effects of mangifera pajang kostermans juice on plasma antioxidant status and liver and kidney...

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Effects of Mangifera pajang Kostermans juice onplasma antioxidant status and liver and kidneyfunction in normocholesterolemic subjects

1756-4646/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jff.2013.09.011

* Corresponding author at: Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra43400 Serdang, Selangor, Malaysia. Tel.: +60 3 89472435; fax: +60 3 89426769.

E-mail addresses: aminis@upm.edu.my, amin@medic.upm.edu.my (A. Ismail).

Muhammad Ibrahima,b, Amin Ismailb,c,*, Sadeq Hasan Al-Sherajib,d, Azrina Azlanb,c,Azizah Abdul Hamide

aDepartment of Nutrition Sciences, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia, Jalan Istana, Bandar Indera

Mahkota, 25200 Kuantan, Pahang, MalaysiabDepartment of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor,

MalaysiacLaboratory of Halal Science Research, Halal Products Research Institutes, Universiti Putra Malaysia, UPM, 43400 Serdang, Selangor,

MalaysiadDepartment of Food Science, Faculty of Agriculture, Ibb University, Ibb, Republic of YemeneDepartment of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

A R T I C L E I N F O A B S T R A C T

Article history:

Received 17 February 2013

Received in revised form

10 September 2013

Accepted 17 September 2013

Available online 11 October 2013

Keywords:

Mangifera pajang

Total antioxidant status

Enzymes

Function test

The effects of a bambangan juice powder (BJP) drink on plasma vitamin and antioxidant

enzyme levels and liver and kidney function were investigated. Thirty-two healthy subjects

(12 male and 20 female) ages 24–28 years were recruited from the Faculty of Medicine and

Health Sciences of University Putra Malaysia, Malaysia. Compared with consuming the

placebo, consumption of the BJP drink daily for 9 weeks significantly increased the concen-

tration of plasma b-carotene and ascorbic acid. Plasma total antioxidant status was

increased, but liver and kidney functions were unaffected after consumption of the BJP

drink. The consumption of a BJP drink resulted in a significant improvement in certain car-

diovascular biochemical parameters and thus reduced the risk of cardiovascular disease.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The term antioxidant refers to any molecule capable of stabi-

lizing or deactivating free radicals before they attack cells.

Antioxidants can be enzymatic or non-enzymatic. Enzymatic

antioxidant defenses include superoxide dismutase, which

accelerates the dismutation of superoxide; glutathione perox-

idase, which catalyzes the reduction of hydrogen peroxide

and lipid hydroperoxides to water and lipid alcohols at the ex-

pense of oxidizing reduced glutathione (Bahorun, Soobrattee,

Luximon-Ramma, & Aruoma, 2006); and others, such as

catalase. Non-enzymatic antioxidants include ascorbic acid

(vitamin C), carotenoids, polyphenols and other phytochemi-

cal antioxidants. A balance exists between both the activities

and the intracellular levels of these antioxidants under

normal conditions. Indeed, this balance is essential for the

survival of organisms and their health (Bahorun, Soobrattee,

Luximon-Ramma, & Aruoma, 2006; Valko et al., 2007).

Malaysia,

J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 9 0 0 – 1 9 0 8 1901

Dietary antioxidants have attracted considerable attention

as agents that protect cells or molecules from oxidative

stress. The consumption of 5 servings of fruits and vegetables

per day has been recommended by the American Dietetic

Association to ensure an adequate antioxidant intake starting

in early childhood (Nicklas & Johnson, 2004). High intake of

fruits and vegetables provides natural antioxidant com-

pounds with health-promoting properties that are associated

with the increase of plasma antioxidants and inversely asso-

ciated with the risk of cardiovascular diseases, according to

several intervention studies (Costa, Garcia-Diaz, Jimenez, &

Silva, 2013; John, Ziebland, Yudkin, Roe, & Neil, 2002; Samman

et al., 2003; Shahidi & Chandrasekara, 2013). Furthermore,

antioxidant compounds have important roles in the preserva-

tion of plasma antioxidant status (Wu et al., 2013). The results

from a cohort study indicated that a lower risk of myocardial

infarction incidence is inversely associated with a higher in-

take of fruits and vegetables in women (Liu et al., 2000). More-

over, high intakes of vegetables have been shown to have an

inverse association with coronary heart disease risk in men

(Liu et al., 2001).

