Effects of Phenol-Depleted and Phenol-Rich Diets on Blood Markers of Oxidative Stress, and Urinary Excretion of Quercetin and Kaempferol in Healthy Vo

anggi bitho dalam Journal of the american college of Nutrition

  1. Hwa-Young Kim, MSc,
  2. Ok-Hee Kim, PhD and
  3. Mi-Kyung Sung, PhD

+ Author Affiliations

  1. Department of Food and Nutrition, Sookmyung Women’s University (H.-Y.K., M.-K.S.), Seoul, KOREA
  2. National Institute of Toxicological Research, Korea Food and Drug Administration (O.-H.K.), Seoul, KOREA
  1. Address reprint requests to: Mi-Kyung Sung, Ph.D., Associate Professor, Department of Food and Nutrition, Sookmyung Women’s University, 53-12 Chungpa-dong 2-ka, Yongsan-ku Seoul, 140-742, KOREA. Email: mksung@sookmyung.ac.kr


Objective: Epidemiological studies have suggested beneficial effects of dietary polyphenols in reducing the risk of chronic diseases. This study was performed to investigate the effects of polyphenol-depleted and polyphenol-rich diets on blood oxidative stress markers and urinary excretions of major phenols.

Methods: Nineteen healthy female non-smokers 19 to 21 years of age took part in the study, which consisted of two dietary intervention periods separated by three days. Experimental diets were composed of common foods selected to comply with low contents of polyphenols for phenol-depleted intervention and high contents of polyphenols for phenol-rich diets. Blood and urine samples were collected on day 0, 3 and 6 of each intervention. Duplicate portions of foods provided to the subjects were also collected. Blood oxidative stress markers included plasma antioxidant vitamins, erythrocyte superoxide dismutase (SOD) activity and lymphocyte DNA damage. Urinary excretions of major phenols were measured to affirm bioavailability of dietary phenols.

Results: Plasma α-tocopherol and β-carotene concentrations were slightly decreased on day 3 and 6 of the phenol-depleted dietary intervention period, although no change was observed with phenol-rich diets. The erythrocyte SOD activity was also slightly decreased during phenol-depleted dietary intervention. However, at day 6 of the phenol-rich intervention, the activity of SOD was significantly increased by 41%. Tail moment and tail length of lymphocyte DNA as markers of DNA damage were higher on day 6 of phenol-depleted intervention, although only tail moment showed a statistical significance. The average intakes of quercetin and kaempferol during the phenol-rich intervention were 21 mg/day and 9 mg/day, respectively. The average urinary excretion rates during phenol-rich intervention were 2.06% for quercetin and 0.46% for kaempferol. There were positive correlations between erythrocyte SOD activity and urinary concentration of quercetin or kaempferol.

Conclusions: These results suggest that polyphenol-rich diets may decrease the risk of chronic diseases by reducing oxidative stress.


Polyphenols occur ubiquitously in most foods of plant origin and can be categorized into flavonoids, phenolic acids and tannins. Recently, polyphenolic compounds have attracted much attention as potent antioxidants due to their ability to scavenge free radical and form relatively inert phenoxy radical intermediates [1]. A number of in vitro studies have shown individual food polyphenol possesses antioxidant activity, and the proposed mechanisms by which polyphenols exert their effects were summarized as 1) scavenging free radicals, 2) inhibiting the oxidation of α-tocopherol in LDL, 3) recycling oxidized α-tocopherol and 4) scavenging metal ions [2]. Free radicals, generated in vivo, are responsible for the oxidative damage of biological molecules such as carbohydrate, lipid, protein and DNA. This damage is involved in the pathogenic processes of various chronic diseases including cancer, cardiovascular heart diseases and arthritis [3].

A number of epidemiological studies have suggested that the consumption of polyphenol-rich foods reduces the risk of developing chronic diseases. For example, red wine consumption might prevent cardiovascular heart disease [4,5], while frequent green tea consumption has been suggested to have an association with a lower incidence of cardiovascular diseases and cancer [6]. Similarly, fruit and vegetable consumption prevents different cancers [7].

However, the physiological significance of dietary polyphenols needs to be evaluated more carefully since bioavailability of polyphenols is relatively low [8]. A limited number of human intervention studies have been conducted to investigate the significance of polyphenol-rich foods in reducing oxidative stress [9,10]. Although increases in urinary excretion of polyphenols followed by intervention were observed, the effects of polyphenol intakes on oxidative stress markers were not conclusive.

The objective of this study was to evaluate the effects of polyphenol-depleted and polyphenol-rich diets on blood oxidative stress markers by a randomized dietary controlled intervention trial. Indicators of oxidative stress included erythrocyte SOD activity, plasma α-tocopherol and β-carotene concentrations and lymphocyte DNA damage. To affirm the availability of major dietary polyphenols, dietary intake and urinary excretion of major dietary polyphenols were assessed.



The subjects, 19 healthy, non-smoking females, between 19 and 21 years of age, were recruited through an in-campus advertisement. None of the subjects was taking any medication including oral contraceptives or supplements at the time of the study. The average BMI was 21.5 (range 18.1–28.8) kg/m2. Details of the study were fully explained to the subjects who gave their written consents. The study was approved by the University Research Ethics Committee.

