J Nutr Health Aging

THE JOURNAL OF NUTRITION, HEALTH & AGING©

IN VIVO ANTIOXIDANT PROPERTIES OF LOTUS ROOT AND CUCUMBER: A PILOT COMPARATIVE STUDY IN AGED SUBJECTS L. JI, W. GAO, J. WEI, L. PU, J. YANG, C. GUO Department of Nutrition, Tianjin Institute of Health and Environmental Medicine, Tianjin 300050, P R China. Corresponding author: Dr. Changjiang Guo, Department of Nutrition, Tianjin Institute of Health and Environmental Medicine, Tianjin, 300050, P. R. China, Tel.: +86 22 84655429; fax: + 86 22 84655020, E-mail: [email protected]

Abstract: Objectives: To compare the effects of lotus root and cucumber on antioxidant function in aged subjects. Design: Pilot comparative study. Setting: Research setting with vegetable intervention. Participants: Healthy aged subjects over the age of sixty. Intervention: 30-day supplementation of lotus root or cucumber powder. Measurements: Plasma value of ferric reducing antioxidant power assay, activity of antioxidant enzymes, contents of some antioxidants, oxidation products, hemolysis, blood mononuclear cell DNA damage and urinary excretion of 8-hydroxy-2’-deoxyguanosine were measured before and after the intervention. Results: Plasma glutathione peroxidase activity, contents of vitamin C, total phenolics were significantly increased, while plasma uric acid content significantly decreased in both groups at the end of the intervention. Meanwhile, hemolysis was significantly reduced in both groups and DNA injury rate of blood mononuclear cells in lotus root group and the ratio of comet tail length to total length in cucumber group were also declined significantly postintervention. However, plasma value of ferric reducing antioxidant power assay, contents of reduced glutathione, vitamin E, malondialdehyde, oxidized low density lipoprotein, carbonyls and activity of superoxide dismutase and catalase were not changed significantly in both group after the intervention. Conclusion: These results suggest that lotus root and cucumber are not remarkably different in improving antioxidant function in aged subjects, though they are significantly different in antioxidant capacity in vitro. The benefits observed in this study may come from the additive or synergistic combinations of antioxidants contained in vegetables. Key words: Lotus root, cucumber, antioxidant function, aged subjects.

Introduction Vegetables vary in antioxidant capacity in vitro and may provide protection against oxidative damage differently in vivo. The protective effect has been attributed frequently to the antioxidants contained in vegetables, such as vitamin C (VC), vitamin E (VE), phenolics (including flavonoids) and carotenoids (1-4). Previously, we measured the antioxidant capacity of different vegetables using ferric reducing antioxidant power assay (FRAP assay) and found that the lotus root ranked the highest in FRAP value among the 36 vegetables tested, while several vegetables were found to be relatively lower in antioxidant capacity, such as cucumber, pumpkin, celery and romaine lettuce (5). Isabelle et al. analyzed the antioxidant capacity of the vegetables sampled in Singapore employing oxygen radical absorption capacity (ORAC) assay. The results showed that lotus root was nearly 9 times higher than cucumber in ORAC value (6). Du et al. also reported that lotus root had a high antioxidant capacity as measured by a l,ldiphenyl-2-picryl-hydrazyl (DPPH) radical scavenging assay and protected significantly against hepatic damages in high fat diet–induced obese rats (7). Antioxidant analysis by a highperformance liquid chromatography procedure coupled with coulorimetric array detection demonstrated that the vegetables high in antioxidant capacity contained more antioxidants than those low in antioxidant capacity (8). Aging is an inevitable biological process in life. Although the fundamental mechanisms are poorly understood, a growing body of evidence indicates that oxidative damage is an Received April 28, 2014 Accepted for publication August 7, 2014

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important contributing factor to the aging process and agerelated diseases. Therefore, it is suggested that antioxidant supplementation may play a potential role in the protection against the oxidative damage and thereby slow down the aging process (9-11). However, it was reported that interventions by synthetic antioxidants, such as VC, VE or carotenoids, were not effective in improving the antioxidant function in humans. Supplementation of antioxidants at high level may be harmful to health, especially in well-nourished population (12-15). Since some epidemiological studies indicated that high intake of vegetables and fruits was generally associated with low incidence of oxidative stress related diseases, it is strongly recommended that regular consumption of the natural antioxidants from vegetables, fruits, tea and herbs may be beneficial in improving antioxidant function in aged subjects (16-17). Therefore, we have hypothesized that different vegetables might provide protection differently against oxidative damages because they are different in antioxidant capacity in vitro. Vegetables high in antioxidant capacity should be more effective than those low in antioxidant capacity in reducing oxidative damage associated with the aging process. However, the results obtained from a previous study in aged rats were not supportive in our laboratory. The lotus root, rape and cucumber, which were high, moderate and low in antioxidant capacity relatively, were compared in an attempt to demonstrate that vegetables high in antioxidant capacity were more protective against the aging-related oxidative damage than those low in antioxidant capacity. It was surprising that the three vegetables tested were not different remarkably in

