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RESEARCH ARTICLE
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A Comparative Study of Antioxidant and Antiplatelet Activity of
Pomelo Juice Extracted from Three Cultivars
Jindaporn Janprasit1, Paveena Yamanont1, Rataya
Noppawan Phumala Morales1
Luechapudiporn2,
1
Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok,
10400, Thailand
2
Department of Pharmacology and Physiology, Faculty of Pharmaceutical
Sciences, Chulalongkorn University, Bangkok, 10330, Thailand
Abstract
Several studies have reported the protective effect of polyphenol-rich diet to
cardiovascular diseases (CVD) thru antioxidant and antiplatelet activity. This study
aimed to evaluate antioxidant activity and inhibitory effect of pomelo on human
platelet aggregation. The nitrite content, total phenolic content, total flavonoid
content and antioxidant activity were determined in juice extracted from three
cultivars of pomelo; Thong Dee, Khao Tang Kwa and Khao Yai. All of pomelo
juice contained phenolic content approximately 6 mM of gallic acid equivalence
while contained nitrite content in range of 3-6 μM. The in vitro effect of pomelo
juice on ADP-induced human platelet aggregation was investigated. Pomelo juice
affected the maximum aggregation in dose-dependent manner and also altered the
response curve in to biphasic shape. The juice extract from Thong Dee showed
highest antioxidants and antioxidant activity and highest effect on platelet
aggregation. The mechanism of action in related to active compounds in various
cultivars of pomelo will be further investigated.
Keywords: pomelo juice, antioxidant, antiplatelet
Address correspondence and reprint request to: Noppawan Phumala Morales, Department of
Pharmacology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand. E-mail address:
noppawan.phu@mahidol.ac.th
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Introduction
Platelets are one of blood cells that are necessary for physiologic processes
of hemostasis. Conversely, they also involve in atherothrombosis, which is the
major cause of cardiovascular diseases (CVD). Atherothrombosis, the leading
cause of mortality in developed countries, is explained as the formation of thrombi
overlaid on atherosclerotic lesion disruption1.
Several epidemiological studies have reported the protective effect of diets
in the prevention of CVD2-5. Thus, the food or food component, which maintain or
improve health, is regarded as functional food. This fact has brought the enormous
interest on the antiplatelet activity of dietary components, including polyphenols
and flavonoids. Several natural components that found in grape, onion, and tomato
have been reported to exert the preventive role against CVD thru inhibition of
platelet aggregation6-7.
Interestingly, some of natural polyphenols presenting in diet affected to the
platelet function, even if it is not well defined that their native compounds,
metabolites, or combination of both exerted their in vivo antiplatelet activities8. The
natural form of polyphenols mostly found as glycoside.
The pomelo (Citrus maxima Merr.) is the largest of all nature citrus fruits
that is widely consumed in Thailand. There are many cultivars that are cultivated in
Thailand, namely Thong Dee, Khao Tang Kwa and Khao Yai. Pomelo enrich with
vitamin C, antioxidant and flavonoids (i.e. kaemferol, myricetin, apigenin, luteolin
and hesperetin). The nutritive composition and content depend on cultivar9.
Previous study have confirmed that pomelo juice enrich with flavonoids in
glycoside form10-11
The aim of this study is to evaluate antioxidant activity of pomelo juice
extracted from 3 different cultivars, including Thong Dee, Khao Tang Kwa and
Khao Yai. Furthermore, the inhibitory effect of pomelo juice on human platelet
aggregation is investigated.
Materials and Methods
Pomelo juice preparation
Three cultivars of pomelo were studied: Thong Dee (TD), Khao Yai (KY)
and Khao Tang Kwa (KT). The pomelo pulps were blended and filtered to obtain
pomelo juice. The samples of pomelo juice were centrifuge 10000g before
performing assays.
Griess assay
The griess assay was performed using sulfanilamide and N-(1Naphthyl)ethylenediamine dihydrochloride (NED) to determine total nitrite (NO2-).
The juice or standard (50 l) was mixed with 1% sulfanilamide in 5% phosphoric
acid (50 l) for 5 minutes and 0.1% NED (50 l) was then added and mixed well.
