Journal of Ethnopharmacology 135 (2011) 797800

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Ethnopharmacological communication

Antimicrobial properties of stem bark extracts from Phyllanthus muellerianus (Kuntze) Excell
G. Brusotti a,b, , I. Cesari a,b , G. Frass a,b , P. Grisoli a,b , C. Dacarro a,b , G. Caccialanza a,b
a b

Department of Drug Sciences, University of Pavia, Pavia, Italy Center for Studies and Researches in Ethnopharmacy (C.I.St.R.E.), University of Pavia, Pavia, Italy

a r t i c l e

i n f o

a b s t r a c t
Ethnopharmacological relevance: The plants of the genus Phyllanthus (Euphorbiaceae) are widely distributed in most tropical and subtropical countries, and have long been used in folk medicine to treat several diseases. Particularly, Phyllanthus muellerianus (Kuntze) Excell, commonly called mbolongo in Cameroon, is used by pygmies baka as a remedy for tetanus and wound infections. Aim of the study: To investigate the antimicrobial properties of Phyllanthus muellerianus (Kuntze) Excell (family Euphorbiaceae) stem bark used in Cameroon by baka pygmies as a remedy for wound healing and tetanus. Materials and methods: Aqueous and methanol extracts with and without defatting treatment, were prepared and their activity against Clostridium sporogenes ATCC 3584, Staphylococcus aureus ATCC 6538, Streptococcus mutans ATCC 25175, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 10536, Candida albicans ATCC 10231, was evaluated on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal-fungicidal concentration (MBC-MFC) by the macrodilution method. Results: Water extract showed a weak activity against Clostridium sporogenes (MIC 900 g/mL) and resulted inactive at the tested concentrations against all the other microorganisms. The defatted methanol extract, inactive against Staphylococcus aureus, Escherichia coli, Candida albicans, exhibited a very interesting activity against Clostridium sporogenes and Streptococcus pyogenes (MIC 100 g/mL and 300 g/mL, respectively), which seems to validate the use of this plant in pygmies traditional medicine for the treatment of tetanus and wound infections. The activity found against Streptococcus mutans (300 g/mL), aetiological agent of caries, may suggest a possible use of this plant as natural remedy to prevent dental diseases. Conclusions: The activity against streptococci and Clostridium sporogenes ATCC 3584, showed by stem bark extracts of Phyllanthus muellerianus, traditionally used by baka pygmies to treat wound infections and tetanus, is reported for the rst time. 2011 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 21 December 2010 Received in revised form 25 February 2011 Accepted 18 March 2011 Available online 4 April 2011 Keywords: Phyllanthus muellerianus Bark extract Ethnomedicine Antibacterial activity

1. Introduction The plants of the genus Phyllanthus (Euphorbiaceae) are widely distributed in most tropical and subtropical countries, and have long been used in folk medicine to treat kidney and urinary bladder disturbance, intestinal infections, diabetes and hepatitis B (Calixto et al., 1998). Phyllanthus muellerianus (Kuntze) Excell is a medicinal plant widespread in the tropical region of West Africa. Commonly called mijiriyar kurumi, ogu azu and nkanga in Nigeria and mbolongo in Cameroon, it has been used as an herbal remedy in many parts of the world. Leaves, twigs and fruits possess antibac-

Corresponding author at: Department of Drug Sciences, University of Pavia, Viale Taramelli 12, Pavia 27100, Italy. Tel.: +39 0382987788; fax: +39 0382422975. E-mail address: gloria.brusotti@unipv.it (G. Brusotti). 0378-8741/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.03.042

terial activity (Breytanbach and Malan, 1989). In Guinea the leaves are boiled with palm fruits and administered to women undergoing labor. In Ghana roots are used for treating chronic dysentery (Fowler, 2006). In Nigeria the plant can be used for the treatment of gastroenteritis, urethritis and wound infections, especially fresh leaves and stem bark (Doughari and Sunday, 2008). The baka pygmies are famous in Cameroon as traditional healers (Betti, 2004); their traditional medicine is an empirical knowledge based on the use of the forest plants for therapeutic applications. In particular, the baka pygmies prepare a water decoction of Phyllanthus muellerianus stem bark which is used as a drink and body lotion, as a remedy for tetanus and wound infections (Brisson, 1999). The micro-organisms which are often associated with these infectious diseases belong to the genus Clostridium. These organisms are present in water, soil, sewage and in the gastrointestinal tract of animals, included humans (Murray et al., 1998).

