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Journal of Medicinal Plants Research Vol. 5(10), pp. 2006-2010, 18 May, 2011
Available online at http://www.academicjournals.org/JMPR
ISSN 1996-0875 ©2011 Academic Journals
Full Length Research Paper
Phytochemical screening and evaluation of cytotoxic,
antimicrobial and cardiovascular effects of Gomphrena
globosa L. (Amaranthaceae)
Daniel Dias Rufino Arcanjo1*, Ingrid Virgínia de Oliveira Sena4, Adonai Carvalho Medeiros de
Albuquerque1, Bernardo Melo Neto2, Lorena Citó Lopes Resende Santana2, Náiguel Castelo
Branco Silva3, Maria Marilza Moita1, Maria das Graças Freire de Medeiros2, Maria José dos
Santos Soares3, Nilza Campos de Andrade4 and
Antônia Maria das Graças Lopes Citó4
1Department of Biophysics and Physiology, Federal University of Piauí, Brazil.
2Department of Biochemistry and Pharmacology, Federal University of Piauí, Brazil.
3Department of Veterinary Morphophysiology, Federal University of Piauí, Brazil.
4Department of Chemistry, Federal University of Piauí, Brazil.
Accepted 19 January, 2011
Gomphrena globosa L. (Amaranthaceae) is used in folk medicine in the treatment of high blood
pressure and other diseases. To confirm this popular use, ethanol extract from leaves of G. globosa L.
was prepared by maceration and analyzed by some phytochemical and biological assays, including
cardiovascular activity. The phytochemical screening detected the presence of saponins, alkaloids,
reducing sugars and coumarins. The extract did not induce either lethality in the brine shrimp (Artemia
salina Leach) bioassay or antimicrobial activity. The negative results for cytotoxic and antimicrobial
assays indicated possible low cytotoxicity to the extract. In other hand, it promoted a hypotensive
activity by significant reduction in arterial blood pressure without change heart rate, confirming the
therapeutic use as antihypertensive for this plant.
Key words: Gomphrena, Amaranthaceae, antihypertensive, cytotoxicity, Artemia salina.
INTRODUCTION
The knowledge about medicinal plants often symbolizes
the only therapeutic option for many communities and
ethnic groups. The comments about the popular use and
*Corresponding author. E-mail: daniel.arcanjo@ufpi.edu.br.
Tel/Fax: +55 (86) 3215-5871.
Abbreviations: Gg-EE, Ethanol extract from leaves of
Gomphrena globosa L.; LC50, lethal concentration that induced
50% from death; MAP, mean arterial pressure; HR, heart rate;
ATCC, American type culture collection.
efficacy of medicinal plants contribute significantly to the
dissemination of therapeutic properties of plants,
commonly used by medical effects they produce,
although chemical constituents sometimes are not known
(López, 2006). Phytochemical screening provides a
better choice of material to be studied and gives the
possibility of adapting extracts fractionation techniques
and isolation and characterization of pure substances,
according to the constituents previously detected by
qualitative tests. Thereby, it facilitates the subsequent
work of isolation and purification of the constituents
biologically actives (Farnsworth, 1966). Specimens of
Artemia salina Leach (brine shrimp), a marine
microcrustacean, are being used as target organisms to
detect bioactive compounds in plant extracts and the
toxicity test against these animals has shown a good
correlation with antitumor activity (McLaughlin and
Rogers, 1988). This bioassay provides an advantage in
the standardization and quality control of botanical
products, and in evaluation of new drugs (Lieberman,
1999; Citó et al., 2003).
The species Gomphrena globosa L. (Amaranthaceae)
is popularly known in Brazil as "perpétua” or “perpétua-
roxa”. Its leaves and flowers have application in folk
medicine, and have been used in the treatment of
hypertension, diabetes, kidney problems (Agra et al.,
2007; Lans, 2006), hoarseness, cough, bronchitis and
other respiratory diseases, mainly due expectorant action
(Camejo-Rodrigues et al., 2003), and reproductive
problems, due estrogenic activity (Lans, 2007). Extracts
of aerial parts of some species of the Gomphrena genus
have the following biological activities reported: larvicide
(Dadang and Ohsawa, 2001) for G. globosa;
antimicrobial for G. martiana and G. boliviana (Pomilio et
al., 1992), antitumor to G. martiana (Pomilio et al., 1994)
and estrogenic to G. demissa Mart. (França et al., 2008).
