Indian Journal of Experimental Biology
Vol. 38, May 2000, pp. 509-511
Effect of eugenol and tincture of crataegus (TCR) on in vitro oxidation of LDL +
VLDL isolated from plasma of non-insulin dependent diabetic patients
Rajalakshmi K
1
,
2
Prema Gurumurthi & S Niranjali Devara{*
'Department of Biochemistry and Molecular Biology, University of Madras Guindy Campus, Chennai 600 025,lndia
2
Department of Biochemistry, Tuberculosis Research Centre, Chetpet, Chennai,India
Received 21 Apri/1999; revised 18 January 2000
The present study was carried out to study the effect of antioxidants on oxidised LDL + VLDL and found that vitamin
E. eugenol and tincture of crataegus (antioxidants) inhibited oxidation of (LDL+ VLDL) si mil ar to standard antioxidant
(bu tylated hydroxy toluene). Vitamin C acted as an antioxidant at lower concentration, and prooxidant at higher
concentration.
Hyperlipidemia is a metabolic abnormality associated
with diabetes. Low density lipoprotein (LDL) in noninsulin dependent diabetes mellitus (NIDDM) patients
leads to abnormal metabolism and is associated with
an increase in very low density lipoprotein (VLDL)
secretion
and
impaired
VLDL
catabolism'.
Ultimately, this leads to atherosclerotic plaques.
Elevated blood lipid concentrations may lead to
elevated blood lipid peroxides contributing to
endothelial injury and acce lerating the process of
.2
at herogenests .
High lipid peroxide concentration in serum is
found in diabetic patients who are a high
cardiovascular ri sk group. A recent study has found
that NIDDM patients have higher level of plasma
thiobarbituric acid (TBA) reactivity and conjugated
dienes than normal individuals'. The end products of
these complex metal ion-catalyzed breakdown of lipid
4
hydroperoxides are cytoxic aldehydes . It is well
establi shed that oxidative stress, ie. an imbalance
between generation of free radical and antioxidant
defenses, increases in diabetes 5 . LDL isolated from
subjects with poorly controlled IDDM and NIDDM
are most susceptible to oxidative modification than
LDL from control subjects without diabetes6 .
Antioxidants play a major role in preventing freeradical mediated processes. Therefore, thi s study is
aimed to assess the role of antioxidants in protecting
LDL lipids against certain oxidative processes.
All chemicals were purchased from Sig ma
Chemical Company , USA. Non-insulin dependent
*Correspondent author:e-mail: niranj al i @yahoo.com
diabetes mellitus (NIDDM) patients who were regul ar
out-patients at Kilpauk Medical College, Madras were
selected for the study. Venous blood samples were
drawn from the patients in fasting condition, in tubes
containing ethylene diamine tetraacetic acid (EDTA)
as an anticoagulant.
Lipidperoxidation products (as malondialdehyde;
MDA) were determined by thiobarbituric acid
reaction(TBARS) 7 . The plasma samples collected
from diabetic subjects were pooled and LDL +VOL
were isolated by precipitation with heparin,
magnesium chloride and sucrose8 . Effect of
antioxidants, butylated hydroxy toluene, vitamin C,
vitamin E, eugenol and TCR in LDL + VLDL fraction
was studied 9 . Conjugated dienes were estimated
spectrophotometrically 10 . The results were analysed
statistically by Student's t-test.
Both TBARS and conjugated diene were
significantly elevated in LDL+VLDL isolated from
the plasma of diabetic samples (Fig. 1). According to
Siekmier 11 , there is an increase in concentration of
conjugated dienes and TBARS on in vitro oxidation
of LDL. Oxidised LDL has six fold hi gher level of
lipid peroxides , and four fold higher level of
conjugated dienes 12 than native LDL.
