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Acute toxicity and anti inflammatory effects of supercritical extracts of Ilex paraguariensis

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This work reports the in vivo evaluation of acute toxicity and anti-inflammatory activity of supercritical carbon dioxide (CO 2) extracts of Ilex paraguariensis. For the acute toxicity study, the extracts diluted in sunflower oil were administered to adult male wistar rats in single doses of 250 mg/kg by the intraperitoneal route. Adverse effects and mortality were monitored along 14 days. In the case of anti-inflammatory activity, the animals were submitted to acute induced-peritonitis assays by injection of oyster glycogen and a single dose of 500 mg/kg of supercritical I. paraguariensis extract diluted in sunflower oil administered by subcutaneous injection. Results obtained in this work suggest the incidence of acute toxicity due to the presence of cells with hyperchromatism, vacuolization and tumefaction in the livers and the presence of cells with tumefaction in the cortical region of the kidneys of the treated group. It was also experimentally observed that treatment inhibited the neutrophil recruitment in the circulating blood, hence demonstrating the anti-inflammatory effect of the I. paraguariensis extracts. The results obtained may be very promising since they open new perspectives for the therapeutic use of supercritical extracts of I. paraguariensis or its preparations as anti-inflammatory agents.
African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1162-1169, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.234
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Acute toxicity and anti inflammatory effects of
supercritical extracts of Ilex paraguariensis
Tiago R. Pasquali1, Sandra M. D. Macedo1, Silvane S. Roman1, Valéria Dal Prá1, Rogério L.
Cansian2, Altemir J. Mossi3, Vladimir J. Oliveira2 and Marcio A. Mazutti4*
1Department of Clinical Pharmacy, URI - Campus de Erechim, Brazil.
2Department of Food Engineering, URI - Campus de Erechim, Av. Sete de Setembro, 1621, Erechim, 99700-000, RS,
Brazil.
3Department of Agronomy, Universidade Federal da Fronteira Sul, Av. Dom João Hoffmann, Erechim - RS, 99700-000 –
Brazil.
4Department of Chemical Engineering - Federal University of Santa Maria, Av. Roraima, 1000, Santa Maria, 97105-900,
Brazil.
Accepted 4 June, 2011
This work reports the in vivo evaluation of acute toxicity and anti-inflammatory activity of supercritical
carbon dioxide (CO2) extracts of Ilex paraguariensis. For the acute toxicity study, the extracts diluted in
sunflower oil were administered to adult male wistar rats in single doses of 250 mg/kg by the
intraperitoneal route. Adverse effects and mortality were monitored along 14 days. In the case of anti-
inflammatory activity, the animals were submitted to acute induced-peritonitis assays by injection of
oyster glycogen and a single dose of 500 mg/kg of supercritical I. paraguariensis extract diluted in
sunflower oil administered by subcutaneous injection. Results obtained in this work suggest the
incidence of acute toxicity due to the presence of cells with hyperchromatism, vacuolization and
tumefaction in the livers and the presence of cells with tumefaction in the cortical region of the kidneys
of the treated group. It was also experimentally observed that treatment inhibited the neutrophil
recruitment in the circulating blood, hence demonstrating the anti-inflammatory effect of the I.
paraguariensis extracts. The results obtained may be very promising since they open new perspectives
for the therapeutic use of supercritical extracts of I. paraguariensis or its preparations as anti-
inflammatory agents.
Key words: Acute toxicity, Anti-inflammatory, Supercritical fluid extraction, Ilex paraguariensis.
INTRODUCTION
Mate (Ilex paraguariensis St. Hill., Aquifoliaceae) a world-
famous tea consumed in Brazil, Paraguay, Uruguay and
Argentina, was used by the Indians of South America
before the European colonization. The mate tea leaves
has remarkable importance in the economy and in the
*Corresponding author. E-mail: marciomazutti@gmail.com Tel:
+55-55-3220-9591.
