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Journal of the American College of Nutrition, Vol. 22, No. 4, 277-282 (2003)
Published by the American College of Nutrition

Original Research

An in Vivo Study of the Antioxidant Potentials of a Plant Food Concentrate

Vincent E. Ziccarelli, MSc and Tapan K. Basu, PhD, FACN

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, CANADA

Address reprint requests to: Tapan K. Basu, Ph.D., FACN, Department of Agricultural, Food & Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, CANADA. E-mail:tapan.basu{at}ualberta.ca


   ABSTRACT
TOP
 FOOTNOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: A plant food concentrate (PF) is a source of antioxidants. Its influence on antioxidant status has never been studied. The present longitudinal study investigated the antioxidant and lipidemic responses in 15 moderately hypercholesterolemic (>5.2 mmol/L) male subjects to the supplemental intakes of PF.

Methods: The participants underwent a two-week period where any previous supplemental intakes were withdrawn. This was followed by a two-week baseline period at entry (control). The baseline period was followed by taking PF concentrate (8.5 g twice daily) for two weeks followed by a washout period for two weeks. All subjects completed food frequency questionnaires at pre-supplemental (baseline) and post-PF period. Fasting heparinized and EDTA blood samples were collected at the end of each period. Erythrocyte superoxide dismutase (SOD), whole blood glutathione peroxidase (GPX) and plasma concentrations of zinc and copper, along with plasma levels of lipids, were determined.

Results: The PF supplement contributed significantly to the daily intakes of total dietary fiber. The zinc and copper-dependent SOD but not GPX activity were significantly elevated. The total and LDL-cholesterol concentrations in the plasma were significantly decreased while the ratio of HDL/LDL cholesterol was increased post-PF intake.

Conclusions: These results indicate that the antioxidant and cholesterol status of moderately hypercholesterolemic subjects can be potentially improved with the supplemental intake of PF concentrate.

Key words: antioxidants, superoxide dismutase, glutathione peroxidase, plasma cholesterol, plant food concentrates


   INTRODUCTION
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Increased levels of oxidative stress may be implicated in the etiology of many pathological conditions [1–3]. Antioxidants are thought to quench free radicals and include the endogenous enzymes such as selenium dependent glutathione peroxidase (GPX), copper-zinc dependent superoxide dismutase (SOD) and dietary antioxidants such as vitamins C, E, zinc and phenolic compounds [1–5]. Indeed, there appears to be a substantial amount of evidence suggesting that the increased intake of antioxidant rich fruit and vegetables decreases the risk for developing many chronic conditions, such as malignant and cardiovascular diseases [6–8]. Unfortunately, surveys indicate that the general public is far from achieving the recommended intake of the 5 to 10 servings/day of fruit and vegetables [7,9]. In recent years, there have been many food products that are available over the counter as supplements to fruit and vegetables [10]. One of these products consists of a variety of air-dried plant juice extracts (PF) and is widely available in North American health food stores as a supplement powder. The present study was undertaken essentially to investigate the in vivo potential effects on antioxidant defenses and hypolipidemic effects of plant food (PF) concentrate in a select group of moderately hypercholesterolemic male subjects.


   MATERIALS AND METHODS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
PF Concentrate
PF concentrate includes young barley and wheat grasses, composing 40% of the total mixture. These cereal grasses are known to be sources of antioxidants [11,12]. The remaining ingredients of the concentrate includes antioxidant-rich extracts of ginkgo biloba, grape seed and milk thistle. According to the proximate analysis, carried out by the General Laboratories Division of SGS (General Society of Surveillance) Canada Inc. (Vancouver, B.C., Canada), the PF concentrate is a significant source of micronutrients, particularly antioxidants such as beta-carotene, ascorbic acid and vitamin E (Table 1). The total daily intake of PF (17 g) contributed 1.6 and 3.4 times the RNI of the antioxidant vitamins C and E, respectively (Table 1). In addition, minerals such as copper, zinc and magnesium are present in appreciable quantities in the supplement. These inorganic elements are the constituents of SOD, which catalyses the removal of superoxide radical.


