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Allergy 2000: 55: 1184±1189 Printed in UK.

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Copyright # Munksgaard 2000
ALLERGY ISSN 0105-4538

Short communication

Reduction of exercise-induced asthma oxidative stress by lycopene, a natural antioxidant
Background: Lycopene has previously been shown to have high antioxidative activity. In view of the controversy regarding the bene®cial effect of antioxidants on asthma, the acute effects of lycopene (LYC-O-MATOTM) on airway hyperreactivity were assessed in patients with exercise-induced asthma (EIA). Methods: Twenty patients with EIA participated in our study to verify the antioxidative effects. The test was based on the following sequence: measurement of baseline pulmonary function, 7-min exercise session on a motorized treadmill, 8-min rest and again measurement of pulmonary function, 1-week, oral, randomly administered, double-blind supplementation of placebo or 30 mg/day of lycopene (LYC-O-MATO), measurement of pulmonary function at rest, 7-min exercise session, and 8-min rest and again measurement of pulmonary function. A 4-week washout interval was allowed between each protocol. Results: All patients given placebo showed signi®cant postexercise reduction of more than 15% in their forced expiratory volume in 1 s (FEV1). After receiving a daily dose of 30 mg of lycopene for 1 week, 11 (55%) patients were signi®cantly protected against EIA. Serum analyses of the patients by high-pressure liquid chromatography detected in the lycopene-supplemented patients an elevated level of lycopene compared to the placebo group, with no change in retinol, tocopherols, or in the other carotenoids. Conclusions: Our results indicate that a daily dose of lycopene exerts a protective effect against EIA in some patients, most probably through an in vivo antioxidative effect.

I. Neuman, H. Nahum

Department of Allergy, Hasharon Hospital, Golda Medical Center, Petach Tivka and Sackler School of Medicine, Tel-Aviv University, Tel-Aviv

A. Ben-Amotz

National Institute of Oceanography, Israel Oceanographic and Limnological Research, Haifa, Israel

Key words: asthma; exercise; lycopene; LYC-O-MATOTM. A. Ben-Amotz Israel Oceanographic and Limnological Research P.O.B. 8030, Haifa 31080 Israel Accepted for publication 6 July 2000

The view that exercise training in children with asthma has a bene®cial effect on aerobic conditioning and psychosocial behavior warrants consideration from a general health perspective (1). Recent evidence shows that strenuous exercise increases the production of reactive oxygen species (ROS) associated with depletion of the antioxidant defense in vital tissues of the body (2). Strenuous exercise may promote free radical production, leading to lipid peroxidation and tissue damage. On the other hand, exercise training seems to reduce the extent of oxidative damage (3), and dietary supplementation with antioxidant vitamins has been shown to be bene®cial in combating oxidative stress (2±6). There is evidence that dietary supplementation with antioxidants, such as vitamin C and b-carotene, protects against exercise-induced asthma (EIA) (7, 8). Studies on the use of vitamin C in antigen-inhalation challenges, histamine inhalation, and methacholine-inhalation tests have yielded contradictory results. Proper assessment of the vitamin C effect on airways requires measurement of alterations in airway tone and airway obstruction, such as occurs after exercise-induced bronchospasm, or in 1184

NO2-induced airway hyperresponsiveness. In asthmatic patients, pretreatment with ascorbic acid has been shown to prevent the signi®cant alterations in pulmonary functions induced by exercise (7). A natural isomer mixture of b-carotene from the alga Dunaliella bardawil was recently shown to prevent EIA (8). The present study was undertaken in order to evaluate the prevention of EIA by lycopene supplementation. Recent studies have indicated that lycopene and other carotenoids play an important role in human health and resistance to degenerative conditions (9). LYC-OMATOTM is a new enriched lycopene product of tomato containing other bioactive ingredients such as tocopherols, carotenoids, phytoene, and phyto¯uene. The effect of LYC-O-MATO on hyperreactive airways was examined at rest and following the physical provocation of exercise.