J Nutr Health Aging

IN VIVO ANTIOXIDANT PROPERTIES OF LOTUS ROOT AND CUCUMBER

improving antioxidant function in aged rats (18). The present study was designed to further compare the effects of lotus root and cucumber on several markers of antioxidant function in aged subjects. The objective of this study is to validate whether the antioxidant capacity of vegetables in vitro is correlated with their potentials in improving antioxidant function in aged subjects. The possible effective components were also investigated.

al. (20), Hansen and Warwich (21), Singleton et al. (22), respectively. Plasma reduced glutathione (GSH) was assayed spectrophotometrically by the reaction of 5,5’-dithiobis2-nitrobenzoic acid with thiols. The reagent kit was purchased from Jiancheng Bioengineering Institute (Nanjing, China). Plasma uric acid was measured using a reagent kit from BHKT Company (Beijing, China).

Materials and methods

Table 1 Characteristics of Study Subjects in Two Groups

Subjects and study design Sixteen men and nine women over the age of sixty, living freely in a district of Tianjin, China were recruited by advertisement through the local media and personal contact. They were non-smokers and basically healthy without cancers, hypertension or active conditions of cardiovascular, hepatic, gastrointestinal, or renal diseases and volunteered to participate in the study. A written informed consent was signed by every subject. Approval for this study was granted by the ethical committee of the institute. The subjects were randomly assigned to lotus root group and cucumber group by selection of random number after a regular health check was passed. During the 30-day intervention period, all subjects were asked to abstain from any antioxidant supplements. At the beginning, all subjects were given instructions for balanced diet based on the dietary guidelines recommended by Chinese Nutrition Society. During the 1st and last week of the study, a 3-day dietary survey was carried out using a 24h recall method by experienced interviewers in order to make comparison in daily nutrient intakes between the two groups. Fasting blood samples were collected at the beginning and end of the intervention. Samples of plasma and red blood cells were separated by centrifugation. Fasting urine samples were also collected at the end of the intervention. Preparation of lotus root and cucumber powders Lotus root and cucumber were purchased freshly from a local market and washed in distilled water and squeezed into juices. Lyophilized powders were obtained by freeze-drying under vacuum and stored at -20℃ before being used. The FRAP values, major antioxidant contents of the two vegetable powders were measured and the results were presented in Table 2. During the intervention period, the subjects received daily 25g of lotus root powder or 12.5g of cucumber powder, which was equivalent to 300g of fresh lotus root or cucumber, respectively. The powders were dissolved in warm water and consumed every morning. Plasma antioxidant capacity and contents of VC, VE, reduced glutathione, total phenolics and uric acid Plasma antioxidant capacity was measured by the FRAP assay (19). Plasma contents of VC, VE and total phenolics were determined by the methods described by Brewster et

Parameters

Lotus root Male

n

Cucumber

Female

Male

Female

8

4

8

5

Age (years)

66.6±5.7

66.8±3.3

67.4±5.5

65.8±4.6

Height (m)

1.71±0.04

1.60±0.02*

1.69±0.07

1.56±0.03*

Body weight (kg)

70.0±5.3

60.3±8.5*

70.2±16.0

59.2±8.8

Body mass index (kg/m2)

23.8±1.8

23.5±3.3

24.4±4.2

24.2±2.9

Data are expressed as mean±standard deviation. * P<0.05, compared with the male (twosample t test).

Table 2 FRAP Value and Content of Antioxidants of the Vegetable Powders Group

FRAP mmol/100g

VC mg/100g

TP mg GA/100g

Flavonoids mg Rutin/100g

Lotus root

14.5±2.4

351.7±76.7

692.5±9.1

1054.1±31.7

Cucumber

1.8±0.1*

208.1±43.1*

147.2±0.8*

23.5±5.4*

Data are expressed as mean±standard deviation, n=5. * P<0.05, compared with the lotus root (two-sample t test). FRAP, ferric reducing antioxidant power. GA, gallic acid. VC, vitamin C. TP, total phenolics. The analytical methods used were described in reference 32.

