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composed of flavonoids including tannins,
polyphenols,anthocyanidins, and oligomeric proanthocyanidins.
Apolysaccharide isolated and purified from T. indica seedsshowed
immunomodulatory activities, like the enhance-ment of phagocytosis
and inhibition of leucocyte migrationand cell proliferation,
suggesting possibly interesting bio-
logical applications of this plant (Sreelekha et al., 1993).A
recent study has shown the beneficial effects of the con-sumption
of T. indica fruit extract in an experimentalmodel of
atherosclerosis in hamsters, including decreasedlevels of serum
cholesterol and triglycerides (Martinelloet al., 2006).
Cardiovascular diseases (CVD) are the leading cause ofmorbidity
and mortality in developed countries (Libby,2002). Classic major
risk factors for CVD include hyperlip-idemia, elevated levels of
LDL cholesterol, decreased HDLcholesterol and smoking.
Atherosclerosis is an importantpathologic manifestation underlying
CVD, and has beensuggested to be an inflammatory disease (Ross,
1999). Data
from the literature indicate it to be a consequence of achronic
inflammatory process induced by the activationof macrophages, the
complement system (CS) and T-lym-phocytes. Several studies have
implicated elements of thehumoral-mediated immune response in
atherogenesis(Geertinger and Sorensen, 1973; Hollander et al.,
1979;Hansson et al., 1984; Vlaicu et al., 1985a,b). Among
them,activation of CS has been associated with the
pre-lesionalstages, as well as with the progress of
atherosclerosis(Torzewski et al., 1997).
The CS is a complex cascade of enzymes and regulatoryproteins
that normally participate in host defenses against
microorganisms through opsonization, chemoattraction
ofleucocytes, cell lysis and cell activation (Wallport,
2001).Several studies showed activated complement componentsin the
atherosclerotic plaque, as well as membrane attack,the complex
(MAC, C5b-9) that promotes cellular activa-tion, upregulates
adhesion molecules, stimulates chemo-kine secretion and can cause
cell lysis (Yasojima et al.,2001).
The potential activity of tamarind extracts on the CShad not
been previously investigated; it may be of interestfor application
in research and therapy.
The aim of the present study was to evaluate the effect ofExT on
the activity of the CS in vitro, and on complementactivity in an
experimental model of hyperlipidemia inhamsters submitted to a
cholesterol-enriched diet and trea-ted or not with ExT. Components
of the classical/lectin(CP/LP), or alternative pathways (AP) of the
CS (Wall-port, 2001) and complement lytic activity
wereinvestigated.
2. Materials and methods
2.1. Buffers
Complement fixation diluent (CFD) containing 0.1% gelatin, was
usedfor hemolytic assays of CP/LP, according to Harrison and
Lachamnn
(1986). Triethanolamine (TEA) 0.02 M buffer, pH 7.2,
containing
0.0005 M of azide, 0.08 M of ethyleneglycol-bis-(b-aminoethyl
ether)
N,N,N0,N0,-tetraacetic acid and 0.002 M magnesium
(TEA/EGTA/Mg2+),was used for the evaluation of AP activity.
Phosphate buffered saline(PBS) pH 7.4, maintained on ice, was used
to stop hemolytic reactions.
2.2. Plant material
Ripe fruits of Tamarindus indica were collected in the region
ofRibeirao Preto, SP, Brazil and peeled to obtain the pulp employed
for theextract preparation.
2.3. Preparation of hydroethanolic extract of the pulp of
Tamarindus indica (ExT)
Approximately 100 g of the fruit pulp were placed in a conical
flaskand soaked at room temperature for 5 days, in 400 mL of 70%
ethanol inwater. The resulting extract was filtered through a sieve
and rotoevapo-rated twice, until complete alcohol evaporation had
occurred.
The composition of this ExT, previously determined by
Martinelloet al. (2006), is shown inTable 1. Total sugars were
determined by thephenolsulfuric acid method according to Dubois et
al. (1956); the reac-
tion mixture contained, respectively, 500lL of the sample (a
solution at0.1 mg/mL of crude ExT (w/v)), the blank or a glucose
standard (VetecQumica Fina LTDa, Rio de Janeiro, Brazil)
(20100lg/mL solution),respectively, 500lL of an aqueous phenol 5%
(w/v) solution and 2.5 mLof fuming sulfuric acid. After mixing and
chilling, absorbance was mea-sured at 490 nm in a Beckman DU-70
spectrophotometer to determinehexose content.
