Journal of Efhnopharmacology,

Elsevier Scientific

Publishers

35

( 199 I ) 165 17 I

Ireland

165

Ltd.

Screening of plants used in south Brazilian folk medicine

C.B. Alice, V.M.F. Vargas, G.A.A.B. Silva, N.C.S. de Siqueira, E.E.S. Schapoval,
J. Gleyed, J.A.P. Henriquesb and A.T. Henriques
UCurso Pos GraduaGao, Facultade de Fat-maria. Universidade Federal do Rio Grande do Sul, Porro Alegre, hDepartamento de
Fisiologia, Farmacologia y Biofisica, Institute de Biociencias, Universidade Federal do Rio Grande do Sul. Porro Alegre, Secretaria
da Saude e do Meio Ambienre, Deparramento do Meio Ambiente, Porro Alegre (Bra:il) and FaculrP des Sciences Pharmaceutiques.
UniversU Toulouse 111, 31 All&es Jules Guesde. F 31047 Toulouse (France)
(Accepted

Thirty-seven

species of medicinal

plants

used in folk phytotherapy

anthraquinones,
saponins, sterols and/or triterpenes
activity using the Ames test (Salmonellalmicrosome).
Key words: folk medicine;

south

August

Brazil; medicinal

and tannins.

plants;

27, 1991)

were chemically

mutagenic

The plant kingdom constitutes a source of new

chemical compounds which may be important due
to their potential use in medicine or for their other
biological properties. The growing interest in natural products obtained from Brazilian plants is responsible for this phytochemical survey, and this
paper, particularly, deals with certain species
found in the southern part of Brazil. It is a continuation of a previous study (Alice and Silva,
1985). Plants of the Apocynaceae, Lauraceae and
also the Compositae are not fully represented here
because they are the subject of more in-depth
chemical investigation.
Many commercially sold medicinal plants contain chemical substances known to be mutagenic
and/or carcinogenic (Bjeldanes and Chang, 1977;
Brown and Dietrich, 1979; Weisburger, 1979;
Goggelmann and Schimmer, 1986) and their constant use has been correlated with the occurence of

Ireland

coumarins,

for the presence

flavonoids,
of mutagenic

activity

Materials and Methods

Plant material

Plants were acquired in south Brazil (Rio

Grande do Sul). Some of these were collected from
their natural habitat, while others were purchased
from herb shops. Each species was authenticated
by comparison
with identified
herbarium
specimens and a voucher was deposited in the
Department of Botany, Universidade Federal do
Rio Grande do Sul.
Extract preparation

The shade-dried plants were milled into a tine

powder. Extracts were prepared by refluxing
about 30-35 g of each sample with 100 ml of 90%
ethanol. After 1 h they were filtered while hot. The

Correspondence to: J. Gleye. Faculte des Sciences Pharmaceutiques. hiversitt

Toulouse III. 31 Allees Jules Guesde. F 31047
Toulouse, France.

Publishers

for alkaloids,

diseases and tumors in the population (Morton,

1980). Greater evaluation of the mutagenic activity
of these plants is needed in order to estimate the
long-term risk to humans that could result from
prolonged use. Thus, the genotoxicity of some
aqueous extracts of these plants was evaluated by
means of the Ames test.

Introduction

0378-8741/$03.50
0 1991 Elsevier Scientific
Published and Printed in Ireland

screened

Seven of these were also screened

Ltd.

+++-i

+llS

II

-i

ii1+4+4

lll-l+l

I
+44-i-+

Ill+

4
+

ill-1-I

lil$

I I+>+

vuginutum

campestris Gris.

against

hyemulis Camb.

xanthocarpu

tenet/u (DC.)

St. Hil.

Wultheriu dourudinhu

STERCULIACEAE

ex Riss.

Iudinu rh(~rnh~u~~uAm.

SANTALACEAE

Berg.

Myrikiu

Eugeniu unifrura L.

Eqeniu

Berg.

Cumpomanesia

Berg.

against
diseases

digestive

vulnetaty.