In order to assess in vitro effec t of antioxidants on
oxidised LDL + VLDL isolated from plasma of
NIDDM patients, dibutyl hydroxy toluene (BHT) was
used as the reference antioxidant. BHT has been
found to cause a decrease in TBARS formation in
oxidized LDL + VLDL.
According to the present study, LDL oxidation was
inhibited by vitamin Cat 25f..lg and SOf..lg concentration,
INDIAN J EXP BIOL, MAY 2000
510
it increased at 100 flg concentration (Fig. 2) .
Ascorbate is ab le to protect plas ma LDL lipids from
13
damage •
At
physiological
peroxidative
concentration , ascorbate inhibits LDL oxidation by
copper ions and helps to preserve the endogenou s
lipid soluble antioxidants of LDL. Ascorbic acid
66t
70
..J
g 60
0
Non diabetic
5I
Diabetic
Ol
E so
~
~
700 ca
E
600 ~
c..
500 ..J
<(
0
800
697t
40
E
2
......
400
30
c
::J
E
300
c 20
~
200 ....
..c....
100 <(
10
~
0
TBARS
CONJUGATED DIENES
Fi g. I - LDL+YLDL ox idation in non-diabeti c and diabetic
subj ects.
scavenges free radicals or dehydro-ascorbate may
modify LDL such that copper binds to LDL partic le,
14
thus increasing its resistance to oxidation by copper .
Ascorbate also acts as a prooxidant at a hi gher
15
concentration . In such a condition ascorbate results
in increased lipid peroxi dati on products and
16
macrophage uptake of moderately oxidized LDL .
Vitamin E decreased in vitro and in vivo oxidation
of LDL+ VLDL (Fig. 2) . Increased oxidati ve stress
and/or decreased co-anti ox idant concentrati on appears
to promote the antioxidant acti vity of a-tocopherol. In
accordance with all these studies, we have also found
a defi nite reduction in in vitro oxidation of
17
LDL+ VLDL on vitamin E suppl ementati on •
Eugenol also exhibited antioxidant property,
th ough its effec t was lesser than other two
antioxidants used . Eugenol counteracts the deleterious
action of CCI 4 on membrane lipids and a complete
inhibition has been observed on accu mul ation of lipid
peroxidation products durin g eugenol treatment in
18
CCI 4 treated rats .
Tincture of crataegus (TCR) is a known
9
hypolipidemic and antiatherogenic agene . It
inhibited in vitro oxidation of LDL + VLDL. This
120
100
_J
0
_J
0>
E
<!
0
~
80
60
C/)
<l)
0
E
40
c
20
0
25
50
100
150
Concentration of antioxidants ( }J g)
Fig. 2- ln vitro effect of antioxidants on oxidised LDL+YLDL, isolated from plasma of NIDDM pati ents [(A)-ox idi sed LDL+YLDL
(B) oxidised LDL+YLDL + BHT; (C)- oxidised LDL+YLDL +vitamin C; (D)- oxidised LDL + VLDL + vitamin E; (E)- oxidised LDI
+ VLDL + eugenol; and (F)- oxidi sed LDL + VLDL + TCR]
511
NOTES
effect may be attributed to its flavanoid content.
Flavanoids can donate hydrogen or react with
superoxide anions, thereby, scavenging free radicals
20
an d ot her react1ve oxygen species .
In diabetes, there is a profound alteration in lipid
and lipoprotein metabolism with changes in
antioxidant level. Therefore it is concluded that
vitamin E, eugenol, TCR and vitamin C, at
physiological concentration, are effective in maintaining the level of antioxidant under diabetic condition.
The authors would like to thank Dr. Sundaram,
Head, Dr. Ambedkar Institute of Diabetology,
Kilpauk Medical College, Chennai for permitting us
to collect blood samples from known NIDDM
patients.
0
9
0
I0
II
12
13
14
References
I Howard B V, Lipoprotein metabolism in diabetes mellitus, J
Lipid Res , 28 (1987) 613 .
