Guohua Cao, Robert M. Russell, Neal Lischner, Ronald L. Prior, Serum Antioxidant Capability Is Elevated by Consumption of Strawberries, Spinach, Pink Wine or Vitamin C in Aged Girls, The Journal of Diet, Quantity 128, Concern 12, December 1998, Pages 2383–2390, https://doi.org/10.1093/jn/128.12.2383
Summary
Appreciable epidemiological proof suggests an affiliation between consumption of diets wealthy in vegatables and fruits and a decreased danger of heart problems, hypertension, and sure types of most cancers (Ames et al 1993, Ascherio et al 1992; Doll 1990, Gillman et al 1995, Kohlmeier et al 1995, Rimm et al 1996, Steinmetz et al 1996, Willet 1994). It’s not identified for sure which energetic dietary constituents contribute to those protecting results, however it’s usually assumed that antioxidant vitamins contribute to this protection. Nevertheless, outcomes from intervention trials on the protecting impact of the supplementation with antioxidants akin to β-carotene and vitamin E are usually not conclusive (Hennekens et al 1996, Omenn et al 1996, Rapola et al 1996, Stephens et al 1996). It was lately reported that two months of supplementation with α-tocopherol acetate (100 mg, twice day by day), ascorbic acid (250 mg, twice day by day), α-tocopherol acetate (100 mg, twice day by day) plus ascorbic acid (250 mg, twice day by day) or coenzyme Q10 (30 mg, 3 times day by day) in human topics didn’t lead to vital adjustments within the urinary excretion fee of 8-oxo-7,8-dihydro-2′-deoxyguanosine, a restore product of oxidative DNA injury (Priemé et al 1997); whereas intervention with Brussels sprouts (300 g day by day) for 3 wk resulted in a big 28% lower within the 8-oxo-7,8-dihydro-2′-deoxyguanosine excretion fee (Verhagen et al 1995). Due to this fact, the helpful impact of a excessive consumption of vegatables and fruits on the danger of heart problems and most cancers might rely not on the impact of the properly characterised antioxidants, akin to nutritional vitamins E and C and β-carotene, however somewhat on another antioxidants or nonantioxidant phytochemicals or a concerted motion of various compounds current in these meals.
We (Cao et al. 1996, Wang et al. 1996) have discovered that, usually, greater than 80% of the full antioxidant capability in vegatables and fruits comes from elements apart from vitamin C, indicating the presence of different probably necessary antioxidants in these meals. In these research, whole antioxidant capability was quantitated by utilizing the oxygen radical absorbance capability (ORAC4) assay (Cao et al. 1993 and 1995). ORAC varies significantly (20–30-fold) from one type of fruit or vegetable to a different. Brussel sprouts are one of many greens that present excessive ORAC exercise. Garlic, kale and spinach are notably excessive, as are strawberries and plums (Cao et al. 1996, Wang et al. 1996).
Flavonoids and different phenolic compounds look like antioxidants that contribute to the excessive antioxidant capability noticed in sure vegatables and fruits (Guo et al 1997). There are a number of thousand totally different flavonoids current in crops, and plenty of of them have antioxidant actions (Cotelle et al. 1996, Hanasaki et al. 1994, Salah et al. 1995, van Acker et al. 1996). The antioxidant capacities, measured as ORAC, of some flavonoids had been discovered to be a number of occasions stronger on the premise of molar focus than nutritional vitamins E and C (Cao et al. 1997, Wang et al. 1997). Such phenolic compounds have already been implicated as enjoying a task within the safety that vegatables and fruits have in opposition to persistent ailments (Hertog et al. 1993 and 1995, Keli et al 1996, Knekt et al 1996). Nevertheless, the extent to which these probably necessary antioxidants might be absorbed isn’t clear, though early proof signifies that substantial portions of the flavonoids are absorbed (Hollman et al 1995 and 1996). For instance, absorption of quercetin (a standard flavonoid) outlined as oral consumption minus ileostomy excretion and corrected for the degradation throughout the ileostomy bag was 52 ± 15% for quercetin glucosides from onions (Hollman et al, 1995). It’s vital to offer proof {that a} weight loss plan containing excessive antioxidant exercise from vegatables and fruits can both enhance the general antioxidant capability or change the relative balances between particular person antioxidant parts within the human physique; in any other case, any antioxidant hypotheses associated to the safety from vegatables and fruits in opposition to ailments wouldn’t be sustained. The goals of this examine had been to find out if antioxidant compounds apart from vitamin C are absorbed from spinach, strawberries and crimson wine in enough quantities to alter the full antioxidant capability of human serum. Vitamin C was used to show the impact of a single antioxidant on the full antioxidant capability.