Abbreviations: CO2, Carbon dioxide; PBS, phosphate-
buffered saline; NH4Cl, ammonium chloride; ANOVA,
analysis of variance; G, glomerullus; TCD, distal
convoluted tubules; TCP, proximal convoluted tubules;
CV, centrolobular vein; C, control; T, treated.
cultural life of South America, with an average of 300,000
t produced each year (Alikaridis, 1987; Tormen, 1995). In
the southern region of Brazil, for example, one can find
more than 40 mates processing industries and about
180,000 medium and small rural properties dedicated
almost exclusively to cultivation of this raw material
(Mosele, 2002).
The mate raw material, as leaves and green stalks, is
processed as dye herb for the classical “Chimarrão”,
“Mate” or “Tereré”, as fine or soluble powder for teas, or
as essences used for different industrial purposes.
“Chimarrão” or “mate” is prepared by steeping dry leaves
and twigs of mate in hot water, while “tereré” is prepared
with cold water (Cansian et al., 2008a).
Mate is considered as a stimulant drink that eliminates
lassitude through increasing physical and mental activities.
Biological and therapeutical activities on the
cardiovascular, respiratory, muscular, gastrointestinal,
renal and neurological systems have been attributed to
the presence of xanthines, caffeine, theobromine, tannic
substances, flavonoids, vitamins, and other substances
present in mate extracts (Heck and Mejia, 2007; Cansian
et al., 2008b). In folk medicine, mate infusion has been
used for the treatment of arthritis, rheumatism and other
inflammatory diseases, constipation, hemorrhoids,
headache, obesity, fatigue, fluid retention, hypertension,
slow digestion and, hepatic and digestive disorders
(Cansian et al., 2008b; Silva et al., 2008; Gorzalczany et
al., 2001). Recent findings have shown that the aqueous
extracts of I. paraguariensis can inhibit the progression of
atherosclerosis in cholesterol-fed rabbits (Mosiman et al.,
2006).
The literature is somewhat vast regarding the extraction
and chemical characterization of I. paraguariensis
(Esmelindro et al., 2004; Esmelindro et al., 2005;
Jacques et al., 2006; Jacques et al., 2007a; Jacques et
al., 2007b; Jacques et al., 2008) and there are several
reports dealing with the major constituents of Ilex
species, like the presence of saponins (Taketa et al.,
2004; Taketa et al., 2000; Ouyang et al., 1998; Pires et
al., 1997), the occurrence of flavonoids (Martínez et al.,
1997), xanthines (Saldaña et al., 2002; Reginatto et al.,
1999; Saldaña et al., 1999), aldehydes (Don-Xu and
Zhong-Liang, 1996), hemiterpene glycosides (Fuchino et
al., 1997), triterpenes and alkanes (Vangenden and
Jaarsma, 1990), anthocyanins (Ishikura, 1975), pentyl
esters, hexyl esters, and other lipophylic compounds
(Vangederen et al., 1988).
Some phytochemical or pharmacological investigations
related to the use of alcoholic or aqueous extracts of I.
paraguariensis have also been reported in the literature
(Prediger et al., 2008; Alves et al., 2008; Silva et al.,
2008; Strassmann et al., 2008; Cansian et al., 2008b).
Nevertheless, to our knowledge, no reports are available
concerning the use of mate extracts obtained from
supercritical extraction in pharmacological studies.
The present report is part of a broader project aiming at
characterizing and developing new processes and
products from mate tea leaves. In this context, the main
objective of this work is to evaluate the acute toxicity and
the anti-inflammatory activity of the crude mate extracts
obtained from supercritical carbon dioxide extraction. The
extracts were administered via subcutaneous injections in
rats with the aim to obtain information on the safety of
extracts of I. paraguariensis and provide guidance to the
development of new pharmaceutical products from this
plant.