The fruit of bambangan (Mangifera pajang Kostermans) is

edible and is one of the most common fruits consumed in Sa-

bah and Sarawak, Malaysia. The fruit is brown-skinned, ovoid

in shape and possessed of a pungent smell. The bambangan

is a type of wild mango from the Anacardiaceae family and

can be found in its native areas, such as East Kalimantan

(Indonesia) and Borneo Island (Malaysia-Sabah and Sarawak,

Brunei). The nutritional composition (Al-Sheraji et al. 2011,

2012a; Hassan, Ismail, Abdul Hamid, Azlan, & Al-sheraji,

2011a) and several antioxidant actions (Al-Sheraji et al.

2012b; Hassan, Ismail, Abdul hamid, & Azlan, 2011b) of vari-

ous parts of the bambangan fruit have been studied previ-

ously. A study by Ibrahim, Prasad, Ismail, Azlan, and Abdul

Hamid (2010) reported that BJP contained ascorbic acid

(1321 lg/g), b-carotene (3556 lg/g) and total phenolics (19 mg

gallic acid equivalent/100 g) and possessed antioxidant capac-

ity (40 mM/100 g and 53% by ferric radicals antioxidant power

and DPPH), as shown in Table 1. Abu Bakar, Mohamed, Rah-

mat, Burr, and Fry (2010) reported on the identities and con-

tents of some polyphenols in bambangan flesh, namely

phenolic acids, including p-coumaric acid [29 lg/g dry weight

(dw)], caffeic acid (27 lg/g dw) and chlorogenic acid (6 lg/

Table 1 – Bioactive compounds of flesh of Mangifera pajang Ko

Compound Am

Phenolic acids p-Coumaric acid 29.

Caffeic 26.

Chlorogenic 5.8

Flavanones Naringin 145

Hesperidin 930

Flavonols Quercetin 165

Kaempferol 182

Flavone Luteolin 292

Carotenoids b-Carotene 355

a-Carotene 79.

Cryptoxanthin 11.

Ascorbic acid Ascorbic acid 132

g dw). Flavanones found in the flesh included naringin

(1450 lg/g dw) and hesperidin (930 lg/g dw), while the re-

ported flavonols were quercetin (165 lg/g dw) and kaempferol

(183 lg/g dw). A flavone compound, luteolin, was reported at

292 lg/g dw. A study by Khoo, Prasad, Ismail, and Esa (2010)

showed that bambangan flesh also contained a-carotene

(80 lg/g) and cryptoxanthin (12 lg/g) (Table 1). To the best of

our knowledge, no study has reported on the effect of this

fruit on the risk of degenerative disease in vivo. Therefore,

our study was considered the first to examine potential

health-promoting properties of this fruit in human subjects.

In addition to the positive benefits of high fruit and vege-

table intake, the potential adverse effect of the high levels

of antioxidant compounds present in these fruits and vegeta-

bles should be evaluated. Few studies have documented the

effect of fruits and vegetables on parameters of liver and kid-

ney function that reflect the possible toxicity of an excessive

intake of the antioxidants present in these plants. A study by

Takahashi (1995) suggested that consumption of 0.5% natural

b-carotene has a tendency to cause hemorrhage in male rats.

Another study among 29,133 Finish male smokers showed an

increased incidence of cardiac events, hemorrhagic stroke

and the risk for major coronary events after 6 years of

b-carotene supplementation (Heinonen & Albanes, 1994). Fur-

thermore, in the Health Professionals Follow-Up Study involv-

ing 43,738 men, supplemental vitamins E and C or specific

carotenoids did not reduce the risk of stroke (Ascherio et al.,

1999).

In this study, we conducted a controlled dietary interven-

tion to investigate the effects of the consumption of a BJP

drink on selected enzymatic and non-enzymatic antioxidants

and plasma antioxidant status in humans. Parameters of liver

and kidney function were assessed as toxicity indicators.

2. Materials and methods

2.1. Preparation of samples

Fresh M. pajang Kosterm fruits at their commercial ripening

stage were collected from Bau, Sarawak, Malaysia. The fruits

were then wrapped in paper, placed in boxes and transported

via airmail to the Nutrition Laboratory, Faculty of Medicine

and Health Sciences, University Putra Malaysia, Serdang,

st.

ount (lg/g d.w) Ref.