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  		Table 1. Nutrient Composition of Plant Food (PF) (per 100 g)

 
Subjects
A total of 15 male participants (age range: 30 to 68) with hypercholesterolemia (>5.2 mmol/L) were recruited within the Edmonton area. The study was a total of six weeks in length and consisted of three experimental periods. All subjects completed food frequency questionnaires (pre-intervention and post-intervention periods) and followed the same experimental protocol, thus acting as their own controls. In addition, all participants were instructed to maintain their usual dietary intakes, with the exception of required supplementation, throughout the study. No supplementation was taken during the first two weeks (control). This baseline period was followed by two weeks of taking the plant food (PF) concentrate. The latter was consumed twice daily with meals, as a dietary supplement mixed with water (250 mL); each dose level was 8.5 grams. At the end of the supplemental period, there were two further weeks whereby study participants continued to comply with the criteria required for the study. This period was called "washout period." Fasting heparinized and EDTA blood samples were collected from each participant at the end of each experimental period.

Methods
Estimates of energy and macronutrient intake were obtained by using a comprehensive, semiquantitative food-frequency questionnaire (FFQ) validated previously for the determination of the individual dietary intakes [13] and were analyzed by a nutrition software program (Version 6.0, The Food Processor; ESHA Research, Salem, OR).

Copper and zinc dependent SOD and selenium dependent GPX activities were determined using kits (Cat. No. SD 125 and RS 505, respectively), which were obtained from Randox Laboratories, Montreal, Canada. The SOD activity was measured in hemolysate using an appropriate whole blood SOD control. This method employs xanthine and xanthine oxidase to generate superoxide radicals which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red dye. The enzymatic activity was then measured by the degree of inhibition of this reaction. The GPX activity in whole blood was measured using a whole blood control (Cat. No. SC 692) as described by Paglia and Valentine [14]. Plasma trace element status, including copper and zinc, was determined using an atomic absorption spectrophotometry method [15]. The plasma lipid levels including triglycerides, total cholesterol and HDL cholesterol were measured by the respective enzymatic kits supplied from Sigma Diagnostics (Cat. No. 336, Cat. No. 352-20, Cat. No. 352-3). LDL-cholesterol was determined by a simple calculation that was previously validated [16].

Statistical Analysis
The statistical significance of the PF concentrate-associated changes in erythrocyte SOD, whole blood GPX, plasma trace elements, dietary intakes and blood lipids was assessed by analysis of paired differences using one way ANOVA. The latter included Duncan’s multiple range test, whereby each subject was compared to his own control values using SAS software (Version 6.12 Institute, Cary, NC). The differences were judged to be statistically significant if the associated p value was <0.05.


   RESULTS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
During the initial intake of PF concentrate, two out of the 15 subjects reported mild gastrointestinal discomfort, including lose stools and an increase in bowel motility, which subsided within the first two to three days of intake. No other adverse reactions were reported with the intake of the vegetable supplement. At baseline, the mean body mass index (BMI) of the participants was 29 ± SEM 1. The latter remained unchanged following two weeks of the supplemental intake of PF concentrate (not tabulated). The mean baseline level of cholesterol was 6.03 ± SEM 0.11 (Table 2), suggesting that this group of subjects was moderately hypercholesterolemic [17]. Routine clinical blood chemistry measurements were conducted so as to determine the health status of the subjects at each treatment period. Basal measurements of hemoglobin were found to be within the expected normal range for males. The supplemental intake had no effect on these parameters (Table 3).


