Amazonian plant crude extract screening for activity against multidrug-resistant bacteria

12 Pages·2008·0.13 MB·English

European Review for Medical and Pharmacological Sciences 2008; 12: 369-380
Amazonian plant crude extract screening for
activity against multidrug-resistant bacteria
A.F. CORREIA1, J.F.O. SEGOVIA2, M.C.A. GONÇALVES3, V.L. DE OLIVEIRA1,
D. SILVEIRA1, J.C.T. CARVALHO3, L.I.B. KANZAKI1
1Laboratory of Molecular Pharmacology, Faculty of Health Sciences, University of Brasília,
Brasilia (DF Brazil)
2The Brazilian Agricultural Research Corporation, Rodovia Juscelino Kubitschek, Fazendinha,
Macapá (AP Brazil)
3Laboratory of Drugs, Federal University of Amapá Campus Universitário Marco Zero do Equador,
Jardim Marco Zero, Macapá (AP Brasil)
Abstract. – Antimicrobial resistance is a Introduction
subject of great concern in public health and
also in the designing of strategies for current
therapeutic protocols all over the world. New
The species Calophyllum brasiliense (Clusi-
drugs, including those necessary for a reserve
aceae/Guttiferae) also known in the tupi lan-
armamentarium and exhibiting less side ef-
guage as “jacareúba”, is popularly employed in
fects deserve special attention. In rural areas,
particularly in Brazil, a huge number of natural the treatment of different clinical conditions. The
products, in different artisanal preparations, micromolecular composition analysis of extracts
mainly from plants, have been used by tradi- from different parts of this plant revealed the
tional populations to cure diseases. Despite presence of fatty acids, xanthones, cumarins,
some of these plants have been studied, many
flavonoids and triterpens1-7 exhibiting activity
of them are awaiting to have their compounds
against fungus and gram positive bacteria8, in-
chemically characterized and investigated
cluding Bacillus cereus, Bacillus subtilis, Staphy-
their pharmacodynamics properties. Further,
as well known, the environment plays a cru- lococcus epidermidis and Staphylococcus
cial role in the metabolism of these plants, aureus7-10. Isolated compounds, mainly cumarins,
yielding different and varied molecular com- exerted antiviral3,11 and significative molluscicide
plexes depending on the period of collection,
activity against Biomphalaria glabrata5. Three
climate conditions, kind of soil and also the
xanthones, jacareubin, 6-deoxyjacareubin, and
plant speciation.
1,3,5,6-tetrahydroxy-2-(3-methyl-2-butenyl) xan-
In this report, ethanol crude extract of 10
different botanical specimens from the Ama- thone showed trypanocidal activity12. The
zon region of Brazil, in the Amapa State, were coumarin-type mammea purified from a
screened for antibacterial activity of 7 clinical dichloromethane crude extract of leaves of Calo-
resistant microorganisms utilizing as control phyllum brasiliense potently inhibited the growth
ATCC bacterial species by the Kirby-Bauer
of Leishmania amazonensis and caused impor-
method. Plant extracts of Geissospermum ar-
tant changes in the parasite’s ultrastructure13. The
genteum, Uncaria guianensis, Brosimum acuti-
natural product GUT-70, characterized as a tri-
folium, Copaifera reticulate, Licania macro-
phylla, Ptycopetalum olacoides and Dalbergia cyclic coumarin, 5-methoxy-2,2-dimethyl-6-(2-
subcymosa yielded activity against Staphylo- methyl-1-oxo-2-butenyl)-10-propyl-2H,8H-ben-
coccus aureus and Pseudomonas aeruginosa, zo[1,2-b;3,4-b]dipyran-8-one (C23H26O5), iso-
both multidrug resistant, and Staphylococcus
lated from the stem bark of Calophyllum
aureus ATCC strain.
brasiliense, significantly inhibited the growth of
leukemic cells14.
