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Garlic Increases Antioxidant Levels in Diabetic and Hyperten PDF
Garlic Increases Antioxidant Levels in Diabetic and Hyperten PDF
eCAM 2009;Page 1 of 7
doi:10.1093/ecam/nep011
Original Article
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
as nitrotyrosine (8,10). In addition, a number of in vitro applied to the assessment of antioxidant levels in diabetic
spectrophotometric assays have been developed to assess and hypertensive rat models treated with garlic.
antioxidant capacity including the quantitation of Trolox
equivalent antioxidant capacity (8,11). However, many of Materials and Methods
these assays are laborious, involve the use of difficult and
unclear methodologies and are expensive. In a recent Materials
review, Dalle-Donne and coworkers have discussed the
measurement and reliability of various biomarkers in Streptozotocin, myoglobin (equine) and 2,20 -azinobis-
human disease (10). (3-ethylbenzothiazoline-6-sulphonic acid) diammonium
Recent reports have suggested that diabetic complica- salt (ABTS) were purchased from Sigma Chemical
tions and hypertension have a common etiology involving Co. (St Louis, MO, USA). Trolox (6-hydroxy-2,5,7,
oxidative stress and endothelial damage (9,12–13). In fact, 8-tetramethylchronam-2-carboxylic acid) was purchased
the earliest pathological changes in diabetic complications from Aldrich Chemical Co. (Milwaukee, WI, USA).
have been reported to be hemodynamic in nature (14–16). Vitamin C, reduced glutathione and hydrogen peroxide
Thus, the assessment of oxidative stress in both diabetes were obtained from Fluka (Buchs, Switzerland). Glucose
and hypertension has become crucial as an index of pathol- oxidase kits were obtained from Waco Pure Chemicals
Table 1. Protocol for the antioxidant assay groups containing eight animals each: Group 1, the
Solution Blanka (ml) Sample (ml)
diabetic control group, was injected IP daily with saline
for the treatment period; and Group 2, the garlic-treated
PBS (5 mM, pH 7.4, 1000 – (105 þ x) 1000 – (115 þ x)
138mM NaCl) group, was injected IP daily with 500 mg/kg of the garlic
MetMb (as determined above) xb x extract.
Sample (serum or antioxidant) – 10
After a period of 3 weeks, the rats were sacrificed under
sodium pentobarbitone anaesthesia according to the
ABTS (5mM) 30 30
guidelines for euthanasia in the Guide for Care and
Equilibrate for 3 min at 30 C
Use of Laboratory Animals (49). Before sacrifice, blood
H2O2 (10mM) 75 75 was collected, processed and stored at 80 C for analysis
Incubate for 6 min at 30 C within 1 week as described above.
Read absorbance at 734 nm after
exactly 15 s of incubation
period completionc Induction of Hypertension and Assessment
a
Read against PBS buffer.
b
MetMb concentration is determined experimentally and the required Ten male Sprague–Dawley rats with initial body weight
volume (x) is added to obtain 2.5 mM final concentration in the assay. of 50 g were operated on to induce a two-kidney, one-clip
Figure 1. Standard curves for inhibition of ABTSþ absorbance at Figure 3. Antioxidant levels in hypertensive rats. Normal, untreated
734 nm with (A) glutathione, (B) ascorbic acid (vitamin C) and (C) (control) hypertensive and garlic-treated hypertensive rats were assessed
trolox. Glutathione concentrations were between 0.5 and 2.5 mM. for antioxidant status (TEAC) before and 3 weeks after treatment.
