A gan (A gania spinosa) oil lowe s blood p essu e and imp o es
endo helial dys unc ion in spon aneously hype ensi e a s
Hicham Be ougui
1
, Ma ia Al a ez de So omayo
2
*, Concepcio
´nPe
´ ez-Gue e o
2
, Abdelkade E aib
1
,
Mohamed Hmamouchi
1
, Elisa Ma huenda
2
and Ma ia Dolo es He e a
2
1
Depa emen de Chimie-Biochimie, U.F.R. des Subs ances Na u elles, Facul e de Medecine e de Pha macie,
Uni Mohamed V Souissi. Raba , Mo occo
2
Depa amen o de Fa macologı
´a, Facul ad de Fa macia, Uni e sidad de Se illa.C/P o Ga cia-Gonzalez s/n. 41012 Se ille,
Spain
(Recei ed 22 Oc obe 2003 – Re ised 27 July 2004 – Accep ed 6 Augus 2004)
T adi ionally hand-p essed a gan oil, ob ained om A gania spinosa seeds, is ea en aw in sou h-wes Mo occo; i s ich composi ion o
ocophe ols, MUFA and PUFA make a s udy o i s ac ions on isk ac o s o CVD, such as hype ension, in e es ing. The e ec s o 7
weeks o ea men wi h a gan oil (10 ml/kg) on he blood p essu e and endo helial unc ion o spon aneously hype ensi e a s (SHR)
and no mo ensi e Wis a –Kyo o a s we e in es iga ed. Sys olic blood p essu e and hea a e we e measu ed e e y week by he ail-
cu me hod and endo helial unc ion was assessed by ca bachol (10
28
o 10
24
M)-induced elaxa ions o ao ic ings and small mesen e ic
a e ies p e-con ac ed wi h phenyleph ine. A gan-oil adminis a ion educed he mean blood p essu e o SHR a e he i h week o ea -
men (P,0·05) and inc eased (P,0·01) he endo helial esponses o a e ies om SHR. The NO syn hase inhibi o , L-N- -ni oa ginine
(3 £10
25
M) e ealed a g ea e pa icipa ion o NO in he elaxan e ec a e he ea men . When cyclooxygenase (COX) was blocked
wi h indome hacin (10
25
M), an in ol emen o COX p oduc s in he endo helium-dependen esponse was cha ac e ized. Enzyme immu-
noassay o h omboxane B
2
showed a signi ican dec ease (P,0·05) in he elease o h omboxane A
2
in bo h ao a and small mesen e ic
a e y a e a gan-oil ea men o SHR. Expe imen s in he p esence o he h omboxane A
2
–p os aglandin H
2
ecep o an agonis ICI
192,605 (10
25
M) con i med his esul . Resul s a e incuba ion wi h he an ioxidan s supe oxide dismu ase and ca alase sugges ed ha
a dec eased oxida i e s ess migh con ibu e o explain he bene icial e ec s o a gan-oil ea men .
A gan oil: Hype ension: Endo helium: Cyclooxygenase p oduc s: Spon aneously hype ensi e a s
The e is s ong e idence o an associa ion be ween a
Medi e anean-s yle die and p o ec ion om CVD (De
Lo ge il e al. 1999). Such a die lowe s o al choles e ol
and LDL-choles e ol compa ed wi h a die e y ich in
sa u a ed a y acids, hus educing a dominan isk ac o
o he de elopmen o a he oscle osis (Williams, 2001).
Addi ional mechanisms ha e a ou able e ec s on o he
CVD isk ac o s, such as hype ension (Simon e al.
1996) and diabe es (Hannah & Howa d, 1994; G i in
e al. 1996). This s yle o die , consumed by se e al di e -
en popula ions, has a common cha ac e is ic, namely he
high p opo ion o oli e oil ( ich in MUFA, mainly oleic
acid; Keys, 1995).
As well as oli e oil, he e a e o he ege able oils ha
a e sou ces o die a y unsa u a ed a y acids. A gan oil
is adi ionally used pa icula ly in Mo occo o nu i ional
pu poses. T adi ionally hand-p essed a gan oil, ob ained
om A gania spinosa seeds, is ea en aw in sou h-wes
Mo occo, whe e i ep esen s 25 % o die a y a in ake
and 9 % o annual p oduc ion. A gan oil and i s p ep-
a a ions ha e been used in adi ional Mo occan medicine
o cen u ies o cu e skin diseases opically. In addi ion,
a gan oil is used o ally in heuma ology and is adi ionally
p esc ibed as a chole e ic, hepa op o ec i e agen , and
in cases o hype choles e olaemia and a he oscle osis
(Cha ou & Guillaume, 1999). Howe e , i s po en ial
biological ele ance in cosme ic, pha maceu ical o phy o-
p o ec i e ields has ye no been es ablished.
A gan oil is ich in MUFA and PUFA, whe eas sa u a ed
a y acids a e p esen in lowe p opo ions (Cha ou &
Guillaume, 1999). Se e al s udies ha e sugges ed ha
hype ension and CVD a e ela ed o a de iciency in
PUFA, especially o linoleic acid (Das, 1995; Ho obin,
1995), so ha an inc ease in he linoleic acid con en o
die was associa ed wi h a dec ease in sys olic blood p ess-
u e (Aguila & Manda in-de-Lace da, 2000; Yoshioka e al.
2000). A gan oil is also ich in he an ioxidan a- oco-
phe ol (Cha ou & Guillaume, 1999), which educed
*Co esponding au ho : D Ma ia Al a ez de So omayo , ax þ34 54233765, email [email p o ec ed]
Abb e ia ions: COX, cyclooxygenase; L-NOARG, L-N- -ni oa ginine; Phen, phenyleph ine; SHR, spon aneously hype ensi e a ; SMA, small
mesen e ic a e y; SOD, supe oxide dismu ase; TX, h omboxane; WKY, Wis a –Kyo o a .