MATERIALS AND METHODS
Plant materials and chemicals
Samples of mate tea leaves were collected in an experiment
conducted under agronomic control cultivation system, located at
Pasquali et al. 1163
Barão de Cotegipe (Rio Grande do Sul State, Brazil, 27° 37° 15°), a
typical I. paraguariensis open plantation. The leaves were collected
from plants of about 7 years old, growing at complete sunlight
exposure and without any additional fertilization with age of leaves
of 12 months old. For each tree, fractions from the top, middle, and
bottom were sampled and homogenized. The samples were
immediately dried after collection at room temperature, triturated
and sieved, being collected 100 to 200 mesh particles. The final
moisture of all samples was around 2%. The samples were then
stored at room temperature under nitrogen atmosphere prior to the
extraction. The carbon dioxide (CO2) employed in the extractions
(99.9% in the liquid phase) was purchased from White-Martins S.A.
(Brazil) and the reagents were all of analytical grade.
Supercritical extraction procedure
The extraction was performed in a laboratory-scale unit, presented
in detail elsewhere (Esmelindro et al., 2004; Esmelindro et al.,
2005; Dariva et al., 2003; Rodrigues et al., 2004; Jacques et al.,
2007a; Jacques et al., 2007b). Basically, the apparatus consists of
a CO2 reservoir, two thermostatic baths, a syringe pump (ISCO
500D Lincoln, USA), a 100 mL jacketed extraction vessel, an
absolute pressure transducer (Smar, LD301 - Brazil) equipped with
a portable programmer (Smar, HT 201 - Brazil) with a precision of
± 0.3 bar, a collector vessel with a glass tube, and a cold trap.
Typically, amounts of around 25 g of comminuted mate tea
leaves were charged into the extraction vessel. The CO2 was
pumped at a constant mass flow rate of 2 g.min-1 into the bed,
which was supported by two 300 mesh wire disks at both ends of
the extractor. The CO2 was kept in contact with the herbaceous
matrix for one hour to allow the system stabilization. Afterwards, the
extract was collected by opening the micrometric valve. The
extraction was accomplished until no significant mass was
extracted (about 4 h) and at the end of the process the extract was
weighed and transferred to an appropriate vessel (“bulk”, stock
extract vessel). Based on the results obtained by Jacques et al.
(2007a), all the extractions were conducted at constant temperature
and pressure of 40°C and 250 bar, respectively. The extraction runs
performed led to an overall standard deviation of the extraction
yields of about 0.05. A whole experimental run lasted in general 10
h, including all steps involved: sample weighing, temperature
stabilization (baths, extractor), depressurization, etc. Extracts
obtained were dissolved in dichloromethane prior to gas
chromatography/mass spectrometry analysis.
Extract analysis
The extracts were analyzed in a gas-chromatograph coupled with a
mass selective detector (GC/MSD, Shimadzu QP5050A Kyoto,
Japan), using a capillary column DB5 (30 m, 0.25 mm, 25 m).
Column temperature was programmed 70°C/3 min, C/min to
260°C, 2.5°C/min to 300°C/25 min. Helium was the carrier gas and
the injection port and detector temperatures were 290 and 300°C,
respectively. The sample (1 L of 40.000 mg.L-1 in CH2Cl2)
components were identified by matching their mass spectra with
those of Wiley library database. Triplicate measurements were
performed for each sample.
Animals
Forty eight adult male wistar rats (3 months old, 180-220 g) were
obtained from the breeding colony at the Department of Clinical
Pharmacy (URI - Campus de Erechim, Brazil). The animals were
kept in a room under controlled humidity (50 ± 5%) and temperature
(22 ± 2°C) and subjected to a 12 h light cycle with free access to
1164 Afr. J. Pharm. Pharmacol.
food and water. All the procedures used in the present study
comply with the guidelines on animal care of the Ethics committee
on the use of animals of the University (Register number
187/TCA/07).
