The flavonoid content and antiproliferative, hypoglycaemic, anti-inflammatory and free radical scavenging activities of Annona dioica St. Hill.

0.24 MB·English

by  Formagio, Anelise S N

Formagioetal.BMCComplementaryandAlternativeMedicine2013,13:14
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RESEARCH ARTICLE Open Access
The flavonoid content and antiproliferative,
hypoglycaemic, anti-inflammatory and free radical
Annona dioica
scavenging activities of St. Hill
Anelise S N Formagio1*, Candida A L Kassuya2, Frederico Formagio Neto2, Carla R F Volobuff3, Edna K K Iriguchi2,
Maria do C Vieira1 and Mary Ann Foglio4
Abstract
Background: Annona dioicaSt.Hill (Annonacaeae) is a Brazilian plant used infolk medicine for the treatment of
several types ofrheumatisms and diarrhoea.The focus of this work was to evaluate the in vitro antiproliferativeand
antioxidant activityand thein vivo hypoglycaemic and anti-inflammatory activity of A.dioicaand identify the
principal constituents of this plant.
Methods: The crude methanol extract (EAD) and hexane (HF), chloroform (CF), ethylacetate(EAF) and
hydromethanol fractions (HMF) were evaluated for free radical scavenging activityusing theDPPH assay. The EAD
and EAF were assayed for hypoglycaemic activityin rats. The EAD was testedin an antiproliferation assay and for
anti-inflammatoryeffects in paw oedema,inadditionto myeloperoxidase activityinduced bycarrageenan(Cg) in
mice. The EAF was assayed using chromatographic methods.
Results: The fractionation of theEAF through chromatographic methods identifiedderivativesof theflavonoids
quercetin and kaempferol. Among all the testedfractions, theethyl acetate and hydromethanol fractions were the
most potent, exhibiting an IC of8.53 and 10.57 μg/mL, respectively, which is comparableto that of the
50
commercial antioxidant butylated hydroxytoluene(BHT). The oral administration oftheEAD (100 mg/kg) and EAF
(15 mg/kg) inhibited theincreaseof glucose levels, resulting in a hypoglycaemic effect. The EAD(30 to 300mg/kg)
exhibited an anti-oedematogenic effect in Cg-induced paw oedema in a time- and dose-dependentmanner.The
results showed a reduction of MPO activity byA. dioica6h after theinduction of paw oedema at alldoses tested
withmaximalinhibition at300 mg/kg.
Conclusions: Our results reveal for thefirst time that compounds containedin theA. dioicaleaves exert anti-
inflammatory, hypoglycaemic, antiproliferative, and antioxidant effects. The antioxidant activitymay be associated
withthepresence of flavonoids.
Keywords: Annona dioica,Flavonoids, Antioxidant, Hypoglycaemic,Antiproliferative, Anti-inflammatory
Background Annona dioica St. Hill. (Annonaceae) is a shrub found
Plants have been used as a source of new medicinal in Brazil throughout the states of São Paulo, Minas Ger-
compounds throughout history and continue to serve as ais, Paraná and Mato Grosso. It is commonly referred to
the basis for many of the pharmaceuticals used today as “ceraticum”, “arixicum” and “ariticum”. In Brazil, the
[1]. Standard experimental scientific methods are useful fruits and leaves of this plant are used to treat rheuma-
for the validation of ethnopharmacological knowledge tism, and the seeds are used to treat diarrhoea [2]. In
regardingherbalmedicine. Paraguay, the leaves are used to make tea or are gargled
as an anti-catarrhal, while the edible fruits possess seda-
tive properties. The seeds are used as insecticides or in
*Correspondence:[email protected]
1FaculdadedeCiênciasAgrárias,UniversidadeFederaldaGrandeDourados, the treatment of parasitic infections of the skin [3].