SUBJECTS AND METHODS
Topics.
Eight wholesome feminine topics (age: 66.9 ± 0.6; physique mass index: 26.1 ± 0.7 kg/m2; imply ± sem) had been recruited to take part on this examine (one topic didn’t end all experiments). All examine members had been in good well being as decided by a medical historical past questionnaire, bodily examination and regular outcomes of medical laboratory exams. All the topics fulfilled the next eligibility standards: 1) no historical past of cardiovascular, hepatic, gastrointestinal, or renal illness; 2) no alcoholism; 3) no antibiotic or supplemental vitamin and/or mineral use for at the very least 4 wk earlier than the beginning of the examine and 4) no smoking. The examine protocol was authorized by the Human Investigation Evaluate Committee of Tufts College and the New England Medical Middle, and written knowledgeable consent was obtained from every examine participant.
Examine Design.
Every topic was admitted to the Metabolic Analysis Unit on the Jean Mayer U.S. Division of Agriculture Human Diet Analysis Middle on Ageing at Tufts College within the night and fasted in a single day. Within the morning, an intravenous catheter was inserted into one forearm. A ten mL blood pattern (zero baseline pattern) was obtained from fasting topics, following which they got a glass of breakfast beverage (coconut drink, Desk 1) containing one of many following: 1) strawberries (240 g); 2) ascorbic acid (1250 mg); 3) spinach (uncooked, 294 g); 4) crimson wine (300 mL) (the crimson wine was lyophilized to take away alcohol); or 5) management (coconut drink solely). The strawberry, ascorbic acid, spinach and crimson wine therapies had been all formulated to offer an antioxidant capability equal to three.7 mmol Trolox (a water soluble vitamin E analogue) equal within the ORAC assay. Blood samples (10 mL) had been collected once more at 0.25, 0.5, 1, 2 and 4 h after the consumption of the beverage. Following the blood sampling at 4 h, a lunch meal was given (Desk 1). Further blood samples had been obtained at 7, 9 and 11 h following the breakfast drink. A snack was given after the 7 h blood sampling and a dinner was given instantly after the 11 h blood sampling (Desk 1) . The breakfast, lunch and dinner had been designed to be low in meals containing antioxidant exercise (no substantial quantities of vitamin C, α-tocopherol, carotenoids, and flavonoids), however present the Advisable Dietary Allowance for protein and vitality (Desk 1). Every topic acquired every of the 5 therapies (drinks) on a separate day 2 wk aside throughout a interval of 10 wk. Remedies had been assigned to every topic in a random sequence. 4 topics accomplished a therapy every time with 4 of the 5 therapies represented at every time.
After the breakfast beverage, over a 24-h take a look at interval, urine was collected into two separate pooled collections. Assortment 1 was from baseline to 4 h; Assortment 2 was from 4 to 24 h. Pooled specimens had been collected into urine jugs containing HCl as an additive. Volumes had been recorded and 4 separate aliquots had been saved at -80°C.
Antioxidant Capability Analyses.
Three totally different strategies had been used for the antioxidant capability analyses: 1) ORAC assay, 2) Trolox equal antioxidant capability (TEAC) assay and three) ferric lowering capacity (FRAP) assay.
The automated ORAC assay was carried out on a COBAS FARA II spectrofluorometric analyzer (Roche Diagnostic System Inc., Branchburg, NJ) with fluorescent filters (Ex: 540 nm; Em: 565 nm) as beforehand described (Cao et al, 1995). Briefly, within the remaining assay combination (0.4 mL whole quantity), R-phycoerythrin (16.7 nmol/L) (Sigma, St. Louis, MO) was used as a goal of free radical assault, with 2,2′-azobis(2-amidinopropane) dihydrochloride (4 mmol/L) (Wako Chemical substances USA Inc., Richmond, VA) as a peroxyl radical generator. Trolox (Aldrich, Milwaukee, WI) was used as a management normal. Last outcomes had been calculated utilizing the variations of areas beneath the R-phycoerythrin decay curves between the clean and a pattern. The ORAC assay has been utilized by totally different laboratories and supplied necessary info concerning the antioxidant capability of varied organic samples from pure compounds akin to melatonin, dopamine and varied flavonoids, to advanced matrices akin to tea, fruits, greens, herbs and animal tissues (Cao and Prior 1998). Each serum and serum nonprotein fractions extracted with perchloric acid (PCA) and acetone had been used within the ORAC assay. For preparation of the serum nonprotein fraction, serum was diluted with 0.5 mol/L PCA (1:1, v/v) or pure acetone (1:8, v/v). The samples had been then centrifuged at 100,000 x g for 10 min at 4°C, and the supernatants had been eliminated because the serum nonprotein fractions and appropriately diluted for the ORAC assay.