Acute toxicity evaluation
For the acute toxicity evaluation of the I. paraguariensis extracts
obtained from supercritical extraction, the animals were divided into
two groups of six animals each. For the treated group, a single
dose of 250 mg/kg of the mate extract diluted in sunflower oil was
administered by the intraperitoneal route, while the control group
received the vehicle only (sunflower oil). The animals were
continuously observed for general behavior changes, signs of
toxicity, death and latency of death during 1 h after treatment, then,
at each 4 h, and thereafter at each 24 h up to 14 days. During the
whole period, the weight and the consumption of water and food by
the animals were monitored. The tests were carried out in duplicate.
To evaluate the statistical significance of the results the signs of
toxicity were expressed in terms of semi quantitative analysis as
following: 0 - absence, 2 - Low, 3 - moderate, and 4 - Intense.
After the 14th day, the rats were anesthetized with CO2 and
sacrificed with their livers and kidneys carefully dissected out. Small
slices of these freshly harvested tissues were fixed in buffered
formaldehyde solution (10%), dehydrated by serial ethanol solution,
diaphanized with ethanol-benzene and embedded in paraffin.
Micrometer sections, cut by a microtome (Leitz 1512), were stained
with hematoxylin-eosin and examined under a light microscope,
taking photomicrographs of the samples. All the tests concerning
acute toxicity evaluation of the I. paraguariensis extracts obtained
from supercritical extraction were carried out following the
resolution of National Agency of Sanitary Vigilance of Brazil, which
regulates the pre-clinical studies of toxicity using phytotherapy
(ANVISA, 2004).
Anti-inflammatory evaluation
The anti-inflammatory activity of the I. paraguariensis extracts
obtained from supercritical extraction was evaluated using twenty
four animals, which were equally divided into two groups, control
and treated. All the animals were submitted to blood collection
(orbital plexus) the acute induced-peritonitis assay by the injection
of 10 mL of oyster glycogen (1%) dissolved in of phosphate-
buffered saline (PBS) 10 mM, 7.4 pH under light Zoletil 50®
anesthesia (Oktar et al., 2004). For the treated group, it was
administered a single dose of 500 mg/kg of the mate extract diluted
in sunflower oil through subcutaneous injection, while the control
group received the vehicle only (sunflower oil). 4 h later, rats were
re-anesthetized for blood collection (orbital plexus), and then
sacrificed in CO2 chamber, with cells in the peritoneum removed by
washing with 30 mL of PBS containing 1000 U.L-1 heparin. The
peritoneal exudates at 4 h contained >98% neutrophils. The
suspension was centrifuged at 2000 rpm for 10 min and the
erytrocytes were destroyed by lysis buffer containing 0.15 M
ammonium chloride (NH4Cl). Cells were re-suspended in ice-cold
PBS, and the cell counting was performed using a light microscope.
None of the treatments altered the cell viability, which was >95%.
The assays were carried out in duplicate.
Statistical analysis
The values were expressed as the mean value ± standard error.
One-way analysis of variance (ANOVA) followed by Tukey test
applied for the statistical evaluations of the results obtained in the
anti-inflammatory tests, while for the acute toxicity tests it was
applied the ANOVA followed by the Kruskal-Wallis test. Values of
p<0.05 were regarded as significant.
RESULTS AND DISCUSSION
Chemical composition of the extracts
In this work the extraction yield (defined as the weight
percentage of the extract obtained with respect to the
initial charge of the raw material in the extractor) was
about 2.25 wt%, which is of the same order of magnitude
to that found by Jacques et al. (2007a). The main
compounds identified in the supercritical extracts of I.
paraguariensis were (as expressed in terms of percent of
normalized peak area): caffeine 35.5%, theobromine
0.3%, phytol 1.7%, eicosane 11.9%, squalene 3.9%,
pentatriacontane 0.9%, vitamin-E 8.4%, and stigmasterol
1.5%. Other compounds (6 hydrocarbons, 2 aldehydes, 4
alcohols, 1 ketone and 2 aromatic) were also identified by
matching their spectra with those of the Wiley library, but
attempts to check them were not pursued due to the low
confidence level indicated by the chemical analysis. A
similar chemical profile of the supercritical mate extracts
was also obtained by Cansian et al. (2008a) and
Esmelindro et al. (2005). Jacques et al. (2007a) reported
the presence of 51 compounds in the mate extracts
obtained by supercritical CO2 extraction at 250 bar and
40°C.
