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Phytochemical screening of Achillea millefolium harvested at Araraquara – SP
Souza, T. M.1; Rangel, V. L. B. I.;Pietro, R. C. L. Rr.1,2*; Santos, L. E.3; Moreira, R. R. D.3.
1
Departamento de Fármacos e Medicamentos da Faculdade de Ciências Farmacêuticas da Universidade Estadual
Paulista – UNESP – Araraquara, São Paulo. [email protected]
2
Curso de Ciências Farmacêuticas da Universidade de Ribeirão Preto – UNAERP – Ribeirão Preto, São Paulo.
3
Departamento de Princípios Ativos Naturais e Toxicológicos da Faculdade de Ciências Farmacêuticas da
Universidade Estadual Paulista – UNESP – Araraquara, São Paulo.
ABSTRACT: Phytochemical screening of Achillea millefolium harvested at Araraquara – SP. Achillea millefolium
(Asteraceae) is a native plant from Europe, North America, South Australia and North Asia, but perfectly adapted
for Brazil. Its popular use includes anti-inflammatory, astringent and other actions, which are proportionate for
the presence of the secondary metabolites. The objective of this work was search the presence of alkaloids,
flavonoids, tannins, saponins, antraquinones, antraquinonic and cardiac glycosides in leaves of A. millefolium
cultivated at the “Horto de Plantas Medicinais e Tóxicas da Faculdade de Ciências Farmacêuticas de AraraquaraUNESP, São Paulo” through characterization reactions. In A. millefolium leaves the specific characterization
reactions for alkaloids, antraquinonic and cardiac glycosides and antraquinones presented negative results.
Some of the characterizations performed for flavonoids showed positive reactions. The presence of condensed
and hydrolyzable tannins as well as saponins was characterized. Based on the results of phytochemical screening,
the antibacterial activity of the A. millefolium leaves extract was evaluated, but exhibited negative results.
Key words: Achillea millefolium, tannins, flavonoids, saponins, antibacterial.
INTRODUCTION
The Achillea millefolium is popularly known
by “mil-folhas”, “novalgina”, mil-em-rama”, “erva-decarpinteiro”, “milefólio”, “erva-de-cortaduras”, “erva-doscarreteiros”, “aquiléia”, “macelão” and “yarrow”. It is
perennial, erect, aromatical, and a herbaceous plant,
of 30-50cm of height. Its leaves are composed, finely
pinned, of 5-8cm of length; the flowers are white or
rosaceous. It is a plant of subtropical climate, that
appreciates the heat and resists to dry. It occurs of
native form in the Europe, North America, south of
Australia and north of Asia (Mil-folhas, 2003), and
widely cultivated in almost all Brazil for being perfectly
suitable to the climate (Lorenzi et al., 2002; Martins
et al., 2000; Panizza, 1997).
In its chemical composition, literature
describes the presence of essential oil with terpens
(cineol, borneol, pinens, camphor, azulen), terpenics
and sesquiterpenics derivatives, tannins, coumarins,
resins, saponins, steroids, fatty acid, alkaloids and
principles of bitter taste (Lorenzi et al., 2002; Martins
et al., 2000; Panizza, 1997). It is reported the
presence of flavonoids, apigenin, luteolin and its
glycosides, artemetin and rutin in the flowers and
leaves (Guédon et al., 1993; Teske & Trentini et al.,
1997). The leaves and flowers are the utilized parts of
the plant in ornamentation and in traditional medicine.
It is used due the anti-inflammatory and astringent
Recebido para publicação em agosto/2004
Aceito para publicação em julho/2006
activity, for example, against fevers, diarrhea,
hemorrhoids, etc. However, the juice of the fresh plant
in contact with the skin can develop
photosensibilization (Lorenzi et al., 2002; Martins et
al., 2000; Panizza, 1997).
The objective of this work was a
phytochemistry screening of the main classes of
secondary metabolites in leaves of Achillea
millefolium. Based on the obtained results, a study
for evaluate the antibacterial activity of the extract of
A. millefolium was performed due to the presence of
metabolites whose potentially present this property.
MATERIAL AND METHOD
Plant material
The leaves of “mil-folhas”, scientific name
Achillea millefolium Linné (Asteraceae), which
exsiccate is registered in the “Herbário do Instituto
de Biociências da UNESP de Rio Claro – SP” under
Voucher number 35292, was collected in July of 2003
from the “Horto de Plantas Medicinais e Tóxicas da
Faculdade de Ciências Farmacêuticas de Araraquara,
da Universidade Estadual Paulista, UNESP”. These
leaves had been dried in circulating air greenhouse
at 40ºC during two days, and sprayed in mill of knives
and stored in cool place without humidity.
Phytochemical screening of the main
secondary metabolites classes
The characterization for tannins was
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performed by three reactions: gelatin, iron salts
and lead acetate (Costa, 1994). Hamamelis
virginiana was the plant used as positive control.
Cardiotonic glycosides were characterized by the
reactions of Legal, Kedde, Pesez, Keller-Kiliani and
Liebermann-Burchard described in Costa (1994),
using as positive control Nerium oleander. The
alkaloids analysis was done by the precipitation
reactions with the reagents of Dragendorff,
Bouchardat, Mayer and Bertrand (Costa, 1994) and
Atropa belladonna was used as positive control.
