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2
*
Department of Food Health, Chia Nan College of Pharmacy and Science, Tainan;
Department of Physiology, College of Medicine, National Cheng-Kung University, Tainan;
**
and Department of Food Science, National Chung-Hsing University, Taichung, Taiwan
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the stable
prostacyclin metabolite, but not the plasma nitrite level. On the
contrary, boiled Welsh onion juice consumption was totally ineffective.
In conclusion, consuming raw Welsh onion juice, but not boiled juice,
has blood pressure lowering and antithrombotic effects in rats. These
effects may be mediated by PGI2-cAMP pathway.
KEY WORDS: • platelet function • Welsh onion • prostaglandins • bleeding time • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). These beneficial effects have been attributed to their
abilities to lower blood pressure (Loeper and Debray 1921
) or to inhibit platelet aggregation and thromboxane
formation (Bordia 1978
, Makheja el al. 1979
, Srivastava 1986
). Whether Welsh onion
(Allium fistulosum L., Alliaceae), a member of
Allium family which is an important flavoring vegetable in Asian
dishes, modulates cardiovascular function is largely unknown. Recently
we reported that Welsh onion extracts modulate rat aortic vascular tone
in both endothelium-dependent and endothelium-independent
manners (Chen et al. 1999
). Depending on whether these
extracts were boiled, their effects could be either vasorelaxing or
vasoconstricting. Our previous study also demonstrated that exogenous
administration of Welsh onion extracts, especially the raw green
portion, can affect in vitro human platelet functions, including
adhesion, aggregation and thromboxane release (Chen et al. 1999
). To investigate whether the dietary intake of Welsh onion
has antithrombotic effect, we fed rats raw or boiled Welsh onion juice
for 28 d to evaluate its possible antiplatelet effect. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because our previous study indicated that the green-leaf part was
more potent than the white-sheath part in inhibiting human platelet
function in vitro (Chen et al. 1999
), only the
green-leaf part was used. Briefly, the green portion of Welsh onion
was squeezed and filtered by a food processor (National, Model MJ-C85
N; Tokyo, Japan; pore size of the filter = 0.2 mm) ten
times to obtain the juice. Some juice was obtained after 30 min of
boiling. They were freshly prepared every day during the 28-d feeding
period. The yield was about 50% (wt/wt) for either raw or boiled Welsh
onion. A total of 28 batches of Welsh onion was used. To avoid
batch-to-batch differences, raw or boiled juice from the same batch was
used for the entire set of experiments.
Animals and Welsh onion juice feeding.
This study was conducted in conformity with the policies and procedures detailed in the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 86-23 1985). Nine-wk-old male Sprague-Dawley rats were purchased from National Cheng-Kung University Animal Center, Tainan, Taiwan. They were housed in an environmentally controlled room (temperature 25 ± 1°C; 12 h light/12 h dark cycle) and were given free access to a nonpurified rat diet (PMI Feeds Inc., Richmond, IN) and water. Some rats were fed raw or boiled Welsh onion juice (2 g · kg-1 · d-1) for 4 wk, and the remaining were fed water and acted as the control. No significant differences in body weights among studied groups were found before or after the experiments (data not shown). Two days after the feeding period, the following experiments were conducted.
Measurement of resting systolic blood pressure.
To see if Welsh onion had a hypotensive effect, resting systolic blood
pressure of rats was monitored before and after feeding of Welsh onion
juice by using a tail-cuff method (Chen and Chiang 1996
).
Determination of tail bleeding time.
The tail bleeding time test was modified from the method described by De Clerck et al. (1976)
. In brief, the tail was cut at 2 mm from the
tip, when the urethane (5 g/10 mL; 1 mL/300 g body weight;
i.p.)-anesthetized animal was placed on a platform with the tail
hanging down into a beaker containing 100 mL of normal saline at
37°C. The bleeding time was measured from the moment that the tail
was cut until bleeding had stopped completely for 1 min.
Measurement of platelet aggregation.