Depurative,
pulmonary

antiseptic,

Analgesic,

Ants-in~ammatory.
depurative
Antipyretic.
antirheumatic
Anti-inflammatory

LFiST

LF

LF
LF

LF

-i-

--

--

LF

Antirheumatic,
hypoglycemic
Astringent,
against
enteritis and cough

MYRTACEAE

Biephurocu~yx salirifiliu

L.

LF

LF

Anti-inflammatory

MALVACEAE

Cogn.

Leundru atrupurpurea

Sidu wrpinifolia

-/_

-/-

--/-

--

LF/ST

LF

LF/ST

PL

LF
LF
LF/ST

LF

Tihouchitra asperior Cogn.

Anti-inflammatory.
hemostatic.
hyp~giicemjc

Depurative,
diuretic,
skin diseases, antirheumatic

Diuretic, hypogiicemic
Agaisnt enteritis
Diuretic, antiinflammatory

Diuretic, sedative,
urinary diseases

Antispasmodic,
against
enteritis
Diuretic, depurative

MELASTOMATACEAE

van Thieghem

Tripodunthus ucutifolius

LORANTHACEAE

Smibx

LILIACEAE

Desmodium incanum DC.

Cussia corymhosu Lam.

Buuhiniu cundicuns Bent h .

LEGUMINOSAE

Spreflg.

Sisyrinchium

IRIDACEAE

--

-/-

-/_

-J-

-J-

-I-

-/-

44

+
f

i-f-+

++

+/-

++I++

++

++/++

++
+

-/-

-J-

+i+

I-++

4-t

4-J-k

+/-

+
-/-

++J++

4-b

t/t

+
+/+

++

-_

-/-

-I-

-I-

-._

-I-

t/-k

++

++

i-

+I+

++

+/+t

t/+

+
-

PL
LF

Anti-inflammatory,
diuretic, analgesic
Diuretic, depurative,
against skin diseases

-/-

MR

-I-.

WR

Alkaloidsb

LF

LFfST

Plant
part

Against hemorroids

Diuretic

Use in popular
medicine

FL = flower; LF = leaf; PL = whole plant; RT = root; ST = stem.

bfvfR = Mayers reagent; WR = Wagners reagent.

VERBENACEAE
Aloysia gratissima (Gill.
et Hook.)
Stachyrarphefa eayennensis
Vahl.
Vitex montevidensis Cham.

TlLIACEAE
Luehea divaricara Mart.

Species

TABLE 1 (continued)

-I-

Coumarins

++

++

++/++

Flavonoids

++

++I+

Saponins

-I/+

Sterol and/or
triterpenes

--f

Anthraquinones

++

4-f-k-t

Tannins

169

extracts were then evaporated

to dryness under
reduced pressure. Each crude extract was examined according
to the methods of Rizk (1982),
Hussein Ayoub and Kingston (1982) and Krebs et
al. (1969) to detect the presence of alkaloids,
coumarins,
flavonoids,
anthraquinones,
saponins,
sterols and/or triterpenes
and tannins.

~1 of S9 mix) (Vargas et al., 1988). The extracts of

the dried plant material were prepared in boiling
water at a concentration
of 50/ weight/volume.
Samples were sterilized using a Sartorius filter with
0.22 pm pore size (Carl Zeiss, Brazil).

Ames test

A chemical survey of 37 species of medicinal

plants from Rio Grande do Sul, Brazil, was carried
out. Examination
of the concentrated
extracts with
specific reagents and analytical TLC established a
standard chemical screening process for alkaloids,
coumarins,
flavonoids, anthraquinones,
saponins,
sterols and/or triterpenes and tannins (Table 1).
The results demonstrated
that the alkaloids
occur at a low frequency;
this agrees with the
previous finding that they are more common to
species of tropical areas (Lewin and York, 1978).
According to the local populace, the plants containing alkaloids
produce
serious toxic effects;
therefore, they do not have any use. Coumarins
and quinones were also found in a small percentage of the plants studied. These three groups,