Dimorphandra molis was used as positive control
for the research of flavonoids through the reactions
of Shinoda, Taubock, Pew, ferric chloride and
aluminum chloride (Costa, 1994). The extraction
and the characterization for antraquinones was
done according Costa (1994) using Rhamnus
purshiana as positive control. The analysis of
presence of saponins was done by the formation
of foam (Costa, 1994) and Aesculus
heppocastanum was positive control.
Preparation of Achillea millefolium extract
The extract of A. millefolium was prepared
by percolation using ethanol 92,8º as extracting
liquid. The ethanolic extract was placed in rotatory
evaporator to eliminate the solvent, obtaining an
extract, which was weighed and stored in
dissecator.
Evaluation of the antibacterial activity
To evaluation antibacterial activity of A.
millefolium extract, the strains used were
Staphylococcus aureus (ATCC 25923),
Staphylococcus epidermidis (ATCC 10536) and
Escherichia coli (ATCC 1228). The A. millefolium
extract was ressuspended in dimethylsulfoxide
(DMSO) 250mg/mL and diluted (1:1) in brain and
heart infusion broth (BHI). To initiate the exponential
growth prior to the assay, a sample of strains was
incubated at 37°C for 24 h in BHI. The inoculum in
the assay was adjusted to the 0,5 Mc Farland scale
(1-3 x 108 CFU/mL, colony units forming) in BHI.
After that, 100µL of bacteria diluted (1:100) was
added to the tubes containing extract solution,
prepared as above described, in concentrations to
be assayed. All samples were incubated at 37ºC
for 24h and the antibacterial activity was observed
by the presence of turbidity. Then a sample of each
tube was removed and plated in medium MüellerHinton agar which was incubated at 37ºC for 24h to
verify bacterial growth. Controls of bacteria growth,
medium, extracts and solvents were done in parallel.
RESULT AND DISCUSION
Phytochemical screening of the main
secondary metabolites classes
The results of phytochemical screening
performed with dry and powered leaves of A.
millefolium can be observed in the Table 1, showed
following.
In Table 1 was characterized the presence of
tannins in A. millefolium leaves. The formation of
precipitate, green colour and the formation of white
precipitate with the respective reactions are indicative
of tannins presence. It was observed that the results
of all reactions of characterization for cardiotonic
glycosides are negative, which indicate the absence
of this metabolite. Table 1 shows negative results for
the research of alkaloids and antraquinones. To the
research of flavonoids, done by Shinoda, Taubock,
Pew, ferric chloride and aluminum chloride reactions,
only Taubock, ferric chloride and aluminum chloride
reactions showed positive results. Our results showed
positive result for the saponin research in leaves of A.
millefolium.
Evaluation of the antibacterial activity
The phytochemical study demonstrated the
presence of metabolites that potentially present
antimicrobial property like tannins and flavonoids
(Cowan, 1999). The assays were conduced to strains
that have importance in Public Heath. In all tubes of
positive control assay of S. aureus, S. epidermidis
and E. coli there was growth, nevertheless there was
no bacterial growth in controlj35s of medium, extracts
and solvents. Table 2 presents the results of evaluation
of the antibacterial activity obtained for each
concentration of A. millefolium extract tested.
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153
TABLE 1. Phytochemical screening of A. millefolium leaves.
+: positive result; -: negative result
TABLE 2. Evaluation of the antibacterial activity of the extract of A. millefolium
+ : bacterial growth.
As seen in Table 2, there was bacterial
growth of the strains used in all concentrations of the
A. millefolium ethanolic extract. Thus, it was
determined that the ethanolic extract does not present
antibacterial activity at any concentration assayed.
Probably, the absence of antibacterial activity could
have as cause, the amount of metabolites as flavonoids
and tannins in our samples, because they are
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154
compounds described in the literature that presents
antimicrobial property (Cowan, 1999).
studies may obtain different extracts with modified
composition that can exhibit antibacterial activity.
CONCLUSION
ACKNOWLEDGMENT
Based on the results, it was possible
demonstrated the absence of cardiotonic glycosides
and antraquinones in leaves of A. millefolium being in
according to the literature (Lorenzi et al., 2002; Martins
et al., 2000; Panizza, 1997). Also the positive results,
related to the presence of tannins and saponins in
leaves of this plant were described in the literature
(Lorenzi et al., 2002; Martins et al., 2000; Panizza,
1997).
In literature there are described the presence
of alkaloids in A. millefolium leaves, but we cannot
demonstrate this with leaves used in this work (Lorenzi
et al., 2002; Martins et al., 2000; Panizza, 1997). To
flavonoids, literature also reports the presence in
leaves of this specie (Guédon et al., 1993; Teske and
Trentini, et al., 1997), however, of five reactions
performed to characterization of this metabolite, only
in three, the result was positive. These results can be
due to an insufficient sensitivity of the reactions in
front of low concentration of these metabolites, or to
the place where the plant was cultivated, or even, to
the period of collection (Simões et al., 1999).
Through the evaluation of the antibacterial
activity, it can be verified that the ethanolic extract of
the A. millefolium, in the tested conditions, does not
present activity against strains of bacteria. Further
We are grateful to Luciene Araújo for technical
assistance. One of us (TMS) received a fellowship
from FAPESP (Fundação de Amparo à Pesquisa do
Estado de São Paulo). This work was supported by
PADC – Faculdade de Ciências Farmacêuticas –
UNESP (Brazil).
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