B i ish Jou nal o Nu i ion (2004), 92, 921–929 DOI: 10.1079/BJN20041293
qThe Au ho s 2004
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blood p essu e in an expe imen al model o hype ension
(Chen e al. 2001). Al hough a p e ious epo showed
a dec ease in blood p essu e a e inges ion o a gan oil
(Be ada e al. 2000), he mechanism in ol ed emains
unknown. Because o he composi ion o a gan oil and
i s an ihype ensi e ac ion, i was o in e es o ca y ou
a deepe s udy o i s ca dio ascula e ec s, ocusing on
endo helial unc ion. We ha e in es iga ed he e ec o
ch onic ea men wi h a gan oil o 7 weeks on blood
p essu e and endo helial dys unc ion in spon aneously
hype ensi e a s (SHR) compa ed wi h no mo ensi e
Wis a –Kyo o a s (WKY).
Me hods
A gan oil ex ac ion
A gan oil was ex ac ed by a adi ional hand-p essed
me hod (Cha ou & Guillaume, 1999) om esh seeds
collec ed in he same yea in o de o p e en au o-oxi-
da i e eac ions. The ex ac ion was ca ied ou in
Essaoui a (sou h-wes Mo occo).
Animals
Fou -week-old male hype ensi e SHR (n12) and no mo-
ensi e con ol WKY (n12) a s, weighing 100–120 g,
we e pu chased om Ha lan Ibe ica (Ba celona, Spain).
All expe imen s we e pe o med acco ding o guidelines
o he e hical ea men o animals o he Eu opean
Union (86/609/EEC). Bo h SHR and WKY we e di ided
andomly in o wo g oups o six animals each: he i s
g oup was ed wi h s anda d a chow (con ol g oup;
Panlab SRL, Ba celona, Spain). The second g oup was
ea ed wi h 10 ml a gan oil/kg body weigh pe d in agas-
ically o 7 weeks in addi ion o he s anda d die . This
dose has been shown o ha e hypolipidaemic and hypocho-
les e olaemic e ec s (Be ougui e al. 2003). All he ani-
mals we e main ained in a empe a u e-con olled oom
(22 ^28C) wi h a 12 h ligh –da k cycle and wi h ee
access o s anda d a chow and d inking wa e . The
blood p essu e and hea a e o conscious animals we e
measu ed indi ec ly each week by he ail-cu me hod
wi h a digi al p essu e me e (Nip em 645; Cibe ec,
Mad id, Spain).
A e ial p epa a ion and moun ing
The animals we e anaes he ized wi h pen oba bi one
sodium (50 mg/kg in ape i oneally) and exsanguina ed.
The ho acic ao a and b anch II o III o he small mesen-
e ic a e y (SMA) we e ca e ully emo ed and cleaned o
a and connec i e issue. Then, a e y segmen s (2–3 mm
o 1·6–2·0 mm long o he ao a and he SMA espec -
i ely) we e moun ed on myog aphs illed wi h physi-
ological sal solu ion o he ollowing composi ions (mM)
o he ao a and SMA espec i ely: NaCl 119 and 119,
KCl 47 and 47, MgSO
4
1·17 and 1·17, KH
2
PO
4
1·18 and
0·40, NaHCO
3
25·0 and 14·9, CaCl
2
1·8 and 2·5, glucose
11·0 and 5·5. The physiological sal solu ion was kep con-
inuously a 378C and gassed wi h 95 % O
2
–5% CO
2
a pH
7·4. Res ing ension was adjus ed o 2 g o he ao a and
200 mg o he SMA. Mechanical ac i i y was eco ded
isome ically by a o ce ansduce (Pioden UF-1
(Can e bu y, Ken , UK) o he ao a and Mul i Myog aph
Sys em-610M (Aa hus, Denma k) o he SMA) coupled o
a Powe lab
w
da a acquisi ion sys em (AD-Ins umen s,
Cas le Hill, Vic o ia, Aus alia). A e se ing he essel
o i s wo king leng h, challenges wi h 10
25
Mphenyl-
eph ine (Phen)/l o 10
25
Mno ad enaline/l we e pe o med
in ao a and SMA espec i ely o es hei maximal
con ac ile capaci y and o elici a ep oducible con ac ing
esponse.
Relaxa ion expe imen s
A e ies we e p e-con ac ed wi h Phen: 3 £10
27
M o he
ao a and 3 £10
25
M o he SMA. Fo each p epa a ion, i
was ensu ed ha Phen-induced con ac ions we e s able
du ing all he expe imen s. When he con ac ion eached
a pla eau, cumula i e addi ion o ca bachol (10
28
o
10
24
M) was pe o med. In o de o analyse he in ol emen
o endo helial ac o s, concen a ion– esponse cu es we e
cons uc ed in he absence o in he p esence o he indica ed
inhibi o (s): he NO syn hase inhibi o , L-N- -ni oa ginine
(L-NOARG; 3 £10
25
M), he cyclooxygenase (COX)
inhibi o , indome hacin (10
25
M), he h omboxane A
2
–
p os aglandin H
2
ecep o an agonis ICI 192,605
(10
25
M), he supe oxide anion (O
2
2
) sca enge supe oxide
dismu ase (SOD; 1·5 £10
5
U/l), and ca alase (10
6
U/l). All
he inhibi o s we e used a a maximally ac i e concen a ion
and we e incuba ed wi h he issue o 20 min be o e he p e-
con ac ion wi h Phen excep o SOD þca alase (i.e.
10 min be o e p e-con ac ion wi h Phen). The concen-
a ion o Phen a e inhibi o s was adjus ed in o de o
ob ain simila p e-con ac ion le els.
Th omboxane A
2
p oduc ion
Th omboxane (TX) A
2
is ins able and is quickly con e ed
o TXB
2
. In ac ao as and mesen e ic bed om con ol and
a gan oil- ea ed SHR we e incuba ed in physiological sal
solu ion a 378C and bubbled wi h a 95 % O
2
–5% CO
2
gas
mix u e and s imula ed wi h Phen (10
26
M o ao a and
10
25
M o SMA) and ca bachol (10
26
M) o libe a e o
he medium asoac i e p oduc s. The concen a ion o
TXB
2
was assessed by compe i i e enzyme immunoassay
ki s (Cayman Chemical Company, Ann A bo , MI,
USA). TXB
2
p oduc ion was exp essed as pg/mg d y
issue.