Plasma activity of antioxidant enzymes Plasma superoxide dismutase (SOD) activity was measured through the inhibition of nitroblue tetrazolium reduction by the superoxide radicals generated by the xanthine/xanthine oxidase system in the presence of plasma samples. Plasma glutathione peroxidase (GSH-Px) activity was assayed by measuring the reduction of GSH per min per 0.1 ml plasma after incubation with H2O2. Plasma catalase (CAT) activity was determined by measuring the intensity of a yellow complex formed by molybdate and H2O2, which is catalyzed by plasma CAT. All procedures were performed strictly in accordance to the instructions attached to the commercial assay kits obtained from Jiancheng Bioengineering Institute (Nanjing, China). Plasma contents of malondialdehyde, oxidized low density lipoprotein and carbonyls The content of plasma malondialdehyde (MDA) was determined by the thiobarbituric acid reactive species assay

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J Nutr Health Aging

THE JOURNAL OF NUTRITION, HEALTH & AGING© Table 3 Dietary Intake of Energy and Macronutrients Group

n

Energy (MJ)

Protein (g)

Fat (g)

Carbohydrate (g)

Initial

Final

Initial

Final

Initial

Final

Initial

Final

Lotus root

12

7.8± 1.8

7.9± 1.9

71.4± 23.8

76.7± 14.9

69.9±31.5

60.3± 32.3

246.8± 43.9

271.3± 76.2

Cucumber

13

8.2± 2.0

8.7± 2.7

75.9± 21.8

88.4± 18.8

81.1± 35.0

78.9± 33.2

245.1± 70.4

268.2± 102.6

Data are expressed as mean±standard deviation.

Dietary intake of energy and nutrients As indicated in Table 3, there was no significant difference in dietary intakes of energy, protein, fat and carbohydrates either in the 1st week or the last week between the two groups. No significant difference was also found in dietary intakes of vitamins A, B1, B2, C, E and Ca, Fe, Zn, Se between the two groups in the 1st week or the last week (data not shown).

(23). A commercial enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems Inc., MN, USA) was used to measure plasma oxidized low density lipoprotein (ox-LDL). Plasma carbonyls were detected by the reaction with 2,4-dinitrophenylhydrazine as reported by Levine et al. (24). Hemolytic sensitivity The sensitivity of hemolysis was determined spectrophotometrically according to a method described previously by Grinberg et al. (25). Results were expressed as the percentage of hemolysed red blood cells after incubation with H2O2.

Plasma FRAP value and contents of some antioxidants As shown in Table 4, the content of plasma VC in the lotus root and cucumber groups was significantly increased by 56.5% and 67.1% and total phenolics by 14.9% and 18.9%, respectively after the intervention. Meanwhile, the content of uric acid was significantly decreased in the two groups by 41.7% and 45.1%, respectively. No significant difference was found in plasma FRAP value, VE and GSH between the two groups after the 30-day intervention, though an increase trend was noted for plasma GSH in both groups at the end of the intervention.

Urinary 8-hydroxy-2’-deoxyguanosine excretion and blood mononuclear cell DNA damage The 8-hydroxy-2’-deoxyguanosine (8-OH-dG) in urine was quantified by an ELISA procedure. The reagent kit was purchased from Japan Institute for the Control of Aging. Results were expressed as the amount of 8-OH-dG per gram of creatinine in urine. The content of urinary creatinine was measured by the reaction of creatinine with picrate in alkaline medium. Blood mononuclear deoxyribonucleic acid (DNA) damage was analyzed using the single cell micro-gel electrophoresis assay, also known as Comet assay (26). Results were expressed as the injury rate (the percentage of DNA in tail) and the ratio of comet tail length to total length.

Activity of plasma antioxidant enzymes After the supplementation of lotus root or cucumber, plasma GSH-Px activity was significantly increased by 38.7% and 32.8%, respectively. No significant difference was noted for the activity of plasma CAT or SOD between the two groups before or after the supplementations of two vegetables (Table 5). Plasma contents of MDA, ox-LDL, carbonyls and hemolysis There was no significant difference in the contents of plasma MDA, Ox-LDL and carbonyls before or after the treatment of two vegetables in aged subjects. However, hemolysis was significantly reduced by 73.1% and 74.9%, respectively after the treatment of lotus root or cucumber (Table 6).

