ORIGINAL ARTICLE

Role of natural juice in improving the ventilatory functionsof smokers with chronic obstructive pulmonary disease

AMAL SAAD, MD1, AMR MOSTAFA, PhD2 & KHADIGA IBRAHIM, PhD1

1Department of Environmental and Occupational Medicine, National Research Center, Cairo, Egypt,

and 2Biochemistry Department, Faculty of Agriculture, Cairo University, Cairo, Egypt

AbstractPurpose. Chronic obstructive pulmonary disease (COPD) is a common disease related to smoking.Oxidative stress and antioxidant status may play an important role in the pathogenesis of COPD. Theaims of the present study were to determine the pathogenic effects of oxidative stress and antioxidantstatus in COPD smokers, and the role of dietary antioxidant supplementation in the ventilatoryfunction tests (VFT) of the COPD smokers.Design. This study comprised two phases. The first phase explored oxidative stress and antioxidantstatus and its relationship with the ventilatory functions of COPD diagnosed smokers. The secondphase was a self-controlled clinical trial to study the changes in the oxidative–antioxidant status andthe VFT of the COPD smoking patients after dietary supplementation with freshly prepared juice for30 days.Methods. Personal, occupational and medical questionnaires, a clinical examination, VFT, serumantioxidants (vitamin A, C and E and b-carotene), malondialdehyde (MDA) and superoxidedismutase (SOD).Results. There was a significant correlation between vitamin E and the ventilatory functions of theCOPD smokers at baseline. After supplementation, the ventilatory functions were significantlyimproved. Plasma vitamin A, C and E, b-carotene and SOD were also significantly increased. Thelevel of MDA was significantly decreased.Conclusions. Daily fresh juice supplementation provides correction of the antioxidant status andsignificantly improves the VFT of COPD smokers.

Key words: Smoking, fresh natural juice, ventilatory function test, antioxidant vitamins, MDA,SOD

Introduction

Cigarette smoke contains a large number of oxidants, more than 4000 identified

constituents, in addition to large amounts of free radicals, leading to the adverse effects

of smoking [1]. Many studies suggest that oxidants may play a role in the pathogenesis of

lung diseases [2,3]. The epidemiology of chronic obstructive pulmonary disease (COPD) is

Correspondence: Amal Saad, Department of Environmental and Occupational Medicine, National Research Center, Dokki,

Cairo, Egypt. Email: amel_h3@yahoo. com

Journal of Nutritional & Environmental Medicine

February 2007; 16(1): 16–25

ISSN 1359-0847 print/ISSN 1364-6907 online # 2007 Informa UK Ltd

DOI: 10.1080/13590840701211219

complex. It includes both environmental and genetic factors [4]. The major environmental

risk factor for COPD is cigarette smoking, which is one of the most potent oxidants [5,6].

Experimentally, it was proved that exposure of albino rats to cigarette smoke significantly

decreased the activity of the antioxidant enzyme superoxide dismutase (SOD) and

significantly increased malondialdehyde (MDA) levels [7]. It has been suggested that

oxidative stress, as a result of cigarette smoking, is an important factor in the pathogenesis

of COPD. Abdel-Motaleb et al. [8] found a remarkable reduction in the content of

vitamins C and E, and in the activity of SOD in COPD patients compared with their

controls.

In experimental studies, oxidative stress was proved to increase elastase production,

inactivate anti-proteases, impair connective tissue repair, impair the function of the cilia

and increase mucous production [9]. All these changes, in addition to increased levels of

airway inflammatory cells and cytokine production, can ultimately result in airway

obstruction and interstitial damage and the development of COPD [5].

The aim of the present study was to evaluate the pathogenic roles of oxidative stress and

antioxidant status in COPD smoking patients. The study also aimed to evaluate the role of

dietary supplementation with freshly prepared natural juice (mixture of fruits and

vegetables) on oxidative stress, the antioxidant status, and the ventilatory functions of

COPD smoking patients.