The Salmonella typhimurium strains, TA98 and

TAlOO, as described by Maron and Ames (1983)
were kindly provided by B.N. Ames (University of
California,
Berkeley, CA). The microsomal
S9
fraction,
prepared
from Sprague-Dawley
rat
livers pretreated
with 3-methylcholanthrene
was
purchased
from Bionetic
TM (Litton,
Kengsington,
MD). The S9 mix metabolic
activation
mixture was prepared
according
to Maron and
Ames ( 1983).
Mutagenicity
was assayed by the preincubation
procedure of Maron and Ames (1983). Mutations
tests were performed on samples of plant extracts
in the presence or absence of S9 mix (20 ~1 of S9
fraction containing
16.3 mg/ml of protein per 500

TABLE

Results and Discussion

MUTAGENIC
ACTIVITY
OF PLANT EXTRACTS
UTILIZING
WITHOUT
(-S9) AND WITH (+S9) METABOLIC
ACTIVATION

TA98

Strains

testedC

Extract

name

Plant

partb

AND

TAIOO STRAINS

OF

S. TYPHYMURIUM

TAlOO

TA98
.-s9
Iodina rhombifolia
Peltates pelratus
Baccharis anomala
Tibouchina asperior

(W
0-F)
(ST)
(W
WV
(W

Maytem4s ilicifolia

(ST)
(W
(ST)
(W

Desmodium incanum

(W

Luehea divaricata

Sample dose of extract equivalent to 100 mg of dried plant

bPlant part: LF = leaf; ST = stem.
Mutagenic
0.5 &plate

_
-

+s9

-s9

+s9

+
_

+
-

.-

_
_

material/plate.

response: (+) = mutagenic;

(-) = non-mutagenic.
Positive control: (-S9) = 5 &plate
sodium azide (TA 100 strain);
Cnitrequinoleine-l-oxide
(TA98 strain); (+S9) = 0.5 &plate
aflatoxin-B,.
Negative control: sterile distilled water.

170

alkaloids,
coumarins
and quinones,
occurred in
about 10% of the plants investigated. Tannins were
detected in 57% and saponins and sterols in about
70-76X of the species. The group of compounds
which predominates
in the samples investigated
comprises the flavonoids,
with 80%.
The Compositae
provides us with a number of
plants used in traditional
medicine, among which
we could emphasize Baccharis spp. and Mikania
spp. It should be pointed out that, in the study
area, there are more than 400 species of Baccharis
and 10 species of Mikania.
For the 37 species, the range of uses is broad but
they are mainly used for the treatment of cough,
infectious
diseases, inflammation,
skin, hepatic
and urinary diseases.
Only a few of the plants mentioned
have been
subjected to in-depth study for their chemical constituents. For example, Baccharis patens (Silva et
al., 1985) has been found to contain methoxyflavones, while Stachytarpheta
cayennensis (Garnier,
1977) contains
an iridoid
compound,
ipolamide.
Several species used in popular medicine to treat
inflammation
have been
subjected
to pharmacological evaluation to verify this activity. Two
plants, Stachytarpheta
cayennensis and Eugenia
unifora (Ene et al., 1986) are reported
to be
characterized
by the absence of toxic effects and
remarkable anti-inflammatory
activity.
In Table 2, the results of mutagenicity
screening
of seven plant extracts in the Ames test using
Salmonella strains Al00 to detect base/pair substitution mutagens and TA98 to detect frameshift
mutagens, with and without metabolic activation,
are presented.
The positive results obtained
in
genotoxic assay for Baccharis anomala and Luehea
divaricata may be explained due to the presence of
tannins and flavones in the extracts with certain
hydroxylation
patterns
(3,5,7_hydroxyl
substitution),
such
as
quercetin
and
kaempferol
(Macgregor and Wilson, 1988; Vargas et al., 1989,
1990a, b). In the assays, significant
genotoxic
effect was seen only in the presence of microsomal
activation.
The present study demonstrates
that a high
priority must be given to the evaluation
of the
potential risks that the indiscriminate
use of these
plants could bring to human health.