Key Words:
There are few studies related to Geissosper-
Plant crude extract, Biological activity, Antimicro-
mum argenteum (Apocynaceae), also known in
bial, Amapa State, Amazon rain forest.
northern Brazil as “quinarana da fruta pequena”,
Corresponding Author: Luis Isamu Barros Kanzaki, D.Sc.; e-mail: [email protected] 369
A.F. Correia, J.F.O. Segovia, M.C.A. Gonçalves, V.L. de Oliveira, et al.
which is mainly used as antimalarial remedy al- Known by the Amazonian natives as “mururé”
together with other Geissospermum genera like- or “muirapuranga”, the Brosimum acutifolium
wise Geissospermum sericeum and Geissosper- (Moraceae), is a timber species indigenously uti-
mum leave by traditional communities in the lized as anti-inflammatory, anti-rheumatic42and an-
Amazon region of Brazil and Andes, being able ti-anemic natural medicine43. Its chemical composi-
to impair the intrahepatic cycle of Plasmodium tion is diversified, mainly presenting flavonoids and
falciparum15. The indole alkaloid geissoschizo- lignoids44-50, as well an hallucinogen amine50. From
line and two new derivatives, geissoschizoline N- the bark of this species some isolated flavones and
oxide and 1,2-dehydrogeissoschizoline, were ob- flavolignanes compounds exhibited cytotoxic ef-
tained from the bark of Geissospermum sericeum fects against a vincristin resistant P388 leukemic
together with the beta-carboline alkaloid flavop- cell line50 and antienzymatic activity for A and C
ereirine. In spite of antimalarial activity, antitu- proteinkinase respectively49. An identified
moral and antioxidant activities have also been flavonoid exerted dose-dependent activity on
reported16. pheochromocytoma PC-12 cell line50.
The Uncaria guianensis (Rubiaceae-Cop- As a vernacular term “copaíba”, the species
tosapelteae) also known in the amazon region as Copaifera reticulata (Caesalpinaceae) is report-
“unha-de-gato” or cat’s claw and “jupindá”, is ed since the earliest period of Portuguese colo-
commonly used as a popular anti-inflammatory nization of Brazil, as a medicinal oil, used as an-
remedy as already scientifically corroborat- ti-inflammatory and for its cicatricial activity52-55.
ed17,18,21,22 and in some assays the plant is more Maciel et al53 emphasizes the use of copaiba oil
potent than Uncaria tomentosa, which is consid- in the popular medicine as stimulant, diuretic,
ered the real cat’s claw. Herrera et al19. observed laxative, cicatricial, expectorating, anti-tethanic,
the gastrointestinal anti-inflammatory effects of antihemorrhagic, antirheumatic, anti-inflammato-
plant extracts. The aqueous extracts did not ex- ry, anti-ulcerogenic activities and as antiseptic of
hibit cytotoxic activity but showed antimuta- woman urinary tract. Also, it is indigenously
genic and photomutagenic activity induced by used in the treatment of bronchitis, syphilitic dis-
8-metoxypsoralen and UV radiation in Salmo- eases, skin diseases, leishmaniasis, leukorrhea,
nella spp. The indole alkaloids obtained from psoriasis, diarrhea, urticaria and lung diseases53.
these plant extracts presented immunostimulant The copaiba oil was evaluated for its anti-inflam-
and immunoregulatory activity20-22. They also matory and cicatricial effects in oophorectomized
yielded DNA repair action by reducing skin ep- rats and yielded superior results when compared
ithelial cell death, which points out to formulat- to control animals56,57. Also, it presented larvici-
ing potential sources of solar protectors23. In ref- dal activity against Culex quinquefasciatus in all
erence to Uncaria guianensis chemical compo- larval stages58. Aqueous extracts obtained from
sition, a variety of triterpenic glycosides, oxyn- bark of Copaifera reticulata exhibited high an-
dolic alkaloids and phenolic components were tioxidant and free radical neutralization activity,
isolated20,21,24-29. lately attributed to tannins59-61.
The Simaba cedron (sinon. Quassia cedron), The “anauera”, an indigenous Amazonian de-
popularly known in northern Brazil as “pau-de- nomination for Licania macrophylla (Chrysobal-
gafanhoto” and “pau-para-tudo” is employed as anaceae), besides its medicinal utilization by tra-
antimalarial medicine. It is rich in quasinoids30-36, ditional populations in northern Brazil, it is also
and some of them present cytotoxic activity37,38. employed as timber for construction of houses
Cedronin and cedrin, isolated from Simaba ce- and boats62. There are not reports related to the
dron seeds, exhibited antimalarial activity in vi- biological activity and chemical composition of
vo and in vitro against cloroquine resistant and this species, eventhough some other members of
susceptible strains37-39. Cedronin also presented the family Chrysobalanaceae have been studied
cytotoxic activity in human cervical carcinoma concerning their medicinal applications and
KB cells37. In addition, other compounds ob- chemical composition63-65. Among some
tained from the seeds yielded anti-inflammatory amerindian communities in the Amapa, Brazil,
activity40. The methanolic extracts of Simaba “anauera” is employed to treat diseases common-
cedron seeds exerted ovicidal and larvicide ac- ly caused by protozoans as giardiasis and amebi-
tivity in species of the phytoparasite Tuta abso- asis (personal communication).