TEAC was measured in serum utilizing the strategy of Miller et al. (1993) with commercially accessible kits (Complete Antioxidant standing; Lot 21440, Randox Laboratories, Lakewood, CA). The TEAC assay relies on the inhibition by antioxidants of the absorbance of the novel cation of two,2′-azinobis(3-ethylbenzothiazoline 6-sulfonate).
FRAP was decided in serum by the strategy of Benzie and Pressure (1996), which measures the ferric to ferrous iron discount within the presence of antioxidants. The two,4,6-Tripyridyl-s-triazine used on this assay was from Sigma (St. Louis, MO).
The ultimate outcomes of ORAC, TEAC and FRAP assays had been all expressed utilizing μmol Trolox equivalents per L. The relative exercise of Trolox within the FRAP assay was 2.0 (Benzie and Pressure 1996).
Dedication of Glucose, Protein, Urate, Bilirubin and Vitamin C.
Glucose, protein, urate and bilirubin had been measured in serum utilizing a COBAS MIRA spectrophotometric analyzer with reagent kits bought from Roche Diagnostic Techniques, Inc. (Branchburg, NJ). Vitamin C was decided by HPLC evaluation of plasma deproteinized instantly following separation with 0.5 mol/L PCA (Behrens and Madere 1987).
Statistical Analyses.
Serum responses to dietary therapy had been evaluated by calculating the world beneath the curve following the consumption of the therapy beverage (Matthews et al. 1990). The consequences of the therapies had been decided by ANOVA with repeated measures (SYSTAT for Home windows, model 5, Systat, Inc., Evanston, IL). The variations between two teams had been additionally analysed by paired t-test.
RESULTS
Serum antioxidant capability, which was measured as ORACTOTAL, ORACPCA, ORACACETONE, FRAP and TEAC, plasma vitamin C, and serum urate, bilirubin and protein concentrations at time 0 following a 12-h quick are introduced in Desk 2. No vital variations had been noticed amongst totally different therapies on the zero sampling time. An in depth comparability of the relative deserves of the totally different strategies for figuring out whole antioxidant capability is introduced elsewhere (Cao and Prior 1998). ORACPCA was discovered to be the popular methodology for evaluating adjustments in water soluble antioxidants.
The realm-under-curve (AUC) approach was used to evaluate the serum response of antioxidants following consumption of the 5 therapy drinks. The AUC for the totally different measures of antioxidant capability and particular antioxidants is introduced in Desk 3. Information are introduced for the time interval of 0–4 h. The AUC for the time interval of 0–11 h was additionally calculated however not introduced in Desk 3. Usually, the responses had been related for the 2 time intervals, though for some responses the adjustments had been diminished for the 0–11 h interval, as a result of all therapy teams acquired the identical meals after the 4-h sampling time level. AUC for ORACACETONE (knowledge not proven) and bilirubin didn’t change with any of the meal therapies as decided by ANOVA utilizing repeated measures. Will increase within the AUC for the 0–4 h interval for ORACPCA and FRAP had been noticed for all therapies (in comparison with the management, P < 0.05). The most important enhance in these two parameters of ∼25% was noticed following the spinach meal. The will increase within the AUC for ORACPCA and FRAP relative to the management drink had been 13 and 10%, respectively, following the strawberry therapy. The relative will increase had been 11 and seven%, respectively, following the crimson wine therapy, and had been 21 and 20%, respectively, following the vitamin C therapy. The rise within the AUC for the 0–4 h interval for ORACTOTAL (knowledge not proven) and TEAC was solely seen for spinach therapy (P < 0.05). The proportion of change in ORACPCA focus following consumption of the totally different therapy drinks is introduced in Determine 1a. ORACPCA focus modified little or no within the management group throughout the first 4 h. Different therapies reached a most at 1 or 2 h following consumption of the drink. Ranges returned towards baseline at 4 h after the beverage. ORACPCA focus elevated by 9% for spinach at 2 h, 14% for strawberry at 1 and a pair of h, 16.9% for crimson wine at 1 h, and 17.3% for vitamin C at 2 h. Modifications in serum FRAP focus are introduced in Determine 1b. Spinach consumption produced a 12.2% enhance in FRAP at 1 h, which declined towards baseline by 4 h. Maximal responses attributable to strawberry and crimson wine had been 4.8 and 5.5%, respectively. Vitamin C therapy produced a rise in FRAP of 12.1% at 4 h, however a most of 18% was not reached till 7 h after time zero. Nevertheless, plasma vitamin C concentrations reached a most of 118 μmol/L by 4 h following the vitamin C therapy (Determine 2), which was virtually a two-fold enhance