Statistical analysis Data were expressed as mean and standard deviation and checked for normality before subjected to further analysis. Two-sample t test or paired t test was employed for normal distributed data. Rank test was used for non-normally distributed data. The level of significance was set at P<0.05.

Urinary 8-OH-dG excretion and blood mononuclear cell DNA damage The urinary excretion of 8-OH-dG showed a decreasing trend after the treatment of two vegetables, but without statistical significance. Of the blood mononuclear cell DNA damage as determined by Comet assay, the injury rate was significantly lower by 23.5% after the supplementation of lotus root. Meanwhile, the ratio of comet tail length to total length was significantly decreased by 8.9% after the treatment of cucumber (Table 7).

Results Characteristics of the study subjects The general characteristics of the aged subjects are presented in Table 1. There was a significant difference in height in the two groups and body weight in the lotus root group between the males and the females. However, no significant difference was found in age, height, body weight and BMI between the two groups. 3

J Nutr Health Aging

IN VIVO ANTIOXIDANT PROPERTIES OF LOTUS ROOT AND CUCUMBER

Table 4 Plasma FRAP Value and Contents of Some Antioxidants Group

n

FRAP (mmol/L)

VC (mg/100ml)

VE (ug/ml)

GSH (mg/L)

TP (μg/ml)

UA (mg/100ml)

Initial

Final

Initial

Final

Initial

Final

Initial

Final

Initial

Final

Initial

Final

Lotus root

12

1.24±0.21

1.18±0.12

0.92±0.27

1.44±0.87*

15.8±3.6

15.4±4.4

21.2±13.7

40.1±14.5

53.8±10.7

61.8±8.6*

4.8±1.6

2.8±0.8*

Cucumber

13

1.25±0.24

1.24±0.25

0.85±0.18

1.42±0.79*

14.3±4.4

14.6±4.5

23.1±21.6

35.2±12.3

58.1±11.7

69.1±7.7*

5.1±1.1

2.8±0.8*

Data are expressed as mean±standard deviation. * P<0.05, compared with the initial (paired t test).

Table 5 Activity of Plasma Antioxidant Enzymes Group

n

Lotus root

12

Cucumber

Initial

SOD (U/ml)

81.36±7.93

13

CAT (U/ml)

Final

Initial

85.92±5.20

85.52±5.51

0.94±0.44

79.27±8.85

GSH-Px (U/ml)

Final

1.08±0.83

0.77±0.23

1.05±0.63

Initial

300.69±31.59

302.60±35.45

Final

417.18±34.65*

401.84±43.51*

Data are expressed as mean±standard deviation. * P<0.05, compared with the initial (paired t test). SOD, superoxide dismutase. CAT, catalase. GSH-Px, glutathione peroxidase.

Table 6 Plasma Contents of Some Oxidation Products Group Lotus root

Cucumber

n 12

13

MDA (nmol/L)

Initial

7.60±1.43

7.33±1.38

Final

7.57±3.05

8.47±3.37

Ox-LDL (μg/L)

Initial

41.6± 7.3

42.8± 9.2

Carbonyl (nmol/mg protein)

Final

39.8± 6.1 44.2±9.8

Initial

1.07± 0.26 1.29±0.22

Final

1.24± 0.24

Hemolysis (%)

Initial

Final

6.05± 0.68

1.25±0.33

1.63±0.25*

6.19± 1.07

1.55±0.19*

Data are expressed as mean±standard deviation. * P<0.05, compared with the initial (paired t test). MDA, malondialdehyde. ox-LDL, oxidized low density lipoprotein.

Table 7 Urinary 8-OH-dG Excretion and Blood Mononuclear Cell DNA Damage Group

Lotus root

Cucumber

n

12 13

8-OH-dG (μg/g Cr) Initial

83.2± 69.0

131.4±175.4

Final

80.5± 32.8 95.5±55.3

Injury rate (%)

Initial

37.8±23.5 37.7±17.9

Comet assay

Final

28.9±17.9* 33.1±22.4

Tail L/Total L

Initial

0.50±0.06 0.56±0.06

Final

0.45±0.07

0.51±0.05*

Data are expressed as mean±standard deviation; *P<0.05, compared with the initial (paired t test); Cr, creatinine. 8-OH-dG, 8-hydroxy-2’-deoxyguanosine. Tail L/Total L, tail length/ total length.