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As part of our contribution to phytochemical and biological survey and to validation of traditional uses of baka pygmies medicinal plants (Ngueyem et al., 2008) we report herein the study on Phyllanthus muellerianus stem bark antimicrobial activity. In particular, the purpose of this study was to investigate the water extract (WE) for the potential antimicrobial activity against selected available bacterial strains, which may be involved in skin diseases and soft tissue infections. Further bioassay-guided extractions were carried out in methanol (MeOH), with and without defatting treatment in dichloromethane (DCM), in order to obtain the most active extract with the nal aim to identify the chemical classes responsible for the biological activity. 2. Experimental 2.1. Chemicals and reagents HPLC-grade acetonitrile, methanol and dichloromethane were purchased from Carlo Erba (Milan, Italy); water was deionized by ltering through a Direct-Q system (Millipore, Bedford, MA, USA). Phosphate buffered saline (PBS) ampicillin and amphotericin B were purchased from SigmaAldrich S.r.l. (Milan, Italy). 2.2. Plant material The stem bark of Phyllanthus muellerianus was collected in Cameroon in July 2009 in the camps of Abing. The plant was identied at the National Herbarium of Yaound by the Cameroonian botanist Mr. Nana. A voucher specimen (no. BWPV 03) has also been deposited at the Department of Drug Sciences of the University of Pavia. Bark was dried in the dark, in a ventilate room at 2530 C, then grounded and the powder stored at 20 C. 2.3. Extraction procedure According to the traditional use, the rst extraction was carried out in water. The dried powder (100 g) was reuxed in distilled water (700 ml) for 3 h and the crude extract obtained was frozen and lyophilized. Further extractions were performed in methanol (MeOH). 25 g of dried powder were suspended in MeOH (100 ml) in a round bottom ask equipped with a condenser. The mixture was reuxed for 60 min, ltered, re-suspended in fresh MeOH (100 ml) and reuxed for further 60 min. The procedure was repeated for 3 times, the fractions collected and the solvent removed under vacuum. The defatted methanol extract was prepared following the same procedures described above but using dichloromethane (DCM) (3 100 ml) before MeOH (3 100 ml). All dried extracts were stored at 20 C until biological tests. 2.4. Micro-organisms The following strains were used for testing the antimicrobial activity of the crude extracts: Clostridium sporogenes ATCC 3584, Staphylococcus aureus ATCC 6538, Streptococcus mutans ATCC 25175, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 10536, Candida albicans ATCC 10231. Bacteria were cultured in Tryptone Soya Broth (TSB, Oxoid, Basingstoke, UK) at 37 C, under anaerobic atmosphere (80% N2 , 15% CO2 and 5% H2 ) in an anaerobic jar (Oxoid, Basingstoke, UK) for Clostridium sporogenes. The bacteria cultures were centrifuged at 3000 rpm for 20 min to separate cells from broth and then suspended in phosphate buffered saline (PBS, pH 7.3). The suspension was diluted to adjust the number of cells to 1 107 to 1 108 CFU/ml. Candida albicans was grown in Potato Dextrose Broth (PDB) (DIFCO, Detroit, MI, USA) for 24 h at 25 C. The yeast culture was centrifuged at 3000 rpm for 20 min to separate cells from broth and then suspended in PBS (pH 7.3). The

