In vitro incubations

Analysis of variance revealed that the interaction of subject x cooking time was not significant for OIAA (P = 0.40). Thus, the main effect of cooking time averaged over subject was examined (Figure1). Human platelets in whole blood responded significantly different to cooking times (P < 0.0001). The baseline for each subject served as a control for each subject. Percent changes from baseline at 0 min (raw), 1 min, 3 min, 6 min, 10 min and 15 min of steaming were -81.0 ± 14.4, -74.7 ± 14.4, -11.7 ± 5.3, +8.4 ± 3.9, +21.9 ± 1.5, +25.7 ± 1.8, respectively. Raw juice significantly inhibited the collagen-induced platelet aggregation response (P < 0.0001). Steaming onions for 1 min resulted in a significant inhibition of platelet activity (P < 0.0001), however, steaming onions for 3 min did not result in a significant inhibition of platelet activity (P = 0.39). Steaming onions for 10 min and 15 min significantly promoted collagen-induced platelet aggregation (P = 0.02 and P = 0.01, respectively). Platelet aggregation was increased 22% and 25% by steaming onions for 10 and 15 min., respectively.

Figure 1 Effect of cooked onion extracts on human platelet aggregation in vitro. Negative values are platelet inhibitory, the magnitude of the value reflects the amount of inhibition relative to a parallel control. Positive values are platelet stimulatory reflecting the percentage increase in platelet aggregation above a collagen-induced control cause by the addition of the extract. Full size image

Total polyphenolics

Total polyphenolics did not change over cooking time (P = 0.1470). The total phenolic content and percent change in aggregation over cooking time are shown in Figure2. The mean values for 0 min (raw), 1 min, 3 min, 6 min, 10 min and 15 min were 0.50 ± 0.02, 0.52 ± 0.02, 0.43 ± 0.02, 0.46 ± 0.06, 0.54 ± 0.03, and 0.48 ± 0.04 gallic acid equivalents (mg/mL), respectively.

Figure 2 Mean polyphenolic concentration (by Folin-Ciocalteu’s procedure) of six onion extracts. The concentration was expressed as mg/ml of gallic acid equivalents shown on the left side. No significant change in GAE was detected (P = 0.147). Platelet aggregation was significantly different among extracts. Full size image

Soluble solids

Percent total dissolved solids did not change over cooking time (P = 0.2091). Soluble solids content and percent change in aggregation over cooking time are shown in Figure3. The mean values for 0 min (raw), 1 min, 3 min, 6 min, 10 min, and 15 min were 10.58 ± 0.0, 9.95 ± 0.1, 9.93 ± 0.4, 10.03 ± 0.2, 10.45 ± 0.2 and 10.45 ± 0.1, respectively.

Figure 3 Mean percent soluble solids of the six onion extracts. No significant change in soluble solids over time was detected (P = 0.2091). Platelet aggregation was significantly different. Full size image

Antiplatelet activity is not specific to the edible Alliums. Whole foods and beverages such as fish oil [21], flavanol rich chocolate [22], pomegranate juice [23], purple grape juice [23, 24], and red wine [25] have been shown to exhibit antiplatelet activity. Clearly, our results show raw onion juice significantly inhibits platelet activity in vitro, which is consistent with previous studies [11, 26].

To our knowledge, this is the first study examining the temporal effects of steaming onion on OIAA. Other studies have looked at exclusively raw extracts compared to boiled extracts [16]. Cavagnaro et al. showed boiling for 3 min had no significant effect on OIAA, and that significant inhibition of platelet aggregation by boiled onion occurred up to 6 min. Our results showed that steaming for 3 min had large effects on OIAA, and by 6 min, significant pro-aggregatory effects were measured. The proaggregatory response increased linearly following the 6 min period. Cavagnaro et al. found the proaggregatory response to be significant at 20 min of boiling, compared to our findings that 6 min of steaming was enough to reverse the effect. Taken together, these results suggest a much more rapid destruction and reversal of OIAA from steaming.

Our results show that the longer steaming time, the greater loss of antiplatelet activity. All 12 subjects responded similarly to cooked onions. At 10 min steaming, we observed not only a complete loss of antiplatelet activity, but also a stimulatory effect on platelet aggregation compared to controls. This response could be of particular significance to those attempting to consume onions for their platelet-inhibitory properties. Previous work has shown that boiled Welsh onions (Allium fistulosum) caused a proaggregatory response in vitro but not in vivo in rats [9]. Even though this stimulatory effect was not found in rats, the phenomenon remains a concern. If stimulation of platelet activity occurs in vitro in humans, a possibility that this would occur in vivo remains, given the correct amount/type of cooked onion ingested and absorbed. Moreover, there is a possibility that those at risk for atherosclerosis or impaired endothelial dysfunction would be put at a greater risk, if consuming cooked onions.

