|Year : 2012 | Volume
| Issue : 1 | Page : 38-41
Inhibitory effect of some local medicinal plants on in-vitro oxidative modification of low-density lipoprotein
Faten K. Abd El-Hady1, Ahmed G. Hegazi2, El-Sayed M. Mahdy3, Wafaa G. Shousha3, Zeinab A. El-Shahid1
1 Department of Chemistry of Natural Products, National Research Center, Giza, Egypt
2 Department of Zoonosis, National Research Center, Giza, Egypt
3 Chemistry Department, Biochemistry Division, Faculty of Science, Helwan University, Cairo, Egypt
|Date of Submission||03-Nov-2011|
|Date of Acceptance||02-Feb-2012|
|Date of Web Publication||18-Jul-2014|
Faten K. Abd El-Hady
Department of Chemistry of Natural Products, National Research Center, 12622 Giza
Source of Support: None, Conflict of Interest: None
Oxidative modification of low-density lipoprotein (LDL) has been implicated in atherogenesis. Antioxidants that prevent LDL oxidation may reduce atherosclerosis.
The antioxidant activity of 45 extracts from 15 locally used medicinal plants were studied in-vitro employing using three different systems; the DPPH (1,1-diphenyle-2-picryl-hydrazyl) radical scavenging assay, superoxide anion generated in Xanthine–Xanthine oxidase (X-XOD) system and the LDL oxidation induced by cupper ions.
It was observed that the leaf extracts of olive, Jew's mallow, celery, the seed extract of celery and safflower, and ginger extracts had the highest antioxidant activity in the three assays.
It could be concluded that these plant extracts could play an important role in the inhibition of lipid peroxidation in biological systems through their antioxidant, metal chelating, and free radical scavenging activities.
Keywords: antioxidant, DPPH, low-density lipoprotein, medicinal plants, thiobarbituric acid reactive substance, xanthine oxidase
|How to cite this article:|
El-Hady FA, Hegazi AG, Mahdy ESM, Shousha WG, El-Shahid ZA. Inhibitory effect of some local medicinal plants on in-vitro oxidative modification of low-density lipoprotein. Egypt Pharmaceut J 2012;11:38-41
|How to cite this URL:|
El-Hady FA, Hegazi AG, Mahdy ESM, Shousha WG, El-Shahid ZA. Inhibitory effect of some local medicinal plants on in-vitro oxidative modification of low-density lipoprotein. Egypt Pharmaceut J [serial online] 2012 [cited 2020 Nov 29];11:38-41. Available from: http://www.epj.eg.net/text.asp?2012/11/1/38/136968
| Introduction|| |
Recent research has established the role of reactive oxygen species in the pathogenesis of certain human illnesses including cancer, aging, and atherosclerosis. Oxidation of biomolecules, including lipid peroxidation, involves a series of free-radical mediated chain reactions and is associated with several types of biological damages. Therefore, much attention has been focused on the use of antioxidants to inhibit lipid peroxidation and to protect biomolecules from damage by free radicals 1. Antioxidants that inhibit oxidation of low-density lipoproteins (LPLs) have been considered to be potential antiatherogenic agents 2,3. Many synthetic antioxidant components have shown toxic and/or mutagenic effects, which directed most of the attention toward the naturally occurring antioxidants the use of which has mainly centered on the prevention of illness and the maintenance of health 4.
The oxidative modification hypothesis of atherosclerosis predicts that LDL oxidation is an early event in atherosclerosis 5. Therefore, inhibition of LDL oxidation might be an important step in preventing atherogenesis 6.
Numerous natural compounds have been reported to inhibit oxidation of LPLs in vitro 7, such as Gingko biloba extract 8, several garlic compounds 9, 10, mulberry leaf extract 11, buckwheat hall extract 12, Cichorium intybus root 13, piperlactam S, an alkaloid isolated from Piper kadsura 14, edible plant products 15, and chemical constituents of Morinda citrifolia fruits 6.