luta, which represents a potential natural insec- The Ptycopetalum olacoides (Olacaceae)
ticide41. named by the amerindians as “muirapuama” has
370
Amazonian plant crude extract screening for activity against multidrug-resistant bacteria
its root medicinally indicated for stimulant, en- sides its popular use as balsamic, fluidificant and
ergetic and aphrodisiac activity. Their roots have expectorant, astringent, anti-diarrheial, anti my-
been exported for more than 100 years to Europe cotic, and anti-inflammatory. The essential oil is
leading to the partial extinction of this plant66-68. predominantly composed of α-pinene and
Their prices usually reach twice of other medici- limonene93. One of the most important com-
nal plants69. It is the component of a commercial pound of this species is the xyloglucan, isolated
medicinal beverage, “catuama”, which mixed to from many organs94-106. Also, diterpenoids107-111,
other herbs induce the relaxation of cavernosal sesquiterpenoids112, fatty acids and phenolic
smooth muscle of rabbits in a dose-dependent compounds113,114, with special emphasis to proan-
manner70. The vasorelaxation activity was also thocyanidins115were detected in this plant.
observed in isolated organs of other species, The “jatobá” resin presented toxic effect and
confirming the usefulness of the ethanolic prepa- inhibitted appetite in Spodoptera exígua116. The
ration in cardiovascular diseases71. In clinical tri- hydroalcoholic extract prepared from the bark’s
als of healthy volunteers aiming to evaluate the plant exerted activity against Staphylococcus au-
effects of Ptycopetalum olacoides, the hemato- reus117 and Bacillus cereus118. Some diterpenes
logical and biochemical profile were not signifi- isolated from “jatobá” leaves exhibited marked
cantly altered as also there were not adverse ac- fungicide activity119. The plant extracts also pre-
tions on the subjects72. Also, the ethanolic ex- sented in vitro anti-plasmodium120 and mollusci-
tract exerted many effects on the central nervous cidal activity121. The plant pericarp presented an-
system73,74, including acethylcholinesterase inhi- ti-inflammatory activity, 5-lipooxigenase and
bition, cognitive improvement and ansiolythic tirokinase inhibitory action122 and skin inhibitory
activity in rats compared to that observed with pigmentation induced by UV radiation123.
pentylenetetrazol and also, diminished mobility
similarly to the effect of clonidine. The neuro-
protector activity was assessed in the cells of
rats’ hypocampus deprived of oxygen and glu- Materials and Methods
cose, which showed more viability than in the
controls. Besides these actions, antioxidant, anti- Botanical Material
free radicals activity and fungicide properties From Amapa State, Brazil, barks and resin of
were observed in Colletotrichum acutatum and 10 different plants, were collected. Voucher spec-
Fusarium oxysporum75-82. Triterpenic and imens were deposited at the herbarium of the In-
steroidal compounds, flavonoids, methylxanthin stitute of Scientific and Technological Research
and alkaloids were isolated from the plant. The of the Amapa State (IEPA), Brazil, under the
α-pinene represents the principal component of protocols numbers 016603 for Uncaria guianen-
the essential oil83-89. sis, 016595 for Copaifera reticulate, 016602 for
The Dalbergia subcymosa (Fabaceae-Papil- Simaba cedron, 016594 for Licania macrophylla
ionoideae), named by the amazonian indigenous and 016600 for Dalbergia subcymosa. The
population as “verônica da terra-firme”, is popu- species Calophyllum brasiliense, Geissospermum
larly indicated for the treatment of anemia90. Its argenteum, Brosimum acutifolium, Ptycopetalum
activity in the treatment of anemia was corrobo- olacoides and Hymenaea coubaril are provision-
rated by Silva et al91 detecting significant levels ally kept under registration numbers BRM1,
of soluble iron in a concentration of 4.15 p.p.m. BRM2, BRM5, BRM8 and BRM10 respectively,
In order to evaluate the safety use of Dalbergia at the Brazilian Agricultural Research Corpora-
subcymosa in pregnant women, Peters and Guer- tion, Amapá, Brazil (EMBRAPA).