Ascorbic acid concentrations were between 0.005 and 0.02 mM. Trolox (1) Total antioxidants in normotensive rats are significantly higher than
concentrations were between 0.005 and 0.02 mM. All assays were done in hypertensive rats (P50.05). (2) Total antioxidants in hypertensive
in triplicate. rats at time zero are significantly higher than hypertensive rats after
3 weeks (P50.05). (3) Total antioxidants in untreated hypertensive rats
are significantly lower than garlic-treated hypertensive rats after 3 weeks
known antioxidants good reproducibility within the of garlic treatment (P50.05).
concentration ranges was obtained using the assay. The
inter-assay correlation coefficients (R2) were 0.9984, garlic daily recovered antioxidant activity reaching
0.9768 and 0.987 for trolox, glutathione and vitamin C, levels in excess of those observed in normal rats. This
respectively, confirming the reliability and reproducibility increase in serum antioxidant levels was paralleled by a
of the assay. decrease in serum glucose levels as the garlic-treated
diabetic rats returned to an euglycemic state (Table 2).
Effects in Diabetic Rats
Effects in Hypertensive Rats
Induction of a diabetic state with STZ led to marked
hyperglycemia within 3 days of injection (Table 1). This As shown in Fig. 3, 2K-1C hypertensive rats (systolic
hyperglycemic state was accompanied by significantly BP ¼ 200 17 mm) exhibited significantly decreased total
decreased antioxidant levels (about 40% reduction) both antioxidant levels compared to normotensive rats (about
3 days and 3 weeks after STZ-injection (Fig. 2). In 66% reduction). After 3 weeks of garlic treatment, the
contrast, after 3 weeks, the diabetic rats given 500 mg/kg antioxidant levels in the hypertensive rats increased to
eCAM 2009 5 of 7
Table 2. Serum glucose concentration in different animal groups These timing parameters have allowed the assay to be
Initial serum Final serum
conducted with the use of a 30 C water bath and a basic
glucose (mg/dl) glucose (mg/dl) spectrophotometer. The Rice- Evans and Miller manual
Normala (saline treated) – 129 9 assay (46) required the use of a temperature-controlled
Diabetic controla 400 9 431 13b cuvette holder for continuous measurement of absor-
(Saline treated) bance. Thus, in the current modification, the assay
Garlic-treated diabetica 400 9c 237 8c involves pre-incubation of all reagents except H2O2 for
(500 mg/kg garlic) 3 min at 30 C, addition of H2O2 at 15 s intervals followed
a
n ¼ 10 for all groups. by 6 min incubation at 30 C. The absorbance was then
b
Significantly different compared to normal (P50.05). read at exactly 15 s intervals such that all assays were
c
Significantly different compared to initial and final diabetic controls incubated for exactly 6 min. These timing parameters
(P50.001).
allowed six assays to be carried out simultaneously. The
6 min reaction time is in agreement with that of van den
about two-third of the normal levels, while the anti- Berg et al. (59) and Re and coworkers (60), who also
oxidant levels in the hypertensive control rats continued modified the assay to eliminate the use of methemoglo-
to decrease (about 20% of the normotensive level) during bin. In our hands, the modifications described have
high-sucrose diet-induced obesity in rats. NAC treatment 2. Giugliano D, Ceriello A, Paolisso G. Diabetes mellitus, hyperten-
sion and cardiovascular disease: which role for oxidative stress?
resulted in improved glucose tolerance and lipid profile, Metabolism 1995;44:363–8.
as well as decreased LDL-oxidation and serum oxidative 3. Finkel T, Hollbrook NJ. Oxidants, oxidative stress and the biology
stress. Therefore, several studies have suggested that of ageing. Nature 2000;408:239–47.
4. Wiensberger NF. Oxidative stress: the special case of diabetes.
garlic most likely exacerbates diabetes in animal models Biofactors 2003;19:11–8.
through improved antioxidant status. 5. Touyz RM. Reactive oxygen species, vascular oxidative stress and
Therefore, we have shown the applicability of the redox signaling in hypertension. What is the clinical significance?
Hypertension 2004;44:248–52.
TEAC assay to the assessment of antioxidant status 6. Chinopoulous V, Adam-Vizi V. Calcium, mitochondria and
of diabetic rats. It should be noted that the method oxidative stress in neuronal pathology. FEBS J 2006;273:433–50.
described here measures only water-soluble antioxidants. 7. Sies H. Oxidative Stress: Oxidants and Antioxidants. New York:
Academic Press, 1991.