Thus, the aim of this study was to isolate highly effective antioxidants from locally used medicinal plants to protect LDL from copper-induced oxidation in vitro.
| Subjects and methods|| |
Plant processing and fractionation
Raw medicinal plants used locally in Egypt, fruits, seeds, roots, vegetables, and spices were selected. Common names, scientific names, and parts of the plant used are summarized in [Table 1]. Powdered samples (1 g) from each plant were extracted with methanol, 70% methanol, and water for 24 h to give extracts 1, 2, and 3, respectively. Extracts were filtered and stored at 0°C until used.
|Table 1: Assessment of antioxidant activity through inhibition of DPPH, superoxide anion radicals, and copper-induced low-density lipoprotein oxidation|
Click here to view
Determination of DPPH radical scavenging activity
The DPPH (1,1-diphenyle-2-picryl-hydrazyl) radical scavenging activity was determined according to the method of Matsushige et al. 16. The absorbance was measured at 520 nm. Samples and DPPH were dissolved in methanol. The mean of three measurements of each sample was calculated. caffeic acid was used as a positive control [inhibitory concentration (IC 50 )=5.6 μmol/l].
Determination of superoxide anion radical scavenging activity
The superoxide anion radical scavenging activity by generating superoxide anion free radicals in the xanthine–xanthine oxidase (XOD) system was measured following the method of Matsushige et al. 16. The color obtained was measured at 560 nm. The mean of three measurements of each sample was calculated. Caffeic acid was used as a positive control (IC 50 =6.4 μmol/l).
Measurement of copper-induced low-density lipoprotein oxidation in vitro
Isolation of low-density lipoprotein
LDL was isolated according to the method of Gugliucci and Menini 17. LDL (1.019–1.055 g/ml) was separated by sequential ultracentrifugation using a TL-100 ultracentrifuge (Beckman, Abbott Park, Illinois, USA) from plasma to which EDTA (0.1%) had been added previously. LDL was then extensively dialyzed against PBS (10 mmol/l sodium phosphate buffer, pH 7.2, containing 150 mmol/l NaCl) containing 0.01% EDTA at 4°C. LDL which will be used for oxidative modification by Cu2+ was dialyzed against a 1000-fold volume of PBS at 4°C. Samples were stored at 4°C in the dark and used within 24 h. Protein content was determined according to Lowry’s method. LDL was oxidized using 5 μmol/l of CuSO4. Incubation was carried out at 37°C for 1 h with the antioxidant (plant extract) before the addition of CuSO4. Oxidation of LDL was monitored in the presence or the absence of antioxidant by measuring the amount of thiobarbituric acid reactive substance (TBARS) and protein modification.
Thiobarbituric acid reactive substance assay
LDL was oxidized using 5μmol/ml CuSO4 18, oxidation of LDL was monitored in the presence or absence of plant sample by measuring the thiobarbituric acid reactive substances (TBARS). The absorbance was measured at 534 nm using UV Spectrophotometer [UNICAM UV300]. Malondialdehyde-bis-(dimethylacetal), which yields malondialdehyde (MDA) by acid treatment, was used as a standard.
Protein modification analysis
It was carried out according to the method of Visioli et al. 19. LDL (200 μg protein/ml) was incubated with 5 μmol/l CuSO4 at 37°C. After 2 h (120 min), incubation was stopped.
4-Hydroxynonenal–lysine and MDA–lysine adduct formation was analyzed by measuring fluorescence at Ex 360–Em 430 and Ex 354–Em 410, respectively, using a spectrofluorometer (FP-777 Jasco, Japan).
| Results|| |
Fifteen different locally used medicinal plants were extracted with methanol–water in different proportions to produce 45 different extracts. The antioxidant activities of different extracts were assessed with three different assays: DPPH radical scavenging assay, superoxide anion generated in the xanthine–XOD system, and LDL oxidation assay.