ra92 demonstrated in rats, the absence of embryo-
fetotoxicity and no disturbance of postnatal de- Bacteria Strains
velopment of the pups indicating that the bever- Standard American Type Culture Collection
age may be safe for human use as an anti-inflam- strains (ATCC) of Proteus mirabilis (ATCC
matory medicine. There are not reports referring 7022), Klebsiella pneumoniae (ATCC 27853),
to the chemical composition of Dalbergia subcy- Pseudomonas aeruginosa (ATCC 27853), Es-
mosa. cherichia coli (ATCC 25922), Staphylococcus
The Hymenaea coubaril (Fabaceae), named aureus (ATCC 29213), Enterococcus faecalis
by the amazonian indigenous people as “jatobá” (ATCC 29212), Streptococcus pneumoniae
and “jutai”, is utilized by the timber industry be- (ATCC 46619), and also the same species ob-
371
A.F. Correia, J.F.O. Segovia, M.C.A. Gonçalves, V.L. de Oliveira, et al.
tained from hospital collection of multidrug re- cania macrophylla, Ptycopetalum olacoides and
sistant strains in Brasilia, Brazil, were utilized in Dalbergia subcymosa yielded activity against
the Kirby-Bauer assay124. Staphylococcus aureus and Pseudomonas aerug-
inosa, both multidrug resistant, and Staphylococ-
Extraction Procedures cus aureus ATCC strain (Tables I and II). The
The dried ground barks of each plant and resin minimal inhibitory concentration ranged from
of Copaifera reticulatawere weighed and soaked 80.0 µg to 1.25 µg of crude extract in the filter
in ethanol and left for 7 days in maceration at disc, exhibiting the Brosimum acutifolium ex-
room temperature and repeated 3 times after fil- tract, the most strong activity against the mul-
tration. Solvent evaporation was performed in a tidrug resistant Staphylococcus aureus. Except
rotary evaporator under reduced pressure and the for Dalbergia subcymosa, all the other plants ex-
resulting solute was completely dried in a water tracts as in the assay for Staphylococcus aureus
bath at 40oC. The ethanol crude extracts were multidrug resistant, presented activity against
kept frozen until their use. Staphylococcus aureus ATCC strain, even at less
low concentration, ranging from 80.0 µg to 1.25
The embedded Disc Assay µg. Against the multidrug resistant Pseudomonas
Sterile filter discs (Hartman paper no. 1) mea- aeruginosa, the ethanol crude extract of Geis-
suring 6 mm in diameter were embedded in each sospermum argentumwas the solely one to show
ethanol crude extract in concentrations ranging antibacterial activity, in a 80.0 microg concen-
from 80 µg to 1.25 µg and allowed to dry at room tration. In all essays, the positive control repre-
temperature. Frozen stocks of bacterial growth sented by discs embedded in G sodic penicillin
kept in glycerol were grown in semi solid nutri- yielded inhibitory halo measuring from 32 to 13
ent agar and kept in incubator for 24 hs at 37°C. mm, depending on the microorganism being
Typical and homogeneous colonies were trans- tested. Also, the negative control, immersed in
ferred to liquid Mueller-Hinton medium to grow ethanol and dried, did no exhibit any inhibitory
until reaching 2 × 108 cfu/mL (0.5 units in the effect on the bacterial growth.
McFarland scale). Inoculum at this density
growth was spread out in Petri dishes containing
Mueller-Hinton agar. Sequentially, previously
embedded and dried discs in ethanol crude ex- Discussion
tracts were disposed on the agar surface. As con-
trols, dried discs embedded in G sodic penicillin
and ethanol were also displaced in the dishes. All The initial concentration of 80.0 µg of plant
essays were carried out in triplicate, including crude extract in the discs is justified by the
the controls. The dishes were kept in incubator at fact that the complex mixture of molecules
37°C for 24 hours. The formation of halo inhibi- should be in different amounts, therefore the
tion ray of bacterial colony growth was measured highest the initial concentration most probably
and compared around the discs embedded in the detection of any biological activity. Se-
crude extracts and control disc containing G sod- quentially, serial dilution of the initial concen-
ic penicillin. This essay is based on the Kirby- tration allowed us to determine the minimum
Bauer method125. inhibitory concentration exhibiting antimicro-
The experiments were carried out in two steps. bial activity.