46 Abu Bakar et al. (2010)

75 Abu Bakar et al. (2010)

1 Abu Bakar et al. (2010)

0.00 Abu Bakar et al. (2010)

.00 Abu Bakar et al. (2010)

.10 Abu Bakar et al. (2010)

.70 Abu Bakar et al. (2010)

.10 Abu Bakar et al. (2010)

.90 Ibrahim, Prasad, Ismail,

Azlan, and Abdul Hamid (2010)

60 Khoo et al. (2010)

80 Khoo et al. (2010)

1.4 Ibrahim et al. (2010)

1902 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 9 0 0 – 1 9 0 8

Malaysia. Upon arrival, the fruits were cleaned with tap water

and stored at �20 �C until further use.

Prior to processing, all fruits were thawed at room temper-

ature for 4 h. M. pajang peel was removed, and the pulp was

separated from the kernel. Then, the M. pajang pulp was

homogenized with redistilled water (the ratio of pulp to water

was 1:1). The mixture was filtered through a 0.45 lm sieve,

and the juice was collected. M. pajang juice was lyophilized

using a freeze-dryer (Virtis Gardiner, New York, USA). The

lyophilized juice was powdered, passed through a 0.45 lm

sieve and stored in an airtight container at �80 �C until

further use.

2.2. BJP drink preparation

The daily amount (250 mL) of the beverages used in this study

was packed by the Federal Agriculture Marketing Authority

(FAMA), the Ministry of Agriculture and Agro-based Industry

of Malaysia. Each bottle of prepared drink (250 mL) contained

50 g of BJP (containing 66 mg of ascorbic acid and 18 mg of

b-carotene). The placebo was prepared using a mixture of

maltodextrin and artificial mango flavor that contained the

same amount of calories as the BJP drink.

2.3. Subjects

Thirty-two non-smoking (12 male and 20 female) volunteers

were recruited among staff and postgraduate students of

UPM out of 100 subjects who underwent the screening pro-

cess. The participants were screened during an interview for

the following inclusion criteria: no severe obesity (body mass

index, BMI <30 kg/m2); no regular use of any drug or supple-

ment with antioxidant (b-carotene, vitamins C or E) or lipid-

lowering properties; no chronic diseases, such as diabetes,

coronary heart disease, or other major illness; and willing-

ness to consume 250 mL of BJP daily for 9 weeks. A written in-

formed consent was obtained from all participants. A study

protocol was approved by the Research Ethics Committee,

Faculty of Medicine and Health Sciences, University Putra

Malaysia, Malaysia.

2.4. Study design

The study was a 4-week, single-blinded, crossover supple-

mentation trial. Subjects were randomly assigned to consume

daily 250 mL of a study drink, either flavored mango juice

(placebo group, n = 16) or BJP juice (n = 16). All of the juices

contained 30 mg/dL of citric acid as a preservative. All sub-

jects were asked to continue their habitual diet and physical

activity during the study. The subjects were advised to drink

the beverages after the morning meal. The subjects were also

advised to discontinue the use of vitamin C and intake of

carotenoid-containing foods, fruits and juices 1 week prior

to the study and to avoid the use of alcohol and analgesics

3 days before and vigorous physical activity 1 day before the

study visits. A 3-day food and physical activity record was

collected at weeks 0, 4, 6 and 9 to control for possible

confounding factors and to check the compliance with the

given instructions. The instructions for the food and physical

activity records were given, checked with the subjects, and

analyzed using the Nutritionist Pro software, version 4.1.0

(Axxya System, Stafford, TX, USA), and the International

Physical Activity Questionnaire (IPAQ). The compliance was

assessed by asking the subjects to return the used drink

bottles, and the percentage of compliance was calculated by

differentiating the sum of the drink bottles provided to the

participant with the sum of the drink bottles that were

returned to the researcher.

2.5. Collection of blood

Blood samples (10 mL) were drawn after an overnight fast at

week 0 (baseline), week 4 (end of the first phase), week 5

(end of the wash-out period) and week 9 (end of the study)

into 6 mL ethylenediaminetetraacetic acid (EDTA) tubes for

antioxidant enzyme determination and into 4 mL heparinized

tubes for the determination of antioxidant vitamins. The

blood was drawn between 0730 and 1100 h from an antecubi-

tal vein. Plasma was collected by centrifugation of the whole

blood for 10 min at 1006g. The plasma used for the determina-

tion of ascorbic acid was stabilized immediately by adding

metaphosphoric acid (259 g/L) and then storing at �80 �C until

subsequent analysis.