Study Design and Diets

The study had two six-days intervention periods separated by three days (Fig. 1). Subjects were given a list of foods with high polyphenol contents, which they were to avoid for three days prior to the experimental diet. The experimental diet consisted of common foods selected to comply with low total phenol contents for phenol-depleted diets and high total phenol contents for phenol-rich diets. To compose the diet, we selected 30 phenol-rich foods based on previous reports [2,11,12] and analyzed them for their total phenol content. Twelve food items with higher total polyphenol contents (red cabbage, red chicory, onions, red lettuce, mushrooms, apples, grapes, small red beans, kidney beans, sorghum, black rice, wild grape juice) were used to compose parts of phenol-rich diets. Phenol-depleted diets were deprived of any known phenol-rich food and mainly consisted of potatoes, rice, fish, seaweed, low-phenol containing vegetables, low-fat breads, milk and cheese. The calculated average macronutrients contents and selected antioxidants of the phenol-depleted and phenol-rich diets are shown in Table 1. All meals were prepared and consumed at the metabolic kitchen in the department. Subjects were allowed to drink water at any time.

Fig. 1.
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Fig. 1.

Experimental design.

Table 1.

Estimated Daily Intake of Energy, Macronutrients and Selected Antioxidant Nutrients in Phenol-Depleted Diets and Phenol-Rich Diets

Collection of Blood and Urine Samples

Fasting blood samples were taken on day 0, 3, 6 of each intervention period. Four mL of blood samples were used to separate lymphocytes. The rest of the blood sample was centrifuged at 2100 × g for 15 minutes at room temperature, and the plasma was stored at −20°C until analyzed. The washed erythrocytes were resuspended in 1 volume of saline and then stored at −80°C. Twenty-four hour urine samples were collected in 1.5 L bottles containing thymol on day 0, 3, 6 of each intervention period. The total amount of urine was measured, and aliquots were frozen at −20°C until analyzed. Duplicate portions of the foods provided to the participants were collected.

Analysis of Plasma α-Tocopherol

Plasma α-tocopherol was measured by HPLC [13]. In a vial containing 200 μL of plasma sample, tocopherol acetate was added as an internal standard and extracted with hexane twice. The samples were then evaporated under nitrogen and injected into the HPLC. Separation of α-tocopherol was performed on microBondapack C18 column (Waters, Milford, MA). The mobile phase was composed of methanol and water (95:5) and the flow rate was 1.5 mL/minute. Ultraviolet detection was done at 292 nm.

Analysis of Plasma β-Carotene

Plasma β-carotene was measured based on the method of Lee et al. [14]. Briefly, 200 μL of plasma sample was mixed with 200 μL of tocopherol acetate as an internal standard, followed by extraction with 400 μL of butanol/ethyl acetate (1:1). Twenty μL of the sample mixed with sodium sulfate was injected into the HPLC. Separation of β-carotene was performed on HAISIL 100 C18 column (Higgins Analytical, Inc., Mountain View, CA). The mobile phase was composed of methanol/butanol/water (89.5/5/5.5) and the flow rate was 1.5 mL/minute. Ultraviolet detection was done at 450 nm.

Measurement of Erythrocyte SOD Activity

SOD activity was determined based on the method of Flohé et al. [15]. Hemoglobin was removed from the erythrocyte lysate by precipitating with chloroform:ethanol (1:1), and the supernatant was used to measure enzyme activity. Fifty μL of the sample was mixed with 2 mL of phosphate buffer containing 0.5 mM xanthine and 20 μM of cytochrome in an equilibrated 3 ml cuvette. Reaction was started with 50 μL xanthine oxidase (0.2 U/mL), and absorbance changes at 550 nm were recorded using a UV detector.

Lymphocyte DNA Damage Measurement

Four mL of blood was suspended in 4 mL of RPMI 1640 and 4 mL of Histopaque®-1077 (Sigma Chemical Co., St. Louis, MO). The suspension was centrifuged at 700 × g for 30 minutes. The buffy coat layer was collected, washed with RPMI 1640 twice and centrifuged at 500 × g for 5 minutes. Cell pellets were suspended in media (10% DMSO, 40% RPMI, 50% FBS) and stored in a liquid nitrogen tank until analysis. Comet assay was performed based on the method of Singh [16] to measure lymphocyte DNA damage. Damage was assessed as tail length and tail moment (tail length × % migrated DNA) using a fluorescent microscope (Nikon Biophot II) with an image analysis program (Komet 3.1, Kinetic Imaging Ltd., UK).

Analysis of Dietary and Urinary Quercetin and Kaempferol

Dietary quercetin and kaempferol were determined based on the methods of Arai et al. [17]. Briefly, a 0.25 g freeze-dried food sample was extracted with 25 mL of 50% methanol containing 1.2 mol/L HCl and 1.6 g/L tert-butylhydroquinone for 2 hours at 90°C. The extract was diluted to 100 mL with methanol. After centrifugation (1000 × g, 5 minutes, 4°C), a 2 mL aliquot was dried by evaporation under nitrogen gas flow. The residue was dissolved in 100 μL methanol, of which 10 μL was used for HPLC analysis. Mobile phase was acetonitrile/phosphate buffer (pH 2.4, 40/60) and flow rate was 0.6 ml/min. Quantification was made using a UV detector at 370 nm.

Urinary quercetin and kaempferol were determined based on the method of Young et al. [10]. An aliquot of urine samples was centrifuged at 2100 × g for 5 minutes. To 3 mL of supernatant, 1 mL of acetate buffer was added with 100 μg/mL of morin as internal standard, and mixed. Twenty μL of β-glucuronidase and sulfatase (Helix pomatia, 5.5 × 103 U/L and 2.6 × 103 U/L) respectively were added and incubated for 1 hour at 37°C. One mL of ethyl acetate was added and centrifuged at 2100 × g for 15 minutes. The supernatant was subjected to HPLC analysis.