Material and methods

A group of 20 patients with proven EIA participated in

The concentration of the standards was determined by spectral measurement and calculated with the appropriate extinction coef®cients in ethanol. were purchased from Sigma (St Louis. The column was a Vydac 201 TP54 stainless steel column of 25 cm x 4. short-action bronchodilators. All subsequent exercise testing was performed under the same ambient conditions for all patients and with the same workload that produced EIA at the baseline evaluation. Beer Sheva 84102. The workload was the same for each patient exercising up to a submaximal effort of 80% of the theoretical maximal heart rate. USA). Exercise testing as described above was performed before and after a 7-day supplementation period of 30 mg lycopene or placebo. Kyoto. Chromatography Manager. Pulmonary functions were measured at the same time of the day with similar ambient conditions to avoid any variability. and then the internal standard was extracted as described above for injection into the HPLC at 125 ng/5 ml. Elution was performed at 30t0. Serum was obtained by centrifugation at 3000 g at 5uC and stored at x70uC under nitrogen until analysis. run on an IBM-compatible computer connected to an HP DeskJet 1200 plotter (Hewlett Packard.2uC with an isocratic solvent.0 ml/min. LYC-O-MATO contains 6% lycopene. The two hexane extracts were combined. which is a well-documented system for distinguishing the different carotenoids and their isomers (12. a-tocopherol. as indicated by an Omeda 3700e pulse oxymeter. Each patient underwent this protocol twice. 1185 . all patients discontinued their regular medications. and after vigorous mixing the lipophilic fraction was extracted with 5 ml n-hexane through phase separation and centrifugation at 2000 g at 5uC for 5 min. as described previously. This was a Waters HPLC system (Millipore. b-apo-8k-carotenal. Version 2. 28. UK). Statistical analysis Comparisons of meanstSD between the groups supplemented with either or lycopene or placebo were made by the paired t-test. a Guard-Pak. An amount of 2. USA). Twelve hours before evaluation of the pulmonary functions. Lycopene. and dried by a stream of nitrogen before being analyzed and injected into the HPLC system in methylene chloride. a-carotene. which consisted of antihistamines.05 was considered to be signi®cant.25% b-carotene. Excel (Microsoft. and then to dryness by a stream of nitrogen. retinol. Blood samples were taken after overnight fasting from 7 to 9 am. receiving either lycopene or placebo. Jaeger Compactransfer. nevertheless.5 mg of standard were added to the serum with THF:MeOH.6% tocopherols.Lycopene and asthma the study. PA.6 mm (internal diameter) packed with C-18 reversedphase material with particle size of 5 mm and a pore size of 30 nm (The Separation Group. The system included pumps 501 and 510 and a Waters 996 photodiode array detector attached to Waters Millennium 2010. Ethanol (2. usually within 7 days. inserted with a C-18 mBondapak cartridge (Waters Chromatography. LYC-O-MATO was introduced in the form of soft capsules of 15 mg (6%) lycopene. at a constant ¯ow of 1. Peak responses were measured and assessed at their maximum wavelengths with the photodiode array and detected by the Millennium 3-D ``Max'' absorption. MA.. and inhaled steroids. The lycopene preparation used was LYC-O-MATO. There were 13 males and seven females ranging in age from 10 to 43 years (mean age 23). USA). lutein. an oleoresin product manufactured by LycoRed Natural Products Industries Ltd. Blood was separated for routine blood tests and for vitamin analysis. Cotati. Inc. P<0. and other minor phytochemicals extracted from tomato. Samples were injected with a 7725i syringe-loading sample injector ®tted with a 5-ml loop (Rheodyne. b-apo-8k-carotenal was used as internal standard in all runs. Avondale. Germany. USA). 30).5 ml) was added to 1 ml serum. 1. with a 4-week interval between each protocol to allow washout. Hysperia. we ensured linearity by using three concentrations of the standard. USA). evaporated to 1 ml for spectrophotometric quantitative analysis. Glamorgan. Milford. Peak responses of carotenoids were measured at 450 nm. The standards. CA. and lycopene. USA). The column was maintained at 30t0.2uC in an HPLC Column 7955 Heater/Chillier (Jones Chromatography. CA. The dose of inhaled steroids was constant for several weeks before each challenge. Challenge was not done within several weeks of a respiratory tract infection. administered randomly in a double-blind fashion. Japan) and with a small preguard column. Israel. 0. The upper phase was removed and the water-ethanol phase extracted a second time with 3 ml n-hexane. PA.10. MO. b-cryptoxanthin. We did not note any loss in the quantity of the internal standard along the extraction process. 1% phytoene and phyto¯uene. The mobile phase was ¯ushed with nitrogen to avoid air gassing in the solvents. USA) was used for quanti®cation of the HPLC data. Patients avoided allergen exposure for several weeks before each challenge. All standards were kept at x70uC under nitrogen. and tocopherol serum concentrations were measured on blood samples collected after supplementation of placebo or lycopene. Marlborough. The column was protected by a 5-cm C-18 ODS guard column (Shimadzu. All patients underwent an initial measurement of pulmonary function by Spirograph. in which all of them showed a reduction of at least 15% in their forced expiratory volume in 1 s (FEV1) after a 7-min run on a motorized Quinton treadmill followed by an 8-min rest. The dried residue was dissolved in 100 ml methylene chloride before being injected into the HPLC system. all-trans-b-carotene. HPLC grade methanol:acetonitrile (9:1 by vol). carotenoids.