Platelet aggregation plays a key role in the development of atherosclerosis, a process that begins in adolescence [27]. Atherosclerosis is a systemic proliferative and inflammatory disease of the vascular wall of arteries [28]. One of the first stages of atherosclerosis is damage to the endothelial lining of arteries [29]. When endothelial cells become damaged or dysfunctional, platelets are stimulated. Platelets are an essential element in normal hemostasis, however, when they become hyperactive, they can contribute to the progression of atherosclerosis [30]. If endothelial cells are damaged or dysfunctional and platelets are activated, platelets will aggregate and adhere to the lining of arteries [30]. Activated platelets secrete growth factors that cause luminal narrowing and intimal thickening, a major part of atherosclerosis [30]. A diet rich in natural platelet inhibitors may reduce an individual’s risk of developing or reversing the progression of atherosclerosis, however this work indicates that the method of culinary preparation of such inhibitors may be important.

Extensive work has been reported on specific beverages and foods inhibiting platelet aggregation, whereas, limited work has been reported on beverages and foods stimulating platelet aggregation. Certain fractions of grape skin have been shown to promote aggregation, however, when both stimulatory and inhibitory fractions were tested collectively, grape skin remained inhibitory as a whole [31]. In addition, epinephrine is reported to promote platelet aggregation in whole blood [32, 33], however, discrepancies in reports on caffeinated beverages promoting platelet aggregation exist [34, 35].

Briggs et al. [10] suggested that the thiosulfinates, other organosulfur compounds and flavonoids found in onions act synergistically to inhibit platelet activity. In this study, after examining antiplatelet activity, raw and steamed onion extracts were assayed for total phenols by Folin-Ciocalteu reagent (FCR). The Folin-Ciocalteu antioxidant capacity assay allowed an examination of polyphenols without introducing the reducing capacity of organosulfur compounds into the assay [36]. Our results demonstrated that total polyphenolics remained at similar concentrations over cooking time. Other studies have revealed diminished antioxidant activity of onion after cooking [37, 38]. Agostini et al. [36] used the Trolox equivalent antioxidant capacity assay (TEAC) and found 0.223 equivalent uM Trolox in the raw onion and 0.146 equivalent uM Trolox in the boiled onion. TEAC is unspecific for polyphenols yet excellent linear correlations have been observed between TEAC and total phenolic profile using the FCR assay [36]. Differences in cooking method (steaming versus boiling) could be the source of inconsistency between the present study and previous work. Our hypothesis on why we detected no significant change in total polyphenolics is that steaming may have removed water, thus preserving the remaining polyphenols in a more concentrated form. Therefore, the constant polyphenolic concentration throughout the cooking process is plausible.

Discrepancies exist among cooking studies and quercetin content. In the present study, quercetin was not measured because it was not a primary objective of the study and total polyphenolic concentration was a greater interest than concentration of a specific polyphenol. Nevertheless, quercetin is the main polyphenolic compound found in onion; therefore many studies have measured quercetin loss during cooking. Data show that 150 mg and 300 mg quercetin-4’-O-beta-D-glucoside ingested orally in humans resulted in platelet inhibition 30 min and 120 min after ingestion [39]. Subjects given a diet containing onion slices three times a day (260-360 g/day) for one week resulted in an equivalent of 67.6-93.6 mg/day of quercetin ingestion and it’s concentration in the plasma increased from 0.04 ± 0.04 uM before the trial to 0.63 ± 0.72 uM after the trial [40]. These studies suggest that onion quercetin conjugates are accumulated in the plasma and may provoke antiplatelet effects if ingested at a high enough dose.

The effect on quercetin content is probably contributed by many factors including, cultivar differences, sample preparations, processing techniques, and assessment techniques [41]. Lombard et al. found that baking quartered onions for 15 min in a pre-heated rotisserie oven at 176°C and sautéing sliced onions in sunflower oil for 5 min on an electric skillet at 93°C resulted in an increase of flavonols and quercetin conjugate contents. Twenty five percent and 7% gains of the two major glucosides (3,4’-Qdg + 4’-Qmg) were found after sautéing and baking, respectively. Additionally, the ratio of 3,4’-Qdg to 4’-Qmg conjugates decreased slightly during cooking compared to raw onions. This overall gain was attributed to concentrating the onion by removing water during cooking. Lombard et al. found an 18% decrease in the two conjugates in boiled onion compared to raw onion. Other studies examining boiling for 3–60 min have reported between 20%-75% losses of quercetin [41–46] and these losses were attributed to leaching of quercetin into the cooking water.