2.6. Measurement of blood pressure and anthropometricparameters

For height and weight measurements, subjects were lightly

dressed but had their shoes removed. Height was recorded

during their first visit using a SECA Bodymeter 206 (SECA

Vogel and Halke Gmbh & Co., Hamburg, Germany). At the first

visit and subsequent visits, their weight was measured using

a portable SECA Alpha Model 770 (SECA Vogel and Halke

Gmbh & Co., Hamburg, Germany), and blood pressure was

measured using an automated blood pressure machine

(Omron, West Midlands, UK) (using an average of 2

measurements). Blood pressure was measured in the fasting

state between 0800 and 1000 h after the subjects had rested

in the sitting position for at least 10 min. An appropriately

sized cuff was placed on the nondominant arm supported

at the level of the heart. Blood pressure was measured 3 times

at 5 min intervals at weeks 0, 4, 6 and 9.

2.7. Biochemical analyses

Before analysis, all frozen samples were thawed and

centrifuged at 1006g for 10 min at 4 �C. Samples were

analyzed in batches at the end of the study.

2.7.1. Determination of plasma antioxidant enzymes2.7.1.1. Glutathione peroxidase (GPx). To heparinized whole

blood (0.05 mL), 1 mL of diluting reagent was added and

incubated for 5 min; next, 1 mL hemoglobin reagent was

added, and the sample was mixed for 10 s with a vortex

mixer. The mixture was assayed enzymatically using a

RANDOX kit (Randox Laboratories, Antrim, United Kingdom)

within 20 min of adding the hemoglobin reagent using a

chemistry analyzer (Selectra E, Dieren, The Netherlands).

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2.7.1.2. Superoxide dismutase (SOD). An aliquot of 0.5 mL of

whole blood was placed into a test tube and centrifuged for

10 min at 1006g at 4 �C. The plasma was then aspirated off

the centrifuged blood to leave only erythrocytes. The erythro-

cytes were washed 4 times with 0.9% NaCl. Then, the volume

of the washed erythrocytes was increased to 2.0 mL with cold

redistilled water, mixed and left to stand at 4 �C for 15 min.

The lysate was stored at 4 �C until further quantification,

which was performed using a chemistry analyzer (Selectra

E, Dieren, Netherlands).

2.7.2. Determination of plasma total antioxidant status (TAS)Plasma TAS was assayed through the incubation of 2,2-azin-

obis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS�+) with a

peroxidase (metmyoglobin), which results in the production

of the radical cation ABTS�+. This species is blue-green in color

and can be detected at 600 nm. Antioxidants in the added

sample cause inhibition of this color production to a degree

that is proportional to their concentration.

2.7.3. Determination of plasma non-enzymatic antioxidants2.7.3.1. Ascorbic acid. The extraction was performed accord-

ing to the method described by Esteve, Frıgola, Martorell,

and Rodrigo (1998). In brief, the proteinization was conducted

by adding 0.5 mL plasma to 10% metaphosphoric acid in an

amber test tube, and the solution was mixed and centrifuged

at 3300g for 10 min at 4 �C. The supernatant was filtered

through a 0.22 lm membrane filter and placed in an amber

vial. An aliquot of 25 ll was immediately injected into the

HPLC system.

The HPLC conditions were determined according to the

protocol described by Tanishima and Kita (1993). The follow-

ing mobile phases were used: (I) 2% methanol in 0.1 mM so-

dium phosphate monobasic, pH 4.4, and (II) 10% methanol

in 0.1 mM sodium phosphate monobasic, pH 4.4. Mobile

phase I was pumped through the column at room tempera-

ture at a flow-rate of 0.5 mL/min followed by the injection of

the 25 ll samples. The determination was made at 265 nm

and 1 cm/min chart speed. The column was purged after run-

ning each sample with mobile phase II for 7 min to remove

other plasma components before re-equilibration with mobile

phase I.

2.7.3.2. b-Carotene. The determination of plasma b-carotene

was performed according to the procedure described by

Rajendran, Pu, and Chen (2005), with slight modifications.

The analysis of plasma by RP-HPLC involved 3 steps: denatur-

ation of the protein, extraction with organic solvent, and dry-

ing and reconstitution. Deproteinization was performed by

adding 0.5 mL plasma to 0.5 mL ethanol; the solution was

mixed for 10 s and then centrifuged for 10 min at 1006g.