Discussion

higher in the lotus root powder than in the cucumber powder. After the 30-day intervention, the results showed that both two vegetables were effective in improving some aspects of antioxidant function in aged subjects. Although the content of plasma VC, total phenolics were increased significantly in both groups, the plasma FRAP value was not changed significantly after the intervention. It is explainable because plasma uric acid, an important antioxidant, was significantly reduced after two vegetable treatments, which may diminish the impact of increased VC and total phenolics on plasma FRAP value. Plasma GSH-Px activity was increased significantly in both groups, whereas the activity of SOD or CAT was not

Lotus root is well-known in Asian countries and widely used as a medicinal plant in China, Japan and Korea, because it has several potential benefits, such as antimicrobial and antifungal activities, anti-hypertension, hypo-cholesterolemic and glycaemic effects (6,27,28). Cucumber is cultivated in many countries and sometimes used as a demulcent in antiacne lotions or for relieving headaches (29). In this study, the lotus root powder we prepared was 8 times higher than the cucumber powder in FRAP value. The contents of VC and total phenolics or flavonoids were also significantly 4

J Nutr Health Aging

THE JOURNAL OF NUTRITION, HEALTH & AGING© changed significantly. Plasma contents of MDA and Ox-LDL, two markers for oxidative damages on lipids were also not changed significantly by the treatment of either the lotus root or cucumber. However, hemolysis was significantly declined after the supplementation of lotus root or cucumber, indicating that the membranes of blood red cells were more resistant to the attack by free radicals with the antioxidants derived possibly from the treatment of two vegetables. The urinary excretion of 8-OH-dG, a marker for DNA damage, was not significantly decreased in the two groups, whereas the injury rate was lower significantly in the lotus root group and the ratio of comet tail length to total length was reduced significantly in the cucumber group. Based on the changes of several biomarkers for antioxidant function mentioned above, it is strongly indicated that though lotus root is remarkably higher than cucumber in antioxidant capacity in vitro, they are not significantly different in providing the protection against the oxidative damage in elderly people. The results obtained in this study are basically consistent to that we found in aged rats previously (18). Vegetables are rich in vitamins, minerals and fibers, as well as natural antioxidants such as phenolics (including flavonoids) and carotenoids. Positive correlations between the content of phenolics and antioxidant capacity in vitro were frequently demonstrated in vegetables and the phenolics had been considered one of the important antioxidants in vegetables (30,31). Previously, we also found a significant correlation between the content of phenolics and FRAP value in the vegetables commonly consumed by Chinese (32). Although the lotus root is high in antioxidant capacity and contains more phenolics, they may be absorbed not as efficiently as those in the cucumber in the aged subjects. This point is supported by the results obtained in this study, in which no significant difference was found in fasted plasma content of total phenolics between the two groups after the treatment of two vegetables in the aged subjects. In a preliminary study, we monitored the changes of serum FRAP value 1, 3, 5 h after oral administration of 5 ml of fresh lotus root juice in rats. However, no significant increase in serum FRAP value was found, indicating that the antioxidants contained in lotus roots were not rapidly absorbed (data not shown). It has been demonstrated that most phenolics are not absorbed efficiently and metabolized extensively post-absorption. The metabolites generated in vivo are relatively lower in antioxidant activity (33,34). Therefore, the phenolic compounds presented in vegetables may not act as an important antioxidant in vivo after absorption. On the other hand, the antioxidant composition varies greatly among different vegetables and the possible roles of other antioxidants present in vegetables, such as carotenoids and GSH, also should be fully investigated, because some vegetables are rich in carotenoids and GSH (35,36). It should be mentioned that some phenolic compounds in vegetables had been demonstrated to be effective in inducing the expression of some antioxidant enzymes, which may contribute to the improvements of antioxidant function indirectly (37,38). Therefore, these