Methodology

Design

This study comprised two phases. The first phase was a cross-sectional study to select

smokers with COPD (volunteers), after excluding those who developed COPD secondary

to occupational exposure. Moreover, the relationships of oxidative–antioxidant status with

the ventilatory functions of the volunteers were studied at baseline. The second phase was a

self-controlled clinical trial to evaluate the role of dietary supplementation with freshly

prepared natural mixed fruit and vegetable juice on the oxidative–antioxidant status and

ventilatory function tests (VFT) of COPD smoking patients.

Subjects

The present study was conducted between July 2004 and March 2005. In the first phase of

the study, 76 randomly selected volunteers, clerk males from the National Research

Centre, who had been smokers for at least 10 years, were included. They were all of the

same education and socio-economic status. Their age ranged between 45 and 50 years. All

participants were informed about the study and were asked to maintain their current

lifestyle habits. In the second phase of the study, 15 out of 21 COPD diagnosed cases

accepted to complete the study with the dietary supplementation. Written informed

consent was obtained from all the included volunteers.

Questionnaire

Face-to-face interviews were conducted using the respiratory illness questionnaire adapted

according to the American Thoracic Society Epidemiology Standardization Project [10],

with the addition of demographic characteristics, occupational history and working

conditions.

Role of natural juice in improving the ventilatory functions of smokers 17

Clinical examination

A physical examination was completed for all the included smokers (76 smokers), after the

exclusion of smokers occupationally exposed to chemicals or a dusty environment. Physical

signs of COPD were considered to be diminished breath sounds and prolonged expiration

with generalized wheezing, according to the Egyptian guidelines for the management of

COPD [11].

Spirometric measurements

Spirometric measurements were performed in the sitting position using a portable

spirometer, according to the criteria of the American Thoracic Society [12]. VFT were

carried out for 76 smokers to diagnose COPD cases at zero time. After 30 days of dietary

supplementation with the natural freshly prepared juice, VFT were carried out again for the

15 COPD cases accepted to complete the study. All ventilatory function parameters in the

form of forced expiratory volume in 1 sec (FEV1), forced vital capacity (FVC) and peak

expiratory flow rate (PEFR) were expressed as a percentage of the predicted value for each

person after adjustment for age, gender, race and height.

COPD was diagnosed only if there was a combination of abnormal VFT and

symptoms of chronic cough and sputum production on most days for more than 2

years. According to the Egyptian guidelines for the management of COPD [11],

moderate COPD characteristics were FEV1/FVC,70%, FEV1 30–50%, with or without

symptoms.

Cotinine and creatinine analysis in urine

Urine samples from the diagnosed COPD smokers who accepted to complete the study

were collected at the beginning and at the end of the study, and kept frozen at 220uC until

the end of the study. Cotinine concentration was assayed in the collected urine samples

with a radioimmunoassay kit using a gamma counter instrument [13]. To standardize the

variations in urinary volumes, urinary creatinine was determined by the Jaft method

without deproteinization. The cotinine/creatinine ratio (CCR) was calculated for each

smoker and expressed in mg mg21.

In the present study, the CCR was used to define the acute exposure to cigarette

smoking, whereas the smoking index (SI) was used to define the cumulative exposure to

cigarette smoking.

Analysis of plasma antioxidants

Blood samples were drawn from each one of the smokers before and after juice

supplementation into heparinized tubes. Red blood cells were separated from plasma by

centrifugation of the blood samples at 3500 rpm for 10 min. Red blood cells were

washed three times with normal cold saline. Plasma and red blood cells after washing were

kept at 220uC until analysis. Plasma MDA was estimated according to Satoh [14]. Red

blood cell SOD was determined according to Roth and Gilbert [15], plasma vitamin A and

b-carotene were determined according to Neeld and Pearson [16], and plasma vitamin E

was analysed according to Desai et al. [17]. Vitamin C was estimated using the Folin

phenol reagent, as described by Jagota and Dani [18]. Plasma antioxidants were measured

at zero time (baseline of the study) and after 30 days of supplementation for the 15

volunteers.