The supernatant was collected and extracted with 2 mL of

methylene chloride/hexane (1:5 v/v), and the upper layer

was collected. The procedure was repeated twice. Then, the

pooled organic layer was purged with nitrogen gases,

followed by reconstitution with ethanol/tetrahydrofuran (1:1

v/v).

The gradient elution was used to determine the b-carotene

content. Two mobile phases were used: (I) acetonitrile/

methanol in a proportion of 85/15 (v/v) and (II) acetonitrile/

methylene chloride/methanol in a proportion of 70/20/10

(v/v/v). Mobile phase I was pumped through the column for

20 min, followed by mobile phase II for 30 min. Samples

(25 ll) were injected into the column at room temperature

at a 1.0 mL/min flow-rate. The determination was made at a

wavelength of 450 nm and a chart speed of 1 cm/min.

2.7.4. Determination of plasma liver enzymes and kidneyfunctionDeterminations of gamma-glutamyltransferase (GGT), ala-

nine transaminase (ALT), alkaline phosphatase (ALP), aspar-

tate transaminase (AST), albumin, total protein, creatinine

and urea were made using commercial kits provided by

RANDOX Laboratories LTD (Crumlin, Co. Antrim, UK). All liver

enzymes were assayed using a chemistry analyzer (Selectra E,

Dieren, The Netherlands).

2.8. Statistical analysis

Data are presented as the mean ± standard error of the mean.

Statistical analysis was performed using SPSS for Windows

(version 13.0). General linear models were used to examine

differences in post-intervention values compared to baseline

values using Tukey’s procedure for comparison of means. Dif-

ferences were considered significant for all statistical analy-

ses at P < 0.05.

3. Results

Thirty-eight men and women were selected from 100

screened participants to participate in the study. Only 32 sub-

jects completed the trial, and their baseline characteristics,

dietary intake and physical activity records are shown in Ta-

ble 2. The placebo and BJP drink groups did not differ in nutri-

ent intake (Table 2) or in any of the biochemical variables,

such as vitamins A and C. The BJP drink group consumed

more cholesterol and b-carotene compared to the placebo

drink group, but the differences were not significant. More-

over, the energy intake and expenditure were the same for

both groups. The nutrient, energy intake and energy expendi-

ture values were similar in our population compared to data

presented from the 2003 Malaysia Adult Nutrition Survey

(Mirnalini et al., 2008).

Apparent compliance was greater than 90% for both the

placebo and BJP drink groups. The high rate of compliance

was achieved because all of the subjects were asked to con-

sume the drink every day in the presence of the researcher,

except for the weekends or other days that the subjects were

unable to come to the dietetic laboratory.

Plasma total antioxidant status increased significantly after

BJP consumption (P < 0.001, Table 3) by 18% compared to base-

line, which is consistent with the high degree of compliance.

Similarly, plasma b-carotene (1.48 ± 0.12 vs. 2.30 ± 0.05 lM)

and ascorbic acid (72.10 ± 6.07 vs. 92.43 ± 4.58 lM) levels were

increased significantly by 45% and 28%, respectively, after BJP

drink consumption compared to baseline values (Figs. 1 and 2).

Plasma antioxidant enzymes (glutathione peroxidase and

superoxide dismutase) were not changed by supplementation

(Table 3). There were no changes in antioxidant enzymes or

Fig. 1 – Changes in plasma ascorbic acid levels of the

participants before and after 4 weeks of the consumption of

placebo and BJP drinks. a an = 32 in the placebo and BJP drink

groups. MNValues in the same row with different

superscripts are significantly different, p < 0.05. *Values are

significantly different compared to week 0, p < 0.05.

Week 0 (baseline data), Week 4 (end of study data).

Table 2 – Baseline characteristics, dietary intakes and physical activity record of the participant recruited in the presentstudy.a

Parameter Placebo drink group (mean ± S.E.M.b) BJP drink group (mean ± S.E.M.)