Statistical Analysis

Statistical analysis was performed using SAS package (SAS Institute Inc. 2001). Data was expressed as mean ± standard deviation. Duncan’s multiple range test was used for comparisons. Regression analysis was performed to evaluate the relationship between SOD and urinary concentrations of phenol compounds.


Plasma α-Tocopherol and β-Carotene

As shown in Table 2, plasma concentrations of α-tocopherol and β-carotene were slightly decreased on day 3 and day 6 of the phenol-depleted diet period compared to the baseline concentrations, although no statistical significance was found. Polyphenol-rich dietary intervention did not affect the levels of plasma antioxidants.

Table 2.

Plasma Antioxidant Vitamin Concentrations

Erythrocyte SOD Activity

The erythrocyte SOD activity was slightly decreased after the phenol-depleted diet was introduced (Table 3). However, at day 6 of phenol-rich dietary intervention, erythrocyte SOD activity was significantly increased by 41%.

Table 3.

Erythrocyte SOD Activity

Lymphocyte DNA Damage

Tail moment and tail length of lymphocytes were measured on day 0 and day 6 of both phenol-depleted and phenol-rich diet periods (Table 4). Both tail moment and tail length were higher on day 6 of phenol-depleted dietary intervention compared to those on day 0. However, only tail moment showed a statistical significance. Phenol-rich diet did not affect both damage markers although a slight decrease in tail moment was observed with phenol-rich diets.

Table 4.

Lymphocyte DNA Damage of Study Subjects on Phenol-Depleted and Phenol-Rich Diets

Dietary and Urinary Phenols

Dietary concentrations of quercetin and kaempferol were measured on day 3 and 6 of phenol-depleted dietary intervention and on day 0, 3 and 6 of phenol-rich dietary intervention (Fig. 2). Urinary concentrations of quercetin and kaempferol were measured on day 0, 3 and 6 of each intervention period. The average intakes of quercetin and kaempferol during phenol-rich dietary intervention were 21 mg/day and 9 mg/day, respectively, which were significantly higher than 0.2 mg quercetin/day and 0.7 mg kaempferol/day during phenol-depleted dietary intervention.

Fig. 2.
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Fig. 2.

Dietary and urinary concentrations of quercetin (a) and kaempferol (b) in study subjects.

Urinary excretions of both quercetin and kaempferol during the phenol-depleted diet period were not significantly different from baseline excretion values. Also, kaempferol excretion was not detected in a few samples during phenol-depletion. However, quercetin and kaempferol excretions were increased during phenol-rich dietary intervention compared to the baseline values. Urinary excretion rates (excretion/intake × 100) of unmodified quercetin and kaempferol were 2.06% and 0.46%, respectively, during phenol-rich dietary intervention. As shown in Fig. 3, regression analysis indicates that positive relationships are present between erythrocyte SOD activity and urinary excretion of quercetin (p = 0.057) or kaempferol (p = 0.0005) indicating bioavailable phenols possibly scavenge radicals and spare SOD. However, urinary quercetin possessed a much weaker relationship with erythrocyte SOD activity compared to that of kaempferol. R2 value between erythrocyte SOD activity and the sum of urinary quercetin and urinary kaempferol was 0.075 (p = 0.006).

Fig. 3.
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Fig. 3.

Regression of erythrocyte SOD activity and urinary excretion of quercetin (a) and kaempferol (b).


Dietary phenols have been suggested as potent antioxidants to delay pathogenic processes originating from oxidative damage in vivo. Early studies hypothesized that flavonoids would not enter circulation, but would be cleaved by intestinal bacteria to produce metabolites with no antioxidant activity [18,19]. However, recent studies showed urinary excretion of dietary polyphenols in humans either as free aglycones or as conjugates [20]. Our study used urinary quercetin and kaempferol as biomarkers of dietary phenol absorption, although the urinary content of phenols does not reflect absolute absorptive efficiency because absorbed phenols may be metabolized, stored or excreted through different routes. The average concentrations of urinary phenols were two to three times higher during phenol-rich dietary intervention compared to the baseline values indicating dietary phenols are absorbed and utilized in the body. Although urinary quercetin does not reflect the total quercetin absorbed, Young et al. [10] stated urinary excretion reflects the absorption and, therefore, can be used as a better marker of bioavailability than dietary intake. De Vries et al. [21] also showed urinary flavonols as biomarkers of dietary consumption.

Previous studies indicated the elimination half-life of apple quercetin, onion quercetin and parsley apigenin was 23 hours, 28 hours and 12 hours, respectively [22,9]. Bourne and Rice-Evance [20] also showed urinary excretion of ferulic acid ceased 8 to 10 hours after 360–720 g of tomato consumption. Therefore, we decided to choose six days of phenol-rich intervention, which would be enough to raise and maintain the body pools of phenols. However, unlike with quercetin, urinary excretion of kaempferol was not completely equilibrated in this study, indicating longer intervention may be required to observe definite effects of dietary kaempferol on oxidative stress markers.

