Platelets (2004) Aug; 15 (5): 287-292. © 2004 Taylor & Francis Ltd. (condensed)
Effects of kiwifruit consumption on platelet aggregation and plasma lipids in healthy human volunteers
Asim K. Duttaroy, Aud Jψrgensen
Department of Nutrition, Faculty of Medicine, University of Oslo, Oslo, Norway.
Fruits and vegetables have been thought to be beneficial in cardiovascular disease. The beneficial effects of fruits and vegetables may be explained by the antioxidants and other components contained therein. These nutrients may function individually or in concert to protect lipoproteins and vascular cells from oxidation, or by other mechanisms such as reducing plasma lipid levels (LDL cholesterol, triglycerides), and platelet aggregation response. Kiwifruit which contain high amounts of vitamin C, vitamin E and polyphenols may be beneficial in cardiovascular disease; however very little is known about its cardioprotective effects. Platelets are involved in atherosclerotic disease development and the reduction of platelet activity by medications reduces the incidence and severity of disease. To this end, we evaluated whether consuming Kiwifruit modulated platelet activity and plasma lipids in human volunteers in a randomized cross-over study. We report that consuming two or three Kiwifruit per day for 28 days reduced platelet aggregation response to collagen and ADP by 18% compared with the controls (P<0.05). In addition, consumption of Kiwifruit lowered blood triglycerides levels by 15% compared with control (P<0.05), whereas no such effects were observed in the case of cholesterol levels. All these data indicate that consuming Kiwifruit may be beneficial in cardiovascular disease.
Many risk factors for cardiovascular disease (CVD) including platelet hyperactivity, high blood cholesterol, triglycerides, obesity and diabetes are substantially influenced by dietary factors. Since these risk factors are modifiable therefore primary preventive efforts through dietary means hold much promise.1 High intake of fruit and vegetables is such an important preventive measure by which the risk of cardiovascular disease can be reduced. The American Heart Association and other national agencies recommend a diet that includes 5 servings of fruit and vegetables daily.2,3 These recommendations are based primarily on the studies indicate that fruit and vegetable intake may reduce CVD risk through the beneficial combinations of micronutrients, antioxidants, phytochemicals, and fiber in fruits and vegetables.47 A decreased oxidative modification of LDL may be one of the mechanisms by which fruits and vegetables reduce the risk of CVD and atherosclerotic progression. However, hyperactivity of platelets, and their adhesion and aggregation at the site of injury in atherosclerotic vessel walls, is also critically important in the pathogenesis of CVD.810 There is also an increasing evidence that acute clinical manifestations of coronary atherosclerotic disease are caused by plaque disruption and subsequent plateletthrombus formation.1113 Coronary atherosclerotic lesions may appear in early life and, towards the end of the second decade of life, asymptomatic atherosclerotic lesions are present in most people living in industrialised societies.12,14 Therefore, platelet activity may play a major role in the development as well as in the stability of atherosclerotic plaques. In support of the pathophysiological role of platelets, platelet inhibitory drugs such as aspirin reduce the incidence of myocardial infarction, stroke and death from CVD in secondary prevention trials.1518 Flavonoids are polyphenolic compounds, widely distributed in fruit and vegetables, many of which have antioxidant as well as anti-thrombotic properties.1922 Certain polyphenols have been shown to be inhibitory to platelet aggregation than others, and consequently dietary studies involving flavonoids on anti-platelet effects are not conclusive.2325 The difference in platelet inhibitory effects among polyphenols could be due to several reasons. Variations in digestion, absorption, and hepatic processing may limit the bioavailability of these compounds. It is also possible that compounds other than the polyphenols may be responsible for the platelet inhibitory activity.
We recently examined the anti-platelet activity of several fruits and this activity was not related to the antioxidants potential of fruits.9,26 Despite the fact that Kiwifruit contain very significant amounts of antioxidants such as polyphenols and vitamins C and E,27,28 it was not included in the study. Recently it has been shown that Kiwifruit provide a dual protection against oxidative DNA damage, enhancing antioxidant levels and stimulating DNA repair and may be beneficial in cancer;29 however, no information is available on the effects of Kiwifruit on platelet activity and plasma lipids, these two important risk factors of CVD, in human volunteers. In this paper we report that consuming of two or three Kiwifruit per day for 28 days significantly reduced platelet aggregation in human volunteers in a randomized cross-over design. Moreover, plasma triglycerides levels were also reduced in these volunteers. Our study indicates that consuming kiwifruit (two or three per day) may be beneficial in CVD.