Acute toxicity evaluation
The acute toxicity of the supercritical extracts of I.
paraguariensis was performed in wistar rats treated with
a unique dose of 500 mg/kg. Neither mortalities nor
significant changes in the general behavior or other
physiological activity were observed during the monitored
period of the animals. In addition, the food and water
consumption was not altered during the evaluation.
These results indicate that the supercritical extracts of I.
paraguariensis did not produce any toxic symptoms on
rats at the concentration level employed in the assays.
After 14 days treatment, the animals were sacrificed
and their livers and kidneys were analyzed. Pathological
examinations of the tissue indicated the existence of
detectable abnormalities. Figure 1 presents the histo-
logical changes in the livers (1a) and kidneys (1b) of the
animals tested. The liver cells of the treated animals
showed considerable increase in the cells with hyper-
chromatism, vacuolization and tumefaction in comparison
with the control group (Figure 1a). The cellular alteration
in the hepatic tissue can be better visualized in Figure 2
that presents the photo-micrographs of the sections of
the livers (magnification of 100 times). It is shown that the
supercritical extracts of I. paraguariensis in a single dose
of 250 mg/kg caused several alterations in this organ of
the treated animals compared with the control group
Pasquali et al. 1165
a
Figure 1. a, Effect of acute toxicity of supercritical I. paraguariensis extracts on histopathological parameters of liver; b, kidneys
tissues of rats. C, control; T, treated. *p<0.007 vs control.
(Figure 2a) as the presence of hyperchromatism and
vacuolization (Figure 2b) tumefaction and hepatocytes
with hyperchromatism (Figure 2c) suggests the presence
of hepatocellular necrosis.
The histological analysis of the renal tissue revealed an
increase of cells with tumefaction for the treated group,
1166 Afr. J. Pharm. Pharmacol.
(a)
(b)
(c)
Figure 2. a, Photomicrographs of the sections of the livers showing
normal features in control group; b, the liver of animals treated with a
single dose of 250 mg/kg of supercritical I. paraguariensis extracts
after 14 days showing the presence of hyperchromatism (black arrow)
and vacuolization (white arrow); c, the presence of tumefaction
(double black arrow) and hepatocytes with hyperchromatism (single
black arrow). CV, centrolobular vein. (Hematoxylin-eosin, 100x).
while the cells with vacuolization decreased for this
group. There are no signals of cells with hyper-
chromatism for both control and treated groups (Figure
1b). The cellular alteration in the renal tissue can be
better visualized in Figure 3 through photomicrographs of
the sections of the kidneys (magnification of 100 times).
The supercritical extracts of I. paraguariensis caused an
increase in the cellular tumefaction in the cortical region
of the kidneys of treated group (Figure 3b) when
compared with control group (Figure 3a), which suggests
the presence of renal necrosis. It was not verified the
presence of cells with hyperchromatism and vacuo-
lization, possibly due to the advanced necrosis stage of
the renal tissue.
Anti-inflammatory evaluation
Initially, the anti-inflammatory activity of the supercritical
extracts of I. paraguariensis on acute peritonitis induced
by oyster glycogen was investigated in a single dose of
250 mg/kg, but no significant differences were verified in
the cell recruitment. However, it was verified the
tendency to inhibit the inflammatory process. Then it was
decided to inject a single dose of 500 mg/kg. The results
obtained are presented at Figure 4.
