Antimicrobial Ellagitannin of Punica granatum Fruits

Thelma de B. Machado, Ivana C. R. Leal , Ana Claudia F. Amaral , Kátia R. N. dos Santos, Marlei G. da Silva and Ricardo M. Kuster* Núcleo de Pesquisas de Produtos Naturais, Bloco H, Centro de Ciências da Saúde, Ilha do Fundão, Universidade Federal do Rio de Janeiro, 21921-590 Rio de Janeiro RJ, Brazil Laboratório de Química de Produtos Naturais e Central Analítica, Far-Manguinhos FIOCRUZ, Rio de Janeiro RJ, Brazil Instituto de Microbiologia Prof. Paulo de Góes, Bloco J, Centro de Ciências da Saúde, Ilha do Fundão, Universidade Federal do Rio de Janeiro, 21921-590 Rio de Janeiro RJ, Brazil


Introduction
As part of our effort to identify the substances responsible for the pharmacological activities attributed to plants utilized in Brazil in popular medicine we have studied the epicarp of pomegranate fruits to identify the components with antimicrobial activity.Punica granatum Linn.(Punicaceae) is a shrub or small tree native to Asia 1 where its several parts have been used as an astringent, haemostatic, as a remedy for diabetes, as an anthelmintic specifically against tapeworms and for diarrhoea and dysentery. 2In Brazil the fruits are known as "romã" and are used for the treatment of throat infections, coughs and fever.There are several commercial phytopreparations in Brazil containing extracts from pomegranate.For the validation of such products it is necessary to define chemical markers, substances that when present in the preparations attest their quality. 3Although many reports on the antimicrobial activity of pomegranate exist in the literature, none of them relates such activity with its chemical composition.We describe here for the first time the isolation and identification of the tannin responsible for the activity against a bacterium of medical importance (Staphylococcus aureus).Interest in plants with antimicrobial properties have been revived due to current problems associated with the use of antibiotics.With the increasing prevalence of methicillinresistant Staphylococcus aureus (MRSA) strains as pathogens in both hospital and the community, the investigation of plant extracts active against this organism provides an example of prospecting for new compounds which may be effective against infections currently difficult to treat. 4

General experiments procedures
All reagents were of analytical grade.The NMR spectrum of the compound was taken on a Varian-Gemini 200 ( 1 H: 200 MHZ spectrometer).Column chromatography was performed using Sephadex LH-20 (Pharmacia) and XAD-16 resin (Sigma).Thin layer chromatography was performed on cellulose plates (Merck), HPLC/UV on a Shimadzu instrument equipped with a diode array detector and RP-18 column (5 µm, 20 X 5 mm, Merck) and preparative HPLC on a Shimadzu instrument equipped with UV detector and RP-18 column (10 µm, 25 X 2 cm).Antibacterial assays were performed on Mueller Hinton agar medium (Oxoid).

Extraction and isolation of the constituents
Fresh fruit pericarp (240 g) was exhaustively extracted with EtOH.The dried ethanolic extract was suspended in water and successively partitioned with hexane, chloroform, ethyl acetate and butanol.The most active fraction on bioassay (ethyl acetate) was chromatographed on a XAD-16 column using a water -methanol gradient.The active fraction eluted from the column with H 2 O:MeOH (1:1) was submitted to chromatography on a Sephadex LH-20 column using a gradient H 2 O:MeOH and the active fraction was purified on a preparative HPLC column to afford the active compound punicalagin and the inactive ones, ellagic acid and punicalin.

Bacterial strains
Brazilian prevalent clone methicillin-resistant Staphylococcus aureus strains (6 isolates -Table 1) were obtained from hospitalized patients in two Brazilian hospitals (Hospital Universitário Clementino Fraga Filho -RJ and Hospital de Clínicas da Universidade Federal de Uberlândia -MG) and identified at the Institute of Microbiology, Federal University of Rio de Janeiro. 5 Furthermore, as a comparison parameter, 10 MRSA strains from other clones were tested, as well as 8 MSSA (methicillin-sensitive S. aureus) and 2 reference strains [S.aureus ATCC 29213 (MSSA) and ATCC 33591 (MRSA)].

Assay for antibacterial testing
Disc diffusion method 6 Petriplates containing 20 mL of Mueller Hinton agar medium were seeded with a 24 h old culture of the bacterial strains.The extracts, fractions and pure compounds were tested in concentration of 25 or 50 mg mL -1 , applying 10 µL of each sample to sterile filter paper discs (5 mm in diameter) and placed on the surface of the medium.The inoculum size was adjusted so as to deliver a final inoculum of approximately 10 8 colony-forming units (CFU)/mL. 7Incubation was made at 37 °C for 24 h.The assessment of antibacterial activity was based on the measurement of diameter of the inhibition zone formed around the disc.

Dilution method
The minimum inhibitory concentration (MIC) was determined by dilution according to NCCLS. 8Mueller Hinton agar is prepared and sterilized in the usual fashion   by autoclaving.Before solidification, 20 mL of agar medium is added to each of the Petri dishes containing the samples and the Petri dishes are swirled carefully until the agar begins to set.Final concentration from 250 to 0,97 µg mL -1 were used for each plant sample.The bacterial inoculum size was adjusted so as to deliver a final inoculum of approximately 10 4 colony-forming units (CFU/mL 8 and was added to the medium using a Steers replicator.

Results and Discussion
The active fraction when analyzed by TLC on cellulose plates (H 2 O:CH 3 COOH, 4:1) showed initially the presence of two orange colored spots with NaNO 2 reagent, turning purple after some minutes.This is a characteristic reaction for ellagitannins. 9However, these two substances show up as four when analyzed by analytical HPLC/UV (Figure1A).Each one of the four peaks was obtained by preparative HPLC (H 2 O:CH 3 CN, 9:1) and after liophilization was reanalyzed by analytical HPLC/UV. Figure 1C shows the presence of two substances for each peak.This is in fact a characteristic phenomenon of hydrolysable tannins with an unsubstituted anomeric hydroxyl group. 10The substances were identified as punicalin α 1 and punicalin β 2, punicalagin α 3 and punicalagin β 4, the major ellagitannins from the pomegranate. 11Figure 1B shows the UV spectra of these substances (λ max = 218, 260 and 379 nm).These values are characteristic of a gallagyl cromophore 12 conferring to the compounds a bright yellow colour.Acid hydrolysis of punicalin afforded gallagyldilactone (identified by HPLC/UV, RT 3.2 min) and glucose (identified by co-chromatography on silica gel plates).Acid hydrolysis of punicalagin afforded gallagyldilactone, ellagic acid (HPLC/UV, 3.2 min and 4.9 min) and glucose.

Conclusion
Our results show that the ellagitannin punicalagin is the substance responsible for the antimicrobial activity of the pomegranate.Furthermore, this compound presented an activity 10 fold higher than the one found by Burapadaja and Bunchoo. 13For the standardization of phytopharmaceuticals it could be the chemical marker of choice.

Table 1 .
Susceptibility to antimicrobial agents of Brazilian prevalent clone A methicilin-resistant Staphylococcus aureus strains and antimicrobial activity of punicalagin anomers