Age (year) 24 ± 2 24 ± 2

Weight (kg) 62.02 ± 4.12 61.73 ± 4.06

Height (cm) 162.05 ± 1.41 162.05 ± 1.41

Body mass index 23.46 ± 1.43 23.36 ± 1.41

Systolic blood pressure (mm Hg) 116.92 ± 2.86 110.19 ± 2.62

Diastolic blood pressure (mm Hg) 72.08 ± 2.06 70.00 ± 1.57

Energy (kcal) 1650.78 ± 172.40 1612.91 ± 151.85

Protein (g) 57.03 ± 5.22 53.50 ± 2.94

Carbohydrate (g) 239.48 ± 16.51 232.03 ± 19.70

Fat (g) 51.39 ± 3.27 52.46 ± 5.09

Cholesterol (mg) 173.97 ± 28.65 180.43 ± 23.01

Vitamin A (lg RAEc) 343.56 ± 42.51 373.16 ± 29.71

b-Carotene (IU) 162.20 ± 21.05 181.33 ± 32.12

Vitamin C (mg) 45.94 ± 1.25 47.24 ± 2.59

Energy expenditured 1614.83 ± 210.67 1640.10 ± 195.62

a n = 32 in the placebo drink group and n = 32 in the BJP drink group.b S.E.M. = standard error of the mean.c RAE = retinol activity equivalents.d Calculated through physical activity diaries assessment.

Table 3 – Plasma antioxidant enzymes and total antioxidant status of the participants before and after 4 weeks of theconsumption of placebo and BJP drinks.a

Parameter Placebo drink group BJP drink group

Week 0(mean ± S.E.M.b)

Week 4(mean ± S.E.M.)

Week 0(mean ± S.E.M.)

Week 4(mean ± S.E.M.)

Antioxidant enzymes

GPxc (U/L) 284.79 ± 7.76 241.67 ± 6.29 264.46 ± 5.64 239.18 ± 14.78

SODd (U/mL) 7.72 ± 0.19 7.14 ± 0.21 7.10 ± 0.27 7.53 ± 0.17

Total antioxidant status (mM) 1.29 ± 0.05M 1.04 ± 0.03M 1.12 ± 0.44M 2.16 ± 0.15N, *

Values in the same row with different superscript are significantly different, p < 0.05.a n = 32 in the placebo drink group and n = 32 in the BJP drink group.b S.E.M. = standard error of the mean.c Glutathione peroxidase.d Superoxide dismutase.

* Values are significant different as compared to week 0, p < 0.05.

1904 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 9 0 0 – 1 9 0 8

non-enzymatic antioxidants for the placebo group following

4 weeks of supplementation.

Compared to the baseline values, plasma gamma-glutam-

yltransferase (25.85 ± 2.73 vs. 20.50 ± 2.56 U/L), alkaline phos-

phatase (336.54 ± 11.76 vs. 307.87 ± 10.23 U/L), alanine

transaminase (42.08 ± 0.98 vs. 40.58 ± 1.04 U/L) and aspartate

transaminase (30.23 ± 1.14 vs. 30.12 ± 1.28 U/L) were unaf-

fected by BJP drink consumption at the end of study. Similarly,

plasma total protein, creatinine, urea and albumin were not

changed (Table 4).

4. Discussion

The present study showed that consumption of the study

drink prepared from bambangan fruit juice powder, which

provided vitamins, b-carotene and other phytochemicals, re-

sulted in increased plasma total antioxidant status, b-caro-

tene and ascorbic acid compared to the consumption of a

placebo drink.

Fig. 2 – Changes in plasma b-carotene levels of the

participants before and after 4 weeks of the consumption of

placebo and BJP drinks. a an = 32 in the placebo and BJP drink

groups. MNValues in the same row with different

superscripts are significantly different, p < 0.05. *Values are

significantly different compared to week 0, p < 0.05.

Week 0 (baseline data), Week 4 (end of study data).

J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 9 0 0 – 1 9 0 8 1905

Ascorbic acid is an important contributor to the oxidant

defense system and has been shown to protect against the

in vivo oxidation of lipid and DNA in humans (van den Berg

et al., 2001). Two large cross-sectional studies conducted by

the United States, the National Health and Nutrition Exami-

nation Survey (NHANES) II and III, showed an association be-

tween high serum ascorbic acid concentration and a

decreased prevalence of stroke (Simon & Hudes, 1999; Simon,

Hudes, & Browner, 1998). The present study was conducted to

determine if the consumption of a BJP drink could increase

plasma ascorbic acid levels in healthy human subjects. Other

investigators have reported that the consumption of 500 mL

of fruit and vegetable blends (Broekmans et al., 2000) and 4

capsules of dehydrated fruit and vegetable juice (Samman

et al., 2003) increased plasma ascorbic acid levels by 64%

and 32%, respectively.