in plasma vitamin C relative to the baseline focus of 41.6 μmol/L (Desk 2). Apart from the vitamin C drink, the strawberry drink was the one therapy that produced a big response in serum vitamin C. For strawberry therapy, the rise within the vitamin C AUC throughout the 4 h was 25%, and the vitamin C focus reached a most of 58 μmol/L (40% enhance) at 4 h. Serum urate AUC elevated by 30% throughout the 4 h after the spinach drink, and 14% after the drink containing strawberries. The proportion contribution of vitamin C, urate and different antioxidants to the serum ORACPCA is introduced in Desk 4. Vitamin C accounted for about 7% of the exercise measured by ORACPCA. The proportion contribution from vitamin C roughly doubled with the vitamin C therapy (P < 0.001). Urate accounted for approximately 38% of the antioxidant activity measured by ORACPCA and did not change with dietary treatment. The percentage contribution by other antioxidants was ∼52% for ORACPCA and was not significantly altered by dietary treatment. These calculations also included contributions to the antioxidant capacity measures from protein, bilirubin, vitamin E and carotenoids. These contributions were not altered by dietary treatment (P > 0.05).
A measure of whole antioxidants excreted within the urine as mirrored within the ORAC measure is introduced in Desk 5. Vitamin C therapy produced a 44.9% enhance in urine ORAC in comparison with the management, whereas the ORAC enhance with spinach was 27.5%. Strawberry and crimson wine produced a lot smaller urinary ORAC will increase of 9.6 and eight.2%, respectively (Desk 5). As a result of 3,700 μmol Trolox equivalents got with every of the weight loss plan therapies, the proportion of this dose that would contribute to the elevated ORAC exercise within the urine collected throughout the 24 h interval was 69, 42.6, 14.8 and 12.7% for vitamin C, spinach, strawberry and crimson wine therapies, respectively.
Responses in serum glucose concentrations are introduced in Determine 3. Dietary therapies didn’t alter responses in glucose. A 20–40% enhance in serum glucose inside 1 h of the breakfast drink was noticed, which declined to fasting ranges by 4 h. One other peak was noticed at 9 h, which adopted the afternoon snack.
DISCUSSION
The outcomes of this examine demonstrated that the consumption of strawberries, spinach, crimson wine or vitamin C may enhance the antioxidant capability of serum in aged ladies. Will increase in ORACPCA and FRAP had been noticed for all these therapies in comparison with the management. As a result of serum proteins had been eliminated within the ORACPCA assays and protein produced negligible exercise within the FRAP assay, the numerous enhance of serum antioxidant capability following these drinks was primarily because of the nonprotein antioxidants within the serum. The rise of whole antioxidant capability of serum after consumption of strawberries, spinach, or vitamin C was additional supported by the numerous enhance of urine ORAC following these therapies.
The elevated antioxidant capability in serum (ORACPCA and FRAP) and urine (ORAC) following the consumption of strawberries or spinach indicated a direct absorption and/or an enhanced manufacturing of antioxidants. The absorbed antioxidants included vitamin C, and the produced antioxidants might embody uric acid from purine metabolism; plasma vitamin C degree was considerably elevated after the strawberry drink, and serum urate ranges had been elevated by each the strawberry and spinach therapies. Based mostly on the info from USDA handbooks (USDA 1986), we calculated the strawberry drink contained about 120 mg vitamin C. Nevertheless, the elevated blood vitamin C and urate might be a results of the sparing impact on them by the opposite antioxidants absorbed from strawberries or spinach. Additionally, different antioxidants, somewhat than vitamin C and urate, contributed to half of the elevated serum antioxidant capability. The elevated serum ORACPCA AUC and FRAP AUC (1–4 h) after the strawberry drink had been 275 and 315 μmol ⋅ h/L Trolox equivalents, respectively; whereas the elevated vitamin C AUC following the strawberry drink throughout the identical time interval was solely 44 μmol ⋅ h/L, or 23–44 μmol ⋅ h/L Trolox equivalents within the ORACPCA assay and 44 μmol ⋅ h/L Trolox equivalents within the FRAP assay. The antioxidant capability (Trolox equivalents) of vitamin C was 0.52–1.0 within the ORAC and FRAP assays. Due to this fact, the contribution of vitamin C to the elevated serum ORACPCA and FRAP after the strawberry drink was solely 8–14%. Equally, it was calculated that the contribution of urate to the elevated serum ORACPCA and FRAP following the strawberry therapy was 39–42%, and to the elevated serum ORACPCA and FRAP following the spinach therapy (1–4 h) was 36–44%.