Total soluble phenolic derivatives were determined according
toFolinand Ciocalteau (1927), using a reaction medium containing 50
lL of thesample (a solution of 20 mg/mL of crude ExT (w/v)), the
blank or a gallicacid standard (Sigma) (1001000lM), respectively,
50lL of 7% (v/v)acetic acid, 50lL of the FolinCiocalteaus reagent,
50lL of 35% (w/v)sodium carbonate and 800lL of distilled water.
After mixing, the reac-tants were incubated for 90 min at room
temperature in the dark. Lightabsorbance was measured at 725 nm in
a Beckman DU-70 spectropho-
tometer, and the total phenolic content expressed as gallic acid
equivalentsper mg of extract.
Protein concentration was determined by Bradfords (1976),
calori-metric method, using a commercially available kit (BIO-RAD
ProteinAssay) and a bovine serum albumin (BIO-RAD standard II).
Fiftymicroliter of sample (undiluted ExT) blank or albumin standard
(2.525lg/mL), respectively, were added of 40lL of BIO-RAD reagent.
Aftermixing, the reaction mixture was incubated for 5 min, at room
tempera-ture. Its absorbance was measured at 600 nm in a Beckman
DU-70spectrophotometer. SomogyiNelsons method was employed to
evaluatethe concentration of reducing sugar (Kidby and Davidson,
1973). Briefly,1 mL of the sample (0.1 mg/mL of crude ExT (w/v)),
of the blank or of thegalactose standard (Sigma) (20100lg/mL) were
added to 1 mL ofSomogyis reactant and incubated for 10 min at 100
C. After cooling,1 mL of Nelsons reagent was added and the mixture
made up to 10 mL
with distilled water. Absorbance was measured at 540 nm in a
BeckmanDU-70 spectrophotometer.
For the uronic acid determination (Blumenkratz and
Assboe-Hansen,1973), 200lL of sample solution of 0.1 mg/mL of crude
ExT (w/v), of theblank or the glucuronic acid standard (Sigma)
(2.5100lg/mL) weremaintained in ice and then 1.2 mL of 0.0125 M
sodium tetraborate wereadded. After mixing and incubation for 5 min
incubation at 100 C, 20lL
Table 1Components of the ExT (%)a
Totalsugars
Reducingsugars
Phenoliccompounds
Uronicacid
Protein
61.5 27.6 0.25 3.70 0.57
a Determined as referred to in Section 2.
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strated, suggesting that complement activation in dyslipide-mia
may be induced by this mechanism. Another impor-tant factor is the
decrease of HDL in subjects under ahypercholesterolemic diet. Since
complement regulatoryproteins such as CD59 are transported in serum
by HDL(Vakeva et al., 1994), this decrease could in some way
affect
serum complement activity. The evidences available in
theliterature about complement activation in established
ath-erosclerosis, and our data showing that when
activated,complement action is high in hyperlipidemia, may be
ofinterest and as shown here, the effect of ExT under
theseconditions justifies additional studies. In the CE group,lytic
activity showed values similar to those of controls.The mechanism
through which diets containing high levelsof cholesterol affect
complement activity, and throughwhich ExT prevents this effect will
be subjected to investi-gation.
Similar results were obtained in assays using treated ani-mals
serum samples to restore the lytic activity of serum
deficient of C2 [R(2)] and of factor B [R(B)]. When serumfrom
the Col (hypercholesteremic only) group was used,lysis was higher
than in controls, but serum from the CEgroup restored lytic
activity to values similar to those ofthe controls. These data
point to alterations of CP/LPs atthe level of C2 and of AP at the
level of factor B, possiblyinvolving the components themselves
and/or regulators ofcomplement activation at these points of the
cascade. Suchalterations were not observed when ExT was furnished
toanimals in association with a diet containing high levelsof
cholesterol, and to our knowledge, components of ham-ster
complement or their respective antibodies are not com-
mercially available. In order to enable us to presentlyfurther
investigate the mechanism of these effects, the useof the R(2) and
R(B) reagents for (indirect) evaluation ofthese components effects
appears justified.