2 Halliwell B & Gutteridge J M C, Free radicals, ageing and
disease in Free radicals in biology and medicine (Clarendon
Press, London) 1989, 421 .
3 Steer K A, Wallace T M, Bolton C H & Hartog M, Aspirin
protects low density lipoprotein from oxidative modification,
Heart, 77 (1997) 333.
4 Baynes J W, Role of oxidative stress in the development of
complications in diabetes, Diabetes, 40, ( 1991 ).
5 Tsai A C, Lipid peroxidation and glutathione peroxidase activity
in the liver of cholesterol fed rats, J Nutr, I 05 (!975) 946.
6 Wojcicki J, Rozewicka L, Barcew-Wiszniewska B,
Samochowie L, Juzwiak S, Kodlubowska D, Tustanowski S
& Juzyszyn Z, Effect of selenium and vitamin E on the
development of experimental atherosclerosis in rabbits,
Atherosclerosis, 87 (1991) 5.
7 Santos M T, Valles J, Aznar J & Vilches J, Determination of
plasma malondialdehyde-like material and its clinical
implication in stroke patients, J Clin Pathol, 33 (1980) 973 .
8 Burstein M, Antisera of rabbits immunized against human low
15
16
17
18
19
20
density lipoproteins (I ,006-1 ,063), Nouv Rev Fr Hematol, 12
(1972) 251.
Wallin B, Rosengren B, Shertzer H G & Camejo , G,
Lipoprotein oxidation and measurement of thiobarbituric acid
reacting substances formation in a single microliter plate: its
use for evaluation of antioxidants, Anal Biochem, ( 1993) 208.
Klein R A, The detection of oxidation in liposome
preparations, Biochem Biophys Acta, 210 (1970) 486.
Seikmier R, Wulfroth P, Wieland H, Gross W & Marz W,
Low density lipoprotein su.sceptibility to in vitro oxidation in
healthy smokers and nonsmokers, Clin. Chem, 42 ( 1996) 524.
Sevanian A, Bittolo-Bon G, Cazzolato G, Hodis H, Hwant
J.Zamburlini A, Mairino M & Ursini F, LDL- is a lipid
hydroperoxide-enriched circulating lipoprotein, J Lipid Res,
38 (1997) 419.
Frei B, Ascorbic acid protects lipids in human plasma and
low-density lipoprotein against oxidative damage, Am. J. Clin .
Nutr, 54S (1991) 1113S.
Jialal I, Norkus E P, Cristo! L & Grundy S M, Beta-carotene
inhibits the oxidative modification of low density lipoprotein ,
Biochim Biophys Acta, I 086 ( 1991) 134.
Hunt J V, Smith C C & Wolff S P, Autooxidative
glycosylation and possible involvement of peroxides and free
radicals in LDL modification by glucose, Diabetes, 39 (1990)
1420.
Esterbauer H, Dieber-Rothender M, Striegl G, Waeg G, Role
of vitamin E in preventing the oxidation of low density
lipoprotein,Am J Clin Nutr, 53 (IS) (1991) 314S.
Kontush A, Finckh B, Karten B, Kohlschutter A, Beisiegel U,
Antioxidant and prooxidant activity of alpha-tocopherol in
human plasma and low density lipoprotein, J.Lipid Res, 37
(1996) 1436.
Parasakthy K, Shanthi S, Deepalakshmi P D, Niranjali S D,
The antioxidant effect of eugenol on CC1 4- induced
erythrocyte damage in rats, Nutr. Biochem. 7 (1996) 23 .
Shanthi R, Parashakthy K, Deepalakshmi P D, Niranjali S D,
Protective effect of tincture of Crataegus on oxidative stress in
experimental atherosclerosis, J Clin Biochem Nutr, 20 ( 1996)
211.
Robak J, Grugglewski R J, Flavanoids are scavengers of free
radicals, Biochem. Pharmacal, 37 (1988) 83 .