Chemical eagen s and d ugs
Ace ylcholine chlo ide, indome hacin, L-NOARG, phenyl-
eph ine hyd ochlo ide, ca bachol chlo ide, ca alase and
SOD we e pu chased om Sigma Chemical Co. (S
Louis, MO, USA). ICI 192,605 was pu chased om
Toc is (Biogen Cien i ica S.L., Mad id, Spain). The d ugs
we e dissol ed in dis illed and deionized wa e excep o
indome hacin and ICI 192,605, which we e dissol ed in
dime hylsul oxide. The inal concen a ion o dime hylsul -
oxide in he issue ba h was 0·1 g/l, which was shown o
H. Be ougui e al.922
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ha e no e ec on he basal onus o he p epa a ion. All
concen a ions o he d ugs used a e exp essed as inal
concen a ion in he o gan chambe .
S a is ical analysis
Resul s we e exp essed as a pe cen age om he ini ial p e-
con ac ion le el and as mean alues wi h hei s anda d
e o s o six de e mina ions ob ained om di e en ani-
mals. The p e-con ac ion le els o he a e ies om he
ou g oups o animals a e summa ized in Table 1. A eas
unde concen a ion– esponse o ca bachol cu es we e
calcula ed in he absence o inhibi o s and in he p esence
o indome hacin o L-NOARG. In o de o e alua e he
app oxima e pa icipa ion o COX p oduc s and NO, he
sub ac ion a ea unde con ol cu e minus a ea in he p e-
sence o indome hacin o L-NOARG was calcula ed. A
posi i e sign (þ) in he esul was in e p e ed as he p e a-
lence o a elaxan ac o and he nega i e (2) sign as a
g ea e in ol emen o con ac ing p oduc s. ANOVA ol-
lowed by Tukey’s mul iple compa isons es was used o
s a is ical analysis. P alues ,0·05 we e conside ed o
show a signi ican di e ence. S a iew so wa e package
( e sion 5.0; SAS Ins i u e Inc., Ca y, NY, USA) was
used o ca y ou s a is ical analysis.
Resul s
Blood p essu e
Daily a gan-oil adminis a ion induced a p og essi e
educ ion in mean blood p essu e in SHR; his educ ion
was signi ican om he i h week o ea men
(P,0·05; Fig. 1). Howe e , no change was obse ed in
blood p essu e o no mo ensi e WKY du ing he 7 weeks
o ea men wi h a gan oil. Despi e he dec ease in blood
p essu e in SHR, he hea a e was no a ec ed by ea -
men in ei he g oup (377·4 (SEM 11·6) and 388·8 (SEM
14·5) bea s pe min in WKY con ol and ea ed g oups
espec i ely; 429·6 (SEM 10·9) in SHR con ol and 406·1
(SEM 9·11) bea s pe min a e ea men ).
Al hough animals we e ea ed wi h 1 ml a y com-
pound/d, body weigh was no a ec ed (303·7 (SEM 6·8)
and 284·7 (SEM 18·2) g o WKY con ol and ea ed
g oups espec i ely; 276·2 (SEM 6·8) and 258·7 (SEM
18·2) g o con ol and ea ed SHR g oups espec i ely).
Endo helium-dependen elaxa ion
Endo helial unc ion was assessed in wo di e en ascula
beds by elaxa ion induced by ca bachol in a e ies p e-
con ac ed by Phen. A e he 7-week ea men wi h
a gan oil, he concen a ion– esponse cu es o ca bachol
o ao as and SMA om no mo ensi e WKY we e no sig-
ni ican ly a ec ed (Fig. 2(A and B)). In con as , he endo-
helium-dependen elaxa ion o ao ic ings om a gan
oil- ea ed SHR was signi ican ly inc eased (P,0·01) com-
pa ed wi h ha o he SHR con ol g oup (Fig. 2(C)). The
maximal elaxan esponse eached in SHR a e ea men
(83·7 (SEM 2·8) %) was e en g ea e han ha ob ained in
no mo ensi e WKY (65·7 (SEM 8·6) %, P,0·001).
In SMA om SHR, he elaxa ion– esponse cu e o
ca bachol had a biphasic p o ile wi h con ac ion induced
by ca bachol a concen a ion .10
26
M. Al hough maxi-
mal elaxa ion o ca bachol was signi ican ly inc eased
by a gan-oil ea men , his biphasic p o ile o he cu e
was no al e ed (Fig. 2(D)).
Cha ac e iza ion o endo helial ac o s in ol ed
The e ec o he NO-syn hase inhibi o L-NOARG was
s udied in o de o ind ou whe he NO was in ol ed in
he imp o emen o endo helial elaxa ion in ao ae and
SMA. In bo h ypes o a e ies L-NOARG (3 £10
25
M)
p oduced a s a is ically signi ican blockade o he elax-
a ion in non- ea ed a s (Fig. 3(A and C), ao a; Fig. 4(A
and C), SMA) and a gan oil- ea ed a s (Fig. 3(B and D),
ao a; Fig. 4(B and D), SMA).
Table 1. Con ac ile e ec o phenyleph ine (g) in ao ic
ings and supe io mesen e ic a e ies (SMA) om con ol
and a gan (A gania spinosa) oil- ea ed Wis a –Kyo o and
spon aneously hype ensi e a s†
(Mean alues wi h hei s anda d e o s)
WKY SHR
Mean SEM Mean SEM
Ao a Con ol 2·10 0·04 1·41* 0·09
A gan oil 1·54 0·10 1·54 0·10
SMA Con ol 0·93 0·11 1·29 0·23
A gan oil 1·18 0·04 1·28 0·15
WKY, Wis a –Kyo o a s; SHR, spon aneously hype ensi e a s.