Dourados,MS,Brazil Chemical studies of the wood of A. dioica resulted in
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the isolation and characterisation of six alkaloids [4], over anhydrous sodium sulphate and concentrated
while analysis of the leaves identified four flavonoids [5]. under reduced pressure at a temperature not exceeding
The cytotoxic activities of the flavonoids 3-O-[30,60-di-O- 35°C. This resulted in the n-hexane (HF, 3.8 g), chloro-
p-hydroxycinnamoyl]-β-galactopyranosyl-kaempferol, 60- form (CF, 1.6 g) ethyl acetate (EAF, 6.2 g), and hydro-
O-p-hydroxycinnamoyl-β-galactopyranosyl-kaempferol and methanol (HMF, 4.6 g) fractions. The EAF (3.2 g) was
theflavonoidrichfractionofA.dioicawereassayedinvitro fractionated on Sephadex LH-20 using H O, H O:
2 2
againstmurineEhrlichcarcinomaandwereshownto have MeOH (75:25,50:50, 25:75),and MeOHas thesolvents
significant antiproliferative effects [5]. The extracts of to give ten sub-fractions (AD-1 to AD-10). The purifi-
A. dioica Stem bark, heartwood subterranean, stem cation of sub-fraction AD-3 (505.2 mg) on Sephadex
bark subterranean and of leaves were assayed in vitro LH-20 using H O, H O: MeOH (75:25, 50:50, 25:75),
2 2
against HCT-8 (human colon carcinoma), SF-295 and MeOH as the solvent yielded quercetin-3-O-galacto-
(glioblastoma) and MDA-MB-435 (melanome) tumor side (hyperoside) (32.4 mg) and mixtureof quercetin-3-O-
cell presented high cytotoxic activity, over 75% [6]. galactosideandkaempferol3-O-galactoside(42.6mg).
The data indicate the importance of the continuity of
phytochemical studies and especially in the evaluation Identificationoftheisolatedcompounds
against several human cancer cell lines, in addition other The isolated compounds were identified analysis of their
biologicalactivities. NMR data. The NMR measurements were carried out
Flavonoids are naturally occurring phenolic compounds on aVarian Mercury Plus BB spectrometer operating at
that are found in plants and are commonly consumed in 300 MHz for 1H and 75.5 MHz for 13C using CD OD as
3
the human diet [7]. Some flavonoids have been shown to the solvent and tetramethylsilane (TMS) as the internal
display a number of interesting biological activities, such standard.
as antioxidant [8,9], antiviral, antifungal [10], anticancer- The structures of quercetin 3-O-β-galactoside and the
ous [11,12], anti-angiogenic [13] and anti-inflammatory mixture of quercetin 3-O-β-galactoside and kaempferol-
activities[14,15]. 3-O-β-galactoside were elucidated using 1D and 2D
The traditional use of medicinal plants is common in NMR spectral data and by comparing their 1H and 13C
Brazil as an alternative to primary health care. Several NMRdata with data reported intheliterature[16,17].
plants, including Annona species, are used in Brazilian Quercetin 3-O-β-galactoside: 1H NMR δ (300 MHz,
H
folk medicine. However, these plants are typically used CD OD): 6.20 (1H, d, J=2.1Hz), 6.39 (1H, d, J=2.1Hz,),
3
without considering the toxicity and pharmacological 7.66 (1H, dd, J=8.7 e 2.4Hz,), 6.80 (1H, d, J=8.7Hz),
aspects. Thus, it is essential to confirm the activity of this 7.51 (1H, d, J=2.4Hz,), 5.37 (1H, d, J=7.8Hz,); 13C
plant, especially the anti-inflammatory, hypoglycaemic NMR δ (75,5 MHz, CD OD): 177.7, 164.4, 161.5, 156.5,
c 3
and antiproliferative effects, in addition to identifying the 156.3, 148.7, 145.1, 133.7, 122.2, 121.1, 116.2, 115.3,
specific mechanisms of action. In the present study, we 102.0,104.2,98.9,93.7, 76.1,73.4,71.4, 68.2,60.4.
evaluated the free radical scavenging, antiproliferative, Kaempferol-3-O-β-galactoside:1HNMRδ (300MHz,
H
hypoglycaemicandanti-inflammatoryactivityofthecrude CD OD): 6.19 (1H, d, J=2.1Hz), 6.42 (1H, d, J=2.1Hz,),
3
methanol extract (EAD) and fractions of A. dioica and 8.06 (2H, d, J=8.7Hz,), 6.85 (2H, d, J=8.7Hz), 5.39 (1H,
identifiedtheflavonoidsfoundintheleavesofthisplant. d, J=7.8Hz). RMN 13C δ (75,5 MHz, CD OD): 177.8,
c 3
164.4, 161.5, 160.2, 156.6, 156.5, 133.4, 131.3, 131,3,
Methods 121.3, 115.4, 115.3, 104.2, 101.9, 98.9, 93.9, 76.1, 73.4,
Plantmaterial 71.4,68.2,60.4.