88 3.12 2.28 3.9 % of predic. signi®cantly increased in the lycopene-supplemented patients.88 3.64 2. 86 105 117 89 138 86 75 82 91 76 80 76 73 113 102 93 90 97 86 73 84. Epidemiologic studies show associations among oxidant exposure. vitamin C. affect airway vasculature. Of the 11 patients that bene®ted from the lycopene.92 3.08 2.20 3. and carotenoids in the patient sera after the supplementation of placebo or lycopene.0 x38 x21 x50 x15 x23 x28 x31 x34 x19 x17 x50 x14 x19 x25 x14 x50 x21 x14 x24 x23 x26.34t0.72 4.37 2.36 1.24 2.12 2.84 4.84 1. Intake of the three antioxidants.84 2.1 MeantSD. 26).09 2.64 4.76 2. If reactive oxygen species participate in the in¯ammatory response in airway disease. tocopherols. the radical scavengers or antioxidants could play a useful role in therapy.64 2.56 3. 69 113 103 94 138 99 76 77 86 93 87 104 62 108 104 97 83 95 84 66 92t18 After exercise Liter 2. who did not report any change.84 3.24 2. and asthma. while the lycopene-supplemented patients had a mean reduction after exercise of x14.02 1. The patients that responded more favorably to lycopene reported a subjective feeling of well-being as opposed to the less responsive patients.16 3. 53 84 58 75 105 62 52 54 74 63 40 62 59 84 88 46 72 83 65 56 67t16 Change % above median level tended to be positively associated with pulmonary function (14±19).7at11. and b-carotene Table 1.72 3.71t0.68 2.5% DFEV1.18t0.56 1.52 3.50 1.92 3.36 3.80 4.76 3.93 2. Lycopene-treated Before exercise Liter 2.76 2. the placebo-supplemented patients had a mean reduction after exercise of x26.00 2.84 3.88 1.2 2. 55 111 72 94 126 77 73 69 78 83 66 99 45 84 81 63 83 88 78 48 79t20 Change % x20 x2 x31 0 x8 x22 x4 x10 x9 x11 x24 x4 x27 x22 x22 x35 0 x8 x7 x27 x14.76 2.96 4.8 After exercise Liter 2. and blood monocytes release higher quantities of reactive oxygen species in asthmatic patients than in healthy subjects (23.7% DFEV1. as a consequence of enhanced reactive oxygen species metabolism of airspace cells.79 % of predic.4 3.48 2. FEV1 values before and after exercise in EIA patients protected by lycopene Placebo-treated Patient age (years) 29 10 33 26 17 17 26 25 12 22 11 39 25 15 25 25 43 25 16 22 23t9 Before exercise Liter 3.32 2.68 3. six were males and four females. vitamin E. Eleven patients out of the 20 (55%) that were supplemented with a total dose of 210 mg of the tomato lycopene in 7 days showed a postexercise reduction of less than 14% in FEV1 (Table 1).68 2.86 % of predic.64 4. The role of reactive oxygen species in airway disease has been largely neglected.08 1. a Signi®cant improvement.24 1.48 3. Results All 20 patients taking placebo revealed a signi®cant postexercise reduction of more than 14% in their FEV1.95 % of predic.08 1.48 2.40 2. in part. but not the other components.88 2. Discussion Several studies have shown a bene®cial association between fruit and vegetable intake and lung function (10±12). Lycopene.88 4. 24). In previous clinical evaluations (7±9).92 3.88 2. These reactive oxygen species produce many of the pathophysiologic changes associated with asthma and may contribute to its pathogenicity (24±26).64 3.19t0. There is evidence that oxidants produced endogenously by overactive in¯ammatory cells can contribute to ongoing asthma (20±22).96 4.24 1.96 3. 1186 . It has been observed that mononuclear phagocytes.Neuman et al. Antigen bronchial challenge produces airspace in¯ammation that may develop. respiratory infections. As a group. Patients with asthma are known to generate increased amounts of reactive oxygen species from peripheral blood cells and from cells recovered from bronchoalveolar lavage.96 2.84 2.23 1. alveolar macrophages.52 1.96 0.64 3. Reactive oxygen species may cause bronchial constriction and mucus secretion.60 1. we have shown that both vitamin C and natural b-carotene may be bene®cial to the exercising asthmatic in the prevention of EIA.64 2.76 2.16 2. and increase airway responsiveness (25. Table 2 summarizes quantitatively the content of retinol.00 1.28 3.00 2.76 3.24 3. Intake of both fruits and vegetables above the median level was positively associated with pulmonary function in three European countries (13).5t12.84 4.88 1.40 2.8t30.