* Mean alue was signi ican ly di e en om ha o he con ol
WKY g oup: *P,0·05.
† Fo de ails o ea men s and p ocedu es, see p. 922.
Fig. 1. E ec o a gan (A gania spinosa)-oil ea men on blood
p essu e (mmHg) in no mo ensi e Wis a –Kyo o a s (WKY; O) and
spon aneously hype ensi e a s (SHR; †). Resul s we e compa ed
wi h age- and s ain-ma ched con ol animals (WKY (K) and SHR
(W)). Fo de ails o ea men s and p ocedu es, see p. 922. Values
a e means wi h hei s anda d e o s shown by e ical ba s. Mean
alues we e signi ican ly di e en om hose o he con ol g oup:
*P,0·05, **P,0·01. Mean alues o he no mo ensi e WKY we e
signi ican ly di e en om hose o he hype ensi e SHR wi hou
ea men : ††P,0·01, †††P,0·001.
E ec o a gan oil on blood p essu e 923
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Fig. 2. Endo helial unc ion assessed by elaxan esponse o ca bachol (CCh; 10
28
o 10
24
M) o isola ed a ao ic ings (A and C) and small
mesen e ic a e y (B and D) om no mo ensi e Wis a –Kyo o a s (A and B) and spon aneously hype ensi e a s (C and D). Resul s ob ained
om a gan (A gania spinosa) oil- ea ed animals (†) we e compa ed wi h age- and s ain-ma ched animals (W). Fo de ails o ea men s and
p ocedu es, see p. 922. Values a e means wi h hei s anda d e o s shown by e ical ba s. Mean alues o a gan-oil ea ed animals we e
signi ican ly di e en om hose o he con ol g oup: *P,0·05, **P,0·01.
Fig. 3. Cha ac e iza ion o endo helial ac o s eleased a e s imula ion wi h ca bachol (CCh; 10
28
o 10
24
M) o ao ic ings om non- ea ed
Wis a –Kyo o a s (WKY) (A), a gan (A gania spinosa) oil- ea ed WKY (B), non- ea ed spon aneously hype ensi e a s (SHR) (C) and a gan
oil- ea ed SHR (D). Concen a ion– esponse cu es cons uc ed in he absence o inhibi o s we e conside ed as con ol cu es (W) and
compa ed wi h hose made in he p esence o L-N- -ni oa ginine (L-NOARG; 3 £10
25
M)(†), indome hacin (10
25
M)(O) o indome hacin plus
L-NOARG (K). Fo de ails o ea men s and p ocedu es, see p. 922. Values a e means wi h hei s anda d e o s shown by e ical
ba s. Mean alues we e signi ican ly di e en om hose o he con ol cu e: *P,0·05, **P,0·01. Mean alues o SHR we e signi ican ly
di e en om hose o WKY: †††P,0·001. Mean alues o L-NOARG we e signi ican ly di e en om hose o L-NOARG plus indome hacin:
‡P,0·05.
H. Be ougui e al.924
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In o de o s udy he in ol emen o COX p oduc s in
endo helial elaxa ion, a e ies we e incuba ed in he p e-
sence o a non-selec i e COX inhibi o , indome hacin
(10
25
M). This d ug did no modi y ca bachol-induced
elaxa ion o ao ic ings om WKY. In ao as om con-
ol SHR, indome hacin inc eased he elaxan esponse,
al hough his e ec was no signi ican . Wi h ega d o
SHR ea ed wi h a gan oil, he p esence o indome hacin
did no a ec he concen a ion– esponse cu e o ao ic
ings. In esis ance a e ies om SHR, indome hacin sig-
ni ican ly inc eased ca bachol-induced elaxa ion
(P,0·01) and abolished he biphasic p o ile o he con ol
cu e (Fig. 4(C and D)).
Exposu e o indome hacin (10
25
M) plus L-NOARG
(3 £10
25
M) comple ely abolished (P,0·01) he ca ba-
chol-induced elaxa ion cu e in a ao a and SMA
(Figs 3 and 4). In ao ic ings om ea ed SHR, he inhi-
bi ion ob ained a e incuba ion wi h indome hacin plus
L-NOARG was signi ican ly g ea e han ha achie ed
in he p esence o L-NOARG (P,0·05; Fig. 3(D)).
The p esence o a elaxan COX-p oduc in ao ic ings
a e a gan-oil adminis a ion could well explain his
esul . Howe e , in SMA om non- ea ed SHR, he
g ea e elaxa ion in he p esence o L-NOARG plus indo-
me hacin han in he p esence o L-NOARG (P,0·05;
Fig. 4(C)) could be a ibu ed o he p esence o COX-
de i ed con ac ing p oduc s in hype ensi e a s.
In o de o illus a e be e he con ibu ion o endo-
helium-de i ed ac o s in he elaxa ion induced by ca ba-
chol, a eas unde concen a ion– esponse cu es we e
calcula ed in he absence and in he p esence o L-NOARG
and indome hacin, ep esen ing NO and COX p oduc s
espec i ely. The calcula ed pa icipa ion o NO in he
ca bachol-induced elaxa ion o isola ed ao a om a gan
oil- ea ed a s was g ea e (P,0·05 in WKY; P,0·001 in
SHR) han in ao ic ings om un ea ed a s. Wi h ega d
o he COX p oduc s eleased in isola ed ao a, he esul ing
con ac ing e ec was ound in SHR, bu no in WKY, whe e
elaxan ac o s de i ed om COX had a g ea e in ol e-
men (Fig. 5(A)). The ea men wi h a gan oil dec eased
he pa icipa ion o COX p oduc s in ao ic ings om
SHR (P,0·05) wi hou a ec ing he sign o he calcula ed
alue (Fig. 5(A)).
