Upload
arun-kp
View
219
Download
0
Embed Size (px)
Citation preview
7/31/2019 Antimicrobial Phyllanthus Spp
1/4
Journal of Ethnopharmacology 135 (2011) 797800
Contents lists available at ScienceDirect
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: [email protected] (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:[email protected]://dx.doi.org/10.1016/j.jep.2011.03.042http://dx.doi.org/10.1016/j.jep.2011.03.042mailto:[email protected]://www.elsevier.com/locate/jethpharmhttp://www.sciencedirect.com/science/journal/03788741http://dx.doi.org/10.1016/j.jep.2011.03.0427/31/2019 Antimicrobial Phyllanthus Spp
2/4
798 G. Brusotti et al. / Journal of Ethnopharmacology 135 (2011) 797800
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
7/31/2019 Antimicrobial Phyllanthus Spp
3/4
G. Brusotti et al. / Journal of Ethnopharmacology 135 (2011) 797800 799
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.
References
Baradkar,V.P., Patwardhan, N.S.,Deshmukh, A.B.,Damle,A.S., Karyakarte, R.P.,1999.Bacteriologicalstudyof clinicallysuspected casesof gasgangrene. Indian Journalof Medical Microbiology 17, 133134.
Betti, J.L., 2004. An ethnobotanical study of medicinal plants among the baka pyg-mies in the DJA biosphere reserve, Cameroon. African Study Monographs 25,127.
Breytanbach, J.C., Malan, S.F., 1989. Pharmaco-chemical properties of Cambretumzeyhari. South African Journal of Science 85, 372374.
Brisson, R., 1999. Etudes Pygmes. SELAF n 376. Peeters, Paris.Brusotti, G., Ngueyem, T.A., Biesuz, R., Caccialanza, G., 2010. Optimum extraction
process of polyphenols from Bridelia grandis stem bark using experimentaldesign. Journal of Separation Science 33, 16921697.
Calixto, J.B., Santos, A.R.S., Filho,V.C.,Yunes, R.A., 1998. A reviewof theplants of thegenus Phyllanthus: their chemistry, pharmacology, and therapeutic potential.Medicinal Research Reviews 18, 225258.
Chaudahry, R., Dhawan, B., 1998. Gas gangrene and related infections ina tertiary care hospital. Indian Journal of Medical Microbiology 16,165168.
Chaudhari, M., Mengi, S., 2006.Evaluationof phytoconstituents ofTerminalia arjunafor wound healing activity in rats. Phytotherapy Research 20, 799805.
Cowan, M.M., 1999. Plant products as antimicrobial agents. Clinical Microbiology
Reviews 12, 564582.
7/31/2019 Antimicrobial Phyllanthus Spp
4/4
800 G. Brusotti et al. / Journal of Ethnopharmacology 135 (2011) 797800
De, A., Varaiya, A., Mathur, M., Bhesania, A., 2003. Bacteriological studies of gasgangrene and related infections. Indian Journal of Medical Microbiology 21,202204.
Doughari, J.H., Sunday, D., 2008. Antibacterial activity of Phyllanthus muellerianus.Pharmaceutical Biology 46, 400405.
Fowler, D.G., 2006. Traditional fever remedies: a list of Zambian plants. Fowler,D.G, Available from: http://www.giftshealth.org/ritam/news/traditionalfever remedies1.pdf.
Miskew, D.B., Pinzur, M.S., Pankovich, A.M., 1979. Clostridial myonecrosis in apatient undergoingoxacillintherapy for exacerbation of chronic footulcers andosteomyelitis. A case report. Clinical Orthopaedics and Related Research 138,
250253.Murray, R.P., Rosenthal, K.S., Kobayashi, S.G., Pfaller, A.M., 1998. Medical Microbiol-
ogy, vol. 37. Mosby, St. Louis, MO, pp. 296298.NCCLS, 1999. Methods for Determining Bactericidal Activity on Antimi-
crobial Agents. Approved Guideline, vol. 19. NCCLS, Wayne, PA,USA.
NCCLS,2003. Methods fordilutionantimicrobial susceptibilitytests forbacteriathatgrow aerobically. Approved standard M7-A6, 6th edition. NCCLS, Wayne, PA,USA.
Ngueyem, T.A., Brusotti, G., Marrubini, G., Grisoli, P., Dacarro, C., Vidari, G., VitaFinzi, P., Caccialanza, G., 2008. Validation of use of a traditional remedy fromBridelia grandis (Pierre ex Hutch) stem bark against oral Streptococci. Journal ofEthnopharmacology 120, 1316.
Okoro, I.O., Osagie, A., Asibor, E.O., 2010. Antioxidant and antimicrobial activities ofpolyphenols from ethnomedicinal plants of Nigeria. African Journal of Biotech-nology 9, 29892993.
Raimondi, P.J., 1978. The Clostridium syndromes. The Western Journal of Medicine129, 114118.
Rao, S.R.,Natarajan, M.K., Ramesh, I.,1995.An eightyear bacteriologicalstudyof gasgangrene in Pondicherry. Indian Journal of Medical Microbiology 13, 151154.
Sampaio, F.C., do Socorro, V., Pereira, M., Celidarque Dias, S., Costa, V.C.O., Conde,N., Buzalaf, A.R.M., 2009. In vitro antimicrobial activity of Caesalpinia fer-rea Martius fruits against oral pathogens. Journal of Ethnopharmacology 124,289294.
Udgaonkar, U.S., Dharmadhikari, C.A., Kulkarni, R.D., Kulkarni, V., Pawar, S.G., 1990.Clinicobacteriological study of gas gangrene. The Journal of the Indian MedicalAssociation 88, 810.
Wagner, H., Bladt, S., Zgainsk, E.M., 1984. Plant Drug Analysis. A Thin Layer Chro-matography Atlas. Springer-Verlag, Berlin.