Beruflich Dokumente
Kultur Dokumente
eCAM 2009;Page 1 of 7
doi:10.1093/ecam/nep011
Original Article
Introduction
Oxidative stress, an excessive production of reactive
oxygen species (ROS) above the bodys antioxidant
capacity, has been implicated in the development of
many pathophysiological conditions including hypertension, diabetes, atherosclerosis and cancer, as well as the
process of aging (16). ROS are normal products of
cellular metabolism which are usually removed by endogenous antioxidants (7). However, it has become increasingly clear that overproduction of ROS can lead to a
damaging cycle of lipid peroxidation, depletion of natural
For reprints and all correspondence: Martha Thomson, Department of
Biological Sciences, Faculty of Science, Kuwait University, P.O.
Box 5969, 13060-Safat, Kuwait. Tel: 965-2498-5710; Fax: 965-4847054; E-mail: mmtkuwait2003@hotmail.com
Oxidative damage by free radicals has been implicated in the pathogenesis of vascular disease in
diabetes and hypertension. In the present study, the total antioxidant status in diabetic and
hypertensive rats before and after treatment with garlic (Allium sativum) was determined. The
total serum antioxidants were measured by a modified method reported earlier by Miller and
coworkers. The reproducibility of the assay was confirmed by determining standard curves for
the known antioxidants: trolox (a stable analog of vitamin E), glutathione and vitamin C with
interassay correlation coefficients (R2, n 10 in triplicate) of 0.9984, 0.9768 and 0.987, respectively, confirming the reliability and reproducibility of the assay. This assay was then used to
determine total serum antioxidant levels of streptozotocin-induced diabetic rats and two-kidney
one-clip hypertensive rats both before and after 3 weeks of treatment with an aqueous extract
of garlic (500 mg/kg IP daily). The serum antioxidant levels of rats after 3 weeks of treatment
were significantly higher (P50.001) than the pretreatment levels in both diabetic and hypertensive rats. The increased serum antioxidant levels were paralleled by a decrease in serum
glucose in the garlic-treated diabetic rats and lowered systolic blood pressure in the garlictreated hypertensive rats. We conclude from our study that (i) total antioxidants can be
measured by a simple, reproducible, reliable assay and (ii) the total antioxidant status can be
significantly improved by treatment with garlic.
2 of 7
eCAM 2009
3 of 7
Solution
Blanka (ml)
Sample (ml)
1000 (105 x)
1000 (115 x)
xb
10
ABTS (5mM)
30
30
75
75
Results
Assessment of the Assay
The reproducibility of the antioxidant assay was assessed
by determination of the inhibitory effects of three known
antioxidants, vitamin C, glutathione and trolox (a stable
analog of vitamin E). The standard curve for each
antioxidant was performed 810 times in triplicate. The
standard curves in Fig. 1ac indicate that for all three
4 of 7
eCAM 2009
Final serum
glucose (mg/dl)
129 9
Diabetic controla
(Saline treated)
400 9
431 13b
Garlic-treated diabetica
(500 mg/kg garlic)
400 9c
237 8c
Discussion
During the last decade, it has become increasingly evident
that many chronic diseases are accompanied by increased
levels of oxidative stress exacerbated by decreased antioxidant levels (10,17,5254). These observations have
precipitated much interest in study of the correlations
between oxidative stress, antioxidant potential and
development of chronic diseases in both humans and
animal models. Of particular interest are the correlations
between oxidative stress and development of diabetes and
hypertension (45,9,5557).
With the surge in research in this area, the development
of simple, reliable and consistent methods for quantitation of oxidative stress has become imperative. In
particular, assays for serum antioxidant levels have been
developed including measurement of total peroxy radical
trapping as described by Wayner et al. (58) and RiceEvans and Miller (4547). Whereas the original peroxy
radical trapping parameter assay of Wayner and coworkers (57) required an oxygen electrode endpoint,
Rice-Evans and Miller developed this assay to use a
spectrophotometric endpoint (45). As described above,
this assay involves utilization of the peroxidase activity of
methemoglobin, generation of the radical cation,
ABTS, and quantitation of the quenching of this
radical cation by antioxidants. Results are expressed in
Trolox equivalent antioxidant capacity using the vitamin
E analog, Trolox, as the reference antioxidant.
In the present study, we have modified the RiceEvans and Miller assay (46,47) to include more precise
timing parameters including a 3-min pre-incubation, a
6-min reaction time followed by absorbance measurement exactly 15 s after stopping the inhibition reaction.
about two-third of the normal levels, while the antioxidant levels in the hypertensive control rats continued
to decrease (about 20% of the normotensive level) during
the 3-week experimental period. This increase in antioxidant levels occurred simultaneously with a 50%
decrease in blood pressure (to 100 22 mm systolic) in
the 2K-1C hypertensive rats.
5 of 7
6 of 7
Summary
From this study, the following conclusions can be stated.