Additionally, for species from Gomphrena genus,
chemical identifications were cited: betacyanins (Heuer,
1992; Minale, 1967), hydroxycinamamides (Martin-
Tanguy et al., 1992), and flavonoids, including flavones
(Buschi, 1980; Pomilio, 1994) and flavonols (Bouillant,
1978).
The aim of this study was to characterize the
phytochemical profile, assess the toxicity against brine
shrimp (A. salina Leach) bioassay and determine the
antimicrobial and cardiovascular effects of ethanol extract
from leaves of G. globosa L.
MATERIALS AND METHODS
Botanical identification
G. globosa L. was cultivated and collected from the garden of
medicinal and aromatic plants center (NUPLAM) from Federal
University of Piauí, Brazil. The botanical identification was carried
out at Graziela Barroso Herbarium of Federal University of Piauí,
Brazil (voucher specimen no. 20901).
Extraction and phytochemical screening
Leaves (211.6 g) were dried at room temperature and then
powdered. The powder was exhaustively extracted with 99.8%
ethanol by maceration at room temperature (Gg-EE). The ethanol
extract solvent was evaporated to dryness under reduced pressure
and lyophilized to yield 34.7 g (16.4%). Evaluation of main
phytochemicals groups from Gg-EE was carried out according to
Farnsworth (1966) by application of qualitative tests: saponins,
steroids and terpenoids, alkaloids, flavonoids, catechins, tannins
Arcanjo et al. 2007
and polyphenols, reducing sugars, lactones, coumarins and
quinones. In all tests, results were observed and compared with
published data in respect of the presence or absence of each
phytochemical group and therapeutic uses.
Establishment of toxicity in brine shrimp bioassay
The toxicity bioassay on A. salina Leach was performed according
to the methodology proposed by Meyer et al. (1982), with some
modifications (Nunes et al., 2009). From 20 mg of Gg-EE, 1, 10,
100 and 1000 µg/mL solutions were prepared in triplicate. Then, 10
specimens with 48 h of hatching in sea water and distilled water
(1:1) were placed in each tube and three negative control tubes
(saline solution and DMSO 1%). Appropriate volumes of the saline
solution in tubes were added until 5 mL of saline solution containing
10 nauplii each to afford the final sample concentrations. After 24 h,
the number of deaths was counted and results were tabulated and
analysed.
Evaluation of antimicrobial activity
The Gg-EE was subjected to antibacterial assay using the agar
diffusion plate method as described by Alves and colleagues
(2000), with some changes and guided by NCCLS/CLSI (2005).
Tests were performed against following standard microorganisms:
Staphylococcus aureus ATCC 25923, Staphylococus epidermidis
ATCC 12228, Enterococcus faecalis ATCC 29212, Escherichia coli
ATCC 25922, E. coli ATCC 35218, Klebsiella pneumoniae ATCC
700603 and Pseudomonas aeruginosa ATCC 27853. In addition,
four multiple-drug resistant clinical isolated microorganisms were
also tested: methicillin-resistant S. aureus MRPI 98 (strain typed as
belonging to the Brazilian epidemic clone – BEC; Soares et al.,
2000); methicillin-resistant S. epidermidis MRSE H111 (methicillin-
resistant); E. faecalis 7426 (strain vancomycin resistant and positive
for gene vanA; Soares et al., 2000) and Stenotrophomonas
maltophilia EM 012004 (strain only sensible to Trimethoprim +
Sulfametoxazole).