Table 4 – Plasma liver and kidney function of the participants bBJP drinks.a

Parameter Placebo drink group

Week 0 (mean ± S.E.M.b) Week 4 (mean ±

Liver function

GGT (U/L) 25.04 ± 1.84 24.85 ± 1.92

ALP (U/L) 316.27 ± 26.31 327.48 ± 9.16

ALT (U/L) 42.38 ± 1.31 42.73 ± 1.60

AST (U/L) 30.42 ± 1.28 33.38 ± 1.16

Kidney function

Total protein (g/dL) 5.70 ± 0.08 4.92 ± 0.11

Creatinine (mg/L) 0.58 ± 0.03 0.51 ± 0.03

Urea (mM) 6.27 ± 0.11 7.16 ± 0.12

Albumin (g/L) 31.34 ± 0.48 29.66 ± 0.82

Values in the same row with different superscript are significantly differ*Values are significant different as compared to week 0, p < 0.05.a n = 32 in the placebo drink group and n = 32 in the BJP drink group.b S.E.M. = standard error of mean.

At the beginning of the study, the plasma ascorbic acid lev-

els of all groups ranged from 72 to 78 lM. A similar mean plas-

ma ascorbic level of approximately 80 lM was documented in

a previous study (Samman et al., 2003), and slightly lower lev-

els of approximately 41 and 63 lM were reported by van den

Berg et al. (2001) and Duthie et al. (2006), respectively. A study

by John, Ziebland, Yudkin, Roe, & Neil (2002) reported that the

plasma ascorbic acid level was much lower, at approximately

34 lM. In the present study, the BJP drink provided 66 mg

ascorbic acid/day and resulted in a mean plasma ascorbic

acid level of 92 lM, a mean increase of 20 lM. This change

may have been due to the higher ascorbic acid content in

the BJP drink, as the dietary intake and physical activity levels

were not significantly different between the groups (Table 2).

This magnitude of change was consistent with previous trials

that utilized mixed fruit and vegetable concentrates (Samman

et al., 2003; van den Berg et al., 2001), cranberry juice (Duthie

et al., 2006) and at least 5 daily portions of fruits and vegeta-

bles (John, Ziebland, Yudkin, Roe, & Neil, 2002). A study in el-

derly subjects indicated that the consumption of 250 mL

apple juice and 250 mL pomegranate juice for 4 weeks signif-

icantly increased their plasma ascorbic acid levels (Guo et al.,

2008). The increase in plasma ascorbic acid levels might have

contributed to the antioxidant capacity of the plasma, as

demonstrated by a significant increase in the plasma total

antioxidant status (Table 3). Carotenoids are biologically

important micronutrients with a large range of functions,

and their supplementation has been recommended for the

prevention of degenerative diseases related to oxidative stress

(Butalla et al., 2012).

After 4 weeks of dietary intervention, the plasma b-caro-

tene level was significantly higher in the BJP-supplemented

group than in the placebo group. Plasma b-carotene increased

by a mean of 0.8 lM, a fourfold increase over the baseline val-

ues. The high bioavailability of b-carotene, as reported from

trials that used a mixture of fruit and vegetable concentrates

in healthy men (Samman et al., 2003), could be a possible rea-

son for the increase. Phospholipids are known to modulate

efore and after 4 weeks of the consumption of placebo and

BJP drink group

S.E.M.) Week 0 (mean ± S.E.M.) Week (mean ± S.E.M.)

25.85 ± 2.73 20.50 ± 2.56

336.54 ± 11.76 307.87 ± 10.23

42.08 ± 0.98 40.58 ± 1.04

30.23 ± 1.14 30.12 ± 1.28

5.32 ± 0.12 4.84 ± 0.15

0.56 ± 0.03 0.55 ± 0.03

6.55 ± 0.13 6.56 ± 0.14

30.84 ± 0.70 29.43 ± 1.01

ent, p < 0.05.

1906 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 9 0 0 – 1 9 0 8

the absorption of carotenoids. Therefore, the high bioavail-

ability of b-carotene may be explained in part by a slightly

higher intake of fat and cholesterol in the BJP drink group that

might have facilitated the higher absorption rate of b-caro-

tene into the blood circulation (Table 2). Moreover, several

previous studies have documented an enhanced uptake of

b-carotene and lutein when rats and mice were fed with

micellar phospholipids (Baskaran, Sugawara, & Nagao, 2003;

Lakshminarayana, Raju, Krishnakantha, & Baskaran, 2006).