A reliable and reproducible assay for determination of total
antioxidant levels in serum has been developed and applied.
Treatment of diabetic rats with garlic resulted in significantly increased antioxidant and lowered glucose levels
compared to untreated diabetic animals. Treatment of 2K1C hypertensive rats with garlic increased the total level of
antioxidants to about two-third of the normal level. Garlictreated 2K-1C hypertensive rats had significantly higher
total antioxidant levels and lower systolic blood pressure
than untreated 2K-1C hypertensive rats.
Acknowledgement
This work was supported by the College of Graduate
Studies, Kuwait University for which the authors express
gratitude.
References
1. Halliwell B. Free radicals, antioxidants and human disease: cause or
consequence? Lancet 1994;344:7214.
2. Giugliano D, Ceriello A, Paolisso G. Diabetes mellitus, hypertension and cardiovascular disease: which role for oxidative stress?
Metabolism 1995;44:3638.
3. Finkel T, Hollbrook NJ. Oxidants, oxidative stress and the biology
of ageing. Nature 2000;408:23947.
4. Wiensberger NF. Oxidative stress: the special case of diabetes.
Biofactors 2003;19:118.
5. Touyz RM. Reactive oxygen species, vascular oxidative stress and
redox signaling in hypertension. What is the clinical significance?
Hypertension 2004;44:24852.
6. Chinopoulous V, Adam-Vizi V. Calcium, mitochondria and
oxidative stress in neuronal pathology. FEBS J 2006;273:43350.
7. Sies H. Oxidative Stress: Oxidants and Antioxidants. New York:
Academic Press, 1991.
8. Shishehbor MH, Hazen SL. Antioxidant studies need a change of
direction. Cleveland Clinic J Med 2004;71:285334.
9. Bayraktutan U. Free radicals, diabetes and endothelial dysfunction.
Diabetes Obes Metab 2002;4:22438.
10. Dalle-Donne I, Rossi R, Colombo R, Giustarini D, Milzani A.
Biomarkers of oxidative damage in human disease. Clin Chem
2006;52:60123.
11. Cao G, Prior RL. Comparison of different analytical methods for
assessing total antioxidant capacity of human serum. Clin Chem
1998;44:130915.
12. Hadi HAR, Al Suwaidi J. Endothelial dysfunction in diabetes
mellitus. Vasc Health Risk Manag 2007;3:85376.
13. Nicolls MR, Haskins K, Flores SC. Oxidant stress, immune
dysregulation, and vascular function in type I diabetes. Antioxid
Redox Signal 2007;9:87989.
14. Brownlee M. Biochemistry and molecular biology of diabetic
complications. Nature 2000;414:81320.
15. Sheetz MJ, King GL. Molecular understanding of hyperglycemias
adverse effects for diabetic complications. JAMA 2002;288:257988.
16. Engerman R, Bloodworth MJ, Nelson S. Relationship of microvascular disease in diabetes to metabolic control. Diabetes
1985;26:7609.
17. Halliwell R, Gutteridge JMC. Free Radicals in Biology and
Medicine. New York: Oxford University Press, 1999.
18. Ceriello A. Oxidative stress and diabetes-associated complications.
Endocr Prac 2006;12:602.
19. Ceriello A. New insights on oxidative stress and diabetic complications may lead to a causal antioxidant therapy. Diabetes Care
2003;26:158996.
20. Vasdev S, Gill VD, Singal PK. Modulation of oxidative stressinduced changes in hypertension and atherosclerosis by antioxidants. Exp Clin Cardiol 2006;11:20616.
21. Ali M, Thomson M, Alnaqeeb MA, Al-Hassan JM, Khater SH,
Gomes SA. Antithrombotic activity of garlic: its inhibition of the
synthesis of thromboxane-TXB2 during infusion of arachidonic acid
and collagen in rabbits. Prostaglandins Leukot Essent Fatty Acids
1990;41:959.
22. Kiesewetter H, Jung J, Pindur G, Jung EM, Mrowietz C, Wenzel E.
Effect of garlic on thrombocyte aggregation, microcirculation, and
other risk factors. Int Clin Pharmacol Ther Toxicol 1991;29:1515.
23. Ali M, Thomson M. Consumption of a garlic clove a day could be
beneficial in preventing thrombosis. Prostaglandins Leukot Essent
Fatty Acids 1995;53:2112.
24. Bordia T, Mohammed N, Thomson M, Ali M. An evaluation of
garlic and onion as antithrombotic agents. Prostaglandins Leukot
Essent Fatty Acids 1996;54:1836.
25. Thomson M, Mustafa T, Ali M. Thromboxane-B2 levels in serum of
rabbits receiving a single intravenous dose of aqueous extract of
garlic and onion. Prostaglandins Leukot Essent Fatty Acids
2000;63:21721.