The microorganisms were cultured overnight at 35°C in Mueller-
Hinton Broth (Difco) before use. Suspension of bacterial strains
with optical density of McFarland 0.5 (1x108 CFU/mL) were
made in isotonic sodium chloride 0.9% solution. After inoculation
and drying for 10 min, four wells (6 mm diameter) were made and
aseptically filled up with different concentrations (1,10, 50 and 100
mg/mL) of Gg-EE, whereas equal volume of dimethyl-sulfoxide
(DMSO) was used as negative control and Mueller-Hinton agar only
as positive control for microbial growth. All plates were incubated at
35°C for 24 h. The antibacterial activity was measured at the
diameter (mm) of clear zone of growth inhibition, and it was
interpreted as: 9 to 13mm, moderate activity; > 13 to 17 mm, active;
> 1 7mm, very active. Each test was performed in triplicate.
Effects of Gg-EE on cardiovascular parameters
Male dogs (Canis familiaris) (5 to 10 kg) were adequately
anesthetized with thiopental solution (25 mg/kg, i.v.). Then, inferior
cava vein through femoral vein was cannulated for administration of
Gg-EE. To measure the mean arterial pressure (MAP) and heart
rate (HR) before and after application of substances, a heparinized
catheter inserted into abdominal aorta through femoral artery of
each animal was coupled to a pressure measurement device (AVS
Projects, São Paulo, SP, Brazil). After cardiovascular parameters
2008 J. Med. Plant. Res.
Table 1. Main phytochemical groups identified from Gg-EE.
Phytochemicals groups
Qualitative tests
Result
Saponins
Foam test
+
Steroids and Terpenoids
Liebermann-Buchard reaction
-
Alkaloids
Dragendorff, Bouchardat and Bertrand reactions
+
Flavonoids
Shinoda reaction
-
Catechins
Na2CO3 reaction
-
Tannins e Polyphenols
Ferric chloride 1 %
-
Reducing sugars
Benedict reaction
+
Lactones
Baljet reaction
-
Coumarines
NaOH/Ethanol (w/v), UV
+
Quinones
NH4OH reaction
-
Legend: (+) Present; (-) Absent.
Figure 1. Hypotensive effect in anaesthetized dogs before (control) and after acute
administration of Gg-EE (10, 20 and 40 mg/kg). MAP (mean arterial pressure) values
are mean ± SEM of six experiments. **p<0.01, ***p<0.001 vs. control.
had stabilized, the MAP and HR were recorded before (control) and
after administration of successive doses of Gg-EE (10, 20 and 40
mg/Kg). Successive injections were separated by a time interval
sufficient to allow full recovery of haemodinamic parameters.
All experimental procedures were performed in accordance with
the National Research Council’s guidelines and approved by the
Animal Research Ethics Committee of the Federal University of
Piauí. Values are shown as mean ± standard error of the mean
(s.e.m). Experimental results are expressed as percentage
decreases in arterial blood pressure and heart rate. ANOVA one-
way followed by Student-Newman-Keuls post-test were used in the
data analysis and results were considered significant when p<0.05.
GraphPad™ Prism 5.0 (GraphPad Software, Inc., CA, USA) was
used to perform the data analysis.
RESULTS
The phytochemical screening of G. globosa ethanol
extract (Gg-EE) detected the presence of saponins,
alkaloids, reducing sugars and coumarins (Table 1). In
turn, the extract did not induce mortality in the A. salina
bioassay. Then, it was not possible to calculate the LC50.
The Gg-EE did not promote antimicrobial activity against
microorganisms tested. In other hand, it induced a
significant reduction in arterial blood pressure (Figure 1)
without change heart rate (Figure 2), confirming
hypotensive activity for this extract.
Figure 2. Bradycardic effect in anaesthetized dogs before
(control) and after acute administration of Gg-EE (10, 20 and 40
mg/kg). HR (heart rate) values are mean ± SEM of six
experiments.