In SMA, he pa icipa ion o NO was signi ican ly
g ea e a e a gan-oil ea men in SHR (P,0·01) bu
no in no mo ensi e WKY. Acco ding o he esul s
obse ed in he ao a, he in ol emen o con ac ing
COX p oduc s was also dec eased in SMA om SHR
a e ea men (P,0·05). Howe e , he p e alence o con-
ac ing COX p oduc s was ound in SMA om ea ed
WKY (P,0·05; Fig. 5(B)).
To e i y he na u e o endo helial asocons ic o p o-
duc s om he COX in ol ed, he e ec o he TXA
2
–p os-
aglandin H
2
ecep o an agonis , ICI 192,605 (10
25
M)on
ca bachol-induced elaxa ion in a e ies om SHR was
in es iga ed. Though he elaxa ion o a e ies om non-
ea ed SHR was enhanced by incuba ion wi h ICI 192,605
(Fig. 6(A and C)), his inc ease was no s a is ically di e -
en . In ao ic ings om a gan-oil ea ed SHR, he p esence
o ICI 192,605 did no a ec he elaxan esponse o ca ba-
chol. This an agonis signi ican ly inhibi ed he elaxa ion in
SMA om ea ed SHR (P,0·05; Fig. 6(D)).
Fig. 4. Cha ac e iza ion o endo helial ac o s eleased a e s imula ion wi h ca bachol (CCh; 10
28
o 10
24
M) o small mesen e ic a e ies om
non- ea ed Wis a –Kyo o a s (WKY) (A), a gan (A gania spinosa) oil- ea ed WKY (B), non- ea ed spon aneously hype ensi e a s (SHR)
(C) and a gan oil- ea ed SHR (D). Concen a ion– esponse cu es cons uc ed in he absence o inhibi o s we e conside ed as con ol cu es
(W) and compa ed wi h hose made in he p esence o L-N- -ni oa ginine (L-NOARG) (3 £10
25
M;†), indome hacin (10
25
M;D) o indo-
me hacin plus L-NOARG (K). Fo de ails o ea men s and p ocedu es, see p. 922. Values a e means wi h hei s anda d e o s shown by e -
ical ba s. Mean alues we e signi ican ly di e en om hose o he con ol cu e: *P,0·05, **P,0·01. Mean alue o L-NOARG was
signi ican ly di e en om ha o L-NOARG plus indome hacin: †P,0·05.
E ec o a gan oil on blood p essu e 925
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Th omboxane B
2
p oduc ion
The TXB
2
le els eleased by s imula ed ao ic ings and
mesen e ic bed om non- ea ed SHR we e 312·66 (SEM
39·47) (n4) and 157·49 (SEM 22·93) (n4) pg/mg espec -
i ely. A e 7-weeks ea men wi h a gan oil, he elease
o TXB
2
dec eased signi ican ly in ao ic ings (193·61
(SEM 22·10) pg/mg (n4), P,0·05) and mesen e ic bed
(92·84 (SEM 2·34) pg/mg (n4), P,0·05).
In ol emen o oxygen ee adicals in he e ec o a gan
oil on endo helial unc ion
Finally, o in es iga e whe he an augmen ed p oduc ion o
O
2
2
was in ol ed, he e ec o SOD was s udied in SHR.
In bo h isola ed a ao a and SMA he p esence o SOD
plus ca alase signi ican ly inc eased (P,0·05) he ca ba-
chol-induced elaxa ion in un ea ed a s, since SHR a e
ich in ee adicals de i ed om O
2
. A e a gan oil
adminis a ion, he endo helium-dependen elaxa ion o
ao ic ing was no inc eased by he p esence o an ioxidan
enzymes (Fig. 7(B)). When he same expe imen was ca -
ied ou on SMA om ea ed animals, he p esence o
SOD plus ca alase inc eased he con ac ion phase o he
concen a ion– esponse cu e (Fig. 7(D); P,0·01).
Discussion
Die a y a y acids ha e been epo ed o in luence he
de elopmen o hype ension and ascula eac i i y o
bo h esis ance and la ge conduc ance a e ies (Schmid ,
1997; Ange e & Von Shacky, 2000). The p e en i e
e ec s o PUFA such as linoleic and g-linolenic acid on
hype ension a e well known, whe eas sa u a ed a y
acids ha e been shown o p omo e hype ension (Aguila
& Manda in-de-Lace da, 2000; Yoshioka e al. 2000).
Changes in lipid me abolism caused by a die ich in mono-
unsa u a ed oleic acid ha could be a ou able in he p e-
en ion o a he oscle osis and h ombosis ha e been
obse ed (Williams, 2001). In he p esen s udy, he e ec s
o a gan-oil inges ion on blood p essu e and endo helial
unc ion we e e alua ed.
In ela ion o he chemical composi ion o a gan oil,
unsa u a ed a y acids a e he majo componen s (oleic
plus linoleic acids cons i u e 80 g/100 g o al a y acids)
and linolenic acid is only p esen as a ace (Cha ou
& Guillaume, 1999). A gan oil is abou wice as ich in
ocophe ol as oli e oil (620 . 320 mg/kg); ocophe ol is
p esen mainly as a- ocophe ol (69 %). This compound, a
known an ioxida i e agen , makes a gan oil a e y impo -
an sou ce o i amin E and is p obably esponsible o he
good keeping quali ies o he oil (Chimi e al. 1994). In
addi ion o PUFA, an ioxidan s such as a- ocophe ol and
i amin C a e known o p e en de elopmen o hype en-
sion (Newaz & Nawal, 1998; Newaz e al. 1999) and endo-
helial dys unc ion in SHR (Abeywa dena & Head, 2001;
Chen e al. 2001).
We ha e shown ha ch onic ea men wi h a gan oil
p e en ed he de elopmen o hype ension in his animal
model (SHR), subs an ially modi ying mean blood p essu e
om he i h week o ea men wi hou al e ing hea a e
and body weigh . Taking in o accoun he composi ion o
a gan oil, wo hypo heses could be pu o wa d. Fi s , he
high p opo ion o he PUFA linoleic acid p esen in
a gan oil could play a ole in blood p essu e egula ion.