Results from the present study showed phenol-depleted diets slightly reduced plasma concentrations of β-carotene and α-tocopherol without statistical significance. In spite of the high content of β-carotene in phenol-rich diets during the intervention period, plasma β-carotene and α-tocopherol concentrations were not affected. Van het Hof et al. [23] showed consumption of the spinach-supplemented meal did not affect plasma levels of β-carotene, while broccoli and green pea consumption induced significant increases in plasma β-carotene levels, indicating the bioavailability of β-carotene is substantially different among different vegetables. The conversion of β-carotene to retinol is another possible explanation for no observable change in β-carotene levels as observed in children fed yellow green vegetables [24]. A carrot supplementation study also failed to increase plasma β-carotene concentrations [25]. Also, the consumption of red wine-derived phenol compounds did not improve blood β-carotene levels, while plasma LDL oxidation was decreased [26]. Similar results were observed when healthy subjects consumed 400 mL/day of red wine [27]. A recent human intervention study failed to increase erythrocyte antioxidant enzyme activities and antioxidant vitamins with β-carotene supplementation [28]. Wang and Russell [29] suggested contradictory epidemiological study results on the effectiveness of β-carotene intake to prevent oxidative stress-induced chronic disease are due to the prooxidant property of β-carotene. Further studies on the metabolism and interactions with other nutrients are required to elucidate their precise physiological roles.

Phenol-rich diets also contained higher levels of vitamin C (84 mg/day) compared to 37 mg/day of vitamin C consumed during phenol-depleted period. Since vitamin C is another potent dietary antioxidant, we cannot rule out its possible confounding effects. Since oxidative stress is an important cause of many chronic diseases, there are suggestions that vitamin C supplementation may lower the risk of chronic diseases. However, clinical trials of vitamin C supplementation with blood oxidative stress markers, including LDL oxidation and lymphocyte DNA damage, showed mixed results [30,31], and Carr and Frei [30] suggested that an intake of 90–100 mg/day vitamin C is required for optimum reduction of chronic disease risk by decreasing oxidative stress. In the present study, high-phenol diets contained 84 mg/day of vitamin C, which may not be enough to reduce oxidative stress. Jacob et al. [32] also showed no difference in lymphocyte oxidative damage between low (5 mg/day) and high (250 mg/day) vitamin C interventions indicating low vitamin C intake did not cause increases in oxidative damage. More studies are required to fully understand the level of dietary vitamin C supplementation to control oxidative stress under different circumstances.

The present study also showed changes in erythrocyte SOD activity. The phenol-depleted diets slightly decreased erythrocyte SOD activity, while phenol-rich diets significantly increased the SOD activity by 41%. Since polyphenols effectively remove oxygen radicals, diminished enzyme degradation partly explains increased SOD activity during phenol-rich intervention [28]. Similar results were observed with parsley and fruit juice intakes [9,10], while lowered intake of fruits and vegetables may lead to an overall decreasing trend in antioxidant enzymes [9]. Since β-carotene supplementation studies did not show changes in enzyme activities [28], phenols may be a good candidate to reduce radical attack by increasing antioxidant enzyme activities.

Results from this study also indicate that phenol-rich diets reduce oxidative DNA damage, although the effects are not large. At present, a number of in vitro studies have confirmed the antioxidant effects of different polyphenols. Only recently, Boyle et al. [33] showed the decreased DNA damage after 100 g of fried onion consumption in human subjects. Also, three weeks of brussels sprouts consumption reduced the level of oxidized guanosine [34]. The phenolic hydroxyl group is shown to donate electrons to oxygen radicals and also to reduce ferrous ion to ferric ion, thereby suppressing oxidation [35].

The present study indicates phenol-rich diets decrease oxidative stress as assessed by different blood markers. Since phenol-rich diets also contain high levels of antioxidant vitamins, we cannot exclude the possible role of the combination of phenolic and vitamin antioxidants in the observed decrease in oxidative stress. However, positive relationships between urinary excretion of phenols and plasma radical scavenging SOD activity indicates phenols may act as radical scavengers.


This work was supported by Korean Ministry of Health and Welfare Grant HMP-00-B 22000-0155.

  • Received June 14, 2002.
  • Accepted October 7, 2002.



oleh anggibitho dalam cyberforum.com

Contoh antioksidan yaitu vitamin E, vitamin C, kelompok karetonoid (beta karoten, likopen, dan lutein), serta kelompok flavonoid. Sedangkan contoh mineral antioksidan yaitu selenium dan seng.

Secara alami, antioksidan dapat diperoleh dari sayur dan buah yang kita konsumsi setiap hari. Namun, bagaimana mekanisme kerjanya?

Vitamin antioksidan yang cukup terkenal adalah vitamin C dan E. Vitamin C mencegah oksidasi pada molekul yang berbasis cairan, misalnya plasma darah dan mata. Sedangkan vitamin E yang larut dalam lemak bekerja pada sel lipid dan sirkulasi kolesterol.

Sebuah studi yang diterbitkan dalam New England Journal of Medicine menunjukkan bahwa vitamin E dapat memperlambat gejala Azheimer. Journal of the American Medical Association juga menyatakan bahwa vitamin E dapat mencegah penyakit jantung koroner. Sedangkan menurut Journal Ophtalmology, vitamin E dapat menurunkan risiko terjadinya katarak.

Cara kerja vitamin E sebagai antioksidan adalah dengan menyumbangkan elektron kepada radikal bebas. Karena itu, vitamin E yang kaku akan berubah menjadi vitamin E yang radikal. Untuk menjinakkannya, diperlukan vitamin C yang akhirnya akan membuat vitamin C juga menjadi radikal. Di sinilah, glutation akan muncul untuk menetralkan vitamin C.