Effect of different plant extracts on DPPH free radical scavenging activity
[Table 1] summarizes the results of the free radical scavenging activity in the DPPH radical scavenging assay. The antioxidant activities in the DPPH scavenging assay ranged from 44.87 to 75.26%. Extract 2 of olive leaves showed the highest antioxidant activity, followed by extract 3 of celery seeds, extract 1 of Jew’s mallow leaves, extract 2 of turnip seeds, extract 3 of olive leaves, and extract 3 of Jew’s mallow leaves. Other extracts with relatively high antioxidant activities were the extract 2 of Jew’s mallow leaves, extract 2 of safflower, extract 1 of celery leaves, extract 3 of ginger, extract 1 of olive leaves, extract 1 of ginger, extract 2 of celery leaves, and extract 3 of black seeds. The moderate effect ranged from 29.68 to 43.46% and the lowest activity was less than 29% (at a concentration of 100 µg/ml of the plant extract).
Effect of different plant extracts on the superoxide anion radical
The free radical scavenging activity on the superoxide anion radical generated by an enzymatic method was evaluated. The results are shown in [Table 1]. Fourteen different plant extracts showed the highest activities that ranged from 79.6 to 42.6%; the moderate antioxidant activities ranged from 37.4 to 22.8%. From these data it was clear that all the extracts of celery leaves and the extract 3 of Jew’s mallow showed the highest activity, followed by extract 3 of black seeds, ginger, and extract 2 of parsley seeds.
Antioxidant activity of plant extracts on copper-induced low-density lipoprotein oxidation
The plants showed the highest free radical scavenging activity against the DPPH radical and/or the xanthine–XOD system, which was assessed by measuring the inhibition of human LDL oxidation in vitro.
TBARS, an index of lipid peroxidation, were undetectable in control LDL, with the level rising slightly only after 3 h of incubation. Incubation with the oxidant resulted in a marked elevation of TBARS. After 24 h of incubation in the presence of the oxidant, the level TBARS did not further increase significantly (data not shown). Preincubation of LDL with any of the plant extracts that showed the highest free radical scavenging activity in DPPH and/or the xanthine–XOD system resulted in significant inhibition of TBARS accumulation. From the data shown in [Table 1] it was clear that extract 2 of celery, olive, Jew’s mallow leaves, safflower, and ginger and extract 3 of celery seeds, ginger, and olive leaves had the highest antioxidant activities against copper-induced LDL oxidation.
Effect of different plant extracts on protein modification
Values of fluorescence at Ex 360–Em 430 and Ex 354–Em 410 increased 120 min after the addition of CuSO4 to the LDL samples, indicating the formation of 4-hydroxynonenal–lysine and MDA–lysine adducts, respectively. Preincubation of the samples with the plant extracts markedly reduced protein modification [Table 1].
| Discussion|| |
LDL lipid peroxidation is considered to be essential in the pathogenesis of atherosclerosis 20,21. Although data concerning the mechanisms by which lipid peroxidation occurs in vivo are scarce, several lines of evidence suggest that some endogenous and exogenous compounds with antioxidant activities could have some beneficial effects in the prevention of the disease. Many plant phenols and flavonoids may be important dietary antioxidants 22,23.
In this study, we set out to demonstrate the antioxidant properties of 45 different extracts using three different assays: the DPPH radical scavenging assay, superoxide anion generated in the xanthine–XOD system, and the LDL oxidation assay.
In the DPPH radical system, antioxidants directly react with the DPPH radical. In the xanthine–XOD system, a superoxide anion radical is enzymatically generated. The harmful effect of superoxide is reduced by the XOD present in the animal body. Plant extracts also showed activities similar to those of the superoxide dismutase enzyme. The activities of some plant extracts in these two systems showed similar trends; thus, in the present study, DPPH free radical (chemical) and xanthine–XOD (enzymatic) systems were selected to isolate plant extracts that have high antioxidant activities. It is believed that finding a new property in locally used medicinal plants that have withstood the test of time in terms of lack of toxicity may prove extremely relevant.
