Research ArticleIn Vitro Antimicrobial and Modulatory Activity ofthe Natural Products Silymarin and Silibinin
Dayanne Rakelly de Oliveira,1 Saulo Relison Tintino,2
Maria Flaviana Bezerra Morais Braga,2 Aline Augusti Boligon,3
Margareth Linde Athayde,3 Henrique Douglas Melo Coutinho,2
Irwin Rose Alencar de Menezes,4 and Roselei Fachinetto1,5
1Programa de Pos-Graduacao em Ciencias Biologicas, Bioqumica Toxicologica, Universidade Federal de Santa Maria,97105-700 Santa Maria, RS, Brazil2Laboratorio de Microbiologia e Biologia Molecular, Universidade Regional do Cariri (URCA), 63100-000 Crato, CE, Brazil3Departamento de Farmacia Industrial, Universidade Federal de Santa Maria, 97105-700 Santa Maria, RS, Brazil4Laboratorio de Microbiologia e Biologia Molecular, Departamento de Qumica Biologica, Universidade Regional do Cariri,63100-000 Crato,CE, Brazil5Programa de Pos-Graduacaoo em Farmacologia, Universidade Federal de Santa Maria, 97105-700 Santa Maria, RS, Brazil
Correspondence should be addressed to Henrique Douglas Melo Coutinho; firstname.lastname@example.org
Received 26 September 2014; Revised 12 February 2015; Accepted 15 February 2015
Academic Editor: Glen Jickling
Copyright 2015 Dayanne Rakelly de Oliveira et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.
Silymarin is a standardized extract from the dried seeds of the milk thistle (Silybum marianum L. Gaertn.) clinically used asan antihepatotoxic agent. The aim of this study was to investigate the antibacterial and antifungal activity of silymarin and itsmajor constituent (silibinin) against different microbial strains and their modulatory effect on drugs utilized in clinical practice.Silymarin demonstrated antimicrobial activity of little significance against the bacterial strains tested, with MIC (minimuminhibitory concentration) values of 512g/mL. Meanwhile, silibinin showed significant activity against Escherichia coli with a MICof 64g/mL. The results for the antifungal activity of silymarin and silibinin demonstrated a MIC of 1024 g/mL for all strains.Silymarin and silibinin appear to have promising potential, showing synergistic properties when combinedwith antibacterial drugs,which should prompt further studies along this line.
Microbial infections have become one of the principalproblems of public health in the world, affecting all coun-tries, developing or developed. It can be related to the processof natural selection in bacterial development or the naturalconsequence of the adaptation of bacteria to exposureto antibiotics in the course of the indiscriminate use ofantibiotics in humans and animals. Various cases related toresistance have been reported, includingmethicillin-resistantStaphylococcus aureus (MRSA) , penicillin-nonsusceptibleStreptococcus pneumoniae (PNSSP), vancomycin-resistant
enterococci (VRE), extended spectrum beta-lactamase-(ESBL-) producing Enterobacteriaceae, and Candida spresistant to imidazoles.
Staphylococcus aureus is a common cause of cutaneousand soft tissue infections, as well as invasive illness, suchas bacteremia, septic arthritis, osteomyelitis, and necrotizingpneumonia [2, 3]. Escherichia coli is a Gram-negative bacillusthat causes infections, especially neonatal, such as meningitisand septicemia, and even diarrheal diseases, in the wholeworld, particularly affecting children up to 5 years old. E.coli is typical of the intestinal flora and commensal of thevaginal flora [4, 5]. Also, Pseudomonas aeruginosa has often
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015, Article ID 292797, 7 pageshttp://dx.doi.org/10.1155/2015/292797
2 BioMed Research International
been associated with occurrence of hospital infections andantibiotic resistance events [6, 7].
Microbial resistance is thus problematic for public health,especially coupled to virulence potential of these multire-sistant pathogens. Accordingly, we should emphasize therelevance of the discovery of new drugs with antimicro-bial capacity and/or showing synergism with drugs alreadyemployed in clinical practice .Thus, in the last years, therehas been increased use of plants and their derivatives asan alternative modality in the treatment of various diseases,including infections caused by microorganisms .
Infections by yeasts of Candida occur on cutaneous andmucosal surfaces, but, in some cases, they become severeby causing systemic infection, especially in immunocompro-mised patients. Moreover, therapeutic options are still lim-ited, particularly in treating resistant pathogens . Addi-tionally, the indiscriminate use of broad spectrum antibioticshas contributed to the development of fungal infections .