The leaves of A. dioica were collected in May 2009 in
Dourados, in the state of Mato Grosso do Sul, Brazil. A Determinationoftotalphenolcontent
voucher specimen (DDMS 4598) was deposited at the The total phenolic content in the EAD was determined
Herbarium of the Federal University of Grande Dourados, using the Folin-Ciocalteu method [18]. Specifically, 100
Dourados,MS,Brazil. μLofEADinmethanol(1g/L)wasmixedwith1.0mLof
’
distilledwaterand0.5mLofFolin-Ciocaleus(1:10v/v)re-
Extraction,fractionationandisolationprocedure agent. After 3 min, 1.5 mL of a saturated solution of
The air-dried and powdered aerial parts of A. dioica Na CO (2%) was added. After 30 min, the absorbance
2 3
(850 g) were successively extracted via maceration with was measuredat 765 nm usinga spectrophotometer.The
methanol. The extract was filtered and concentrated in a quantification was carried out using a standard curve of
vacuum to obtain dry, crude methanol extract (EAD) gallic acid prepared in 80% methanol, and the results are
(24.5 g). A portion of this extract (17.8 g) was dissolved expressed in milligrams of gallic acid equivalent per gram
in MeOH:H O (1:1) and partitioned with n-hexane, of extract. The equation for the gallic acid curve was y=
2
chloroform and ethyl acetate. Each extract was dried 6.8502x+0.0148, with a correlation coefficient of R=
Formagioetal.BMCComplementaryandAlternativeMedicine2013,13:14 Page3of8
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0.9946.Themethanolsolutionwasusedasablank.Allof accordance with the guidelines of the U.S. National In-
theassayswerecarriedoutintriplicate. stitute of Health and were approved by the ethics com-
mittee on laboratory animal use of the Centro
Determinationoftotalflavonoids Universitário da Grande Dourados (UNIGRAN) (Nbr.
To determine the level of flavonoids, 500 μL of EAD 118/2010).
wasmixed with 1.50 mLof95%ethanol, 0.10 mLof10%
aluminiumchloride(AlCl .6H O),0.10mLofacetateso- Theoralglucosetolerancetestinnon-diabeticrats
3 2
dium (NaC H O .3H O) (1 M) and 2.80 mL of distilled Thisexperimentwasdesignedtoevaluatethehypoglycaemic
2 3 2 2
water. The tubes were kept at room temperature for 40 potential of the EAD and EAF in normal rats using the
min. The optical density was measured at 415 nm using method described by Al-Awadi et al. (1985) [21]. The
a spectrophotometer. The same procedure was used for rats were orally treated daily for 5 d with crude metha-
the analysis of the blank [19]. To calculate the concen- nol extract (EAD, 100 mg/kg) or the ethyl acetate frac-
tration of flavonoids, we prepared a calibration curve tion (EAF, 15 mg/kg). The reference drug, metformin
(2.5, 5.0, 10.0, 20.0, 25.0, 50.0, 100.0 and 125.0 μg) using (MET, 300 mg/kg), was also administered orally to the
quercetin as the standard. We then used these data to rats once a day. A separate control group of animals
generate a linear regression model, and the line equation were orally administered the vehicle (saline plus tween
was obtained and used for the calculation of the experi- 80 0.5%).
mental samples. The results are expressed in milligrams Theoralglucosetolerancetest(OGTT)fornon-diabetic
of quercetin equivalents per gram of extract. The equa- ratswasperformedaccordingtothestandardmethod[22].
tion of the quercetin curve was y=12.341x+0.009, with Briefly,allgroupswereselectedfortheOGTtestafterfood
a correlation coefficient of R=0.9977. All of the assays deprivation for 16 h. The baseline (B) glucose level was
W
were carriedout intriplicate. measuredusing aglucometer(Accuchek Performa)prior
toglucoseadministration(2g/kgbodyweight).