Jika vitamin C dan E bertindak sebagai antioksidan langsung, mineral sendiri akan berperan sebagai komponen antioksidan tubuh (endogen). Selenium, misalnya, merupakan komponen penting glutation peroksidase. Selenium juga bekerja secara sinergis dengan vitamin E. Sebuah studi yang diterbitkan dalam jurnal The Lancet menyatakan bahwa mereka yang kekurangan selenium akan lebih berisiko menderita kanker dibandingkan mereka yang berkecukupan selenium.

Seng (Zn) juga merupakan mineral antioksidan yang cukup penting. Seng akan membantu mencegah oksidasi lemak dan diperlukan oleh tubuh untuk memproduksi antioksidan superoksida dismutase. Keberadaan seng dibutuhkan juga untuk menjaga kadar vitamin E dalam darah sehingga membran sel darah merah dapat terlindungi dari efek oksidasi mineral lainnya.

Flavonoid dan karotenoid
Zat antioksidan dalam tumbuhan dibedakan menjadi flavonoid yang larut dalam air dan karotenoid yang larut dalam lemak. Flavonoid mampu memperbaiki ketidakseimbangan sistem antioksidan dalam tubuh. Diketahui ada lebih dari 4.000 jenis flavonoid, seperti epigalokatekin dalam teh hijau, isoflavon dalam kedelai, dan lain-lain.

Contoh karotenoid yaitu beta karoten, alfa karoten, likopen, dan lutein. Ada sekitar 700 karetonoid di alam dan sekitar 50 jenisnya dapat diserap oleh tubuh. Beberapa karotenoid dapat berperan sebagai pembentuk (prekursor) vitamin A dan mampu memerangi radikal bebas.

Kombinasi antioksidan
Antioksidan bekerja sebagai sebuah sistem untuk menghentikan kerusakan akibat radikal bebas. Oleh karena itu, para ahli nutrisi menyarankan agar kita sering mengonsumsi produk yang mengandung banyak variasi antioksidan, kombinasi vitamin, mineral, dan zat berkhasiat lainnya.

Meskipun diketahui bersifat baik, antioksidan yang berlebihan juga dapat berbahaya bagi tubuh. Vitamin C yang berlebihan akan berpotensi menjadi vitamin C radikal yang bersifat radikal bebas, sehingga glutation tidak cukup untuk menetralkannya. Selain itu, kelebihan vitamin C (sintetis) akan membuat ginjal bekerja semakin keras.

Begitu juga dengan vitamin E. Sebuah teori menyatakan bahwa kelebihan vitamin E dapat mengganggu proses pembekuan darah. Selain itu, vitamin E juga dapat terakumulasi dalam jaringan tubuh yang mangandung lemak (misalnya organ hati) dan berpotensi dapat meracuninya.
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oleh: anggi bitho lokmanto dalam Dr. Albert GO Sumampow dalam MedikaHolistik.com

Akhir-akhir ini di dalam majalah, surat kabar bahkan menonton iklan di televisi, maupun seminar-seminar ilmiah banyak dibahas mengenai radikal bebas dan antioksidan. Apa sebenarnya radikal bebas dan antioksidan itu, bagaimana bekerjanya sehingga bisa merusak sel-sel tubuh dan apakah ada manfaatnya terhadap kesehatan kita? merupakan fenomena yang relatif baru. Hingga permulaan abad ke 20, tidak seorangpun mengetahui bahwa radikal bebas dapat berwujud dan bekerja secara bebas. Pemahaman ilmiah kita tentang hubungan radikal bebas dengan antioksidan baru muncul pada tiga hingga empat dekade terakhir ini.

Pengetahuan baru ini, kini banyak diterapkan oleh para dokter di ruang praktek dan klinik-klinik di seluruh negeri tetapi sebagian besar hasil penelitian yang ada dalam pustaka ilmiah itu sesungguhnya masih belum diterapkan secara baik bagi pasien.

Jadi agar dapat mendiskusikan berbagai masalah penting tentang masalah penyakit dan kesehatan dikaitkan dengan radikal bebas dan antioksidan ini maka ada perlunya kita mengetahui perkembangan paling mutakhir dalam bidang kesehatan. Hanya dengan informasi itu, kita dapat berharap mengerti semua informasi tentang apa saja yang dapat dilakukan radikal bebas terhadap kita dan apa saja yang dapat dilakukan antioksidan untuk kita.

Para pakar kimia di abad ke 19 semula menggunakan istilah radikal bebas untuk suatu kelompok atom yang membentuk suatu molekul. Pada saat itu, para ilmuwan tidak percaya bahwa radikal bebas dapat berada dalam keadaan bebas.

Terjadi perubahan drastis pada abad ke 20 dari hasil kerja seorang rusia bernama Moses Gomberg yang lahir di Blisavetgrad pada tahun 1866 dan hijrah bersama keluarga ke Amerika serikat pada usia 18 tahun yang mendapat gelar Doktor di universitas Micihigan pada 1894., dia membuat radikal bebas organik pertama dari trifenilmetan suatu senyawa hidrokarbon yang digunakan sebagai bahan dasar berbagai zat pewarna. Sebagai hasil dari penelitian Gomberg dan ilmuwan lain pada tahun-tahun pertama abad ke 20 istilah radikal bebas kemudian diartikan sebagai molekul yang relatif tidak stabil yang mempunyai satu atau lebih elektron yang tidak berpasangan di orbit luarnya. Oleh karena elektron yang tidak berpasangan itu mengitari orbit mereka. Di dalam molekul mereka membentuk semacam efek magnet yang menyebabkan radikal bebas berikatan dengan molekul-molekul di dekatnya.