18 A. Saad et al.

Supplementation

Daily dietary supplementation with one cup (150 ml) of natural freshly prepared juice from

a mixture of apple, cucumber, carrot and parsley was given to the selected 15 COPD

patients by one of the authors.

Analysis of the prepared juice

The concentrations of b-carotene, vitamins C and E in one cup of the natural prepared

juice were measured. The concentration of b-carotene was determined by high-

performance liquid chromatography according to Bushway [19]. The vitamin C

concentration was determined by the 2,4-dinitrophenylhydrazine method [20]. Vitamin

E was measured according to the method of Martinek [21].

Statistical analysis

The collected data were computerized and a statistical analysis was performed using SPSS

version 11. Cumulative exposure to tobacco smoke was calculated in pack-years (SI) for the

current smokers [22]. The correlation coefficient, paired t-test and Wilcoxon signed ranks

test were used for the statistical analysis of the data. Statistical significance was considered

when p(0.05.

Results

The statistical analysis revealed that 27.6% of the examined smokers were diagnosed as

moderate COPD patients (21 cases out of 76 smokers). Phase two of the study was

completed by 15 volunteers out of 21 COPD cases.

At the baseline of the study, the SI of the volunteers (15 cases) ranged from 20 to 45

pack-years (27.04¡4.13 pack-years), and their CCR ranged from 4360.5 to

8716.6 mg mg21 (6841.3¡560.5 mg mg21). After 30 days of dietary supplementation, the

SI and the CCR of the volunteers were not significantly changed (p.0.05).

Table I shows that at baseline there was a significant correlation between plasma

vitamin E and the ventilatory functions of the volunteers, whereas there was a non-

significant positive association of the ventilatory functions of the volunteers with the

plasma levels of vitamins A and C, b-carotene and SOD and an inverse association with

MDA.

Table I. Relationships between serum antioxidants and the pulmonary function tests of the chronic obstructive

pulmonary disease smokers after controlling for the cotinine/creatinine ratio at baseline.

FEV1 FVC FEV1/FVC PEFR

r p r p r p r p

Plasma vitamin A (mg dl21) 0.1 NS 0.1 NS 0.4 NS 0.4 NS

Plasma b-carotene (mg dl21) 0.3 NS 0.2 NS 0.1 NS 0.1 NS

Plasma vitamin C (mg dl21) 0.3 NS 0.3 NS 0.4 NS 0.4 NS

Plasma vitamin E (mg dl21) 0.5 ,0.05 0.6 ,0.05 0.6 ,0.05 0.6 ,0.05

Malondialdehyde (ng ml21) 20.5 NS 20.4 NS 20.2 NS 20.1 NS

Superoxide dismutase (U g.Hb21) 0.3 NS 0.3 NS 0.4 NS 0.3 NS

FEV, forced expiratory volume in 1 sec; FVC, forced vital capacity; PEFR, peak expiratory flow rate.

Role of natural juice in improving the ventilatory functions of smokers 19

Table II illustrates the relationships of the VFT, the plasma oxidant–antioxidants and

both the CCR and the SI. There were significant negative correlations between the plasma

levels of vitamin A and MDA with both the CCR and the SI. Plasma b-carotene and SOD

were significantly correlated with both the CCR and the SI.

The chemical analysis of the prepared juice revealed that the concentrations of b-

carotene, vitamins C and E in 150 ml of juice were 9200 mg, 35.3 mg, and 1.4 mg,

respectively. After 30 days of dietary supplementation, the respiratory symptoms of the

volunteers were slightly improved, without significant difference (p.0.05).

According to the Wilcoxon signed ranks test, after dietary supplementation, vitamins A,

C and E, b-carotene, SOD, MDA and VFT (FEV1, FVC and FEV1/FVC) were improved

in 100% of the volunteers, whereas PEFR was improved in 60% of the volunteers (nine of

15).

Table III shows that after supplementation, the plasma concentrations of vitamins A, C

and E, b-carotene and SOD were significantly increased, whereas MDA was significantly

Table II. Relationships between the smoking index (SI) and the cotinine/creatinine ratio (CCR) and the

pulmonary function tests of the smokers with severe chronic obstructive pulmonary disease at baseline.