36t0.08t0. a de®ciency of vitamin C was recorded in the majority of bronchial asthma patients during exacerbation of their disease. 34).04t0. Furthermore. and tocopherols in EIA patients supplemented with placebo or lycopene (LYC-O-MATO) Serum variable (mg/l) Retinol Placebo (n=20) Lycopene (n=20) 0. may under certain conditions provide a better antioxidative effect than the use of one type of quencher. Such autonomic imbalance might be involved in the genesis of bronchial hyperreactivity during lung in¯ammation (28).04 MeantSD. and cause microvascular leakage (29. (36) found no signi®cant changes in FEV1 and forced vital capacity after ascorbic acid administration as compared to placebo administration. Indeed. Thus. and therefore prevent the destruction of the lipophilic parts of the cell or the membrane.06t0. such as vitamins C and E or b-carotene (32). Nevertheless. However.08t0. as well as of the plasma malondialdehyde content. for example. Conventional treatment has not yet been able to correct either enhanced lipid peroxidation or weak antioxidant defense (35). the free radical presence is more intensive than in nonsteroid-dependent patients (27). Therapeutic action aimed at increasing antioxidant defense mechanisms is still a clinical challenge. elevate mucus secretion. other different minor constituents common to the fruit. If reactive oxygen species participate in the in¯ammatory response occurring in airways disease. antioxidants should prove bene®cial in therapy (30).04 0. or synergistically with polar antioxidants. Recent studies support the thesis that vitamin C has no bronchodilatory effect. hydrophilic and lipophilic. 1187 . carotenoids. it may prevent the consequent in vivo formation of reactive oxygen species.12t0.02a Lutein 0. In patients with steroid-dependent bronchial asthma. and lycopene.28t0. and also signi®cantly reduced the bronchospasm normally observed 5 min after exercise.20 Lycopene 0. the available evidence is contradictory or inconclusive. have been limited. than did patients who received conventional therapy alone.10t0. The present study shows that even within the scope of lycopene itself.02 0. A combination of different antioxidants.and b-Carotenes 0.23t0. If lipophilic lycopene functions as an ef®cient quencher of singlet oxygen and other free radicals (41). aside from a high concentration of lycopene.08 0.26 0. The latter ®nding concurs with our own results. The reactive oxygen species induce bronchoconstriction. bronchial asthma patients who received antioxidants in addition to conventional therapy were found to exhibit a more pronounced lowering of chemiluminescence in the blood. 30).05 0. and reactive oxygen species may induce an autonomic imbalance between the muscarinic receptor-mediated contraction and the b-adrenergic-mediated relaxation of the pulmonary smooth muscle. more attention should be paid to the origin of the supplement and to the mode of its intake. The amount of oxidative damage depends on the exercise intensity and could be reduced through dietary supplementation with antioxidants. a mixture of stereoisomers of b-carotene. The possibility of synergistic effects and the possible bene®cial potency of the plant nutrients are still unknown and warrant further research.05 a. a Signi®cantly different from placebo (P<0. With regard to the use of vitamin C to alleviate asthma and to enhance athletic activity. slow the formation of more radicals. 40). all of which appear to be very good quenchers of activated forms of singlet oxygen and free radicals (33. which contains. It was concluded that ascorbic acid has no acute bronchodilator effect and does not alter bronchial responsiveness in subjects with asthma. ingestion of 1 g of vitamin C or 30 mg of b-carotene diminished the markers of lipid peroxidation at rest and after exercise. Nutritional supplementation that contains lycopene will provide strong protection against damage by free radicals through the effective antioxidation of lycopene.31t0. but did not prevent the exercise-induced increase in oxidative stress (31). Lycopene was administered to the subjects in soft capsules of LYC-O-MATO. retinol. It is evident that lycopene content increases in the serum of the lycopene-supplemented patients.05).06 0. We used LYC-O-MATO lycopene made from the whole tomato.08 b-Cryptoxanthin 0.18t0. as ingestion of ascorbic acid had no detectable effect on the degree of bronchodilation of the resting asthmatic (36±38). and such research is in its infancy. such as vitamin C. Serum measurements of values for lycopene. It has been suggested that ascorbic acid exerts its effect by altering arachidonic acid metabolism (7). Experimental nutritional and medical studies with natural carotenoids originating from different plant sources speci®cally. Lycopene seems to suppress the attack of cellular and reactive oxygen species. Asthmatic subjects represent a classical case study where the cellular level of free radicals is basically high and increases upon strenuous exercise. pretreatment with ascorbic acid obviated the signi®cant alteration in airway geometry which is induced in asthmatic patients by exercise (39. Malo et al.05 Tocopherols 23t12 25t11 Carotenoids 0.14t0.Lycopene and asthma Table 2.

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