DISCUSSION
The major finding of this study is the ethanol extract from
leaves of G. globosa L. showed hypotensive effect in
anesthetized dogs. The study of Gg-EE cardiovascular
effects was motivated by the use of this species in folk
medicine to treat high blood pressure (Agra et al., 2007;
Lans, 2006). Interestingly, this is the first report of these
pharmacological activities for this specie. The
hypotensive effect promoted by Gg-EE at doses of 10, 20
and 40 mg/kg was significant, in contrast to the
bradycardic effect, which was not observed. This
observation indicates that even in blood pressure
reduction, heart rate remains unaltered, probably due to
the baroreflex response down-regulation, aiming to
normalize cardiovascular parameters (Kawaguchi et al.,
2007). A decrease in arterial pressure reduces
baroreceptor afferent discharge and triggers reflex
increases in heart rate, cardiac contractility, vascular
resistance, and increased venous return (Lanfranchi and
Somers, 2002). Thus, these results represent an
important activity for drugs to treat certain types of arterial
hypertension.
The phytochemical screening is an important step in the
chemical and pharmacological study of a medicinal plant.
It may suggest possible pharmacological effects of its
extracts or fractions in comparison of identified
phytochemicals groups, highlighting a close relationship
with its main therapeutic uses. Thereby, results of
phytochemical screening are in accordance with the
popular use of G. globosa L. species in the treatment of
hypertension. The presence of saponins in Gg-EE was
confirmed by the foam test, observing its formation and
Arcanjo et al. 2009
continuing for about 20 min. In aqueous solution,
saponins form persistent and abundant foam due a
lipophilic portion in its chemical structure, called aglycone
or sapogenin, and a hydrophilic portion, formed by one or
more sugars that provide detergent properties
(Farnsworth, 1966; N’guessan et al., 2009). Additionally,
reducing carbohydrates were identified by ‘‘Benedict
reaction’’. These results are consistent with the report of
saponosides identified for this species (Heuer et al.,
1992; Minale et al., 1967). This group of substances may
promote lower blood pressure and cholesterol levels
(N'guessan et al., 2009).
The presence of alkaloids was confirmed in Gg-EE by
precipitate formation after Dragendorff, Bouchardat and
Bertrand reactions (Farnsworth, 1966). Antihypertensive,
cardiac depressant, and antitussive pharmacological
activities reported for some alkaloids isolated from
several species provide evidence for some therapeutic
uses of G. globosa L. (Cordell et al., 2001; Santos et al.,
2006). The identification of coumarins by reaction with an
ethanol solution of sodium hydroxide had a positive result
under ultraviolet light at 366 nm (Farnsworth, 1966). The
hypotensive activity and myocardium relaxant for some
coumarins may have related to the G. globosa L. use as
antihypertensive (Chiou et al., 2001; Chung et al., 1993).
Substances tested by brine shrimp bioassay which
leads to death half nauplii at a lethal concentration until
1000 µg/mL (LC50), are considered actives, and thus a
good potential for antitumor activity (McLaughlin and
Rogers, 1988; Lieberman, 1999). In our studies, Gg-EE
did not show cytotoxic activity in this bioassay. Similarly,
there was no antimicrobial activity for Gg-EE, in contrast
with results from other species of the Gomphrena genus
(Pomilio et al., 1992). Several studies have also tried to
correlate the toxicity against A. salina with other activities
such as antifungal, virucidal and antimicrobial (MacBae et
al., 1988), parasiticide (Sahpaz et al., 1994), trypanocida
(Zani et al., 1995), among others.
In conclusion, Gg-EE showed hypotensive activity,
related to its popular use for treating hypertension.
Further studies are needed to elucidate what
mechanisms are involved in the antihypertensive activity
for this species. Although Gg-EE has showed no lethality
against various microorganisms, these results indicate
low cytotoxicity to the extract. Thereby, the brine shrimp
bioassay provides an advantage in the standardization and
quality control of botanical products, and in development of
new drugs.
ACKNOWLEDGEMENTS
The authors thank CAPES, CNPq and FAPEPI, for
supporting this research. Thanks to Professor Roseli
Farias Melo de Barros, PhD, from Graziela Barroso
2010 J. Med. Plant. Res.
Herbarium of Federal University of Piauí, for your support
to botanical identification.
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