CCR SI (pack-years)

r p r p

SI 0.3 NS 1.0

FEV1 20.3 NS 20.2 NS

FVC 20.2 NS 20.2 NS

FEV1/FVC 20.2 NS 20.2 NS

PEFR 20.3 NS 20.1 NS

Plasma vitamin A (mg dl21) 20.6 ,0.05 20.7 ,0.01

Plasma b-carotene (mg dl21) 0.9 ,0.0001 0.5 ,0.05

Plasma vitamin C (mg dl21) 0.2 NS 0.3 NS

Plasma vitamin E (mg dl21) 20.5 NS 20.2 NS

Malondialdehyde (ng ml21) 20.7 0.01 20.6 0.01

Superoxide dismutase (U g.Hb21) 0.7 0.01 0.9 ,0.0001

FEV, forced expiratory volume in 1 sec; FVC, forced vital capacity; PEFR, peak expiratory flow rate.

Table III. The effect of juice supplementation on the plasma oxidative stress and antioxidant status of chronic

obstructive pulmonary disease smokers.

Before After Paired t-test

Mean SD Mean SD t-test p

Plasma vitamin A

(mg dl21) (range)

21.8 (19.7–25.2) 1.97 32.2 (30.1–33.2) 1.17 19.545 0.0001

Plasma b-carotene

(mg dl21) (range)

83.8 (79.6–90.1 4.35 107.7 (101.5–111.2) 3.38 31.497 0.0001

Plasma vitamin C

(mg dl21) (range)

0.732 (0.64–0.82) 0.07 1.002 (0.91–1.10) 0.06 12.956 0.0001

Plasma vitamin E

(mg dl21) (range)

1.18 (0.9–1.14) 0.18 1.46 (1.2–1.7) 0.19 7.128 0.0001

Malondialdehyde

(ng ml21) (range)

2.86 (2.2–3.4) 0.44 2.04 (1.9–2.2) 0.12 9.268 0.0001

Superoxide dismutase

(U g.Hb21) (range)

1553.6 (1467–1600) 51.48 1898.6 (180–2005) 90.6 15.409 0.0001

SD, standard deviation.

20 A. Saad et al.

decreased. After dietary supplementation, the VFT of the volunteers were also significantly

increased, except PEFR (Table IV).

After dietary supplementation with the natural juice, the VFT parameters were

significantly correlated with the plasma concentrations of vitamins A and E, b-carotene

and SOD, and inversely correlated with MDA (Table V).

Discussion

Smoking is considered to be the major environmental risk factor for the development of

COPD. Siafakas and Tzortzaki [23] found that less than 20% of smokers developed

COPD. In the present study, 27.6% of the examined smokers were found to have COPD,

according to the guidelines of the Egyptian Society of Chest Diseases and Tuberculosis

[11].

Guenegou et al. [24] found that heavy smokers with low levels of vitamin E and b-

carotene were associated with the steepest decline in FEV1. The second National Health

and Nutrition Examination Survey (NHANES II) (1976–1980) revealed that dietary

vitamin C and serum vitamin C were both found to be inversely associated with chronic

respiratory symptoms [25]. NHANES III (1988–1994) provided evidence that the positive

association of antioxidants (vitamins C, E, b-carotene and selenium) with lung function

differs according to smoking status [26]. However, at baseline in the present study, the

plasma level of vitamin E was the only antioxidant that had a significant correlation with the

VFT of the examined COPD smokers (volunteers).

Several studies have detected through trend analysis that with increasing daily exposure

to cigarettes or with increasing plasma cotinine concentration there were significant lower

Table IV. The effect of juice supplementation on the pulmonary function tests of chronic obstructive pulmonary

disease smokers.