Acknowledgment
This work was supported
cil for Research (Brazil).

by the National

Coun-

References
Alice, C.B. and Silva, G.A.A.B.
(1985) Levantamento
titoquimico de alguns vegetais utilizados na medicina popular
do Rio Grande do Sul (Parte 1). Cudemo Farmaciu I. 83-94.
Bjeldanes, L.F. and Chang, G.W. (1977) Mutagenic activity of
quercetin and related compounds.
Science 197, 577-578.
Brown, J.P. and Dietrich, P.S. (1979) Mutagenicity
of plant
flavonols in the Salmonella/mammalian
microsome test activation of flavonol glycosides by mixed glycosides from rat
cecal bacteria and other sources. Muration
Research 66.
223-240.

Ene, L. (1986) Ensaio para avaliacao

da actividade
farmacologica
de Stuchyturphetu
cayennensis. 111. In: Reunion
Latinoamericana
de Ciencias Furmaceuticas.
Anales. Montevideo, Uruguay, p. 36.
Garnier,
J. (1977) Etude chimique de deux Verbena&es
de
Guyane: Stachyturpheta
guyanensis Vahl et Stachyturphetu
murahilis
Vahl. Plantes Medicinales
et Phytotherapie
I 1~
303-305.

Goggelmann,
W. and Schimmer, 0. (1966) Mutagenic activity
of phytotherapeutical
drugs.
Progress
in Clinical
und
Biological

Research

206. 63-72.

Hussein Ayoub. S.M. and Kingston, D.G. (1982) Screening of

plants in Sudan folk medicine for anticancer
activity (II).
Fitorerupiu
53. I 119-l 123.
Krebs, K.G., Heusser,
D. and Wimmer,
H. (1969) Spray
reagents.
In: E. Stahl (Ed.), Thin-layer
Chromatography.
Springer Verlag, Berlin, pp. 854-909.
Lewin. D.A. and York, B.M. (1978) The toxicity of plant
Biochemical
alkaloids:
an ecogeographic
perspective.
Sysremarics

and Ecology

6, 61-76.

Macgregor, J.T. and Wilson, R.E. (1988) Flavone mutagenicity

in Sulmonellu
typhimurium.
Dependance
on the pkMlOl
plasmid and excision-pair
deficiency.
Environmentcrl
und
Moleculur
Murugenesis
1I. 3 15-322.
Maron,

D.M. and Ames. B.N. (1983) Revised methods for the

mutagenicity
test, Murution
Reseurch
I 13.
173-215.
Morton. J.F. (1980) Search for carcinogenic
principles. Recent
Sulmonellu

Adwnces

in Phytochemisrry

14. 53-73.

Rizk, A.M. (1982) Constituents

of plants growing in Qatar 1.
A chemical survey of sixty plants. Firoterapia 53, 35-39.
Silva. G.A.A.B..
Henriques,
A.. Alice. C.B., Gleye. J. and
Moulis.

C. (1985) Methoxyflavones

Jourmd

oj Natural

Products

from Brrcchuris pawns.

(Lloydiu)

48. 86

Vargas, V.M.F., Motta. V.E.P. and Henriques, J.A.P. (1988)

Analysis of mutagenicity
of waters under the influence of
petrochemical
industrial
complexes
by the Ames test
(Sulnwnell~lmicrosome).
Bruziliun
Journul qf Generics I I.
505-518.

Vargas,

V.M.F..

and Henriques.

Motta.

V.E.P.. Alice, C.B.. Guidobono.

J.A.P. (1989) Mutagenic

activity

R.R.

of plant ex-

171
tracts used in popular
70, 65-67.
Vargas, V.M.F., Motta,

medicine.
V.E.P.,

Rev&a
Leitao,

Brasileira Farmaciu
A.C. and Henriques,

J.A.P. (1990a) Mutagenic and genotoxic effects of aqueous

in
prokaryotic
of
Achyrocline
sarureioides
extracts
organisms. Mutation Research 240, I3- 18.

Vargas, V.M.F.,
Guidobono,
R.R. and Henriques,
J.A.P.
(1990b) Genotoxicity
of plant extracts.
Memorias do Insfituto Oswald0 Cru:, in press.
Weisburger,
E.K. (1979) Natural carcinogenic
products.
Environmental Science Technology 13, 278-28 I.