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Journal of Ethnopharmacology 135 (2011) 797800

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Journal of Ethnopharmacology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j e t h p h a r m

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 Department of Drug Sciences, University of Pavia, Pavia, Italyb 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

Article history:

Received 21 December 2010Received in revised form 25 February 2011

Accepted 18 March 2011

Available online 4 April 2011

Keywords:

Phyllanthus muellerianus Bark extract

Ethnomedicine

Antibacterial activity

a b s t r a c t

Ethnopharmacological relevance: The plants of the genus Phyllanthus (Euphorbiaceae) are widely dis-

tributed in most tropical and subtropical countries, and have long been used in folk medicine to treat

several diseases. Particularly, Phyllanthus muellerianus (Kuntze) Excell, commonly calledmbolongo 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 pre-

pared and their activity against Clostridium sporogenes ATCC 3584, Staphylococcus aureus ATCC 6538,

Streptococcus mutans ATCC 25175, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 10536, Can-

dida 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 900g/mL) and

resulted inactive at the testedconcentrationsagainst all theother microorganisms.The defatted methanol

extract, inactive against Staphylococcus aureus, Escherichia coli, Candida albicans, exhibited a very inter-

estingactivity againstClostridium sporogenes and Streptococcus pyogenes (MIC 100g/mL and300g/mL,

respectively), which seems to validate the use of this plant in pygmies traditional medicine for the treat-ment of tetanus and wound infections. The activity found against Streptococcus mutans (300g/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 ofPhyllanthus muellerianus, traditionally used by baka pygmies to treat wound infections

and tetanus, is reported for the first time.

2011 Elsevier Ireland Ltd. All rights reserved.

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 kidneyand urinary bladderdisturbance, 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-

Correspondingauthorat: Department ofDrug Sciences,Universityof Pavia,Viale

Taramelli 12, Pavia 27100, Italy. Tel.: +39 0382987788; fax: +39 0382422975.

E-mail address: gloria.brusotti@unipv.it (G. Brusotti).

terial activity (Breytanbach and Malan, 1989). In Guinea the leaves

are boiled with palm fruits and administered to women undergo-

ing 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 freshleaves and stem bark (Doughari and Sunday, 2008). The baka pyg-

mies 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 particu-

lar, 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 infec-

tious 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).

0378-8741/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.jep.2011.03.042

http://dx.doi.org/10.1016/j.jep.2011.03.042http://dx.doi.org/10.1016/j.jep.2011.03.042http://www.sciencedirect.com/science/journal/03788741http://www.elsevier.com/locate/jethpharmmailto:gloria.brusotti@unipv.ithttp://dx.doi.org/10.1016/j.jep.2011.03.042http://dx.doi.org/10.1016/j.jep.2011.03.042mailto:gloria.brusotti@unipv.ithttp://www.elsevier.com/locate/jethpharmhttp://www.sciencedirect.com/science/journal/03788741http://dx.doi.org/10.1016/j.jep.2011.03.042

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As part of our contribution to phytochemicaland biological sur-

vey and to validation of traditional uses of baka pygmies medicinal

plants (Ngueyem et al., 2008) we report herein the study on Phyl-

lanthus 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 extrac-

tions were carried out in methanol (MeOH), with and without

defatting treatment in dichloromethane (DCM), in order to obtain

the most active extract with the final 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

filtering 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 iden-

tified at the National Herbarium of Yaound by the Cameroonian

botanistMr. 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 first extraction was carried

out in water. The dried powder (100g) was refluxed in distilled

water (700 ml) for 3 h and the crude extract obtained was frozenand lyophilized. Further extractions were performed in methanol

(MeOH). 25g of dried powderwere suspendedin MeOH (100 ml)in

a round bottom flask equipped with a condenser. The mixture was

refluxed for 60 min, filtered, re-suspended in fresh MeOH (100 ml)

and refluxed 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) (3100ml) before MeOH (3100ml). All dried extracts

were stored at 20 C until biological tests.

2.4. Micro-organisms

The following strains were used for testing the antimicro-bial 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 bacte-

ria 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 1107 to 1108 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

1108 CFU/ml.

2.5. Evaluation of minimum inhibitory concentration (MIC) and

minimum bactericidalfungicidal concentration (MBCMFC)

All the extract weredissolvedin 10% dimethylsulfoxide(DMSO)

aqueous solution at a concentration of 40 mg/ml. These solutions

wereused in the determinationof the antimicrobial activityagainstthe reference strains.