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  		Table 2. The Effect of Supplemental Intake of PF (2 Weeks) on Blood Lipid Status (Mean ± SEM+) in Hypercholesterolemic Male Subjects (n = 15)

 

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  		Table 3. The Influence of PF Intake (2 weeks) on the Plasma Levels of Hemoglobin, Copper (Cu), Zinc (Zn), Whole Blood Glutathione Peroxidase (GPX) and Erythrocyte Superoxide Dismutase (SOD) Activity, in 15 Male Subjects with Hypercholesterolemia

 
Analyses of pre-supplemental and post-supplemental dietary food frequency questionnaires showed no difference in the dietary intake of total energy, lipids, carbohydrates or protein, suggesting no change in dietary intake during the study period and hence the study protocol (Table 4). The supplemental intake of PF concentrate for two weeks significantly (p < 0.05) increased mean erythrocyte SOD activity by 11% in comparison to baseline levels in the hypercholesterolemic subjects (Table 3). Since the blood hemoglobin levels remained unchanged between the experimental periods (Table 3), there was no apparent need to convert these values to SOD activity per g of Hb (hemoglobin). When the supplemental intake was withdrawn for two weeks, the level of SOD activity was decreased to its baseline value (Table 3). In parallel with SOD, the mean plasma concentration of zinc was elevated with the supplemental period versus the baseline period (Table 3). After a washout period for two weeks, plasma concentration of zinc was significantly reduced (p < 0.05) to its baseline level. Plasma copper status on the other hand did not change with supplementation (Table 3). Unlike SOD the mean whole blood GPX activity remained unaffected by the intake of PF concentrate (Table 3).


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  		Table 4. Daily Dietary Intake (Mean ± SEM+) of Study Subjects (n = 15) in Response to Supplemental Intakes of Plant Food (PF)

 
The supplementation of PF concentrate significantly (p < 0.05) reduced the levels of total cholesterol and LDL-cholesterol concentrations in the plasma (Table 2). Plasma HDL-cholesterol and triglyceride levels, however, were not changed with the intake of PF. Although the mean HDL-cholesterol concentration was unresponsive to PF, its ratio with LDL-cholesterol was significantly (p < 0.05) elevated by 8% versus the basal level (Table 2). After the cessation of supplements for a two-week period, the latter ratio was reduced (p < 0.05) back to baseline values while the other lipidemic responses remained unchanged.


   DISCUSSION
 
An altered antioxidant status was evident with the supplemental intake of PF concentrate for two weeks in a select group of moderately hypercholesterolemic male subjects. Since the dietary intakes, as shown by consistent levels of total energy intake that was found between pre- and post-PF concentrate supplemental periods, the changes observed with SOD activity, zinc status and cholesterol status were a true effect of the supplemental intake. A micronutrient analysis of the PF supplement indicated that it contained substantial amounts of antioxidant nutrients, including ascorbic acid, vitamin E, carotenoids, copper and zinc. The PF concentrate supplement can thus be considered as an antioxidant-rich vegetable supplement. These findings are in parallel with other studies in which fruit and vegetables have been found to provide rich sources of antioxidants to the daily diet [18,19].

The SOD activity was increased by 11% after a two week intake of PF concentrate. This association was further supported by the fact that the enzyme activity was reversed to its pre-supplemental level when the supplemental intake was discontinued for two weeks. The SOD catalyzes the removal of superoxide radicals, thus reducing the degree of oxidation [20,21]. The increased activity of this enzyme may, therefore, suggest a greater level of endogenous antioxidant defense associated with the supplemental intake of PF concentrate [22]. It is plausible that the increased SOD activity may also be an adaptive enzymatic response to elevated oxidative stress, as opposed to a sign of increased antioxidant status. Perhaps future investigations should examine the levels of lipid peroxidation in response to this antioxidant supplement, so as to determine its effects on oxidative stress. Nevertheless, long term adherents to a strict uncooked vegan diet were reported to have a significantly higher level of mean erythrocyte SOD activity in comparison to omnivores. The increased activity of the antioxidant enzyme has also been attributed to a high intake of antioxidant-rich food in the vegetarians [19]. Thus the SOD-response to PF concentrate is in accordance with that reported in relation to a fresh fruit and vegetable intake [23].