I has been demons a ed ha plasma concen a ion o
linoleic acid is in e sely associa ed wi h blood p essu e
(G imsgaa d e al. 1999) and ha die s en iched wi h
linoleic o g-linolenic acid a enua ed he de elopmen
o hype ension in SHR (Abeywa dena & Head, 2001;
F enoux e al. 2001). The second hypo hesis o conside
is ha he high p opo ion o a- ocophe ol p esen in
a gan oil could be ela ed o he an ihype ensi e e ec
obse ed. Thus, he dose o a- ocophe ol adminis e ed in
he a gan oil o a s (3·8 mg/kg pe d) was simila o he
dose ha has p e iously demons a ed p e en ion o high
blood p essu e in SHR (Newaz & Nawal, 1998; Newaz
e al. 1999).
Rega ding he imp o emen o endo helial dys unc ion
o SHR, pha macological ools we e used o e alua e he
Fig. 5. Con ibu ion o NO (A) and cyclooxygenase (B) p oduc s o
he endo helial esponse o ca bachol in ao ic ings (A) and small
mesen e ic a e ies (SMA) (B). AUC, a ea unde he cu e; WKY,
Wis a –Kyo o a ; SHR; spon aneously hype ensi e a ; c, con ol;
a gan, a gan (A gania spinosa) oil- ea ed. Values we e calcula ed
by he di e ence be ween AUC in he absence o inhibi o s minus
AUC in he p esence o L-N- -ni oa ginine o indome hacin. AUC
.0 means ha he eleased p oduc s induce a elaxa ion. I AUC
,0, p oduc s p omo e con ac ion. Fo de ails o ea men s and
p ocedu es, see p. 922. Values a e means wi h hei s anda d
e o s shown by e ical ba s. Mean alues o a gan oil- ea ed a s
we e signi ican ly di e en om hose o he non- ea ed g oup o
he same s ain: *P,0·05; **P,0·01, ***P,0·001.
H. Be ougui e al.926
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ela i e con ibu ion o di e en endo helial ac o s o his
e ec . The p esence o a NO syn hase inhibi o e ealed an
inc eased pa icipa ion o NO in elaxa ion induced by ca -
bachol. In o de o explain his inc ease o NO-dependen
elaxa ion, he an ioxidan p ope ies o a gan oil due o
a high concen a ion o he an ioxidan i amin a- oco-
phe ol should be no ed. I has been shown p e iously
ha he oxida i e s ess in SHR could be dec eased a e
ea men wi h i amin E (Newaz & Nawal, 1998). Those
animals ecei ing a- ocophe ol elici ed a lowe elease o
Fig. 6. E ec o he Tp ecep o an agonis , ICI 192,605 (10
25
M) on he ca bachol (CCh)-induced elaxa ion o ao ic ings (A and B) on
small mesen e ic a e ies (C and D) o non- ea ed spon aneously hype ensi e a s (SHR) (A and C) and a gan oil- ea ed SHR (B and D).
†, Concen a ion– esponse cu e made in he p esence o ICI 192,605; W, con ol cu e in he absence o any inhibi o . Fo de ails o ea -
men s and p ocedu es, see p. 922. Values a e means wi h hei s anda d e o s shown by e ical ba s. Mean alue was signi ican ly di e en
om ha o he con ol cu e: *P,0·05.
Fig. 7. E ec o he an ioxidan enzymes supe oxide dismu ase (SOD; 150 U/ml) plus ca alase (1000 U/ml) on he ca bachol (CCh)-induced
elaxa ion o ao ic ings (A and B) on small mesen e ic a e ies (C and D) o non- ea ed spon aneously hype ensi e a s (SHR) (A and C)
and a gan (A gania spinosa) oil- ea ed SHR (B and D). (†), Concen a ion– esponse cu e made in he p esence o SOD plus ca alase; (W),
con ol cu e in he absence o any inhibi o . Fo de ails o ea men s and p ocedu es, see p. 922. Values a e means wi h hei s anda d e o s
shown by e ical ba s. Mean alues we e signi ican ly di e en om hose o he con ol cu e: *P,0·05, **P,0·01.
E ec o a gan oil on blood p essu e 927
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anion supe oxide and consequen ly endo helial esponse
imp o ed (Chen e al. 2001). The anion supe oxide
eac s quickly wi h NO o p oduce pe oxyni i e, which
does no ha e he same asodila o and an i-agg ega ing
p ope ies (G yglewski e al. 1986). Besides his, a- oco-
phe ol is epo ed o inc ease NO syn hase ac i i y by a
mechanism in ol ing ee adicals and concomi an ly
educes he blood p essu e (Newaz e al. 1999). In his
way, a lowe oxida i e s a us o SHR due o he an ioxi-
dan s p esen in a gan oil could imp o e bioa ailabili y
o NO by bo h dec easing i s b eakdown and inc easing
i s syn hesis. To con i m his hypo hesis expe imen s in
he p esence o an ioxidan enzymes we e ca ied ou .
The endo helial elaxa ion o bo h ao ic ings and SMA
a e incuba ion wi h SOD plus ca alase was imp o ed in
con ol animals. This inc ease in endo helial esponse
a e incuba ion wi h he an ioxidan enzymes was due o
an imp o ed oxidan s a us, as p e iously demons a ed
by ou esea ch g oup (Ca neado e al. 2002). Howe e ,
he p esence o SOD plus ca alase did no al e he concen-
a ion– esponse cu e in ao a om ea ed animals, p ob-
ably because o a lowe elease o supe oxide o an
inc ease in an ioxidan de ence a e ea men wi h he
oil. In SMA, he p esence o SOD plus ca alase e en inhib-
i ed elaxa ion. This inhibi o y e ec on endo helium-
dependen elaxa ion could be ela ed o a high concen-
a ion o SOD ha blun ed endo helial NO. Howe e , i
is possible ha eac i e oxygen species we e playing a
ole in he inc ease o endo helium-dependen elaxa ion
o SMA a e ea men wi h a gan oil. In his way, i has
been shown p e iously ha O
2
2
could enhance Ca
2þ
–NO
signalling in endo helial cells (G aie e al. 1996) and
e en s imula e hose cells o p oduce NO (D ehe e al.