Materials and methods
Collagen, ADP and arachidonic acid were obtained from Chrono-Log (Havertown, USA). Uric acid, ascorbic acid, EDTA, acetic acid, sodium hydroxide, metaphosphoric acid and phosphate-buffered saline (PBS) tablets were obtained from Sigma (Poole, UK). All other reagents used were of analytical grade quality.
Thirty (12 males and 18 females) healthy volunteers took part in this study. Volunteers were aged 2051 years and their mean BMI was 22.430.52 kg/m2. The volunteers were recruited by means of advertisements at the Institute for Nutrition Research, University of Oslo, Oslo, Norway. Volunteers were requested to maintain normal diet during the trial.
Subjects were allocated randomly to two groups (nΌ15), each of which was given different order of Kiwifruit doses. Group A took two Kiwifruit per day in the first period and three Kiwifruit per day in the second period, whereas group B took two and three Kiwifruit per day. Each volunteer consumed two and three Kiwifruit per day for successive 28-day periods separated by at least 2-week wash-out periods. The study protocol was approved by the Ethical Committee of Ulleva˚ l University Hospital, Oslo.
Healthy subjects were recruited after assessment of their medical and dietary history. Exclusion criteria were the presence of overt vascular, haematological or respiratory disease, hypertension, infection, frequent consumption of drugs which affect platelet function (e.g., aspirin, paracetamol, ibuprofen, steroid, habitual consumption of omega-3 fatty acid supplements).
Preparation of Kiwifruit extract
In order to investigate the effects of Kiwifruit extract on human platelet aggregation in vitro. Extracts consisting of 100% fruit juice were freshly prepared on the day of the assay from Kiwifruit. To prepare 100% fruit juice, the fruits were peeled and the pulp after being weighed was homogenised. The resulting homogenate was centrifuged at 3000g for 10 min. The supernatant was collected and the pH was adjusted to 7.4 with sodium hydroxide. The pHadjusted Kiwifruit extract (KFE) was then used for in vitro platelet aggregation experiments.
Platelet aggregation study
Overnight fasted blood was collected through siliconized needles into plastic syringes: coagulation was prevented by mixing 9 vol of blood with 1 vol sodium citrate (final concentration, 13 mM). Platelet-rich plasma (PRP) was obtained by centrifugation of citriated blood for 15 min at 180g. The supernatant platelet-rich plasma (23108 cells/ml) was collected and used for platelet aggregation.24
Platelet aggregation was monitored on a platelet aggregometer (Chrono-Log) at a constant stirring speed of 1000 rpm at 37C, as described before.24 The aggregating agents used were ADP (4 and 8 mM), and collagen (4 and 8 mg/ml). Platelet aggregation was followed over 10 min. Platelet aggregation was performed within 2 h after the blood was taken.
Platelet aggregation is expressed as the area under thecurve. To determine the effect of KFE on platelet aggregation in vitro, PRP (450 ml) was incubated with 50 ml of KFE for 15 min at 37C prior to the addition of an aggregating agent. Since maximal amplitude of aggregation of PRP was obtained with either 4 mg/ ml collagen, 8 mM ADP, and 500 mg/ml arachidonic acid, these concentrations of agonists were used and aggregation was followed as described above.
Controls were run in parallel replacing fruit extract with 50 ml of PBS. Inhibition of platelet aggregation is expressed as the decrease in the area under the curve compared with the control.
In order to determine the effects of consumption of Kiwifruit on platelet aggregation response in human volunteers, blood samples were taken at study entry (day 0), and after day 28 of fruit consumption, between 08:00 and 10:00 h after an overnight fast. PRP was prepared for platelet aggregation experiments as described before.