DPPHfreeradicalscavengingassay After measuring the glucose baseline, each group
The free radical scavenging activities of the crude metha- received the specified oral treatment. Serum glucose in a
nol extract,and thehexane, chloroform, ethyl acetate and blood sample from the tail vein was measured using a
hydromethanol fractions were determined using the 1,1- glucometer at 60 min. The data are expressed as the
diphenyl-1-picrylhydrazyl free radical (DPPH) method means±standard error of mean (SEM). Statistical com-
[20].Variousconcentrationsofthesampleswereaddedto parisons were performed using a one-way ANOVA fol-
2 mL of a methanol DPPH solution (0.1 mM) that was lowed by the Student-Newman-Keuls test, and the
prepared daily. The mixture was shaken and left to stand differences were considered statistically significant when
at room temperature in the dark. After 30 min, the ab- P<0.05. All statistical calculations and graphs were pre-
sorbance was measured at 517 nm against a blank con- pared using GraphPad Prism version 5.0 for Windows
taining all of the reagents except for the test samples. All (GraphPadSoftware,SanDiego, CA,USA).
ofthe assays were carried out intriplicate.BHT wasused
as the positive control. The IC (the concentration Carrageenan-inducedpawoedemainmice
50
requiredfor50%inhibitionofDPPH)wascalculatedusing Five groups of mice (n=6), were orally treated (p.o.) with
thegraphof%I(inhibitionpercentage)versustheextract the EAD (30–300 mg/kg). A separate control group was
concentration in mg/mL. The percentage of DPPH inhib- orallyadministeredthevehicle(salineplustween800.5%).
ition (% I) was calculated using the following equation: % Another group of mice was treated subcutaneously with
I=(A - A/A ) x 100, where A is the absorbance of the anti-inflammatory drug dexamethasone (1 mg/kg).
0 0 0
DPPH (control), and A is the absorbance of the sample After 1 h, the animals received a 50-μl subcutaneous (s.c.)
withDPPH. injection of Cg (300 μg/paw) dissolved in sterile 0.9%
saline in the right hindpaw. The contralateral paw
Animals was injected with saline and used as the control. The
The experiments were conducted using 30 male Wistar thickness of paw oedema was measured using a digital
rats (150–230 g) and 30 male Swiss mice (25–35 g) pro- micrometer [23] 1 h before any treatment and at several
vided by the Universidade Federal da Grande Dourados time points (1, 2, and 4 h) after the injection of Cg. The
(UFGD). The animals were maintained under a 12-h resultsareexpressedinμm,andthedifferencebetweenthe
light–dark cycle, with controlled humidity (60–80%) and basalandpost-injectionvaluesarequantifiedasoedema.
temperature (22±1°C). The animals were acclimatised
to the experimentation room for at least 2 h before test- Determinationofmyeloperoxidase(MPO)activity
ing and were used only once throughout the experi- To investigate whether oral treatment with EAD (30 and
ments. All experimental procedures were carried out in 300 mg/kg) or vehicle could affect the cellular migration
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induced by Cg, the myeloperoxidase activity was mea- Chemicals
sured. Animals were euthanised 6 h after Cg injection, 1,1-Diphenyl-2-picrylhydrazyl(DPPH),butylatedhydroxy-
asdescribedpreviously[24].ForMPOactivity,thetissue toluene(BHT)λ-carrageenan(Cg),Tween80,dexametha-
was homogenised in 5% (w/v) of 80 mM phosphate buf- sone,trichloroaceticacidanddoxorubicinwerepurchased
fer (pH 5.4) containing 0.5% hexadecyltrimethylammo- from Sigma Chemical Co. (MO, USA). Analytical-grade
nium bromide. The homogenate was centrifuged at 3200 methanol,hexane,anhydroussodiumsulphateandDMSO
rpm and 4°C for 20 min. Aliquots (30 μl) of each super- wereobtainedfromVetec(RJ,Brazil).
natantweremixedwith100μlof80mMphosphatebuf-
fer, 85 μl of 0.22 M phosphate buffer and 15 μl of Results and discussion
0.017% H O on a 96-well plate. The reaction was trig- The free radical scavenging activity was initially evaluated
2 2
gered with 20 μl of 3,3,3-tetramethylbenzidine (dissolved for the crude methanol extract of A. dioica (EAD).