Before After Paired t-test

Mean SD Mean SD t-test p

FEV1% (range) 40.5 (28–52) 9.6 49.8 (42–53) 4.8 4.740 0.001

FVC % (range) 50.5 (35–64) 11.5 62.5 (53–68) 5.9 4.723 0.001

FEV1/FVC % (range) 69.6 (67–69) 1.19 80 (78–82) 1.05 92.182 0.0001

PEFR % (range) 67.5 (53–92) 15.7 68.8 (53–88) 13.2 1.121 NS

SD, standard deviation; FEV, forced expiratory volume in 1 sec; FVC, forced vital capacity; PEFR, peak expiratory

flow rate.

Table V. Relationships between the levels of serum antioxidants and pulmonary function tests after

supplementation after controlling for the cotinine/creatinine ratio.

FEV1 FVC FEV1/FVC PEFR

r p r p r p r p

Plasma vitamin A (mg dl21) 0.8 ,0.0001 0.9 ,0.0001 0.7 ,0.01 0.9 ,0.0001

Plasma b-carotene (mg dl21) 0.05 NS 0.1 NS 0.2 NS 0.2 NS

Plasma vitamin C (mg dl21) 20.7 0.01 20.6 0.01 20.6 0.01 20.4 NS

Plasma vitamin E (mg dl21) 0.7 0.01 0.7 0.005 0.7 0.005 0.9 ,0.001

Malondialdehyde (ng ml21) 20.8 0.001 20.8 0.001 20.7 ,0.01 20.4 NS

Superoxide dismutase (U g.Hb21) 0.7 ,0.01 0.7 0.005 0.8 ,0.005 0.9 ,0.0001

FEV, forced expiratory volume in 1 sec; FVC, forced vital capacity; PEFR, peak expiratory flow rate.

Role of natural juice in improving the ventilatory functions of smokers 21

plasma ascorbic acid and b-carotene [27–30]. In the present study, there were significant

inverse correlations of plasma vitamins A and E with the CCR and the SI of the volunteers

at baseline. This inverse correlation may be due to the scavenger role of these vitamins.

Vitamins A and E are powerful free radical scavengers [8]. However, for unexplained

reasons, there was a significant positive correlation of plasma b-carotene and both the CCR

and the SI of the examined volunteers, which requires further study.

Dietrich et al. [31] showed the importance of adjusting for the dietary intake of

antioxidants when investigating the effect of active and passive smoking on the antioxidants

status. In Egypt, the traditional therapy for respiratory illness was increasing the dietary

consumption of foods rich in vitamin C. In the present study, all the included individuals

had symptoms of chronic cough with expectoration and wheezes with decline in their VFT

that were increased with the increase in the CCR and the SI. Therefore, it was suspected

that there may be an increase in their consumption of foods rich in vitamin C, as there was a

non-significant relationship of plasma vitamin C and the CCR and the SI. Hu and Cassano

[27] found that the dietary intake of vitamin C and carotenes was highly correlated in part

because some of the food sources of the antioxidants are similar.

In the present study, MDA was used as a biomarker of oxidative stress, and SOD as a

biomarker of the antioxidant status. Abdel-Motaleb et al. [8] found that MDA was

significantly higher in COPD patients than in a control group, and their levels were higher in

moderate and severe disease. They also found a reduction in the activity of SOD in COPD

patients than in a control group, and this reduction was correlated with the severity of disease.

In the present study, there was a significant inverse correlation of MDA and a significant

correlation of SOD with the CCR and the SI, which support the explanation of decreasing

vitamins A and E with increasing CCR and SI. This may indicate that oxidative stress could

play an important role in the pathogenesis and progress of COPD severity. Moreover, it

could be a result of poor antioxidant defences that alter the inflammatory events that may

progress the COPD severity. Bhavarahamurth et al. [32] documented that SOD catalyses

the cell defence reaction against potentially harmful effects of free radicals. Howard et al.

[33] denoted that exposure to environmental tobacco smoke results in increased oxidative

stress.

The COPD smokers included in the study were asked not to change their dietary habits.