Transition metals are powerful initiators of lipid peroxidation. It was observed that several aldehydes are formed, mainly 4-hydroxy-2-nonenal and MDA 24,25. The formation of MDA was monitored by measuring the amount of TBARS. LDL oxidation can also be detected spectroflurometrically by measuring the amount of MDA–lysine or histidine adducts formed. These protein modifications of LDLs alter their charges and configurations, leading to more negatively charged LDLs with atherogenic properties 24,25.
Antioxidants have two basic mechanisms of action, the first is by free radical scavenging (i.e. electron donation) and the second is by chelating transition metal ions 7, 26, 27. In the DPPH system, some plant extracts show high free radical scavenging activities by quenching the stable free radical DPPH [Table 1]. The antioxidant activity of plant extracts was tested on a different radical generated enzymatically, namely, the superoxide anion radical generated in the xanthine–XOD system [Table 1]. The activities of some plant extracts in these two systems showed similar trends. From the two different systems it was observed that plants with high antioxidant activities act by a free radical scavenging mechanism through electron donation.
The data shown in [Table 1] indicate that some plant extracts have high antioxidant activities because of their ability to inhibit LDL oxidation. A mechanism suggested that these plant extracts act by metal chelation. Some of the plant extracts had the ability to protect LDL against protein modification.
| Conclusion|| |
From all of the above mentioned data it could be concluded that the leaf extracts of olive, Jew’s mallow, and celery; seed extracts of celery and safflower; and ginger extracts had the highest antioxidant activities in the three assays. Thus, it was suggested that these plant extracts could play an important role in the inhibition of lipid peroxidation in biological systems through their antioxidant, metal-chelating, and free radical scavenging activities.
| Acknowledgements|| |
The authors are grateful for the financial support by the National Research Center of Egypt (Contract 1/48/5).
| References|| |
|1.||Kumar PS, Sucheta S, Deepa VS, Selvamani P, Latha S. Antioxidant activity in some selected Indian medicinal plants. Afr J Biotechnol. 2008;7:1826–1828 |
|2.||Heinecke JW. Clinical trials of vitamin E in coronary artery disease: is it time to reconsider the low-density lipoprotein oxidation hypothesis? Curr Atheroscler Rep. 2003;5:83–87 |
|3.||Tamura K, Kato Y, Ishikawa A, Kato Y, Himori M, Yoshida M, et al. Design and synthesis of 4,6-di-tert-butyl-2,3-dihydro-5-benzofuranols as a novel series of antiatherogenic antioxidants. J Med Chem. 2003;46:3083–3093 |
|4.||Aviram M, Dornfeld L, Kaplan M, Coleman R, Gaitini D, Nitecki S, et al. Pomegranate juice flavonoids inhibit low-density lipoprotein oxidation and cardiovascular diseases: studies in atherosclerotic mice and in humans. Drugs Exp Clin Res. 2002;28:49–62 |
|5.||Stocker R, Keaney JFJ. Role of oxidative modifications in atherosclerosis. Physiol Rev. 2004;84:1381–1478 |
|6.||Kamiya K, Tanaka Y, Endang H, Umar M, Satake T. Chemical constituents of Morinda citrifolia fruits inhibit copper-induced low-density lipoprotein oxidation. J Agric Food Chem. 2004;52:5843–5848 |
|7.||Pinchuk I, Lichtenberg D. The mechanism of action of antioxidants against lipoprotein peroxidation, evaluation based on kinetic experiments. Prog Lipid Res. 2002;41:279–314 |