phenolic compounds may act on antioxidant function indirectly and the actions are not measurable by the antioxidant capacity assays in vitro. In conclusion, the results of this study indicate that the supplementation of either lotus root or cucumber is effective in improving some aspects of the antioxidant function in aged subjects. The actions are not correlated positively with their antioxidant capacity in vitro. The benefits observed in this study may come directly or indirectly from the additive or synergistic combinations of the antioxidants contained in vegetables. The antioxidant capacity measured in vitro may not be applicable in evaluating the potentials of vegetables in improving antioxidant function in the aged. However, we are aware that this pilot study is limited in the number of aged subjects participated and the intervention period is also relatively short. Large scale and long-term studies are needed to confirm the actions of vegetables and their functional components involved. Acknowledgments: We are grateful to all the volunteers who participated in this study. Our appreciations are also extended to the staffs at Tianjin Dongjiang Food Company for their assistance in preparing vegetable powder. Funding sources: This study was supported financially by a grant from the Research and Education Projects (DIC 2009-02) of Danone Institute of China. Ethical Standards: The experiments in this study comply with the current laws of the country in which they were performed. Conflicts of interest: Dr. Linlin Ji has nothing to disclose. Dr. Weina Gao has nothing to disclose. Dr. Jingyu Wei has nothing to disclose. Dr. Lingling Pu has nothing to disclose. Dr. Jijun Yang has nothing to disclose. Dr. Changjiang Guo reports a grant from China Natural Science Foundation during the conduct of the study.

References 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12.

13.

5

Cao G, Sofic E, Prior RL. Antioxidant capacity of tea and common vegetables. J Agric Food Chem1996;44:3426-3431 Halvorsen BL, Holte K, Myhrstad MCW, et al. A systematic screening of total antioxidants in dietary plants. J Nutr 2002;132:461-471 Song W, Derito CM, Liu MK, He X, Dong M, Liu R. Cellular antioxidant activity of common vegetables. J Agric Food Chem 2010;58:6621-6629 Chao PY, Lin SY, Lin KH, Liu YF, Hsu JI, Yang CM, Lai JY. Antioxidant activity in extracts of 27 indigenous Taiwanese vegetables. Nutrients 2014; 6:2115-2130 Guo CJ, Wei JY, Yang JJ, Li YF, Xu J, Jiang YG. The antioxidant capacity of 66 vegetables and fruits: a comparative study. Acta Nutr Sin 2003;25:203-207 Isabelle M, Lee BL, Lim MT, Koh W, Huang D, Ong CN. Antioxidant activity and profiles of common vegetables in Singapore. J Food Chem 2010;120:993-1003 Du H, Zhao X, You JS, Park J, Kim S, Chang K. Antioxidant and hepatic protective effects of lotus root hot water extract with taurine supplementation in rats fed a high fat diet. J Biomed Sci 2010;17:s39-s44 Guo CJ, Cao GH, Sofic E, Prior RL. High-performance liquid chromatography coupled with coulorimetric array detection of electroactive components in fruits and vegetables: relationship to oxygen radical absorbance capacity. J Agric Food Chem 1997; 45:1787-1796 Harman D. Aging: overview. J Ann N Y Acad Sci 2001; 928:1-21 Matin GM. The biology of aging: 1985-2010 and beyond. FASEB J 2011; 25: 37563762 Sohal RS, Orr WC. The redox stress hypothesis of aging. Free Radic Biol Med 2012; 52: 539-555 Hennekens CH, Buring JE, Manson JE, Stampfer M, Rosner B, Cook NR, Belanger C, LaMotte F, Gaziano JM, Ridler PM, Willett W, Peto R. Lack of effect of longterm supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular disease. N Engl J Med 1996;334:1145-1149 Prieme H, Loft S, Nyyssonen K, Salonen JT, Poulsen HE. No effect of supplementation with vitamin E, ascorbic acid, or coenzyme Q10 on oxidative damage estimated by 8-ox-7,8-dihydro-2’- deoxyquanosine excretion in smokers. Am J Clin Nutr 1997;65:503-507