Banyak radikal bebas sangat tidak stabil sehingga keberadaan mereka hanya sesaat , selama hidup mereka yang sangat singkat itu radikal bebas bertindak seperti katalis yang menjembatani reaksi kimia dan berubah bentuknya dalam molekul lain.

Sebenarnya radikal bebas ini penting artinya bagi kesehatan dan fungsi tubuh yang normal dalam memerangi peradangan, membunuh bakteri, dan mengendalikan tonus otot polos pembuluh darah dan organ-organ dalam tubuh kita. Kunci kerjanya radikal bebas yang aman dan efektif dalam tubuh kita bila tidak dalam jumlah yang berlebihan atau dalam keadaan seimbang, akan tetapi masalahnya adalah mekanisme keseimbangan tubuh kita yang sangat rapuh ini sering sekali keluar jalur sehingga menimbulkan penyakit.

Saat tubuh kita dipenuhi radikal bebas yang berlebihan maka molekul yang tidak stabil yang berada didalam tubuh kita berubah bentuk menjadi molekul pemangsa. Mereka mulai bergerak liar dan menyerang bagian tubuh yang sehat maupun yang tidak sehat sehingga terjadi penyakit.

Berbagai penyakit yang telah diteliti dan diduga kuat berkaitan dengan aktivitas radikal bebas. Penyakit-penyakit tersebut mencakup lebih dari 50 kelainan seperti Stroke, Asma, Pankreatitis, Berbagai penyakit radang usus, Penyumbatan kronis pembuluh darah di jantung, Penyakit parkinson, Sel Sickle Leukemia, Artritis rematoid, Perdarahan otak & tekanan darah tinggi, bahkan AIDS.

Untuk memperbaiki keadaan ini tubuh kita membentuk pembasmi radikal bebas yang dikenal sebagai antioksidan endogen. Antioksidan endogen ini akan menetralisir radikal bebas yang berlebihan itu sehingga tidak merusak tubuh. Antioksidan endogen ini dikemukakan oleh ilmuwan Amerika pada tahun1968 oleh J.M. Mc Cord dan I. Fridovich yang menemukan enzim antioksidan alami dalam tubuh manusia yaitu Superoksid dismutase yang saat ini disingkat SOD.. Hanya dalam waktu singkat setelah teori tersebut disampaikan, selanjutnya ditemukan enzim-enzim antioksidan alami lainnya seperti Glutation peroksidase , Katalase yang siap menetralisir radikal bebas yang berlebihan agar tetap seimbang. Saat ini enzim-enzim antioksidan alami ini sudah dapat diperiksa kadarnya dalam tubuh di laboratorium.

Sedangkan antioksidan yang kita makan dari luar melalui makanan atau melalui food suplemen untuk membantu tubuh melawan kelebihan radikal bebas, kita sebut antioksidan eksogen.

Menurut Phyllis A Balch, Cnc & James F. Balch, MD dalam bukunya Prescription for Nutritional Healing maka yang dapat dimasukkan dalam antioksidan eksogen ini adalah : Alpha lipoic acid ( ALA ), Bilberry ( Vaccinium myrtillus ), Burdock ( Artium lappa ), Carotenoids, Coenzyme Q 10, Curcumin ( Tumeric ), Flavonoids, Garlic, Ginkgo biloba, Glutathione, Grape seed extract, Green tea, Melantonin, Mettthionine, N-Acetylcysteine( NAC ), Nicotinamide Adenine dinucleotide ( NADH ), Oligomeric Proanthocyanidins ( OPCs ), Pycnogenol, Selenium, Silymarin, Vitamin A, Vitamin C, Vitamin E, Seng.

Sayangnya sistem perlindungan dari dalam maupun dari luar tubuh sering tidak memadai karena terlalu banyaknya radikal bebas yang terbentuk seperti polusi udara, asap rokok, sinar ultra violet yang diproduksi sinar matahari, pestisida dan pencemaran lain di dalam makanan kita , bahkan karena olah raga yang berlebihan.

Tampaknya kemanapun kita bergerak berbagai senyawa dan keadaan tertentu senantiasa membayangi kita dengan berbagai radikal bebas akibat ulah kita sendiri.

Ada 4 langkah yang dapat dilakukan menurut Dr. Kenneth H. Cooper yang menjadi pencetus Preventive medicine untuk melawan radikal bebas yang berbahaya dalam tubuh kita yaitu :

1. Berolah raga dengan intensitas rendah
2. Mengkombinasi beberapa antioksidan setiap hari
3. Mengatur diet dan memasak secara benar agar antioksidan dalam makanan tidak rusak
4. Bergaya hidup bebas dari radikal bebas

Berikut merupakan ulasan dan saran-saran bagaimana cara memahami 4 langkah tersebut diatas.

Langkah 1: Lakukan Olah Raga Dengan Intensitas Rendah

Pada keadaan normal radikal bebas terbentuk secara amat perlahan kemudian dinetralisir oleh antioksidan yang ada dalam tubuh. Namun jika laju pembentukan radikal bebas sangat meningkat karena terpicu oleh latihan yang terlalu keras atau berolahraga secara berlebihan sehingga jumlah radikal bebas akan terbentuk melebihi kemampuan sistem pertahanan tubuh, maka molekul pemberontak tambahan yang tidak dapat dicegah ini lalu menyerang membran sel , sehingga terjadi kerusakan pada sel-sel tubuh kita yang mengakibatkan timbulnya penyakit . Sebaliknya dengan meningkatkan ketahanan tubuh kita secara bertahap melalui program latihan olah raga dengan intensitas rendah yang disarankan seperti jalan cepat, jogging, berenang, dan bersepeda statis ini, dapat meningkatkan enzim antioksidan endogen seperti enzim superoksid dismutase, glutation peroksidase dan katalase untuk mencegah kerja setiap radikal bebas yang merusak.