MICs and MBCs were determined by twofold serial broth dilu-

tion method in Iso-Sensitest broth (ISB, Oxoid, Basingstoke, UK)

according to Clinical and Laboratory Standards Institute (CLSI; for-

merlyNCCLS)procedures (NCCLS, 1999). Thestartinginoculum was

1.0107 CFU/ml. Concentrations of plant extracts were tested in

the range 204000g/ml. Solvent blanks were included.

The MIC was the lowest Phyllanthus muellerianus extract solu-

tion concentration inhibiting observable microbial growth after

24 h incubation at 37 C. The MBCMFC was the lowest concen-

tration resulting in >99.9% reduction of the initial inoculum after

24 h incubation at 37 C. All experiments were performed in trip-

licate (NCCLS, 2003). Stock standard solutions of ampicillin and of

amphotericin B were used as a positive control.

2.6. Phytochemical screening

A phytochemical screeningwas performed on all active extracts

by thin layer chromatography (TLC, Merck Kieselgel 60 F254 and

RP-18 F254 S), accordingto 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 first 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 achievedusing 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 bio-

logical 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 micro-

organisms, 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, Can-

dida albicans. ME and DME showed a good bacteriostatic activity

against Streptococcus mutans and Streptococcus pyogenes (MIC val-ues 300g/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 312.5g/mL againstStaphylococcus aureus and MIC values 1250g/mL against Strepto-

coccus 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 hypothe-

sis was made on the results mismatch. Fresh bark could contain

active substances that may be partially (even completely) lost dur-

ing the air drying procedures. Moreover, same species growing in

different countries could show different chemical composition and

accordingly different biological activity.

Experimental data were definitely more interesting for what

concerns the activity ofPhyllanthus muellerianus stem bark against

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Table 1

MIC (minimum inhibition concentration) and MBC (minimum bactericidal concentration) values ofPhyllanthus 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) ME (g/ml) DME (g/ml) AmpAmpha (g/ml)

MIC MBCMFC MIC MBCMFC MIC MBCMFC MIC MBCMFC

Staphylococcus aureus >1000 >1000 >1000 >1000 >1000 >1000 0.5 1

Streptococcus mutans >1000 >1000 300 900 300 900 0.05 0.2

Streptococcus pyogenes >1000 >1000 200 500 300 900 0.02 0.1

Clostridium sporogenes 900 >1000 200 357.1 100 250 0.7 1.4

Escherichia coli >1000 >1000 >1000 >1000 >1000 >1000 5 10

Candida albicans >1000 >1000 >1000 >1000 >1000 >1000 0.5 2

a AmpAmph: ampicillinamphotericin B, antibiotic control.

Clostridium sporogenes (MICand MBC values of 100 and 255g/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 abil-

ity to cause myonecrosis or gas gangrene, a bacterial infection

(alsoknown as Clostridial myonecrosis), thatproduces 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, whichis a progress destructive process caused

by the diffusion of Clostridia through fascial planes (Clostridiumperfrigens, Clostridium septicum) (Murray et al., 1998). In partic-

ular 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 Clostrid-

ium sporogenes (MIC 900g/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 tetaniand may justify the use of Phyllanthus muellerianus stem bark in

the pygmies traditional medicine. Furthermore, the activity found

againststreptococciis referablewith a healinguse of these extracts:

Streptococcus pyogenes, for example, is an important cause of sup-

purative 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

purification steps.

Thus,following describedprocedures,all the extracts wereanal-

ysed on TLC plates (Wagner et al., 1984) and by means of specific

colorimetric and gravimetric test (Chaudhari and Mengi, 2006), for

a qualitative screening of the main secondary plant metabolites,

such as alkaloids, saponins, coumarins, flavonoids, carbohydrates,phenolic compounds and tannins. These preliminary phytochem-

ical screening highlighted the presence of polyphenols. Specific

spectrophotometric methods were carried out, following a pro-

cedure described in our previous work (Brusotti et al., 2010), to

further confirm the presence of this chemical family. Polyphe-

nols (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 hypothe-

size 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. Previ-

ously 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 micro-

organisms suchas 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 apossible use of this plant, as natural remedy to prevent dental dis-

eases, and underlines other antimicrobial properties of this baka

pygmies plant.

Although there is very little difference between the activities

found forME and DME extracts,the defattingtreatment make DME

easier to handle for phytochemical and microbiological analyses.

Thus,the defattingtreatment withdichloromethane, before extrac-

tionwith methanol, maybe the bestmethodologyfor theextraction

of these bioactive compounds.

Preliminary qualitative phytochemical analyses and colorimet-

ric assays, together with spectrophotometric analyses, suggested

the presence of polyphenols as mainconstituents of the phytocom-

plex responsible for the biological activity.

Further studies arenow in progressboth to confirm this hypoth-esis 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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