Blood samples were drawn from rat aorta after urethane anesthesia and laparotomy. These blood samples were anticoagulated with sodium citrate (38 g/L, 1 vol anticoagulant for 9 vol of blood). Platelet-rich plasma (PRP)3 and platelet-poor plasma (PPP) were prepared by centrifugation. Platelet aggregation kinetics in 200 µL of PRP was measured with a platelet aggregometer (Hema Tracer 2; Niko Bioscience, Tokyo, Japan) after addition of various concentrations of ADP. Platelet aggregation was expressed as percentage of the PPP transmission value.
Measurement of platelet adhesion.
A tapered flow chamber which had a linear variation of shear stress,
starting from a predetermined maximal value at the entrance and falling
to zero at the exit, was used to determine platelet adhesiveness
(Jen et al. 1996
, Wang et al. 1994
). A
constant flow rate of 1.62 mL/min through this chamber would provide a
range of shear stress from 55 to 0 dyne/cm2, covering the
entire physiological range in circulation. In this study, the flow
chamber contained a glass slide coated with fibrinogen as the testing
surface. One mL of PRP was gently infused into this chamber and was
then incubated at 37°C for 15 min to let platelets settle onto the
fibrinogen-coated surface. Subsequently the chamber was inverted
and flushed with buffer at 1.62 mL/min for 5 min. The remaining
platelets per unit area were counted at different locations along the
central line under an inverted microscope. The counting area at each
location was 0.16 mm2. The remaining platelets at different
locations were expressed as the percentage of those at the exit of the
flow chamber where the shear stress was near zero.
Measurement of platelet thromboxane release.
Thromboxane extraction from 500 µL of PRP samples, with or without
pretreatment of 2 µmol/L of ADP, was carried out as described
previously (Powell 1980
). Since thromboxane
A2, the active substance released by platelets, has
relatively short half-life, we measured its stable metabolite
thromboxane B2 by using commercial kits (Cayman, Ann Arbor,
MI).
Measurement of platelet cyclic nucleotides.
Platelet cyclic nucleotides were determined by enzyme-linked
immunosorbent assay (EIA) as described in details in our previous study
(Wang et al. 1997
). In brief, citrated PRP containing 2
x 10-4 mol/L of 3-isobutyl-1-methyl-xanthine (IBMX),
a phosphodiesterase inhibitor, was mixed with ice-cold HEPES buffer
and centrifuged at 6,500 x g at 4°C for 5 min.
The pellet was vortexed with ice-cold 60 g/L trichloroacetic acid
(TCA) for 2 min. After centrifugation (10,000 x g,
15 min, 4°C), TCA was removed by washing four times with 5 vol of
water-saturated ether. The extracted samples were stored at
-80°C until measurement. Results were normalized to platelet count
in the specimen.
Measurements of plasma levels of PGI2 and nitric oxide (NO) metabolites.
Plasma levels of prostacyclin (PGI2), indicated by its
stable metabolite 6-keto-PGF1, were measured by an EIA
method as described in details previously (Chen et al. 1993
).
NO metabolites in plasma were determined by the Griess reagent-based colorimetric method, using commercially available assay kits (Catalog # 780001; Cayman). First, plasma nitrate was reduced to become nitrite. Then, nitrite was converted into a deep purple azo compound with an absorbance at 550 nm. Measured values represented the total amount of plasma NO metabolites, i.e., nitrite and nitrate.
Chemicals.
EIA kits were purchased from Cayman. ADP and IBMX were purchased from Sigma (St. Louis, MO). Other chemicals were obtained from Merck (Darmstadt, Germany).
Statistical analysis.
All data were expressed as mean ± SEM With the
Macintosh statistical software, the differences among control, raw
juice feeding and boiled juice feeding groups were analyzed by ANOVA
(Zar 1984
). Unpaired two-tailed Student’s
t test between two groups was used as a posthoc test.
Paired t test was used to analyze the results of pre-
vs. postfeeding in the same group. Dose response relations of
ADP-evoked platelet aggregation and platelet adhesion curves under
various shear stresses were compared by ANOVA with repeated measures
(Zar 1984
). P-values < 0.05 were
considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This treatment also prolonged the tail bleeding time (337 ± 10 s, n = 6; P < 0.01), while the
boiled juice did not affect the bleeding time, compared to the control
(308 ± 12 s vs. 279 ± 8 s).