1995; Hu e al. 1998).
In addi ion o NO, he pa icipa ion o COX-de i ed p o-
duc s in he e ec o a gan oil was s udied. The p esence o
he COX inhibi o , indome hacin, e ealed a dec ease in
he esul ing COX con ac ing componen . To explain
his esul , he mino in ol emen o asocons ic o
TXA
2
and p os aglandin H
2
and/o an inc ease in asodila-
o p os aglandin I
2
should be no ed. The enzyme inmu-
noassay con i med ha a e ea men wi h a gan oil he
TXA
2
eleased by ei he ao ic ings o SMA was signi i-
can ly educed. This esul ag ees wi h hose shown in
he p esence o ICI 192,605 in ao ic ings. In hese
a e ies, he TXA
2
–p os aglandin H
2
Tp ecep o an agon-
is imp o ed elaxa ion only in non- ea ed SHR. Howe e ,
in ao a we canno exclude a g ea e pa icipa ion o a
elaxan de i ed om COX a e ea men wi h a gan
oil, since he p esence o indome hacin plus L-NOARG
was signi ican ly mo e e ec i e han L-NOARG alone in
inhibi ing elaxa ion. Rega ding esis ance a e ies, he
main ac o in ol ed in endo helial dys unc ion does
no ac h ough he ecep o an agonized by ICI 192,605.
Vascula bed he e ogenei y wi h ega d o eicosanoids
eleased by he endo helium is also desc ibed in endo helial
dys unc ion due o ageing (Ma z e al. 2000) whe e COX
p oduc s ela ed o endo helial dys unc ion o SMA we e
di e en om TXA
2
and emain uninden i ied. Al hough
i was no he esponsible o endo helial dys unc ion,
TXA
2
p oduc ion dec eased a e ea men wi h a gan
oil. This could be ela ed o he inhibi ion o he ca ba-
chol-induced esponse a e incuba ion wi h ICI 192,605
in SMA om ea ed SHR.
Ano he ele an ac was ha in ol emen o COX p o-
duc s u ned in o a g ea e con ac ing componen in SMA
om ea ed WKY wi hou inc ease in blood p essu e. I
has been epo ed ha he wo s ains o a di e in
hei a y acid me abolism (Mills e al. 1990) and hei
ascula esponses a e die s en iched in PUFA we e
al e ed in di e en ways (Engle e al. 1992). Those di e -
ences in me abolism be ween he s ains may help o
explain he opposing endo helial esponse a e COX inhi-
bi ion. In addi ion o he p e iously discussed an ioxidan
p ope ies o a gan oil, i s ichness in linoleic acid could
be ela ed o he e ec on endo helial COX p oduc s.
Some s udies sugges ha linoleic acid could bo h inc ease
syn hesis o asoac i e p os aglandins (Calde , 1997) and
dec ease TX p oduc ion (Engle , 1996).
In conclusion, ea men o hype ensi e animals wi h
a gan oil no only p e en ed he inc ease in blood p essu e,
bu also imp o ed endo helial unc ion. A high concen-
a ion o linoleic acid and a- ocophe ol could con ibu e
o explaining his e ec ha was dependen on bo h
COX p oduc s and NO. Howe e , u he s udies should
be done in o de o iden i y he mechanisms o he e ec s
o a gan oil on endo helium as well as i s mechanism o
ac ion. Al hough he p esen s udy suppo s he use o
his oil in he die and as a die a y supplemen , he concen-
a ion used in he p esen s udy was highe han no mal
human consump ion. Clinical esea ch should be done
be o e alida ing i s use o imp o e endo helial dys unc ion
and hype ension.
Re e ences
Abeywa dena MY & Head RJ (2001) Die a y polyunsa u a ed
a y acid and an ioxidan modula ion o ascula dys unc ion
in he spon aneously hype ensi e a . P os aglandins Leuko
Essen Fa y Acids 65, 91–97.
Aguila MB & Manda in-de-Lace da CA (2000) E ec o di e en
high- a die s on he myoca dium s e eology and blood p essu e
in a s. Pa hol Res P ac 196, 841–846.
Ange e P & Von Shacky C (2000) N-3 Polyunsa u a ed a y
acids and he ca dio ascula sys em. Cu Opin Lipidol 11,
57–63.
Be ada Y, Se a A, Baddou i K, Che ah A & Hassa M (2000)
Expe imen al e idence o an an ihype ensi e and hypocholes-
e olemic e ec o oil o a gan, a gania side oxylon. The apie
55, 375–378.
Be ougui H, E aib A, He e a MD, Al a ez de So omayo M,
Benna i-Kabchi N & Hmamouchi M (2003) Hypolipidemic
and hypocholes e olemic e ec o o gan oil (A gania spinosa
L.) in Me iones shawi a s. J E hnopha macol 89, 15–18.
Calde PC (1997) n-3 Polyunsa u a ed a y acids and immune
cell unc ion. Ad an Enzyme Regul 37, 197–237.
Ca neado J, Al a ez de So omayo M, Pe
´ ez-Gue e o C, e al.
(2002) Sim as a in imp o es endo helial unc ion in SHR a s
h ough a supe oxide dismu ase-media ed an ioxidan e ec . J
Hype ens 20, 429–437.
Cha ou Z & Guillaume D (1999) E hnoeconomical, e hnomedi-
cal, and phy ochemical s udy o A gania spinosa (L.) Skeels: a
e iew. J E hnopha macol 67, 7–14.