|8.||Sloley BD, Urichuk LJ, Morley P, Durkin J, Shan JJ, Pang PK, et al. Identification of kaempferol as a monoamine oxidase inhibitor and potential neuroprotectant in extracts of Ginkgo biloba leaves. J Pharm Pharmacol. 2000;52:4–451 |
|9.||Lau BHS. Suppression of LDL oxidation by garlic1,2. J Nutr. 2001;131(3 Suppl):985S–988SS |
|10.||Ide N, Lau BH. Garlic compounds minimize intracellular oxidative stress and inhibit nuclear factor-kappa B activation. J Nutr. 2001;131(3 Suppl):1020S–1026SS |
|11.||Doi K, Kojima T, Fujimoto Y. Mulberry leaf extract inhibits the oxidative modification of rabbit and human low density lipoprotein. Biol Pharm Bull. 2000;23:1066–1071 |
|12.||Mukoda T, Sun B, Ishiguro A. Antioxidant activities of buckwheat hall extract toward various oxidative stresses in vitro and in vivo. Biol Pharm Bull. 2001;24:209–213 |
|13.||Kim TW, Yang KS. Antioxidative effects of Cichorium intybus root extract on LDL (low sensity lipoprotein) oxidation. Arch Pharm Res. 2001;24:431–436 |
|14.||Tsai JY, Chou CJ, Chen CF, Chiou WF. Antioxidant activity of piperlactam S: prevention of copper-induced LDL peroxidation and amelioration of free radical-induced oxidative stress of endothelial cells. Planta Med. 2003;69:3–8 |
|15.||Katsube T, Tabata H, Ohta Y, Yamasaki Y, Anuurad E, Shiwaku K, et al. Screening for antioxidant activity in edible plant products: comparison of low-density lipoprotein oxidation assay, DPPH radical scavenging assay and Folin–Ciocalteu assay. J Agric Food Chem. 2004;52:2391–2396 |
|16.||Matsushige K, Basnet P, Kadota S, Namba T. Potent free radical scavenging activity of dicaffeoylquinic acid derivatives from propolis. J Trad Med. 1996;13:217–228 |
|17.||Gugliucci A, Menini T. Three different pathways for human LDL oxidation are inhibited in vitro by water extracts of the medicinal herb Achyrocline satureoides. Life Sci. 2002;71:693–705 |
|18.||Masaki N, Kyle ME, Farber JL. Tert-butyl hydroperoxide kills cultured hepatocytes by peroxidizing membrane lipids. Arch Biochem Biophys. 1989;269:390–399 |
|19.||Visioli F, Bellomo G, Montedoro GFGC. Low density lipoprotein oxidation is inhibited in vitro by olive oil constituents. Atherosclerosis. 1995;117:25–32 |
|20.||Chisolm GM, Steinberg D. The oxidative modification hypothesis of atherogenesis: an overview. Free Radic Biol Med. 2000;28:1815–1826 |
|21.||Weber C, Erl W. Modulation of vascular cell activation, function and apoptosis: role of antioxidants and nuclear factor-kappa B. Curr Top Cell Regul. 2000;36:217–235 |
|22.||Giugliano D. Dietary antioxidants for cardiovascular prevention. Nutr Metab Cardiovasc Dis. 2000;10:38–44 |
|23.||Fuhrman B, Aviram M. Flavonoids protect LDL from oxidation and attenuate atherosclerosis. Curr Opin Lipidol. 2001;12:41–48 |
|24.||Esterbauer H, Gebicki J, Puhl H, Jürgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic Biol Med. 1992;13:341–390 |
|25.||Uchida K, Toyokuni S, Nishikawa K, Kawakishi S, Oda H, Hiai H, et al. Michael addition-type 4-hydroxy-2-nonenal adducts in modified low-density lipoproteins: markers for atherosclerosis. Biochemistry. 1994;33:12487–12494 |
|26.||Esterbauer H, Jürgens G, Quehenberger O, Koller E. Autoxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes. J Lipid Res. 1987;28:495–509 |
|27.||Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med. 1991;11:81–128 |