Silymarin is a standardized extract from the dried seedsof the milk thistle (Silybum marianum L. Gaertn.), familyAsteraceae . Silymarin contains approximately 7080%flavonolignans and 2030% nonidentified oxidized polyphe-nolic compounds fraction. The mixture of flavonolignansconsistsmainly of silybin (silibinin), themajor bioactive com-ponent of the extract, and isomeric isosilybin, silychristin,and silydianin and two flavonoids (taxifolin and quercetin)[13, 14]. Silibinin currently is recommended for use inalcoholic liver disease. Ethanol induces free radical formationthrough multiple pathways, resulting in steatohepatitis andcirrhosis with chronic use [15, 16].
Silymarin has clinical use as antihepatotoxic agent ,has anti-inflammatory properties , and is antitumor ,antifibrotic, and cytoprotective . Studies have reportedthe synergistic activity of silibinin when combined withampicillin and gentamicin against bacteria that attack the oralcavity . However, there are few works that have evaluatedthe antimicrobial capacity of silymarin and silibinin, demon-strating the need to extend the study of their therapeutic usein this regard.
The objective of this work was to investigate silymarinand its major component, silibinin, for possible antimicrobialeffects and drug-modifying activity when combined withantibacterial and antifungal drugs commonly used in theclinic and also to compare the activity of the two agents.
2. Materials and Methods
2.1. Quantification of Compounds by HPLC-DAD. Reversephase chromatographic analyses were carried out undergradient conditions using C
18column (4.6mm 250mm)
packed with 5 m diameter particles. The mobile phase waswater containing 1% formic acid (A) and methanol (B), andthe composition gradient was 15% of B for 10min and waschanged to obtain 20%, 30%, 50%, 60%, 70%, 20%, and 10%B at 20, 30, 40, 50, 60, 70, and 80min, respectively, followingthe method described by Boligon et al.  with slight mod-ifications. Silymarin extract was analyzed at a concentrationof 2.4mg/mL; the identification of silybin (A and B), gallic
acid, and caffeic acid was performed by comparing theirretention time and UV absorption spectrum with those ofthe commercial standards. The flow rate was 0.6mL/min,the injection volume was 40 L, and the wavelength was254 nm for gallic acid, 280 nm for silybin (A and B), and327 nm for caffeic acid. All the samples andmobile phasewerefiltered through 0.45 m membrane filter (Millipore) andthen degassed by ultrasonic bath prior to use. A stock solutionof standards references was prepared in the HPLC mobilephase at a concentration range of 0.0300.250mg/mL. Thechromatography peaks were confirmed by comparing DAD(DiodeArrayDetector) retention timewith those of referencestandard and by DAD spectra (200 to 500 nm). Calibrationcurve for gallic acid is = 11945 + 1268.4 ( = 0.9997), forcaffeic acid is = 13407 + 1361.8 ( = 0.9992), for silybin A is= 12683 + 1185.9 ( = 0.9999), and for silybin B is= 13045x+ 1376.1 ( = 0.9995). All chromatography operations werecarried out at ambient temperature and in triplicate.The limitof detection (LOD) and limit of quantification (LOQ) werecalculated based on the standard deviation of the responsesand the slope using three independent analytical curves. LODand LOQ were calculated as 3.3 and 10/, respectively,where is the standard deviation of the response and is theslope of the calibration curve.
2.2. Preparation of Stock Solution and Test Solutions. Stocksolutions of silymarin and silibinin were prepared at aconcentration of 10mg/mL in 1mL of dimethylsulfoxide(DMSO). Using this concentration, the compounds werediluted in 1mL of sterile distilled water to obtain a concen-tration of 1024 g/mL (test solution).
2.3. Fungal and Bacterial Strains. The minimal inhibitoryconcentration (MIC) of silymarin and silibinin was deter-mined using the bacterial strains Escherichia coli 25922,Staphylococcus aureus 25923, and Pseudomonas aeruginosa9027 and the fungal strains Candida albicans 62, C. krusei 02,and C. tropicalis 20. All strains were obtained from the Clini-calMycology Laboratory of the Federal University of Paraiba.In the antibiotic-modifying assays, we used the multiresis-tant bacterial strains from the clinical isolates Pseudomonasaeruginosa 03, Escherichia coli 06, and Staphylococcus aureus10 and the standard yeasts Candida albicans, INCQS 40006,C. krusei, INCQS 40095, and C. tropicalis, INCQS 400042.
We utilized the following culturemedia for bacteria: heartinfusion agar (HIA; Difco Laboratories Ltda.) and brain heartinfusion broth (BHI at 10% as indicated by the manufacturer;Acumedia Manufacturers Inc.). Sabouraud dextrose brothwas used for fungi. All culturemedia were prepared followingthemanufacturers instructions. Fungal and bacterial cultureswere maintained at 4C in HIA. Before the tests, the strainswere passaged using the above media and incubated at 37Cfor 24