in N,N-dimethylformamide). The plate was kept at 37°C Theresults(Table1)showthattheEADpossessessignifi-
for 3 min, after which the reaction was stopped by add- cant free radical scavenging activity, with an IC of
50
ing 30 μl of 1.46 M sodium acetate, pH 3.0. The enzym- 17.84 μg/mL. The fractionation of this extract by solvent
atic activity was determined by measuring the optical partition yielded the hexane (HF), chloroform (CF), ethyl
density at 630 nm and is expressed as the mOD per acetate(EAF),andhydromethanol(HMF)fractions,which
milligramofprotein. weretestedfor their antioxidantabilitytowardthe radical
DPPH. A comparison of the obtained IC data (Table 1)
50
Antiproliferativeassay indicated potent activity for the EAF and HMF fractions,
The National Cancer Institute, Frederick MA/USA, withIC valuesof8.53μg/mLand10.53μg/mL,respect-
50
kindly provided nine human cancer cell lines: U251 ively, which was comparable to that of the commercial
(glioma, CNS), UACC-62 (melanoma), MCF-7 (breast), antioxidantBHT.
NCI-ADR/RES (ovarian expressing phenotype multiple After determination of the antioxidant potential, we
drug resistance), 786–0 (renal), NCI-H460 (lung, non- determined the levels of total phenols and flavonoids in
small cells), PC-3 (prostate), OVCAR-03 (ovarian), k-562 the crude methanol extract. The EAD had high levels of
(leukaemia) and HT29 (colon). VERO (green monkey kid- total phenols and flavonoids with values of 187.77 mg/g
neycells),a normal cell line, was also used. The stock and and 733.20 mg/g, respectively. The total polyphenol and
experimentalculturesweregrowninmediumcontaining5 radical scavenging activity have been determined in
mL of RPMI 1640 (GIBCO BRL) supplemented with 5% some fruits of the genus Annona, namely A. muricata
foetalbovineserum(GIBCOBRL). [26], A.cherimolia[27]andA.squamosa[28].
The stock cultures were grown in 5 mL of RPMI-1640 Flavonoidsareveryimportantforthetreatmentofmany
(GIBCOBRL)supplementedwith5%foetal bovineserum diseases due to their potent antioxidant nature. Some
(FBS, GIBCO). A penicillin:streptomycin mixture (1000 authorshavereportedthatflavonoidssuchasrutin(quer-
U/mL:1000μg/mL,1mL/LRPMI,Nutricel)wasaddedto cetin-3-rutinoside) and quercetin show antioxidant activ-
theexperimentalcultures. ity [14,15]. The antioxidant or free radical scavenging
The cells were plated in 96-well plates (100 μL cells/ activity of flavonoids has been related to the number and
well) and exposed to different concentrations of the position of free hydroxyl groups, which could be a result
EAD (0.25, 2.5, 25 and 250 μg/mL) in DMSO/RPMI oftheirhydrogendonatingability[29,30].Theantioxidant
(0.1% v/v) at 37°C and 5% CO for 48 h. The final activity and radical-scavenging activity of flavonoids de-
2
DMSO concentration did not affect cell viability. The pendhighlyontheirstructure,especiallythepresenceofa
cells were then fixed with a trichloroacetic acid solution
(50%, v/v), and cell proliferation was determined via
Table1DPPHfreeradicalscavengingactivity(IC )for
spectrophotometric quantification (540 nm, Molecular 50
EAD,HF,CF,EAF,andHMFfractionsfromA.dioica
Devices Versa Max Microplate Reader) of the cellular
Sample IC μg/mL
protein content using a sulphorhodamine B assay [25]. 50
Doxorubicin (0.025-25 μg/mL) was used as a positive EAD 17.84
control.Threemeasurementswereobtained:firstattime HF 101.66
zero (To, at the beginning of incubation) and then 48 h CF 98.35
post-incubation for both the compound-free (C) and EAF 8.53
tested (T) cells. Cell proliferation was determined using
HMF 10.57
twhaeseoqbusaetriovend1w00hexn[(TT≥-TTo,o)w/Chi-leTao].cyAtoccyidtoasltaetfifcecetffoecc-t BHTa 16.8
curred when T<To. The experiments were performed maBeHtTha(bnuotlyelaxtteradcth);ydHrFox(hyetoxlauneenefr)awctaiosnu);seCdFa(cshploorsoitfiovremcofrnatcrtoiol;nE)A;EDA(Fcr(uedtheyl
intriplicate. acetatefraction);HMF(hydromethanolfraction).