J Nutr Health Aging

IN VIVO ANTIOXIDANT PROPERTIES OF LOTUS ROOT AND CUCUMBER 14. Bjelakovic G1, Nikolova D, Gluud LL, Simonetti RG, Gluud C. Antioxidant supplements for prevention of mortality in healthy participants and patients with various diseases. Cochrane Database Syst Rev 2012;3:CD007176 15. Sadowska-Bartosz I, Bartosz G. Effect of Antioxidants Supplementation on Aging and Longevity. Biomed Res Inter 2014;Article ID 404680, 17 pages. 16. Dauchet L, Amouyel P, Hercberg S, Dallongeville J. Fruit and vegetable consumption and risk of coronary heart disease: a meta-analysis of cohort studies. J Nutr 2006;136:2588-2593 17. Hjartaker A, Knudsen MD, Tretli S, Weiderpass E. Consumption of berries, fruits and vegetables and mortality among 10,000 Norwegian men followed for four decades. Eur J Nutr 2014; Aug 3. [Epub ahead of print] 18. Ji LL, Gao WN, Wei JY, Wu JQ, Yang JJ, Meng B, Guo CJ. No correlation is found for vegetables between antioxidant capacity and potential benefits in improving antioxidant function in aged rats. J Clin Biochem Nutr 2014;54:198-203 19. Benzie IFF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of «antioxidant power»: the FRAP assay. J Anal Biochem1996;239:70-76 20. Brewster MA, Turley CP. Vitamin C. In: Pesce AJ, Kaplan LA (eds) Methods in Clinical Chemistry. The C.V. Mosby Company, St Louis, 1987;pp574-581. 21. Hansen LG, Warwich WJ. An improved assay method for serum vitamin A and E using fluorometry. Am J Clin Pathol 1978;70:922-923 22. Singleton VL, Orthofer R, Lamuela-Raventos RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent. J Methods Enzymol 1999;299:152-178 23. Smith CV, Anderson RE. Methods for determination of lipid peroxidation in biological samples. Free Radic Biol Med 1987;3:341-344 24. Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, Ahn BW, Shaltiel S, Stadtman ER. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 1990;186:464-478 25. Grinberg LN, Newmark H, Kitrossky N, Rahamim E, Chevion M, Rachmilewitz EA. Protective effects of tea polyphenols aganist oxidative damage to red blood cells. J Biochem Pharmacol 1997;54:973-978 26. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175: 184-189 27. Lee JJ, Park SY, LeeMY. Effect of lotus root (nelumbo nucifera G.) on lipid metabolism in rats with diet-induced hypercholesterolemia. Korean J Food Preserv 2006;13:634-642

28. Mukherjee PK, Pal SK, Saha K, Saha B. Hypoglycaemic activity of Nelumbo nucifera gaertn. (Fam. Nymphaeaceae) rhizome (methanolic extract) in streptozotocin induced diabetic rats. Phytother Res 2006;9:522-524 29. Kumar D, Kumas S, Singh J, Narender Rashmi Vashistha BD, Singh N. Free radical scavenging and analgesic activities of cucumis sativus L. fruit extract. J Young Pharmacists 2010; 2: 365-368 30. Jacobo-Velázquez DA, Cisneros-Zevallos L. Correlations of antioxidant activity against phenolic content revisited: a new approach in data analysis for food and medicinal plants. J Food Sci 2009;74: R107-R113 31. Landete JM. Updated knowledge about polyphenols: functions, bioavailability, metabolism, and health. Crit Rev Food Sci Nutr 2012;52:936-948 32. Ji LL, Wu JQ, Gao WN, Wei JY, Yang JJ, Guo CJ. Antioxidant capacity of different fractions of vegetables and correlation with the contents of ascorbic acid, total phenolics and flavonoids. J Food Sci 2011;76:1257-1261 33. Justino GC, Santos MR, Canario S, Borges C, Florencio MH, Mira L. Plasma quercetin metabolites: structure-antioxidant activity relationships. Arch Biochem Biophys 2004;432:109-121 34. Lotito SB, Zhang WJ, Yang CS, Crozier A, Frei B. Metabolic conversion of dietary flavonoids alters their anti-inflammatory and antioxidant properties. Free Radic Biol Med 2011; 51: 454-463 35. Jones DP, Coates RJ, Flagg EW, Eley JW, Block G, Greenberg RS, Gunter EW, Jackson B. Glutathione in foods listed in the National Cancer Institute’s Health Habits and History Food Frequency Questionnaire. Nutr Cancer 1992;17: 57-75 36. Khoo HE, Prasad KN, Kong KW, Jiang Y, Ismail A. Carotenoids and their isomers: color pigments in fruits and vegetables. Molecules 2011;16:1710-1738 37. Rebrin I, Zicker S, Wedekind KJ, Paetau-Robinson I, Packer L, Sohal RS. Effect of antioxidant-enriched diets on glutathione redox status in tissue homogenates and mitochondria of the senescence-accelerated mouse. Free Radic Biol Med 2005;39: 549-557 38. Yeh CT, Yen GC. Induction of hepatic antioxidant enzymes by phenolic acids in rats is accompanied by increased levels of multidrug ressistance-associated protein 3 mRNA expression. J Nutr 2006;136:11-15

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