The animals presented a level of 7.5 leukocytes/mm3
before inflammatory induction, with no variation verified
compared to the treated group after induction with
supercritical extracts of I. paraguariensis, while the
control group decreased the level of leukocytes in the
circulating blood. The statistical analysis of the results
indicated significant differences (p<0.001) among the
treated and control groups for the number leukocytes in
the circulating blood. The mobilization of leukocytes of
the bone marrow to the circulating blood caused a
decrease in the leukocytes recruitment, which demon-
strates an effective decrease in the acute inflammatory
process. The number of total leukocytes observed for the
treated group was very similar to that obtained for the
animals before the induced-inflammatory process. This
result suggests the occurrence of inhibition of the total
leukocytes recruitment in the circulating blood after the
induced-inflammatory process by the supercritical
extracts of I. paraguariensis in a single dose of 500
mg/kg.
The induced-inflammation resulted in an increase of
approximately 2000 segmented neutrophils/mm3 in the
circulating blood for all the animals. For the treated group
with supercritical extracts of I. paraguariensis it was
verified a reduction in the levels of segmented neutrophils
compared with the control group. Such reduction of
neutrophils number in the circulating blood demonstrates
the anti-inflammatory activity of the supercritical extracts
of I. paraguariensis, as an increase in the neutrophils in
the circulating blood is an indicative of inflammatory
process, since these cells are the first defense
mechanism of the body (Male, 2003).
Pasquali et al. 1167
(a)
(b)
Figure 3. a, Photomicrographs of the sections of the kidneys showing normal features in control group; b, the kidneys of animals
treated with a single dose of 250 mg/kg of supercritical I. paraguariensis extracts after 14 days showing the presence of cellular
tumefaction in the cortical region (double black arrow). G, glomerullus; TCD, distal convoluted tubules; TCP, proximal
convoluted tubules. (Hematoxylin-eosin, 100x)
Total leukocytes Neutrophils
Lymphocytes Monocytes
Figure 4. Effect of the supercritical I. paraguariensis extracts on the cell recruitment of the circulating blood. *p<0.001 and
**p<0.05 vs control. Non-inflamed refers to the animals before the induction of inflammation; control refers to the inflamed
animals that have received only the vehicle; treated refers to inflamed animals that have received the extract.
1168 Afr. J. Pharm. Pharmacol.
The mean levels of lymphocytes in the circulating blood
was around 7000 lymphocytes/mm3 for the treated group
with supercritical extracts of I. paraguariensis, which was
very similar to that obtained for the animals before the
induced-inflammation. The control group showed around
2000 lymphocytes/mm3 in the circulating blood after the
induced-inflammation process.
The mean levels of monocytes in the circulating blood
was around 250 monocytes/mm3 for both treated and
control group, which is similar to the value obtained for
the animals before the induced-inflammation. This
occurred due to the fact that the blood was collected 4h
before the induced-inflammation, where the monocyte
level did not change, as previously reported by Male
(2003).
In this work it was verified an increase in the number of
segmented neutrophils in the control group (this group
received sunflower oil only), which indicates that the
experimental model used in this study to evaluate the cell
recruitment was applicable and effective. The segmented
neutrophils are responsible for the initial defense of the
body subjected to an inflammatory process, which
confirms the existence of an inflammation caused by the
oyster glycogen.
The number of cells migrated to the peritoneum of the
treated animals was equivalent to that of control animals
(8760±3301 cells/mm3 for treated and 6280±1647
cells/mm3 for control).
Some of the pharmacological activities of I.
paraguariensis are attributed to the high content of
caffeyol-derivatives and flavonoids (Filip et al., 2001).