Conversely, intake of 40 g/kg body weight of a pure b-caro-

tene suspension in vegetable oil did not improve the plasma

b-carotene concentrations in healthy, non-smoking, normo-

lipidemic men and women compared to subjects who con-

sumed spinach (Castenmiller et al., 1999). The increase in

plasma b-carotene together with ascorbic acid might have

contributed to the antioxidant capacity of the plasma that

was demonstrated in the present study.

The present study determined the effect of BJP drink con-

sumption on plasma GPx. No changes in the individual anti-

oxidant enzymes (GPx and SOD) were detected. These

results are in agreement with other studies reporting no

changes in SOD and GPx when healthy human volunteers

consumed supplemental cranberry juice for 4 weeks (Duthie

et al., 2006) and when 72 normolipidemic, non-smoking

men and women consumed b-carotene and spinach for

3 weeks (Castenmiller et al., 1999).

A possible reason for the unchanged plasma antioxidant

enzymes could be a suppression of GPx and SOD activities

by high levels of b-carotene in circulating plasma. A study

by McGill, Green, Meadows, and Gropper (2003) indicated that

supplementation of 30 mg/day b-carotene for 60 days de-

creased leukocyte SOD activity and serum glutathione perox-

idase concentrations in healthy adults.

Although the consumption of a BJP drink for 4 weeks did

not alter the antioxidant enzymes, plasma total antioxidant

status (TAS) was increased significantly compared to baseline

values. The high plasma TAS in the BJP drink group could be

explained by the high ascorbic acid and b-carotene contents

in the BJP drink. Indeed, the plasma ascorbic acid and b-caro-

tene concentrations were found to be strongly (r = 0.92,

p < 0.001) and moderately (r = 0.73, p < 0.05) correlated, respec-

tively, with plasma TAS in the BJP drink group. Our data are in

agreement with a previous study that reported that the con-

sumption of a vegetable burger and a fruit drink significantly

improved the plasma antioxidant capacity (van den Berg

et al., 2001). Increased intake of fruits and vegetables from 5

to 10 serving per day in 36 healthy nonsmokers significantly

increased plasma antioxidant capacity (Cao, Booth, Sadowski,

& Prior, 1998). Additionally, Kay and Hulob (2002) reported

that the consumption of 100 g of wild blueberries increased

the serum antioxidant status in human subjects, and the con-

sumption of 330 mL/day of a mixture of apple, mango and or-

ange juice for 10 weeks modulated and enhanced antioxidant

status in 27 non-smoking men in Germany (Bub et al., 2003).

Moreover, Guo et al. (2008) showed that the consumption of

250 mL of pomegranate juice for 4 weeks enhanced the anti-

oxidant activity in elderly subjects.

This study clearly showed that BJP drink consumption did

not alter liver function parameters (GGT, ALP, ALT and AST en-

zymes) compared to the baseline values. The results showed

that the consumption of a BJP drink could protect against liver

disease rather than having a cytotoxic effect, as was demon-

strated by increases in liver enzymes in the placebo groups at

the end of the study. The observations align with the effects

of the BJP drink on parameters of plasma kidney function.

Compared to the baseline values, plasma total protein, albu-

min, creatinine and urea concentrations did not differ from

the concentrations measured after 4 weeks of BJP consump-

tion. Most of the studies conducted to demonstrate the car-

dioprotective effects of fruits and vegetables (including their

derived products) did not include toxicity biomarkers in their

assessments (Bub et al., 2003; Duthie et al., 2006; Guo et al.,

2008; Kay & Hulob, 2002; Record, Dreosti, & McInerney, 2001;

van den Berg et al., 2001). Based on the results presented here,

a BJP drink could be considered safe for consumption.

5. Conclusions

Consumption of a BJP drink by healthy human volunteers im-

proved non-enzymatic antioxidant status. Total antioxidant

status was significantly increased due to the consumption

of the BJP drink. This effect could be due to the present of

vitamin C, b-carotene and other health-promoting com-

pounds in M. pajang fruit. No adverse effects were observed

due to the BJP drink consumption, as demonstrated by no

changes in liver and kidney function parameters. Therefore,

the consumption of a BJP drink may improve the antioxidant

status of healthy adults.

Acknowledgments

We would like to acknowledge the financial support provided

by the Ministry of Science, Technology and Innovation of

Malaysia (MOSTI) under an e-Science Fund Grant scheme

titled, ‘‘Development of innovative antioxidant ingredients

from mango’’ (Project No. 05-01-04-SF0048).

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