The SOD enzyme requires zinc and copper for its antioxidant role [19,24]. Although the mean plasma copper level was not changed in the study subjects, a significantly (p < 0.05) elevated level of plasma zinc was evident in association with the intake of PF concentrate. Zinc is thought to have its own antioxidant property. It thus induces endogenous antioxidants, such as the metallothionines [25]. Zinc salts have also been reported to exert radical scavenging properties in vitro [26]. It seems possible that the increased mean plasma Zn levels found with the supplemental intakes of PF may thus improve the antioxidant status of the participants by possibly synthesizing zinc-containing proteins.

Glutathione peroxidase (GPX) is another important antioxidant index, which is active in removing hydrogen peroxide, thereby preventing the generation of free hydroxyl radicals [27]. Unlike SOD activity, dietary intake of the plant food supplement did not alter the activities of whole blood GPX. In agreement with these results, it was reported that a higher intake of fruit and vegetables altered the activities of SOD but not GPX [19]. However, other studies have found significant changes in the levels of GPX with the higher intake of antioxidant rich food which was attributed to the improved status of selenium [28–30]. GPX activity is dependent on plasma selenium status. The enzyme activity and the trace element have been reported to be positively correlated [30,31]. Hence, given the lack of selenium content within the supplement, it is of no surprise that GPX status was not significantly altered. In support of these findings, most fruit and vegetables are not particularly high sources of selenium, which would explain the lack of this trace element in the PF concentrate [30].

Elevated plasma cholesterol is one of the risk factors for cardiovascular disease (CVD) [32]. Dietary intake of the PF supplement for two weeks resulted in significant reductions in plasma total cholesterol and LDL-cholesterol and was accompanied by an increased ratio of HDL to LDL-cholesterol. The mean plasma levels of triglycerides were >2.4 mmol/L for all three treatments. Considering the fact that the study subjects were hypercholesterolemic/hypertriglyceridemic, the high fasting TG values were not out of line. However, these values are not high enough to affect the Friedwald calculation for LDL-cholesterol [16]. Furthermore, the reduction of LDL-cholesterol has been shown to be in parallel with the decrease in total cholesterol associated with PF intake. In agreement with these findings, epidemiological evidence reveals that populations that consume greater quantities of plant food have lower plasma cholesterol concentrations [33]. Foods of plant origin, such as fruit and vegetables, are generally considered to contain substantial amounts of dietary fiber and phytosterol [34,35]. Diets high in fiber content are known to modify blood lipid status [36]. Two weeks of supplemental intakes of PF increased significantly (p < 0.05) the intake of dietary fiber by 20% of that provided by the basal diet. It is, therefore, possible that the greater intake of dietary fiber associated with the PF supplementation was responsible for its hypolipidemic effects [37,38]. Phytosterols are also known to modify cholesterol status with particular reference to LDL-cholesterol [35]. The reduction of LDL-cholesterol, observed in the presence of the PF supplement, may also have been caused by its phytosterol content. Unfortunately, the phytosterol content of the PF concentrate was not measured.


   CONCLUSION
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
ACKNOWLEDGMENTS
 REFERENCES
 
The results of this study indicate that PF concentrate may be a dietary supplement with the potential properties of improving antioxidant and lipid status. However, long term studies with a larger study population are required to further explore the antioxidant and lipidemic responses to PF before conclusions can be made about its use as a dietary supplement. The latter should also examine other antioxidant parameters such as serum catalase activity, plasma concentrations of uric acid, bilirubin and albumin as well as plasma total antioxidant status in response to this dietary supplement. The phytosterol content of the PF concentrate should also be determined as a possible factor linking to the modifying effects of the concentrate on cholesterol status.


   ACKNOWLEDGMENTS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Natural Factors Nutritional Products Ltd, Burnaby, BC and Alberta Agriculture Research Institute (AARI) for providing financial support for this study.


   FOOTNOTES
TOP
 FOOTNOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Natural Factors Nutritional Products Ltd, Burnaby, BC and Alberta Agriculture Research Institute (AARI) provided financial support for this study.

Received August 24, 2001. Accepted September 27, 2002.


   REFERENCES
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 

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