suspension was diluted to adjust the number of cells to 1 107 to 1 108 CFU/ml. 2.5. Evaluation of minimum inhibitory concentration (MIC) and minimum bactericidalfungicidal concentration (MBCMFC) All the extract were dissolved in 10% dimethyl sulfoxide (DMSO) aqueous solution at a concentration of 40 mg/ml. These solutions were used in the determination of the antimicrobial activity against the reference strains. MICs and MBCs were determined by twofold serial broth dilution method in Iso-Sensitest broth (ISB, Oxoid, Basingstoke, UK) according to Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) procedures (NCCLS, 1999). The starting inoculum was 1.0 107 CFU/ml. Concentrations of plant extracts were tested in the range 204000 g/ml. Solvent blanks were included. The MIC was the lowest Phyllanthus muellerianus extract solution concentration inhibiting observable microbial growth after 24 h incubation at 37 C. The MBCMFC was the lowest concentration resulting in >99.9% reduction of the initial inoculum after 24 h incubation at 37 C. All experiments were performed in triplicate (NCCLS, 2003). Stock standard solutions of ampicillin and of amphotericin B were used as a positive control. 2.6. Phytochemical screening A phytochemical screening was performed on all active extracts by thin layer chromatography (TLC, Merck Kieselgel 60 F254 and RP-18 F254 S), according to the procedure described in the TLC atlas Plant Drug Analysis (Wagner et al., 1984) and by using appropriate tests (Chaudhari and Mengi, 2006). 3. Results and discussion The rst extraction of Phyllanthus muellerianus stem bark was carried out in water (water extract, WE). Since, as previously reported (Ngueyem et al., 2008), better results could be achieved using MeOH as solvent, two further extractions were performed in MeOH, with (defatted methanol extract, DME) or without (methanol extract, ME) defatting treatment in dichloromethane. The defatting treatment was made to remove lipidic compounds that may hinder the extraction of bioactive components (especially polyphenols) from vegetable matrices; to avoid any loss of biological activity, the DCM extract was also tested and, as expected, resulted completely inactive (data not shown). The MIC and MBCMFC values against all the tested microorganisms, in comparison with those related to antibiotic control ampicillin and amphotericin B, are reported in Table 1. No activity was found against Staphylococcus aureus, Escherichia coli, Candida albicans. ME and DME showed a good bacteriostatic activity against Streptococcus mutans and Streptococcus pyogenes (MIC values 300 g/mL for both the extract) while WE was still inactive. These results are in contrast with data reported by Doughari and Sunday (2008) since they found MIC values 312.5 g/mL against Staphylococcus aureus and MIC values 1250 g/mL against Streptococcus pyogenes, for both WE and ME extracts. Although it is quite unusual that plants collected from a botanic garden could show higher biological activity than wild growing plants, an hypothesis was made on the results mismatch. Fresh bark could contain active substances that may be partially (even completely) lost during the air drying procedures. Moreover, same species growing in different countries could show different chemical composition and accordingly different biological activity. Experimental data were denitely more interesting for what concerns the activity of Phyllanthus muellerianus stem bark against

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Table 1 MIC (minimum inhibition concentration) and MBC (minimum bactericidal concentration) values of Phyllanthus muellerianus barks water (WE), methanol (ME) and defatted methanol (DME) extracts against Staphylococcus aureus, Streptococcus mutans and Streptococcus pyogenes, Clostridium sporogenes, Escherichia coli and Candida albicans. Bacteria WE ( g/ml) MIC Staphylococcus aureus Streptococcus mutans Streptococcus pyogenes Clostridium sporogenes Escherichia coli Candida albicans
a

ME ( g/ml) MBCMFC >1000 >1000 >1000 >1000 >1000 >1000 MIC >1000 300 200 200 >1000 >1000 MBCMFC >1000 900 500 357.1 >1000 >1000

DME ( g/ml) MIC >1000 300 300 100 >1000 >1000 MBCMFC >1000 900 900 250 >1000 >1000

AmpAmpha ( g/ml) MIC 0.5 0.05 0.02 0.7 5 0.5 MBCMFC 1 0.2 0.1 1.4 10 2

>1000 >1000 >1000 900 >1000 >1000

AmpAmph: ampicillinamphotericin B, antibiotic control.