Their smoking habit was also not changed, as there was no significant difference in the CCR

and the SI between before and after dietary supplementation. The plasma concentrations of

b-carotene, vitamins A, C and E were significantly increased after the dietary supple-

mentation by the natural juice for 30 days. Goraca and Skibska [25] suggested that the

supplementation of antioxidants could be useful for the enhancement of antioxidant status,

especially in the elderly and smokers. Fruit and vegetables contain many phytonutrient

compounds, including antioxidants, vitamins, trace elements, and fibre, and they can

protect against oxidative stress damage and improve antioxidant status [34].

In a previous experimental study, administration of the natural prepared juice (the same

components and preparatory method as used in the present study) caused an increase in the

activity of SOD and a significant decrease in the MDA level of male albino rats exposed to

cigarette smoke [7]. The same results were also found in the present study, as SOD was

significantly increased, whereas MDA was significantly decreased. Bamonti et al. [34]

proved almost the same; they found that MDA was significantly higher in smokers than in

non-smokers, and significantly decreased after 1 month phytonutrient supplementation.

Many studies support the suggestion that dietary antioxidants may protect lung tissue

against reactive oxygen species-induced injury, adverse respiratory effects and reduced

22 A. Saad et al.

pulmonary function [33–36]. In the present study, there was significant improvement in the

VFT of the volunteers after supplementation with the natural prepared juice, which

contained high doses of dietary antioxidants (b-carotene 9200 mg, vitamin C 35.3 mg and

vitamin E 1.4 mg). Because the different antioxidants may have different pathophysio-

logical ways of protecting lung tissue against oxidant-induced damage, each antioxidant

may exhibit different effects across categories of smoking [26]. Ochs-Balcom et al. [37],

using multiple linear regression analysis, showed that dietary b-carotene and vitamin C

were positively associated with FEV1. They denoted that dietary habits and/or oxidative

stress play contributing roles in the severity of chronic airflow limitation. Tujague et al. [36]

also supported the positive association between dietary vitamin C and lung function of non-

smokers (males and females).

After supplementation by the natural juice for at least 1 month, plasma vitamin A

became significantly correlated with the VFT, and the significant correlations of vitamin E

with pulmonary function tests were increased. However, plasma b-carotene was still non-

significantly associated with VFT. Hu and Cassano [27] found that higher levels of

antioxidant nutrients were associated with better lung function. They also found that serum

antioxidants were significantly associated with FEV1, but serum b-carotene was less

positively associated with FEV1 in smokers. However, there are some reports that

supplementation by high doses of b-carotene may be dangerous for smokers, and may have

had an adverse effect on the increase in lung cancer and on the risk of death from lung

cancer and cardiovascular diseases [38].

However, in the present study, the plasma vitamin C concentration was significantly

increased after supplementation, but unexpectedly plasma vitamin C was inversely

correlated with the VFT. This needs further study to determine an explanation for this

unexpected relationship. Previously, Marangon et al. [1] found that cigarette smoking is

associated with decreased plasma vitamin C concentrations, even with high vitamin C

diets. There was the suggestion that smoking directly lowers plasma vitamin C

concentrations by mechanisms that do not depend on dietary vitamin C intake, such as

impaired vitamin C absorption or decreased turnover [27,28].

In addition, in the present study, after supplementation by the natural juice, SOD was

significantly correlated and MAD significantly inversely correlated with the VFT. Thus,

daily fresh juice supplementation provided correction of the antioxidant status of the

COPD smokers and improved the VFT of the COPD smokers. Above all, the significant

increase in the plasma antioxidant concentrations, the significant decrease in MDA levels,

and the significant improvement in the lung functions of the COPD smokers after just 1

month of daily supplementation have prompted us to carry out further studies on a larger

population and for a longer period of time to confirm these preliminary findings.

In conclusion, ventilatory functions of COPD smokers were correlated with the serum

levels of vitamin E, and daily fresh juice supplementation provided correction of the

antioxidant status and significantly improved the VFT of the COPD smokers.

Generally, healthy lifestyle habits, such as a prudent diet and not smoking, may help to

prevent pathological conditions. Daily fresh juice increased the plasma antioxidant status

and improved the VFT of the COPD smokers.

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