Vital will increase in serum antioxidant capability had been noticed within the strawberry and crimson wine therapies though whole carotenoids decreased (Paiva et al. 1998). Due to the low plasma carotenoid focus and the comparatively decrease antioxidant exercise of carotenoids as assessed by the ORAC assay (Cao et al. 1993), carotenoids don’t make a significant contribution to the ORAC in serum.
The opposite antioxidants answerable for the elevated serum antioxidant capability following the consumption of strawberries or spinach are possible phenolic compounds together with flavonoids, though it was not clear which particular phenolic compounds had been absorbed. Some phenolic compounds, akin to ellagic acid, catechin, rutin, naringin and anthocyanins have been recognized in strawberries (Daniel et al. 1989, Guo et al. 1997, Wang et al 1997). It was urged that phenolic compounds had been answerable for the excessive antioxidant capability present in strawberries and spinach and likewise for the variations within the antioxidant capacities between vegatables and fruits (Guo et al. 1997).
The absorption of some particular person flavonoids in people was reported by a number of laboratories. Xu et al. (1994) reported that in grownup ladies the common plasma focus of whole isoflavones reached 4.4 μmol/L at 6.5 h after a dose of two mg soybean isoflavones/kg physique weight. Two research by Hollman et al. (1995 and 1996) point out that quercetin glucosides from fried onions had been absorbed in people and that quercetin absorption was enhanced by conjugation with glucose. This was supported by a latest examine that urged quercetin glucosides are able to interacting with the sodium dependent glucose transport receptors within the mucosal epithelium (Gee et al. 1998). The aglycones naringenin and hesperitin had been detected by optimistic chemical ionization-collisionally activated dissociation tandem mass spectrometry (PCI-CAD MS/MS) in human plasma and/or urine after oral administration of naringin and hesperidin (Ameer et al. 1996). Naringenin was additionally detected by HPLC/UV both on or in saline-washed erythrocytes 2 and 4 h after the oral administration of naringin (Ameer et al. 1996).
Pink wine comprises phenolic compounds, akin to quercetin, rutin, catechin and epicatechin; the focus is about 1 g/L, 20-fold the extent discovered within the common white wine (Singleton 1982). Pink wine additionally has a comparatively excessive ORAC exercise of 12.3 mmol/L, which is 5.3-fold the exercise measured in white wine (Cao et al. 1995). Pink wine, or the phenolic compounds within the crimson wine, had been proven to cut back the susceptibility of human plasma and low-density lipoprotein to lipid peroxidation each in vitro (Frankel et al. 1993) and in vivo (Fuhrman et al. 1995), though the in vivo examine has not but been confirmed (Rijke et al. 1996). The outcomes of the current examine assist the antioxidant speculation concerning the function of crimson wine within the ‘French Paradox’ (obvious compatibility of a excessive fats weight loss plan with a low incidence of coronary atherosclerosis); the crimson wine drink considerably elevated the general antioxidant capability as mirrored within the serum ORACPCA and FRAP.
A single antioxidant, vitamin C, additionally affected the general antioxidant standing. It seems that strawberries and spinach are as efficient in enhancing the general antioxidant standing in serum as a big dose of vitamin C.
In conclusion, we discovered that the general antioxidant capability in serum or urine of aged ladies was considerably elevated following the consumption of strawberries, spinach, crimson wine or vitamin C. The elevated serum antioxidant capability after the therapies of strawberries, spinach, and crimson wine indicated the potential absorption of phenolic compounds in these diets.
ACKNOWLEDGMENTS – “vitamin c orac”
We thank John McEwen and Christine O’Brien for his or her technical help; Gayle Perrone and the workers of the Diet Analysis Laboratory for his or her experience in performing lots of the laboratory analyses; Helen Rasmussen for her help within the formulation of the diets; and the nursing workers of the Metabolic Analysis Unit on the HNRC for his or her help within the care of the themes.
LITERATURE CITED
Abbreviations
“vitamin c orac”