Clostridium sporogenes (MIC and MBC values of 100 and 255 g/mL, respectively) and so far as we know no data are available on this matter. As reported by many authors, in addition to their etiologic role in tetanus and botulism, Clostridia are best known for their ability to cause myonecrosis or gas gangrene, a bacterial infection (also known as Clostridial myonecrosis), that produces gas within tissues in gangrene and generally occurs at the site of trauma (Raimondi, 1978; De et al., 2003). Furthermore, they can initiate cellulitis or fasciitis, which is a progress destructive process caused by the diffusion of Clostridia through fascial planes (Clostridium perfrigens, Clostridium septicum) (Murray et al., 1998). In particular Clostridium sporogenes is one of the clostridial species that may cause gas gangrene (Miskew et al., 1979; Udgaonkar et al., 1990; Rao et al., 1995; Chaudahry and Dhawan, 1998; Baradkar et al., 1999). These diseases were a major problem during the two world wars, primarily because of inadequate early surgical management of traumatic wounds. Moreover, these organisms are present, besides in the gastrointestinal tract of animals, in water, soil, sewage. Thus, since baka pygmies live in the forest, there is an high probability to suffer from dirty wounds contaminated with Clostridia. Although WE showed a weak activity against Clostridium sporogenes (MIC 900 g/mL), the antibacterial activity showed by ME and DME (Table 1) allows to assume a similar effect on micro-organisms such as Clostridium perfringens, Clostridium tetani and may justify the use of Phyllanthus muellerianus stem bark in the pygmies traditional medicine. Furthermore, the activity found against streptococci is referable with a healing use of these extracts: Streptococcus pyogenes, for example, is an important cause of suppurative and non suppurative diseases like pyoderma, erysipelas, necrotizing fasciitis and other infections (Murray et al., 1998). Finally, it is worthy to note that WE, ME and DME are crude extracts thus the activity could be improved after the opportune purication steps. Thus, following described procedures, all the extracts were analysed on TLC plates (Wagner et al., 1984) and by means of specic colorimetric and gravimetric test (Chaudhari and Mengi, 2006), for a qualitative screening of the main secondary plant metabolites, such as alkaloids, saponins, coumarins, avonoids, carbohydrates, phenolic compounds and tannins. These preliminary phytochemical screening highlighted the presence of polyphenols. Specic spectrophotometric methods were carried out, following a procedure described in our previous work (Brusotti et al., 2010), to further conrm the presence of this chemical family. Polyphenols (PT) were found in all the extracts in different concentration (WE 12.6%, ME 21.8% and DME 23.7%), while tannins were not detected. Based on our experimental results, we could hypothesize that the biological activity found could be ascribed to PT since higher the content of PT lower the MIC value, higher the activity against micro-organisms, especially Clostridium sporogenes. Previously reported literature data (Cowan, 1999; Sampaio et al., 2009; Okoro et al., 2010) may also support the hypothesis of a direct correlation between antimicrobial activity and polyphenols.

4. Conclusion The antimicrobial properties of Phyllanthus muellerianus stem bark extracts have been demonstrated against different microorganisms such as Clostridium sporogenes, Streptococcus mutans and Streptococcus pyogenes. In particular, the interesting activity found against Clostridium sporogenes and Streptococcus pyogenes seems to validate the use of this plant in pygmies traditional medicine for the treatment of tetanus and wound infections. Moreover the activity found against Streptococcus mutans, aetiological agent of caries, may suggest a possible use of this plant, as natural remedy to prevent dental diseases, and underlines other antimicrobial properties of this baka pygmies plant. Although there is very little difference between the activities found for ME and DME extracts, the defatting treatment make DME easier to handle for phytochemical and microbiological analyses. Thus, the defatting treatment with dichloromethane, before extraction with methanol, may be the best methodology for the extraction of these bioactive compounds. Preliminary qualitative phytochemical analyses and colorimetric assays, together with spectrophotometric analyses, suggested the presence of polyphenols as main constituents of the phytocomplex responsible for the biological activity. Further studies are now in progress both to conrm this hypothesis and to verify the potential activity of all the other chemical classes.

Acknowledgement Authors knowledge the technical support of the National Herbarium of Yaound, Cameroon.

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