H. Be ougui e al.928
Downloaded om h ps://www.camb idge.o g/co e. IP add ess: 88.14.28.68, on 14 May 2020 a 14:57:53, subjec o he Camb idge Co e e ms o use, a ailable a h ps://www.camb idge.o g/co e/ e ms. h ps://doi.o g/10.1079/BJN20041293
Chen X, Touyz RM, Bae Pa k J & Schi in EL (2001) An ioxi-
dan e ec s o i amins C and E a e associa ed wi h al e ed
ac i a ion o ascula NADPH oxidase and supe oxide dismu-
ase in s oke-p one SHR. Hype ension 38, 606–611.
Chimi H, Cilla d J & Cilla d P (1994) Au ooxyda ion de l’hile
d’a gan A gania Espinosa L. du Ma oc (Au o-oxida ion o
a gan oil A gania Espinosa L. om Mo occo). Sci Alimen s
15, 117–124.
Das UN (1995) Essen ial a y acid me abolism in pa ien s
wi h essen ial hype ension, diabe es melli us and co ona y
hea disease. P os aglandins Leuko Essen Fa y Acids 52,
387–391.
De Lo ge il M, Salen P, Ma in JL, Monjaud I, Delaye J &
Mamelle N (1999) Medi e anean die , adi ional isk ac o s
and he a e o ca dio ascula complica ions a e myoca dial
in a c ion: he inal epo o he Lyon Die Hea S udy. Ci cu-
la ion 99, 779–785.
D ehe D, Jo no L & Junod AF (1995) E ec s o hypoxan hine–
xan hine oxidase on Ca
2þ
s o es and p o ein syn hesis in human
endo helial cells. Ci c Res 76, 388–395.
Engle MM (1996) g-Linolenic acid: a po en blood p essu e low-
e ing nu ien . In
g
-Linolenic Acid: Me abolism and i s Roles in
Nu i ion and Medicine, pp. 200–217 [YS Huang and DE
Mills, edi o s]. Champaign, IL: AOCS P ess.
Engle MM, Engle MB, E ickson SK & Paul SM (1992) Die a y
gamma-linolenic acid lowe s blood p essu e and al e s ao ic
eac i i y and choles e ol me abolism in hype ension. J Hype -
ens 10, 1197–1204.
F enoux JMR, P os ED, Belle ille JL & P os JL (2001) A poly-
unsa u a ed a y acid die lowe s blood p essu e and imp o es
an ioxidan s a us in spon aneously hype ensi e a s. J Nu
131, 39–45.
G aie WF, Simecek S, Kuko e z WR & Kos ne GM (1996)
High D-glucose-induced changes in endo helial Ca
2þ
/EDRF
signalling a e due o gene a ion o supe oxide anions. Diabe es
45, 1386–1395.
G i in M, Dimi iadis E, Lenehan K, Owens D, Collins P,
Johnson A & Tomkin GH (1996) Non-insulin-dependen
diabe es melli us: die a y monounsa u a ed a y acids and
low-densi y lipop o ein composi ion and unc ion. Q J Med
89, 211–216.
G imsgaa d S, Bona KH, Jacobsen BK & Bje e KS (1999)
Plasma sa u a ed and linoleic a y acids a e independen ly
associa ed wi h blood p essu e. Hype ension 34, 478–483.
G yglewski RJ, Palme RMJ & Moncada S (1986) Supe oxide
anion is in ol ed in he b eakdown o endo helium de i ed
elaxing ac o . Na u e 320, 454–456.
Hannah J & Howa d B (1994) Die a y a s, insulin esis ance, and
diabe es. J Ca dio asc Risk 1, 31–37.
Ho obin DF (1995) Abno mal memb ane concen a ions o
20 and 22-ca bon essen ial a y acids: a common link be ween
isk ac o s and co ona y and pe iphe al ascula disease? -
P os aglandins Leuko Essen Fa y Acids 53, 385–396.
Hu Q, Co da S, Zweie JL, Capog ossi MC & Ziegel-S ein RC
(1998) Hyd ogen pe oxide induces in acellula oscilla ions in
human ao ic endo helial cells. Ci cula ion 97, 268–275.
Keys A (1995) Medi e anean die and public heal h: pe sonal
e lec ions. Am J Clin Nu 61, 1321S–1323S.
Ma z RL, Al a ez de So omayo M, Scho C, S ocle JC &
And ian si ohaina R (2000) Vascula bed he e ogenei y in
age- ela ed endo helial dys unc ion wi h espec o NO and
eicosanoids. B J Pha macol 131, 303–311.
Mills DE, Wa d RP, Mah M & Huang YS (1990) Fa y acid me ab-
olism in no mo ensi e and hype ensi e a s II. Dis ibu ion and
oxida ion o die a y [1.
14
C]-18:2N-6 and [1.
14
C]-18:3N-3. Nu
Res 10, 675–681.
Newaz MA & Nawal NNA (1998) E ec o alpha- ocophe ol on
lipid pe oxida ion and o al an ioxidan s a us in spon aneously
hype ensi e a s. Am J Hype ens 11, 1480–1485.
Newaz MA, Nawal NNA, Rohaizan CH, Muslim N & Gapo A
(1999) a-Tocophe ol inc eased ni ic oxide syn hase ac i i y
in blood essels o spon aneously hype ensi e a s. Am J
Hype ens 12, 839–844.
Schmid EB (1997) n-3 Fa y acids and he isk o co ona y hea
disease. Dan Med Bull 14, 1–22.
Simon JA, Fong J & Be ne JT (1996) Se um a y acids and
blood p essu e. Hype ension 27, 303–307.
Williams CM (2001) Bene icial nu i ional p ope ies o oli e oil:
implica ions o pos p andial lipop o eins and ac o VII. Nu
Me ab Ca dio asc Dis 11, 51–56.
Yoshioka S, Uemu a K, Tamaya N, Tamagawa T, Miu a H,
Iguchi A, Nakamu a V & Ho a N (2000) Die a y a -induced
inc ease in blood p essu e and insulin esis ance in a s. J
Hype ens 18, 1857–1864.
E ec o a gan oil on blood p essu e 929
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