Ada beberapa pedoman dasar yang dapat kita pergunakan untuk merencanakan program latihan olahraga dengan intensitas rendah ini yaitu berolah raga dengan frekwensi 3 – 5 kali dalam satu minggu dan lamanya kita berolah raga 45 – 60 menit sampai tercapai target denyut nadi yang dapat dihitung dengan rumus yang terdapat dibawah ini :

Angka batas denyut nadi maksimal = 220 – Usia x 0,70
Contoh : Pria berusia 40 tahun, perkiraan laju denjut jantung maksimum adalah 126 detak jantung permenit didapat dari 220 - 40 x 0,70 .
Bila kita dalam berolah raga belum dapat mencapai nadi yang telah ditentukan itu maka olah raga kita belum benar.
Dengan menerapkan pedoman ini maka kita dapat memantau intensitas olah raga kita sehingga tidak melakukan olah raga yang berlebihan.

Langkah 2 : Gunakan Kombinasi Beberapa Antioksidan Setiap Hari

Seperti kita ketahui campuran antioksidan ada beraneka ragam bergantung pada usia, jenis kelamin, dan tingkat kegiatan , serta bobot badan kita.

Banyak pandangan sangat meyakini bahwa kebutuhan semua vitamin dan mineral dapat kita peroleh dari makanan yang kita makan melalui menu harian kita, ternyata tidak semudah itu.

Untuk memperoleh vitamin E dengan dosis 100 IU dimana jumlah dosis itu lebih kecil dari dosis optimum harian rata-rata yang disarankan oleh para ahli nutrisi, kita harus makan dua mangkuk kemiri, atau semangkuk biji bunga matahari dan bila kita memakannya maka pemasukan lemak dan kalori akan luar biasa banyaknya.

Untuk memperoleh 1000 mg vitamin C diperlukan mengkonsumsi 15 buah jeruk, atau 25 buah cabe hijau, atau untuk memperoleh 25.000 – 50.000 IU beta karoten diperlukan makan paling sedikit dua sampai tiga batang wortel atau tiga mangkuk butternut squash. Bila kita melihat contoh diatas maka jalan terbaik untuk dapat mencukupi vitamin atau mineral adalah menyusun dan mengkonsumsi beberapa suplemen yang disesuaikan dengan kebutuhan kita sendiri.

Pengunaan suplemen makanan ini tentunya tergantung dari pada usia, jenis kelamin, tingkat kegiatan, bobot badan serta penyakit yang sedang diderita oleh kita.

Untuk mengetahui jenis apa saja yang dapat dikonsumsi tentunya harus konsultasi dengan dokter atau ahli nutrisi anda.

Langkah 3 : Cara Memasak dan Cara Diet Agar Antioksidan Dalam Makanan Tidak Rusak. Sekalipun kita mengetahui suatu makanan mengandung banyak antioksidan, ini tidak berarti bahwa jika kita memakannya akan memperoleh seluruh keuntungan yang terdapat di dalam makanan tersebut. Nilai gizi makanan dapat hilang banyak selama pegemasan, penyimpanan, pemasakan, atau penyiapan lain .

Sebagai paduan didalam menyiapkan makanan ingatlah hal-hal berikut ini :

. Perubahan nilai PH nya , keasaman, atau kebasaannya makanan dapat terjadi selama proses pembuataannya.
penambahan zat tambahan misalnya vetsin, dll.
. Metode masak terbaik untuk mempertahankan kandungan antioksidan adalah : Microwave, Uap, Tumis.
. Hindari bahan-bahan yang sudah layu dalam mengolah makanan.
. Hindari pemotongan, perajangan, pengirisan, pembilasan, atau perendaman yang berlebihan.
. Cobalah mengkonsumsi air yang kita gunakan dalam merebus bahan makanan mungkin antioksidan ada didalamnya.
. Jangan menyimpan di kulkas makanan yang telah dimasak lebih dari satu hari tanpa mengunakan wadah yang kedap udara.
. Jangan menghangatkan kembali makanan nabati yang telah dimasak satu kali.
. Hindari mempertahankan kehangatan makanan selama lebih dari 30 menit sebelum dihidangkan.
. Jangan menyimpan bahan makanan segar dalam lemari es lebih dari 1 minggu

Langkah 3: Gaya Hidup Bebas Dari Radikal Bebas.

Tidak ada jalan untuk mundur atau melarikan diri ke suatu lingkungan yang betul-betul bebas dari gangguan radikal bebas. Dengan hidup di tengah masyarakat modern kita akan terpapar oleh berbagai pemicu dari lingkungan yang memacu pembentukan molekul radikal bebas yang bisa merusak dalam tubuh kita. Kendati demikian kita dapat meminimalisasi ancaman radikal bebas terhadap kesehatan kita dan membuat hidup kita lebih panjang serta menjadi lebih produktif secara maksimal. Seperti kita ketahui, olah raga yang tidak berlebihan, mengkonsumsi suplemen antioksidan, dan tata menu makanan yang benar dapat meningkatkan daya tahan tubuh terhadap radikal bebas secara bemakna. Akan tetapi untuk memperoleh pertahanan yang betul-betul sempurna perlu juga dilakukan tindakan pencegahan yang memungkinkan hadirnya radikal bebas dalam diri kita. Ini berarti bahwa kita harus belajar mengenali dan mengurangi atau bahkan menghilangkan faktor-faktor yang dapat terus-menerus memacu pembentukan radikal bebas dalam tubuh kita.

