Antimicrobial activities of the methanol extract and compounds from Artocarpus communis (Moraceae)

Background Artocarpus communis is used traditionally in Cameroon to treat several ailments, including infectious and associated diseases. This work was therefore designed to investigate the antimicrobial activities of the methanol extract (ACB) and compounds isolated from the bark of this plant, namely peruvianursenyl acetate C (1), α-amyrenol or viminalol (2), artonin E (4) and 2-[(3,5-dihydroxy)-(Z)-4-(3-methylbut-1-enyl)phenyl]benzofuran-6-ol (5). Methods The liquid microdilution assay was used in the determination of the minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC), against seven bacterial and one fungal species. Results The MIC results indicated that ACB as well as compounds 4 and 5 were able to prevent the growth of all tested microbial species. All other compounds showed selective activities. The lowest MIC value of 64 μg/ml for the crude extract was recorded on Staphylococcus aureus ATCC 25922 and Escherichia coli ATCC 8739. The corresponding value of 32 μg/ml was recorded with compounds 4 and 5 on Pseudomonas aeruginosa PA01 and compound 5 on E. coli ATCC 8739, their inhibition effect on P. aeruginosa PA01 being more than that of chloramphenicol used as reference antibiotic. Conclusion The overall results of this study provided supportive data for the use of A. communis as well as some of its constituents for the treatment of infections associated with the studied microorganisms.


Background
Artocarpus comminis J.R. & G. Forst., commonly known as breadfruit tree because of the "bread-like texture" of its edible fruits, is an equatorial lowland species of flowering tree in the mulberry family (Moraceae) that grows best below elevations of 650 m [1]. Numbers of medicinal uses are assigned to plants of the genus Artocarpus worldwide. This includes treatments of cardiovascular diseases (yellow leaf decoction of A. communis in Bahamas, Haiti, Trinidad and West Indies), chest pain and vomiting from heart problems (Artocarpus spp. in South Pacific), boils, abscess, and skin infections (leaf ash, macerated root, or latex of Artocarpus spp. sap in Dominican Republic, Haiti, Hawai'i, Malaya, Java, Samoa, Tahiti and Tonga), cracked-skin and dermatosis (A. communis in Hawai'i), burns (A. communis in Haiti), rashes (sap of Artocarpus spp. in Tahiti, Tonga); stomach pain (bark of Artocarpus spp. diluted latex in Samoa, Solomon Islands and Tonga), diarrhea or dysentery (diluted latex or roots boiled of Artocarpus spp. in Borneo, Java, Pacific Islands and Samoa), diabetes (yellow leaf as tea of Artocarpus spp. in Trinidad, West Indies), headache (leaves of A. communis in Bahamas, bark in Samoa and Pacific Islands, toothache (toasted flowers of A. communis and A. integra in Java and Malaya), thrush (crushed leaf buds and latex of A. communis on tongue in Bahamas, Trinidad and Pacific Islands), eye problems (A. communis leaf or petiole juice in Futuna and Samoa), ear infections (leaves juice or diluted latex in Pacific Islands), herpes infections (A. communis in Amboina), fever (A. communis leaves in Bahamas, Malaya and Samoa), enlarged spleen (A. communis in Java) [2]. In Cameroon, the fruits of A. communis are used as food; other parts of the plants are traditionally used to treat headache, infectious and associated diseases such as toothache, eye problems, ear infections, herpes, enlarged spleen, sprains, contusions, swelling [3][4][5]. Some scientific evidences of the bioactivity of A. communis were reported on the extract or isolated compounds [6][7][8][9]. However, few reports are related to the antimicrobial activity of this taxon. The present work was therefore designed to investigate the antibacterial and anticandicidal activities of the methanol extract and compounds isolated from the stem bark of Artocarpus communis.

Plant material
The roots of Artocarpus communis J.R. & G. Forst. were collected in Nkolbisson, Center region of Cameroon in March 2010. The plant was identified by Mr. Victor Nana of the National herbarium (Yaoundé, Cameroon) where a voucher specimen was deposited under the reference number 43982/HNC.

Extraction and purification
The air dried and powdered stem bark (700 g) were extracted with methanol (MeOH) for 48 h at room temperature. The extract was then concentrated under reduced pressure to give 170 g of a brown residue that constituted the crude extract (ACB). Part of FPR (150 g) was submitted to silica gel 60 (0.04-0.063 mm, 120 g) vacuum column chromatography using as eluent, hexane, hexane/CHCl 3 1:1 mixture, CHCl 3 [14]. The chemical structures of the isolated compounds are illustrated in Figure 1.

General procedure
Aluminum sheet pre-coated with silica gel 60 F254 nm (Merck) was used for thin layer chromatography; The spots were visualized using both ultraviolet light (254 and 366 nm) and 50% H 2 SO 4 spray reagent. NMR spectra were recorded on a Bruker Avance 300 at 300 MHz ( 1 H) and 75 MHz and Bruker Avance 600 at 600 MHz ( 1 H) and 150 MHz ( 13 C), with the residual solvent peaks as internal references. The melting point (m.p.) were determined using a Kofler microhot stage apparatus. Mass spectra were recorded with API QSTAR pulsar mass spectrometer. The structures of the compounds were confirmed by comparing with reference data from available literature.

