338-Google Scholar
- Savafi L, Duran N, Savafi N, Onlem Y, Ocak S: The prevalence and resistance patterns of Pseudomans aeruginosa in intensive care units in a university Hospital. J Med Sci. 2005, 35: 317-Google Scholar
- Arvind S, Reg FC, Enzo AP: Identification of antimicrobial components of an ethanolic extract of the Australian medicinal plant. Eremophila duttonii. Phytother Res. 2004, 18: 615-View ArticleGoogle Scholar
- You J, Xue X, Cao L, Lu X, Wang J, Zhang L, Zhou SV: Inhibition of Vibrio biofilm formation by a marine actinomycete strain A66. Appl Microbiol Biot. 2007, 76: 1137-1144. 10.1007/s00253-007-1074-x.View ArticleGoogle Scholar
- Swiatlo E, Champlin FR, Holman SC, Wilson WW, Watt JM: Contribution of choline-binding proteins to cell surface properties of Streptococcus pneumoniae. Infect Immun. 2002, 70: 412-415. 10.1128/IAI.70.1.412-415.2002.View ArticlePubMedPubMed CentralGoogle Scholar
- Turi M, Turi E, Koljalg S, Mikelsaar M: Influence of aqueous extracts of medicinal plants on surface hydrophobicity of Escherichia coli strains of different origin. Acta Pathol Microbiol Immunol Scand. 1997, 105: 956-962.View ArticleGoogle Scholar
- Nostro A, Cannatelli MA, Crisafi G, Musolino AD, Procopio F, Alonzo V: Modifications of hydrophobicity, in vitro adherence and cellular aggregation of Streptococcus mutans by Helichrysum italicum extract. Lett Appl Microbiol. 2004, 38: 423-427. 10.1111/j.1472-765X.2004.01509.x.View ArticlePubMedGoogle Scholar
- Prabu GR, Gnanamani A, Sadulla S: Guaijaverin – a plant flavonoid as potential antiplaque agent against Streptococcus mutans. J Appl Microbiol. 2006, 101: 487-495. 10.1111/j.1365-2672.2006.02912.x.View ArticlePubMedGoogle Scholar
- Razak FA, Othman RY, Rahim ZH: The effect of Piper beetle and Psidium guajava extracts on the cell-surface hydrophobicity of selected early settlers of dental plaque. J Oral Sci. 2006, 48: 71-75. 10.2334/josnusd.48.71.View ArticlePubMedGoogle Scholar
- Hall-Stoodley L, Costerton JW, Stoodley P: Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol. 2004, 2: 95-108. 10.1038/nrmicro821.View ArticlePubMedGoogle Scholar
- Lembke C, Podbielski A, Hidalgo-Grass C, Jonas L, Hanski E, Kreikemeyer B: Characterization of biofilm formationby clinically relevant serotypes of group A Streptococci. Appl Environ Microb. 2006, 72: 2864-2875. 10.1128/AEM.72.4.2864-2875.2006.View ArticleGoogle Scholar
- Kannan P, Mohankumar R, Ignacimuthu S, Gabriel paulraj M: Indirubin potentiates ciprofl oxacin activity in the NorA efflux pump of Staphylococcus aureus Scandinavian. J Infect Diseas. 2010, 42: 500-505.Google Scholar
- Jakobs S, Merz KH, Vatter S, Eisenbrand G: Molecular targets of indirubins. Int J Clin Pharmacol Ther. 2005, 43: 592-594.View ArticlePubMedGoogle Scholar
- Meijer L, Shearer J, Bettayeb K, Ferandin Y: Diversity of intracellular mecha-nisms underlying the anti-tumor properties of indirubins. Indirubin, the Red Shade of Indigo. Chapter 24. 2006, 235-246.Google Scholar
- Nam S, Buettner R, Turkson J, Kim D, Cheng JQ, Muehlbeyer S: Indirubinanalogs inhibit Stat3 signaling and induce apoptosis in human cancer cells. Proc Natl Acad Sci USA. 2005, 102: 5998-6003. 10.1073/pnas.0409467102.View ArticlePubMedPubMed CentralGoogle Scholar
- Leclerc S, Garnier M, Hoessel R, Marko D, Bibb JA, Snyder GL: Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer’s disease. A property common to most cyclin-dependent kinase inhibitors?. J Biol Chem. 2001, 276: 251-260.View ArticlePubMedGoogle Scholar
- Perabo FG, Landwehrs G, Frössler C, Schmidt DH, Mueller SC: Antiproliferativeand apoptosis inducing effects of indirubin-30-monoxime in renal cell cancercells. Urol Oncol. 2009, 29: 815-820.View ArticlePubMedGoogle Scholar
- Perabo FG, Frössler C, Landwehrs G, Schmidt DH, von Rücker A, Wirger A: Indirubin-30-monoxime, a CDK inhibitor induces growth inhibition and apoptosis-independent up-regulation of survivin in transitional cell cancer. Anticancer Res. 2006, 26: 2129-2135.PubMedGoogle Scholar
- Marko D, Schätzle S, Friedel A, Genzlinger A, Zankl H, Meijer L: Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin analogs in human tumour cells. Br J Cancer. 2001, 84: 283-289. 10.1054/bjoc.2000.1546.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhen Y, Sørensen V, Jin Y, Suo Z, Wiedłocha A: Indirubin-30-monoxime inhibits autophosphorylation of FGFR1 and stimulates ERK1/2 activity via p38 MAPK. Oncogene. 2007, 26: 6372-6385. 10.1038/sj.onc.1210473.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/12/242/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.