The present study suggests that the polyphenols, mainly quercetin conjugates, in onion juice may have little or no independent effect on platelet aggregation. In addition, onion juice may have too low a total polyphenolic content to contribute to an inhibitory effect. This may explain why platelet aggregation changed, even though no significant difference in total polyphenolic content was detected in raw and treated onions. Polyphenolics are in much higher total concentrations in other vegetables and fruits, and it is likely that the polyphenols from comparatively low polyphenolic onions are not the primary contributors to the inhibitory effect. For example, total phenolic concentration of broccoli and spinach were 80.76 ± 1.17 mg/100 g and 79.55 ± 8.39 mg/100 g [47], whereas the onions used in the present study were 47.8 ± 5.15 mg/100 g. Compared with fruits, vegetables typically have a lower total polyphenolic concentration. Cranberries, apples, red grape were analyzed and exhibited 507.0 ± 21.1 mg/100 g, 272.1 ± 6.2 mg/100 g, and 182.0 ± 2.6 mg/100 g phenolic concentrations, respectively [48]. It is possible that platelet inhibitory effects may be explained better by foods and/or beverages with a much greater antioxidant capacity than the onions used in the present study. Purple grape juice had three times the total polyphenolic concentration of orange and grapefruit juice and it had about three times greater effect on platelet inhibition [24]. More applicable to the present study, varietal differences in polyphenolic concentration exist among onions. An onion with a high value, such as, Western Yellow (104.9 ± 13.0 mg/100 g) was twice as high as the yellow onion used in this study. Polyphenols in Western Yellow could have a greater affect on antiplatelet activity more so than the onion used in the present study [49].

It is well established that organosulfur compounds in onions change dramatically with time and depend on processing method [50]. The chemistry of Allium-derived sulfur compounds is complex because many compounds are volatile, thermally unstable, condense or decompose to form other compounds, and under different conditions, different compounds are generated. Thiosulfinates, initial chemicals formed in freshly macerated tissues, undergo transformation and decomposition. Most flavor compounds are formed from the decomposition of thiosulfinates [50]. With regard to steaming, it has been shown that during steam distillation, oils are generated. During steam distillation, tissues are heated to 100°C and initially formed thiosulfinates are rapidly converted into their corresponding polysulfide. Block et al. found that heating pure diallyl disulfide for 10 minutes at 150°C resulted in the formation of greater than 30 sulfur components [51]. Lawson et al. found steam-distilled garlic cloves were only 35% as active in inhibiting platelets compared to aqueous garlic clove homogenates [52]. Differences in reductions in antiplatelet activity were noted between our study, which involved steaming, and other studies, which have involved boiling. It is possible that the generation of oils or polysulfides is partially responsible for this effect. Because of the unstable nature and difficulty characterizing sulfur composition in onion tissues, we were unable to provide data showing specific sulfur components found in our steamed and raw extracts. It would be of interest to determine whether platelet stimulatory fractions are present in raw onion and if their activity is countermanded by platelet inhibitory compounds in raw onion. Further analysis of sulfur components from domestically prepared onion tissues would be an important and useful measurement. More importantly, it would be useful to determine if the platelet stimulatory response is reflective of a loss of specific sulfur components, formation of different sulfur components, or is reflective of other facets related to cooking.

We sought to examine antiplatelet activity inducement of other vegetables in raw and cooked forms in a pilot study. We found that raw broccoli inhibited platelet activity in three out of the four human subjects (data not shown). Additionally, cooked broccoli did not inhibit platelet activity and became stimulatory after cooking beyond 6 min. This finding is interesting because the change from inhibitory to stimulatory may not be specific for onion, but may encompass other vegetables that are platelet inhibitors in raw form. Causes of this proaggregatory response are beyond the scope of our study, but remain of great interest to our laboratory.

Lastly, we examined soluble solids and found that they did not change significantly during the cooking process. In raw form, OIAA is correlated strongly with high pungency and high soluble solids [53]. In contrast, we found high soluble solids remained when the onion was cooked, yet OIAA changed. Since soluble solids mostly consist of carbohydrates and ASCOs make up a low percentage of total soluble solids, this finding was not surprising. However, cooking decreases pungency, therefore, OIAA and pungency may remain strongly correlated.