We believe that a direct correlation between the resultsobtained
in our in vivo and in vitro studies, is not to beexpected. Factors
such as intake, absorption and metabo-lism of our study material
can affect its bioavailabilityand bioefficiency (Ross and Kasum,
2002). The ExT is amixture of many compounds whose bioavailability
dependson their structure. The activity of compounds present in
thecrude tamarind extract could be modified or/and
destroyedfollowing absorption and metabolism. Nevertheless,besides
its use in research, we consider the possibility thatExT is a
potential source of compounds that if properlyisolated, may
eventually become of therapeutic use. Therationale to use drinking
water containing 5% ExT wasthat this concentration is usually
present in tamarind juicedrinks consumed by the human
population.
In order to rule out an eventual damage to the liver bythe
hypercholestorlemic diet or by the ExT treatment, weinvestigated
possible alterations of serum protein, albuminlevels and
transaminase activity following experimentaltreatments. As shown in
Fig. 5, hamster liver functionwas not affected by them. This is of
importance considering
that the liver is the site of the synthesis of the majority
of
the CS components (Paul, 1999), which could be alteredby hepatic
lesions. However, even if this is occurring at lev-els not
detectable by our analyses, it is important to restatethe
protective action of the ExT in these conditions. Fur-thermore,
besides its known role in the process of athero-sclerosis,
increased activity of CS as a consequence of
hyperlipemic diets could eventually enhance the triggeringof the
inflammatory process that precedes atherosclerosis.Serum levels of
total cholesterol and triacylglycerols
were increased in animals given a high-cholesterol diet. Inthe
CE group, triacylglycerol levels were normal, but totalcholesterol
remained high, although it is worth consideringthat determinations
of total cholesterol may mask varia-tions due to the different
types of components measured,like high (HDL) and low (LDL) density
lipoproteins; thiseffect was already observed in recent studies on
the effectof the ExT on cholesterol levels in our experimental
animalmodel (Martinello et al., 2006). A decrease in serum
triglyc-erides may be associated with the epicatechins present
in
the ExT (Sudjaroen et al., 2005); they promote high
fecalexcretion of total fatty acids, neutral and acidic
sterols,suggesting that the hypolipidemic activity of this
materialmay be mediated by its influence on the absorption
ofdietary fat and cholesterol (Chan et al., 1999). Moreover,the ExT
is rich in total sugars, and it has been describedthat consumption
of b-glucan lowers total and non-HDL-cholesterol in plasma due to
its soluble fiber content(Wilson et al., 2004). Nevertheless, the
mechanism bywhich high-level cholesterol diets affect complement
activ-ity and by which ExT prevents this effect, certainly
deservesadditional investigation.
To conclude, the apparent discrepancy between in vivoand in
vitro results produced by the ExT, deserves furtherstudy of factors
involved in its in vivo actions dependenton the intake, absorption
and metabolism of its compo-nents possibly affecting
bioavailability and bioefficiency.The finding that ExT appears to
contain substances show-ing differing, even opposite effects on the
complement sys-tem, deserves consideration, and justifies
additional effortsto isolate the substances responsible for such
effects and toinvestigate their mechanisms of action.
Acknowledgements
The authors are indebted to the Conselho Nacional
deDesenvolvimento Cientfico e Tecnologico (CNPq) forfinancial
support (Grant Nos. 475267/2003-6 and 306965/2003-8) and Fundacao
de Amparo a Pesquisa do Estadode Sao Paulo (FAPESP) (Grant No.
05/00887-9). Wethank Denise Pimenta da Silva Leitao and Dr.
AdolfoRothschild for manuscript revision, Ieda Maria RazaboniPrado
for ExT components determinations, Alcides Pere-ira for animal
handling, Antonio Zanardo Filho and JoaoJose Franco for biochemical
determinations and the pri-sioners of the Ribeirao Preto, SP
Penitentiary for helping
in the peeling of the fruits ofT. indica.
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