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freeC-3–OH,afreeC-4–OH,adoublebondbetweenC-2 The criteria of the American National Cancer Institute
and C-3, and an O-dihydroxy group in the B-ring [31]. to consider a crude extract promising for further purifi-
The isolated flavonoids were a good match for these cation is a GI lower than 30 μg/mL [33]. A number of
50
requirements; the only exception is the absence of a free studies have demonstrated anticancer activity associated
C-3–OH. The antioxidant effects observed for EAD, EAF with flavonoids [34].
and HMF are most likely due the presence of quercetin Extracts from different species of the Annonaceae
and/orkaempferolandphenoliccompoundsbasedonthe familyhavebeen shown to be active. The strongest cyto-
DPPH free radical activity of these flavonoids reported in toxic activities were detected for the ethanol extracts of
theliterature[32]. theA.crassiflorarootbark[35].
The in vitro antiproliferative assay of the EAD was On the first day of oral treatment, EAF (15 mg/kg) as
evaluated against VERO and nine human cancer cell well as MET (300 mg/kg), but not EAD (Figure 1A) and
lines. Three response parameters (GI , TGI, and LC ) quercetin 3-β-galactoside (results not shown), inhibited
50 50
were calculated for the cell lines tested, and the results the increase in glucose levels with inhibitions of 16±2%
are summarised in Table 2. The GI value (growth in- and 13±2%, respectively (Figure 1A) (results not
50
hibitory activity) refers to the drug concentration that shown). On the fifth day, EAD (100 mg/kg), EAF (15
produces a 50% reduction in cellular growth when com- mg/kg), quercetin 3-β-galactoside (1.5 mg/kg; results not
pared to the untreated control cells. TheTGI (cytostatic shown) and MET (300 mg/kg) significantly reduced the
activity) concentration that produces 0% cell growth or glucose increase, with inhibitions of 22±4%, 14±3%,
cytostatic effect and the LC (cytotoxic activity) refer to 16±3% and 13±2%, respectively (Figure 1C). Together,
50
the drug concentrations necessary for total growth in- these results show that the extract from the leaves of
hibition andforkilling50%ofthe cells,respectively. A. dioica exhibited hypoglycaemic effects and that the
The results demonstrate that the EAD possesses antic- flavonoid derivatives quercetin and kaempferol present
anceractivitywithGI valuesintherangeof0.03–83.47 in EAD and EAF are responsible, at least in part, for the
50
μg/mL. Regarding the sensitivity against particular cell observed hypoglycaemic activity in non-diabetic rats.
lines, the EAD was particularly effective in NCI-H460 We cannot confirm that the flavonoids are the only
lung cells (GI 0.03 μg/mL), HT29 colon cells (GI 0.10 compounds present in the extract responsible for this
50 50
μg/mL and TGI 4.10 μg/mL), NCI/ADR-RES ovarian action because the EAD showed efficacy in relation to
expressing phenotype multiple drug resistance cells (GI allgroups,including themetformimgroup.
50
1.95 μg/mL), OVCAR-3 ovarian cells (GI 6.38 μg/mL) The potential anti-inflammatory or anti-oedema prop-
50
andMCF7breastcells(GI 11.10μg/mL).Lungcanceris erties of pharmacological substances in experimentally
50
among the most common types of cancer throughout the induced rodent models are assessed using the digital
world and shows only a modest response to the che- water plethysmometer or micrometer methodology. The
motherapeutic treatments available. For this type of can- micrometer method was used to measure acute paw
cer, monotherapy presents only a partial response in 15% oedema in order to screen for anti-inflammatory activity
to 20% of cases, and even with combined treatments, the [23]. The injection of Cg into the paw induced an
therapeuticeffectdoesnotexceed40%-50%. oedema that peaked at 2 h (Figure 2A). Oral treatment
Table2GI ,TGIandLC (μg/mL)forEADandDox
50 50
EAD Dox
Cancercelllines GI TGI LC GI TGI LC
50 50 50 50
U251(glioma) 83.47 5.07 23.45
UACC-62(melanoma) >100 0.06 0.86
MCF7(breast) 11.10 >100 0.14 24.52
NCI/ADR-RES(ovarianresistance) 1.95 1.74 23.92
786-0(renal) >100 0.22 1.51
NCI-H460(lung) 0.03 >100 0.05
OVCAR-3(ovarian) 6.38 0.30 1.72
HT29(colon) 0.10 4.10 >100 2.12 7.31 20.86
K-562(leukemia) 31.13 0.90
Vero 1.40 11.36
EAD(crudemethanolextract);dox(doxorubicin);GI (growthinhibitoryactivity);TGI(cytostaticactivity);LC (cytotoxicactivity);IC >100(μg/mL)was
50 50 50
considerednotactive.