Among several important biological activities exerted by
flavonoids, it may be important to highlight its inhibitory
effect on the enzyme systems involved in the initiation
and maintenance of the inflammatory and immune
response (Gorgen et al., 2005). In fact, it has been shown
that I. paraguariensis presents a higher content of
flavonoids, such as quercetine, when compared to other
assayed species (Filip et al., 2001). Although the
flavonoids content was not determined in the supercritical
extracts of I. paraguariensis obtained in this work, based
on literature results (Jacques et al., 2007a Jacques et al.,
2008, Martínez et al., 1997), it seems reasonable to
believe that this class of compounds was also extracted
in the process. In this regard, the anti-inflammatory effect
of the supercritical extracts of I. paraguariensis could be
associated with the flavonoids content.
Conclusions
Results obtained in this work suggest the incidence of
acute toxicity due to the presence of cells with
hyperchromatism, vacuolization and tumefaction in the
livers and the presence of cells with tumefaction in the
cortical region of the kidneys of the treated group.
Toxicity studies in experimental animals may not however
be directly extrapolated to humans since a reasonable
estimate of the self-administered dose may be difficult to
make. Besides, in view of the widespread use of
supercritical extracts of I. paraguariensis, additional
clinical toxicological evaluations need to be performed to
determine a safe dose and protect the population from
possible toxic effects. Regarding the anti-inflammatory
effect of the supercritical extracts of I. paraguariensis, it
was verified that 500 mg/kg inhibited the neutrophil
recruitment in the circulating blood. It is well known that
numerous medicinal plants present significant anti-
inflammatory activities, evaluated in different models, and
several active metabolites which are responsible for
these actions have been identified. Therefore, it is
believed that results found in the present work may be
very promising since they open new perspectives for the
therapeutic use of supercritical extracts of I.
paraguariensis (and other plants) or its preparations as
an anti-inflammatory agents. Further studies are under-
way within our working group to identify the active
compounds responsible for the reported anti-
inflammatory activity as well as to elucidate their
mechanisms of action.
ACKNOWLEDGEMENTS
The authors thank CNPq and CAPES for the financial
support and scholarships.
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... The results also indicate the potential positive effect of this extract on the cardiovascular system [21]. Some of the pharmacological effects of I. paraguariensis are associated with a high content of caffeic acid (and its derivatives), flavonoids, and hydroxylated derivatives of cinnamic acid, which have antioxidant and anti-inflammatory activities [22]. Among the biological activities of Yerba Mate, it is essential to highlight its inhibitory effect on the enzymes involved in the initiation and maintenance of the inflammatory response [23]. ...
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... Ilex paraguariensis is increasingly exported to Europe, the United States, and Japan where it is sold ground or as an extract used in herbal medicines as well as active additives for various foods. According to the Argentine Food Code, yerba mate is defined as a product exclusively consisting of dried, slightly roasted, and ground I. paraguariensis leaves, which can contain fragments of young branches, pedicels, and stalks (Pasquali et al., 2011;Cogoi et al., 2013). Dried yerba mate, also with leaves and green stems, is used as a green dye in the classic "Chimarrao", "Mate", and "Terere" in powdered form to make teas and as an essence for various industrial purposes (Cansian et al., 2008a). ...
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The lipoid-fraction of the surface-wax of young leaves of Ilex aquifolium, mainly consists of alkanes and esters of long-chain fatty acids. One series of pentyl-esters, and two series of hexyl-esters were found. Since the esters disappear as the leaves grow mature, they can be regarded as leaf expansion markers. It seems possible that the alkanes are synthesized from these fatty acid-esters, as they replace them in older leaves.
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Leaf flavonoids of 59 Ilex species were analysed. Three flavonols: kaempferol, quercetin and isorhamnetin, and two flavones: apigenin and luteolin, were identified. This is the first report of isorhamnetin and flavones in the genus Ilex. The possible phylogenetic significance of the compounds found as well as the taxonomic value of the flavones in certain species are discussed.