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). Raw juice feeding suppressed platelet adhesion (P < 0.01).
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). Moreover, this treatment completely suppressed 2 µmol/L of
ADP-stimulated thromboxane release from platelets without altering
their basal thromboxane level. In contrast, the boiled juice did not
have any effect.
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levels (P < 0.01), but did not significantly affect NO metabolites (P
= 0.11) (Table 3).In addition, feeding rats boiled Welsh onion juice had no effect on
either PGI2 or NO metabolites in plasma.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and lowered systolic
blood pressure. Therefore, chronic oral administration of Welsh onion
juice had antiplatelet effects, and these effects were mainly mediated
by the PGI2-cAMP pathway.
Other Allium vegetables, such as onion and garlic, can inhibit platelet
activity (Bordia 1978
, Goldman et al. 1996
, Makheja el al. 1979
, Srivastava 1986
). Our previous in vitro study on human platelet function
supported this (Chen et al. 1999
). In that study,
exogenous administration of the Welsh onion extracts from the odorous
green portion was more potent than extracts from the white portion in
inhibiting human platelet function. Moreover, the extract from boiled
Welsh onion had platelet-stimulating effect in vitro. This is
consistent with previous reports showing that some of the
platelet-inhibiting ingredients were odorous, e.g., ajoene
(Apitz-Castro et al. 1994
).
Whether these platelet-modulating effects of dietary Welsh onion diet will be changed by cooking is still unknown. Our results from this in vivo study indicated that oral administration of raw juice for 28 d could prolong bleeding time, reduce platelet aggregation and elevate cAMP level. However, feeding boiled juice was ineffective. Thus the platelet-stimulating factor(s) in boiled juice seem to be more labile in vivo than the platelet-inhibiting factor(s) in raw juice.
Normal hemostasis relies on the balance between procoagulant and
anticoagulant activities. While platelets play a major role in
procoagulant activities, vascular endothelial cells control the
anticoagulant activities, notably by synthesizing vasorelaxing and
platelet-inhibiting factors, such as NO and prostacyclin
(Miller and Vanhoutte 1992
). Recently we found that
Welsh onion extracts modulate rat aortic vascular responses and in
vitro human platelet function (Chen et al. 1999
). Raw
green extract caused vasorelaxation and antiplatelet effect by
stimulating the release of endothelium-derived NO, which is a
potent inhibitor of platelet aggregation and adhesion (Azuma et al. 1986
, Jen et al. 1995
, Radomski et al. 1987
). On the other hand, the boiled extract stimulates the
release of a constricting factor, most likely to be thromboxane
A2, and potentiates platelet activity. Taken
together with the current results, it is clear that the raw Welsh onion
is antithrombotic.
Although the active compounds in raw Welsh onion have not been
identified, the prostaglandin synthesis pathway appears to be
modulated, as indicated by the reduction of ADP-stimulated
thromboxane release, along with an elevated plasma
PGI2 level and an increase in platelet cAMP
levels. It was reported that ajoene, the garlic-derived principle,
alters arachidonic acid’s metabolism in rat platelets
(Srivastava 1986
). Ali and Thomson (1995)
also
demonstrated that long-term consumption of garlic reduces human
serum thromboxane level and prevents thrombosis.
In conclusion, our results show that oral administration of raw Welsh onion, especially the green portion, inhibits platelet activities, which may be due to a change in PGI2-thromboxane balance.
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FOOTNOTES |
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3 Abbreviations used: EIA, enzyme-linked immunosorbent assay; IBMX, 3-isobutyl-1-methyl-xanthine; 6-keto-PGF
1, 6-keto-prostaglandin F1; NO, nitric oxide; PGI2, prostacyclin;
PPP, platelet-poor plasma; PRP, platelet-rich plasma; TCA, trichloroacetic acid; TXB2, thromboxane B2.
Manuscript received July 12, 1999. Revision accepted September 29, 1999.
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