26. Steiner M, Li W. Aged garlic extract, a modulator of cardiovascular
risk factors: a dose-finding study of the effects of AGE on platelet
functions. J Nutr 2001;131:980S4S.
27. Rees LP, Minney SF, Plummer NT, Slater JH, Skyrme DA.
A quantitative assessment of the antimicrobial activity of garlic
(Allium sativum). World J Microbiol Biotechnol 1993;9:3037.
28. Yoshida H, Iwata N, Karsuzaki H, Naganawa R, Ishikawa K,
Fukuda H, et al. Antimicrobial activity of a compound isolated
from an oil-macerated garlic extract. Biosci Biotechnol Biochem
1998;62:10147.
eCAM 2009
29. Bakri IM, Douglas CW. Inhibitory effect of garlic extract on oral
bacteria. Arch Oral Biol 2005;50:64551.
30. Ali M, Al-Qattan KK, Al-Enezi F, Khanafer RM, Mustafa T.
Effect of allicin from garlic powder on serum lipids and blood
pressure in rats fed with a high cholesterol diet. Prostaglandins
Leukot Essent Fatty Acids 2000;62:2539.
31. Anwar MM, Meki AR. Oxidative stress in streptozotocin-induced
diabetic rats: effects of garlic oil and melatonin. Comp Biochem
Physiol A Mol Integr Physiol 2003;135:53947.
32. Stevinson C, Pittler MH, Ernst E. Garlic for treating hypercholesterolemia. Ann Intern Med 2000;133:4209.
33. Sheela CG, Augusti KT. Antidiabetic effects of S-allyl cysteine
sulphoxide isolated from garlic Allium sativum Linn. Indian J Exp
Biol 1992;30:5236.
34. Sheela CG, Kumud K, Augusti KT. Anti-diabetic effects of onion
and garlic sulfoxide amino acids in rats. Planta Med 1995;61:3567.
35. Augusti KT. Therapeutic values of onion (Allium cepa L.) and garlic
(Allium sativum L.). Indian J Exp Biol 1996;34:63440.
36. Kasuga S, Ushijima M, Morihara N, Itakura Y, Nakata Y. Effect
of aged garlic extract (AGE) on hyperglycemia induced by
immobilization stress in mice. Nippon Yakurigaku Zasshi 1999;
1999:1917.
37. Eisenbarth GS, Kotzin BL. Enumerating autoreactive T cells in
peripheral blood: a big step in diabetes prediction. J Clin Invest
2003;111:17981.
38. Jelodar GA, Maleki M, Motadayen MH, Sirus S. Effect of
fenugreek, onion and garlic on blood glucose and histopathology
of pancreas of alloxan-induced diabetic rats. Indian J Med Sci
2005;59:649.
39. Liu C-T, Hse H, Lii C-K, Chen P-S, Sheen L-Y. Effects of garlic
oil and diallyl trisulfide on glycemic control in diabetic rats. Eur J
Pharmacol 2005;516:16573.
40. Banerjee SK, Maulik SK. Effect of garlic on cardiovascular
disorders: a review. Nutr J 2002;1:417.
41. Mizuguchi S, Takemura S, Minamiyama Y, Kodai S, Tsukioka T,
Inoue K, et al. S-allyl cysteine attenuated CCl4-induced oxidative
stress and pulmonary fibrosis in rats. Biofactors 2006;26:8192.
42. Avci A, Atli T, Erguder IB, Varli M, Devrim E, Aras S, Durak I.
Effects of garlic consumption on plasma and erythrocyte antioxidant parameters in elderly subjects. Geront[ology 2008;54:17376.
43. Al-Qattan KK, Khan I, Alnaqeeb MM, Ali M. Mechanism of garlic
(Allium sativum) induced reduction of hypertension in 2K-1C rats:
a possible mediation of Na/H exchanger isoform-1. Prostaglandins
Leukot Essent Fatty Acids 2003;69:21722.
44. Al-Qattan KK, Thomson M, Al-Mutawaa S, Al-Hajeri DA,
Drobiova H, Ali M. Nitric oxide mediates the blood-pressure
lowering garlic in the rat two-kidney, one-clip model of hypertension. J Nutr 2006;136:774S6S.
45. Miller NJ, Rice-Evans CA. A novel method for measuring
antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci 1993;84:40712.
46. Rice-Evans CA, Miller NJ. Total antioxidant status in plasma and
body fluids. Methods Enzymol 1994;234:27993.
47. Miller NJ, Rice-Evans CA. Spectrophotometric determination of
antioxidant activity. Redox Report 1996;2:16171.
48. Huang D, Ou B, Prior RL. The chemistry behind antioxidant
assays. J Agric Food Chem 2005;53:184156.
49. Veterinary Medical Care. In: Guide for the Care and Use of
Laboratory Animals. Washington:Institute for Laboratory Animal
Research, American Academy of Sciences: 1996, 5670.
7 of 7