The extracts were initially in 80 µg concentration Eventhough standard known antibiotics em-
in the discs and those presenting inhibitory activ- bedded in the discs were used as control, we
ity over bacterial growth were diluted to reach could not compare crude extract activity to the
the minimal inhibitory concentration. one displayed by the utilized antibiotics as it is
not known which molecule or molecules exerted
antibacterial activity either which mechanism is
under way. Therefore, the antibiotic inhibitory
Results activity assess the sensibility of microorganisms
to the drugs rather than establishing any chemical
Ethanol diluted crude plant extracts of Geis- and mechanism comparison between known an-
sospermum argenteum, Uncaria guianensis, tibiotics used in the assay and unknown crude
Brosimum acutifolium, Copaifera reticulate, Li- extracts molecules.
372
Amazonian plant crude extract screening for activity against multidrug-resistant bacteria
use
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The minimum inable I. Plant crude extract Uncaria guianensisCopaifera reticulataSimaba cedronLicania macrophyllaDalbergia subcymosaCalophyllum. brasiliensGeissospermum argenteBrosimum acutifoliumPtycopetalum olacoidesHymenaea coubaril
T
373
A.F. Correia, J.F.O. Segovia, M.C.A. Gonçalves, V.L. de Oliveira, et al.
use
ca yyyyyyyyyy
ococmoni ctivitctivitctivitctivitctivitctivitctivitctivitctivitctivit
reptneu No aNo aNo aNo aNo aNo aNo aNo aNo aNo a
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xtracts by the ains hylococcus aureus 2.5 µg2.5 µg o activity40.0 µg20.0 µg o activity5 µg1.25 µg40 µg o activity
xerted by plant crude e sistant bacterial str scherichiaStapcoli No activityNo activityNo activityNNo activityNo activityNo activityNNo activity≥No activityNo activityNo activityN
e e E
ns g r
ai u
esistant str Multi-Dr omonasginosa ctivityctivityctivityctivityctivityctivityctivityctivityctivityctivity
drug r seudaeru No aNo aNo aNo aNo aNo aNo aNo aNo aNo a
ulti- P
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erial growth of Klebsiellaneumoniae No activityNo activityNo activityNo activityNo activityNo activityNo activityNo activityNo activityNo activity
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ncentration Proteusmirabilis No activityNo activityNo activityNo activityNo activityNo activityNo activityNo activityNo activityNo activity
o
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The minimum inhibitable II. Plant crude extract Uncaria guianensisCopaifera reticulataSimaba cedronLicania macrophyllaDalbergia subcymosaCalophyllum. brasilienseGeissospermum argenteumBrosimum acutifoliumPtycopetalum olacoidesHymenaea coubaril
T
374
Amazonian plant crude extract screening for activity against multidrug-resistant bacteria
Preliminary screening for biological activity of The promising results presented here push us
plant crude extract is the first step in order to de- to initiate studies of chemical characterization of
cide the priorities to carry out chemical and bio- extracts exhibiting antibacterial activity and to
logical assays with promising botanical species. understand the mechanisms involved in their bio-
Development of new drugs, particularly microbi- logical activity. Also, we are presently carrying
cides are mandatory. The uncontrolled use of an- out in vitro screenings to assess antineoplastic
timicrobials, mainly in poor and developing coun- and antiviral activity, mainly against retroviruses,
tries, spread out multidrug resistant microorgan- involved in both neoplasia and immunodeficien-
isms all over the world, mainly carried by human cy pathologies respectively. Our initial studies
subjects and possibly by animal food products. utilizing ethanol extracts will be extended to oth-
Despite estimating the existence of 125,000 er solvents, presenting different polarity, allow-
plants in the Amazon rain forest, a minimum ing us to extract compounds in a wide spectrum
number of them, about 1,250 species have been of molecular composition.
in most of the cases, partially investigated125. In
our research work, all of these plants are current-
ly been utilized to cure a variety of illness by
Amazonian communities and, all of them have References
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