Antimicrobial assays Microbial strains and culture media
The studied microorganisms included reference strains of Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella typhi, Escherichia coli, Candida albicans obtained from the American Type Culture Collection. They were maintained on agar slant at 4°C and sub-cultured on a fresh appropriate agar plates 24 h prior to any antimicrobial test. Nutrient Agar and Sabouraud Glucose Agar were used for the activation of bacteria and fungi respectively. The Mueller Hinton Broth (MHB) was used for the MIC and MMC determinations. The Mueller Hinton Agar (MHA) was also used for the determination of the MMC on these species [15].

MIC and MMC determinations
The MIC determinations on bacteria and C. albicans were conducted using rapid INT colorimetric assay according to described methods [16,17] with some modifications. Briefly, the test sample was first of all dissolved in 10% (v/v) DMSO/MHB to give a final concentration of 512 μg/ ml and serially diluted twofold to obtain concentration ranges. 100 μl of each concentration was added in a well (96-well microplate) containing 95 μl of MHB and 5 μl of inoculum (standardized at 1.5 × 10 6 CFU/ml by adjusting the optical density to 0.1 at 600 nm SHIMADZU UV-120-01 spectrophotometer) [18]. The final concentration of DMSO in the well was less than 3% (preliminary analyses with 3% (v/v) DMSO do not alter the growth of the test organisms). The negative control well consisted of 195 μl of MHB and 5 μl of the standard inoculum [19]. The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a plate shaker and incubated at 37°C for 24 h. The assay was repeated three times in triplicate. The MIC of samples was detected following addition (40 μl) of 0.2 mg/ml p-iodonitrotetrazolium chloride and incubation at 37°C for 30 min [16,17]. Viable microorganisms reduced the yellow dye to a pink colour. MIC was defined as the lowest sample concentration that prevented this change and exhibited complete inhibition of bacterial growth. For the determination of MMC, a portion of liquid (5 μl) from each well that showed no change in colour was plated on MHA and incubated at 37°C for 24 h. The lowest concentration that yielded no growth after this sub-culturing was taken as the MMC [20].
In the present work, the crude extract as well as most of the compounds isolated from the bark of A. communis were tested for their antibacterial activities and against C. albicans. The results are reported in Tables 1 and 2. The MIC results (Table 1) indicated that the crude extract (ACB) as well as compounds 4 and 5 inhibited the growth of all tested microbial species. All other compounds showed selective activities, their inhibitory effects being noted on 3 of the 8 (37.5%) tested organisms for compound 1 and 2. The lowest MIC value (64 μg/ml) for the crude extract was recorded on two of the tested microbial species namely S. aureus and E. coli ATCC8739. Phytochemicals are routinely classified as antimicrobials on the basis of susceptibility tests that produce MIC) in the range of 100 to 1000 mg/mL [24]. Their activity is considered to be significant if MIC values are below 100 μg/ml for crude extract and 10 μg/ml for pure compounds [25]. Therefore, the activity recorded herein can be considered as important, when considering the cutoff point 100 μg/ml required for MIC values of plant extracts with significant activity [25]. Nevertheless, moderate activities  [25] were recorded with compounds 4 and 5 on three (37.5%) and four (50%) of the studied microorganisms respectively. P. aeruginosa is an important nosocomial pathogen highly resistant to commonly used antibiotics, causing a wide spectrum of infections and leading to substantial morbidity and mortality [26]. The lowest MIC value of 32 μg/ml was recorded with compounds 4 and 5 on P. aeruginosa and compound 5 on E. coli ATCC8739, highlighting some medicinal potential for the two compounds, as the activity on P. aeruginosa was better than that of chloramphenicol. However if considered a more flexible stringent criteria indicating that extracts having activities with MIC values below 8 mg/ml [27] are considered to possess some antimicrobial activity and natural products with MIC values below 1 mg/ml are considered noteworthy [28,29], the overall activity recorded therefore with the extracts, compounds 4, and 5 could be considered as important, highlighting the antimicrobial potency of A. communis. However, the tested samples were less active than chloramphenicol and nystatin used as reference antibiotic on most of the microbial strains. The results of Table 2 showed detectable MMC values for some of the studied samples on the tested microbial strains. When analysing carefully the MIC and MMC results for the crude extract, compounds 4 and 5, it can be noted that MMC/MIC ratios lower than 4 were obtained with these samples on most of the tested microbial species, suggesting that a killing effects could be expected [30]. However, all MMC values obtained were greater than the MICs. It can also be noted the reference antibiotics were in most of the case more active than all studied samples, except on P. aeruginosa PA01 where the MIC values obtained with compounds 4 and 5 were two time lower.
To the best of our knowledge, the antibacterial and anti-candicidal activities of the bark extract of A. communis as well as that of compounds 4, and 5 are being reported for the first time. However, the antimicrobial activity of this plant might be due to the presence of both antibacterial and anticandicidal compounds as demonstrated in the present study. The antimicrobial activity of sitosterol-3-O-b-D-glucopyranoside (compound 3) was reported [31,32], and this compound was not tested again in the present work. It's activities were moderate, but sitosterol-3-O-b-D-glucopyranoside as well as the tested compounds might contribute to the overall activity observed with the extract of A. communis.

Conclusion
Finally, the present investigation provides supportive data for the use of A. communis as well as some of its constituents for the treatment of infections associated with the studied microorganisms. However, this will be confirmed with further pharmacological (in vivo activity, bioavailability) and toxicological studies (acute and subacute toxicities using animal models).