Determination of plasma vitamin C
Blood was collected in heparin on ice and was centrifuged at 2000g for 10 min at 4C. Then 600 ml of plasma were mixed with 600 ml of 10% metaphosphoric acid in a tube and was snap frozen The samples were stored at 80C until required for determination of total vitamin C concentration (vitamin C and dehydroascorbic acid) by HPLC, as published before.30
Ferric reducing/antioxidant power (FRAP) assay
The FRAP assay was performed according to Benzie and Strain (1997).31 The FRAP assay was used to measure the concentration of total antioxidants in plasma using Technicon RA 1000 system (Technicon Instruments Corporation, New York, USA). An intense blue colour with absorption maximum at600nm was measured.
Determination of plasma lipids
Plasma triglycerides, HDL and total cholesterol concentrations were determined using reagents from Genzyme diagnostics (UK) following the protocol. LDL-cholesterol was calculated using the Friedwald formula.32
Results are presented as the meanSEM.Results were analysed by the Students t-test. Values for the area under the curve for platelet aggregation in the absence and in the presence of different concentrations of fruit extract were evaluated by a computer-assisted programme. Other statistical analyses were performed using ANOVA where appropriate, values were considered to be significantly different when P<0.05.
Effects of Kiwifruit extract on human platelet
aggregation in vitro
One hundred percent Kiwifruit extract (KFE) (w/v) was used in this study. Incubation of KFE (expressed as weight of pulp used to prepare KFE) inhibited ADP-induced platelet aggregation in a dosedependent manner (Figure 1). ADP-induced aggregation was inhibited by 11% with 5mg KFE, 54% with 10 mg KFE, and 96% with 20 mg KFE, compared with controls. KFE also inhibited collagen-induced platelet aggregation, however, the level of inhibition was lower with the 5- and 10-mg KFE incubations compared with those observed with ADP-induced platelet aggregation (Table 1). Inhibition of arachidonic acid-induced platelet aggregation exhibited a very different profile with only 32% inhibition at the highest KFE level (20 mg) tested, and nothing at all at the lower concentrations of KFE. The IC50 (minimum concentration required for 50% inhibition of platelet aggregation induced by ADP in 500 ml PRP) of KFE extract was around 10 mg KFE. The KFE extract inhibited both collagen- and ADP-induced platelet aggregation to a greater extent, whereas it had very little inhibitory effects on arachidonic acid-induced aggregation.
Effects of consumption of Kiwifruit on plasma
antioxidants in human volunteers
Of these 30 subjects, all except two volunteers completed the two phases of Kiwifruit consumption. During the supplementation period no significant change in their mean BMI was observed. The kiwifruit were well tolerated without any adverse effect. Dietary antioxidants (antioxidant levels, and total vitamin C) were measured in plasma before and after the trial period. The plasma levels of FRAP increased significantly after two or three Kiwifruit per day. FRAP levels were increased very significantly in both the groups after consuming fruit (two or three Kiwifruit per day) (P<0.01), although no statistically significant differences between these two groups were observed. Similar to FRAP values, plasma vitamin C levels in these volunteers were also increased significantly, increasing from 65.929.0 mM (day 0) to 10223.1 mM (day 28) (P<0.01) and from 6815.2 mM to 92.3814.7 mM (P<0.01) after consuming two and three Kiwifruit per day, respectively.
Platelet response to both low and high concentrations of ADP or collagen was inhibited by Kiwifruit consumption. Consuming two Kiwifruit inhibited ADP-induced platelet aggregation significantly (18% in case of 4 mM ADP and 15% in case of 8 mM ADP compared with those at day 0 (P<0.05). Similar reduction in platelet aggregation in response to collagen was observed when volunteers consumed two or three Kiwifruit per day for 28 days. After the wash out period (minimum 2 weeks) platelet aggregation response returned to the base line level (day 0).
Mean total cholesterol, LDL, and HDL cholesterol values in all groups were unchanged from days 0 to 28 in both groups, whereas triglyceride concentrations were significantly lowered on day 28 (1.160.45 mmol/l at day 0 vs. 0.870.29 mmol/l, P<0.05, at day 28 and 1.190.35 vs.0.84 0.35 mmol/l, P<0.05, in the case where volunteers consumed two and three Kiwifruit per day, respectively), compared with those on day 0 in both groups (P<0.05). After the wash-out period (minimum 2 weeks), plasma triglyceride concentrations returned to the base line level.