Therefore, the results reported here do not include fat- 8. Shishehbor MH, Hazen SL. Antioxidant studies need a change of
soluble antioxidant levels which also may be affected by direction. Cleveland Clinic J Med 2004;71:285–334.
garlic treatment. 9. Bayraktutan U. Free radicals, diabetes and endothelial dysfunction.
Diabetes Obes Metab 2002;4:224–38.
In previous studies, we have reported that the 2K-1C 10. Dalle-Donne I, Rossi R, Colombo R, Giustarini D, Milzani A.
rat model is characterized by the development of renal Biomarkers of oxidative damage in human disease. Clin Chem
hypertension accompanied by increased expression of two 2006;52:601–23.
29. Bakri IM, Douglas CW. Inhibitory effect of garlic extract on oral 50. Axler DA. Stability of the diabetogenic activity of streptozotocin.
bacteria. Arch Oral Biol 2005;50:645–51. IRCS Med Sci 1982;10:157–65.
30. Ali M, Al-Qattan KK, Al-Enezi F, Khanafer RM, Mustafa T. 51. Al-Qattan KK. Different levels of hypertension induce opposite
Effect of allicin from garlic powder on serum lipids and blood diuretic behaviors from the nonclipped kidney in the rat two-kidney,
pressure in rats fed with a high cholesterol diet. Prostaglandins one-clip model. Kidney Blood Press Res 2001;24:44–51.
Leukot Essent Fatty Acids 2000;62:253–9. 52. Halliwell B. Role of free radicals in the neurodegenerative diseases:
31. Anwar MM, Meki AR. Oxidative stress in streptozotocin-induced therapeutic implications for antioxidant treatment (Review). Drugs
diabetic rats: effects of garlic oil and melatonin. Comp Biochem Aging 2001;18:685–716.
Physiol A Mol Integr Physiol 2003;135:539–47. 53. Stocker R, Keaney JF. Role of oxidative modifications in
32. Stevinson C, Pittler MH, Ernst E. Garlic for treating hypercholes- atherosclerosis. Physiol Rev 2004;84:1381–478.
terolemia. Ann Intern Med 2000;133:420–9. 54. Klaunig JE, Kamendulis LM. The role of oxidative stress in
33. Sheela CG, Augusti KT. Antidiabetic effects of S-allyl cysteine carcinogenesis. Annu Rev Pharmacol Toxicol 2004;44:230–67.
sulphoxide isolated from garlic Allium sativum Linn. Indian J Exp 55. Baynes JW. The role of oxidative stress in the development of
Biol 1992;30:523–6. complications of diabetes. Diabetes 1991;40:405–12.
34. Sheela CG, Kumud K, Augusti KT. Anti-diabetic effects of onion 56. Touyz RM. Reactive oxygen species in vascular biology: role of
and garlic sulfoxide amino acids in rats. Planta Med 1995;61:356–7. arterial hypertension. Expert Rev Cardiovasc Ther 2003;1:91–106.
35. Augusti KT. Therapeutic values of onion (Allium cepa L.) and garlic 57. Medina LO, Veloso CA, de Abreu Borges E, Isoni CA,
(Allium sativum L.). Indian J Exp Biol 1996;34:634–40. Calsolari MR, Chaves MM, Nogueira-Machado JA. Determination
36. Kasuga S, Ushijima M, Morihara N, Itakura Y, Nakata Y. Effect of the antioxidant status of plasma from type 2 diabetic patients.
of aged garlic extract (AGE) on hyperglycemia induced by Diab Res Clin Prac 2007;77:193–7.
immobilization stress in mice. Nippon Yakurigaku Zasshi 1999; 58. Wayner DDM, Burton GW, Ingold KU, Barclay LRC, Locke SJ.