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Extraction of Ilex dumosa leaves, a material frequently used for the adulteration of erva-maté, resulted in the isolation of 3-O-β-d-glucuronopyranosyl-28-O-β-d-glucopyranosyl-3β-hydroxyolean-12-en-28-oic acid, 3-O-β-d-6-O-methylglucuronopyranosyl-28-O-β-d-glucopyranosyl-3β-hydroxyolean-12-en-28-oic acid, and three new saponins:  3-O-[β-d-glucopyranosyl-(1−2)-β-d-galactopyranosyl]-28-O-β-d-glucopyranosyl-3β,29-dihydroxyolean-12-en-28-oic acid, 3-O-[α-l-arabinopyranosyl-(1−2)-α-l-arabinopyranosyl]-28-O-β-d-glucopyranosyl-3β-hydroxyolean-12-en-28-oic acid, 3-O-β-d-galactopyranosyl-28-O-β-d-glucopyranosyl-3β-hydroxyolean-12-en-28-oic acid. None of these saponins are present in the leaves of Ilex paraguariensis (genuine erva-maté). Keywords: Ilex paraguariensis; Ilex dumosa; erva-maté; saponins
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This work presents a study concerning the chemical characteristics and analytical separation process of the essential oil obtained from high-pressure carbon dioxide extraction of Ilex paraguariensis. The experiments were performed in a laboratory-scale unit in the temperature range of 20–40 °C, from 100 to 250 bar. A blend of the I. paraguariensis extracts was percolated through a preparative chromatographic column, containing silica gel, and successively eluted with 150 mL of each of the following solvents: hexane, toluene, dichloromethane, ethyl acetate, acetone and methanol. The raw extract and its fractions were analyzed by gas chromatography coupled to mass spectrometry detection (GC/MS). The fractionation procedure showed to be a good clean up technique due to the isolation of different classes of compounds in each fraction. Chromatographic analyses allowed the identification of caffeine, fatty acids and esters, phytol, squalene, Vitamin E, stigmasterol derivatives and saturated hydrocarbons.
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This work reports the effects of industrial manufacturing steps on the distribution of chemical components of the extracts obtained from supercritical carbon dioxide (SCCO2) of processed mate tea leaves. For this purpose, samples of mate tea leaves were collected from the actual industrial processing after the fast pre-heating, drying and storage (5 and 21 days) steps. To provide a consistent basis for comparisons, extractions were also conducted for the unprocessed leaves fed in the industrial environment. In order to select the appropriate extraction parameters at high pressure, it was evaluated the effects of temperature from 15 to 55 °C and pressure from 10 to 20 MPa on the liquid yield of unprocessed mate tea leaves. The SCCO2 extraction experiments regarding the effects of industrial processing conditions were then performed at 35 °C and 20 MPa and the extract chemical analyses were carried out in a GC/MSD. Results show that the extraction yield and chemical composition of the extracts are strongly affected by the industrial processing steps. Chromatographic analyses permitted the identification of caffeine, theobromine, hexadecanoic acid, phytol, squalene, octacosanol, 2-heptacosanone, steroids and triterpenes as the main constituents in the extracts.
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A survey of 27 plants of Ilex and Euonymus revealed that the distribution of anthocyanins and cinnamic acid esters in their fruits is correlated with accepted taxonomic classification. In the skin of the fruit, the 3-xylosylglucoside of cyanidin and pelargonidin and the 3-monoglucoside of cyanidin were identified, and the hydrolysed fruit-extracts were found to contain quercetin, kaempferol and caffeic acid. The genus Ilex has been shown to be distinguishable from the genus Euonymus by their anthocyanins; I. micrococca was exceptional in having only chrysanthemin. Additionally, chlorogenic and isochlorogenic acids and caffeylglucose occur in Ilex but not in Euonymus. The microspectrophotometric examination of the pigment cells of the black- and red-Ilex fruits revealed that the position of absorption maxima in the visible region is mainly related to the relative amounts of anthocyanin and flavonol present.