The Kiwifruit extracts inhibited both ADP and collagen-induced platelet aggregation in vitro, but not arachidonic acid-induced aggregation, indicating that the thromboxane pathway does not appear to be involved, as we observed in the case of tomato extract.26 This is quite different than that of aspirins mode of action in platelets. Aspirins anti-platelet action involves inhibition of the cyclooxygenase enzyme in platelets, leading to a decreased formation of prostaglandin G2, a precursor of TxA2.17,18 This blocks the formation of TxA2, a platelet aggregation agonist. Since Kiwifruit extract does not inhibit the thromboxane pathway, it is possible that the mode of action is upstream of platelet activation/aggregation processes. At present we do not know the nature of the anti-platelet factors in Kiwifruit. This study was, therefore, designed to assess the effects of Kiwifruit consumption on the cardiovascular risk profiles in healthy volunteers. Kiwifruit consumption increased plasma antioxidants and vitamin C levels in these volunteers as expected.
Consuming two or three Kiwifruit reduced platelet aggregation to a similar extent. In addition, consumption of Kiwifruit reduced plasma triglyceride levels without affecting cholesterol levels in these volunteers. There were no correlations between individual changes in platelet aggregation response and plasma lipids and vitamin C values. In addition, the magnitude of these effects of Kiwifruit is not related to the number of Kiwifruit consumed. We do not have any explanation for this. The inhibitory effects on platelet aggregation response and lowering effects on plasma triglyceride levels of Kiwifruit disappeared during the wash-out period. This indicates that the effects of Kiwifruit on platelets and plasma lipids are reversible. We restricted our study with relatively small number of Kiwifruit (two, or three), as consuming larger number of fruits is known to have laxative effect. However, further work is necessary to determine the structure and mechanism of action of the active principles in Kiwifruit responsible for lowering platelet aggregation response as well as plasma triglycerides in humans. Epidemiological studies as well as laboratory experiments have provided very strong evidence that fruits and vegetables and specific antioxidants are beneficial for human health.33,34 The protective effects of vegetables and fruit may in part be attributed to antioxidant vitamins and specific polyphenols that display powerful inhibition of oxidative stress. However, we have earlier reported that the anti-platelet potential of the fruits appeared to be unrelated to their antioxidant activity.9,24,26 Although further research on identification of the active compounds requires for definitive conclusions.
Given the role of blood lipids in the development of atherosclerosis and CVD and the positive roles of fruits and vegetables on plasma lipids,3537 we have also assessed the effects of Kiwifruit on plasma lipids. Lowering of plasma triglycerides by Kiwifruit was observed despite these volunteers maintaining their regular diet during the supplementation periods. None of the volunteers reported any loss of appetite or changes in their food intake during the supplementation. The mechanism of action is not known; it is possible that the presence of certain components in Kiwifruit may be responsible for the lipid-lowering effects. However, further work is required to understand the mechanism of action. It must be noted that the lipid-lowering effects of fruits and vegetables were also reported before.3537
In conclusion, consuming Kiwifruit is an effective way of inhibiting platelet aggregation induced by collagen and ADP in human volunteers. Our data thus provide evidence that consuming Kiwifruit has the potential to increase the effectiveness of thrombosis prophylaxis. Modulation of platelet reactivity towards collagen, ADP and plasma triglyceride levels by Kiwifruit could be of potentially prophylactic and therapeutic benefit in preventing and halting pathological processes that lead to CVD.
This work was supported by the International Kiwifruit Organization (IKO). We are thankful to Mr Hamid Shergafi for his technical assistance and Ms Anette Karlson for vitamin C analysis. We are also grateful to BAMA, Norway, and Zespri, New Zealand, for supplying us kiwifruit for this study.
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Asim K. Duttaroy, Aud Jψrgensen, Department of Nutrition, Faculty of Medicine, University of Oslo, Oslo, Norway.
Correspondence to: Professor Asim K. Duttaroy, Department of Nutrition, University of Oslo, P.O. Box 1046 Blindern, N-0316 Oslo, Norway. Tel.: ώ47-22-85-15-47; Fax: ώ47-22-85-13-41;
Platelets (2004) Aug; 15 (5): 287-292. © 2004 Taylor & Francis Ltd. (Condensed)