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A Day 1 A
1000
140 #
ose 120 * m) 800
uc * m
matic gl(mg/dl)100 ema ( 600 * * * **
as 80 ed 400
Pl o
w
60 Pa 200
B V EAD EAF MET
100 15 300 mg/kg 0
B 1 hour after glucose load Vehicle EAD 30 EAD 100 EAD 300 a 1 m g/kg
140 ex
D
e #
s Time (h)
o 120
uc 2 hours after carrageenan injection (300µg/paw)
atic glmg/dl)100 B
m(
Plas 80 O.) 1.0
D.
60 y ( 0.8
B V EAD EAF MET vit
100 15 300 mg/kg cti
a 0.6
1 hour after glucose load se * *
a
d 0.4 **
C Day 5 xi
140 # ero 0.2 ***
p
se elo
Plasmatic gluco(mg/dl)11028000 *** * * My 0.0Vehicle EAD 30 EAD 100 EAD 30D0exa 1 m g/kg
60 Figure2Effectsofcrudemethanolextract(EAD)onoedema
B V EAD EAF MET formationandmyeloperoxidaseactivityinducedby
100 15 300 mg/kg carrageenaninmice.InA,theresultsofsingleoraltreatmentwith
vehicle(controlgroup),EAD(30,100and300mg/kg),and
1 hour after glucose load
dexamethasone(Dexa,1mg/kg,s.c.)inoedemaformationat2hs
Figure1Effectsofcrudemethanolextract(EAD),ethylacetate timepoint(A)andinmyeloperoxidaseactivityat6hsafter(B)
fraction(EAF)onincreasinginglucoseplasmaticlevelsin carrageenan(300mg/kg)inmice.Resultsareexpressesas mg/dL
normoglicemicrats.InA,B,andCtheresultsofdailytreatmentin comparedwithvehicle(V)vstreatedgroup.*P<0.05,**P<0,01,
day1,3and5withvehicle(controlgroup),EAD(100mg/kg),EAF ***P<0,0001,one-wayANOVAfollowedbyStudent-Newman-Keuls.
(15mg/kg),andmetformin(MET,300mg/kg)innomalrats.Results
areexpressesas mg/dLcomparedwithvehicle(V)vstreatedgroup.
EAD contains compounds that exert anti-oedematogenic
*P<0.05,***P<0,0001,one-wayANOVAfollowedbyStudent-
Newman-Keuls. properties.
The marked increase in the myeloperoxidase activity
(indirect evidence for neutrophil influx) into the paw
with EAD (30, 100 and 300 mg/kg) significantly inhib- indicated an inflammatory process induced by Cg, in-
ited oedema formation. The inhibitions for each dose stead of oedema formation only. The injection of Cg
were 39±8% (30 mg/kg), 32±6% (100 mg/kg), and 36± (300 μg/paw) increased MPO activity after 6 h, and oral
10% (300 mg/kg) (Figure 2A). In addition, the inhibition treatment with EAD (30, 100 and 300 mg/kg) inhibited
observed in the dexamethasone-treated group was also the increase in MPO activity induced by Cg. The
significant (Figure 2A). These results suggest that observed inhibitions were 42±6% (30 mg/kg), 42±9%
Formagioetal.BMCComplementaryandAlternativeMedicine2013,13:14 Page7of8
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antiproliferative,hypoglycaemic,anti-inflammatoryandfreeradical
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