Tahap terakhir ini merupakan tahap yang tersulit karena beberapa hal yaitu :

. Berhadapan dengan kebiasaan-kebiasaan pribadi yang sudah berakar kuat misalnya merokok.
. mengatasi berbagai hambatan yang tampaknya sulit teratasi misalnya pencemaran udara di tempat kita hidup atau bekerja.
Kenyataan tersebut diatas jangan menyurutkan semangat kita untuk dapat menghindari dari semua radikal bebas yang berlebihan yang dapat mempengaruhi kesehatan tubuh kita. Memang suatu pekerjaan yang sulit untuk menghilangkan sama sekali radikal bebas yang sangat banyak ini, tetapi paling tidak dengan mengetahui radikal bebas tersebut, dapat kita meminimalkan terpaparnya sehingga rencana dapat kita buat untuk pertahanan seumur hidup terhadap ancaman molekul-molekul pemberontak itu
Sekarang kita telah memiliki 4 pelindung utama untuk mencegah kerusakan akibat radikal bebas yaitu olah raga dengan intensitas rendah, suplemen antioksidan, tatanan menu dengan jumlah antioksidan maksimal, kemudian perlindungan paling akhir bagi kita adalah gaya hidup yang kita pilih sehari-hari untuk menghindari paparan berlebihan berbagai radikal bebas yang mengancam tubuh kita . Langkah tersebut diatas kita sebut revolusi antioksidan jika dipertimbangkan dari berbagai aspek sesungguhnya merupakan suatu cara pendekatan yang menyeluruh dan betul-betul merupakan perubahan baru untuk memperoleh kesehatan dan umur panjang.

Kepustakaan :

- Sehat Tanpa Obat, By Dr Kenneth H. Cooper
- Prescription for Nutritional Healing By Phyllis A. Balch, Cnc. James F. Balch, MD.

Dr.Albert GO Sumampouw / www.medikaholistik.com/ 180102


Pasteurisation of Soursop (Annona Muricata L.) Puree and its Effects on Physico-Chemical Properties


A study was carried out to process soursop puree. The study includes, the establishment of optimum conditions of temperature and time for pasteurisation; shelf life study using different packaging and storage temperature combinations; characterisation and ultrastructural identification of puree and juice cloud at two processing and storage conditions; and characterisation of pectin-protein particulate in the juice cloud. Physico-chemical evaluation of freshly extracted soursop pulp showed high pectin esterase (PE) actMty (32.1 unit/g) and vitamin C content (21 mg/100g). The pH was low (3.7) and the acidity was high (1.02%). These properties were considered advantageous for pasteurisation. A Response surface methodology was used to determine optimum pasteurisation conditions for inactivation of PE with maximum ascorbic acid retention. The results showed that the optimum pasteurisation condition was at 79°C for 69 sec, with predicted nil PE activity and ascorbic acid content of 5.88 mg/100g. The storage stability of the puree was evaluated for 12 weeks and the parameters examined were microbial count (total plate count, yeast & mould, and E. coli), PE activity, cloud stability, colour, viscosity, pH, titratable acidity, °Brix, ascorbic acid and sugar content, as well as sensory properties. The packaging materials used were laminated aluminium foil, general purpose lacquered can and polypropylene bottles and the samples were stored at ambient temperature (28-37°C), 15°C, 4°C, and -20°C. It was observed that natural soursop puree pasteurised at the established optimum conditions of 79°C for 69 sec completely inactivated the PE and stabilised the cloud in juice without affecting nutrients and sensory quality. Samples packed in laminated aluminium foils and stored at 4°C was most stable during the storage period of 12 weeks. It showed decreased loss in cloud, viscosity, colour, nutrient, and lowest microbial growth. Effect of processing and storage studies showed that the puree prepared by maceration process was low in pulp sediment and high in cloud content. Viscosity, °Brix, pulp volume and cloud stability were affected by freezing damage of cloud particles. Scanning electron micrograph of fresh and pasteurised juice cloud showed a continuous matrix of protein filaments. On the other hand, similar observation made on frozen juice cloud showed shrinked protein filaments and collapsed network. This suggest that there was a loss in consistency and cloud due to freezing. The cloud of single strength soursop juice was white, fine, cottony textured and Constituted about 0.103% of the dry solid. The cloud composed of 35.5% protein, 22.5% carbohydrate, 14.3% lipid and 0.64% polyphenol content and a density of 1.08 g/ml. Transmission electron micrograph of stable juice revealed that the cloud particles ranged in size from 0.13µm - 3.0µm and showed an obvious close association of protein-lipid and protein-pectin. Flocculates of insoluble pectates and aggregated particles formed by enzymatic action were evident in unpasteurised juice cloud. Soursop juice cloud contained an average of 10.36% total pectin of which 66.3% soluble pectin, 11% inherently insoluble pectin and 24.4% protopectin. From the 35.5% total cloud protein, 48.7% was inherently insoluble protein, 38.6% complexed with other polymeric constituent and 13% complexed with low molecular weight cloud constituents.

di sunting oleh anggibitho food science and technology UNS dalam Ara, Umme (2000) Pasteurisation of Soursop (Annona Muricata L.) Puree and its Effects on Physico-Chemical Properties. PhD thesis, Universiti Putra Malaysia.