Then, it was incubated at room temperature in dark for 5 minutes. The absorbance
of reaction was measured at 520 nm with microplate multimode reader (Varioskan
Flash, Thermo Scientific, USA).
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Total phenolic content
The total phenolic content was determined using the Folin-Ciocalteau
method compared to standard compound, gallic acid. The juice or standard (20 l)
was mixed with 10%v/v Folin-Ciocalteau reagent (100 l) for 2 minute and 7.5%
sodium carbonate solution (80 l) was then added and mixed well. Then, it was
incubated at room temperature for 60 minutes. The absorbance of reaction was
measured at 765 nm with a microplate multimode reader.
Total flavonoid content
The flavonoids content was determined using aluminum chloride (AlCl3)
compared to standard compound, quercetin. The juice or standard (100 l) was
mixed with 2% AlCl3 in ethanol (100 l). Then, it was incubated at room
temperature for 60 minutes. The absorbance of reaction was measured at 420 nm
with a microplate multimode reader.
DPPH radical scavenging assay
The DPPH radical scavenging assay was performed to evaluate antioxidant
activity compared to standard compound, trolox. The juice or standard (100 l)
was mixed with 500 M DPPH (100 l). Then, it was incubated at room
temperature in dark for 10 minutes. The absorbance of reaction was measured at
520 nm with a microplate multimode reader.
Ferric ion reducing antioxidant power (FRAP) assay
The FRAP assay also was used to evaluate the antioxidant activity
compared to standard compound, ascorbic acid. The juice or standard (20 l) was
mixed with FRAP reagent (180 l). Then, it was incubated at room temperature for
10 minutes. The absorbance of reaction was measured at 593 nm with a microplate
multimode reader.
FRAP reagent is the mixture of 300 mM acetate buffer (pH 3.6), 10 mM
TPTZ, 20 mM Ferric Chloride (FeCl3) and water in 10:1:1:1.2 ratio.
Platelet preparation
The blood sample was obtained from male free-drug healthy volunteer and
anticoagulated with 3.8% sodium citrate. Platelet-rich plasma (PRP) was prepared
by centrifugation at 800 rpm for 10 minutes. After that the rest was centrifuged at
3,500 rpm for 10 minutes to obtain platelet-poor plasma (PPP). PPP was used to
dilute PRP in ratio 1:1.
Platelet aggregation study
The platelet aggregation was studied by turbidimetric method. PRP samples
were stimulated using ADP as agonists. It was monitored in an aggregometer
(AggRAM, Helena Biosciences, UK) at 37°C under continuous stirring at 1,000
rpm.
PRP samples (295 l) was pre-incubated with10 l of 50% or 100% pomelo
juice (TD, KY and KT) for 5 and 30 minutes at 37°C in aggregometer. Then, it was
stimulated with 5 l of 8 M ADP (as shown in final concentration). The extent of
platelet aggregation was estimated by measuring the maximum height above the
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baseline reached by the aggregation curve within 5 minutes following stimulation.
Results
Determination of nitrite content, total polyphenolic content, total flavonoid
contents and antioxidant activities
Results of nitrite content, total phenolic content, total flavonoids content
and antioxidant activities were shown in Table 1. The results showed that TD had
higher phenolic and flavonoids content than others, but the significant difference
was not observed. TD also had higher antioxidant activity than others.
Table 1.
Nitrite content, total phenolic content, total flavonoids content and
antioxidant activities of pomelo juice
Parameters
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TD
Value a
KT
KY
Nitrite content
3.41 ± 0.48
5.53 ± 0.27
4.37 ± 0.62
(μM)
Total phenolic content
2.39 ± 0.02
2.06 ± 0.08
2.00 ± 0.04
(GAEb, mM)
Total flavonoids content
6.85 ± 0.54
6.00 ± 0.33
6.32 ± 0.05
(QEb, μM)
DPPH assay
5.21 ± 0.38
3.98 ± 0.21
4.84 ± 0.62
(TEACb, mM)
FRAP assay
4.59 ± 0.45
3.37 ± 0.07
3.90 ± 0.36
(VCEACb, mM)
a
The value shown in mean ± SD, n=3
b
Abbreviations: Gallic acid equivalence (GAE), Quercetin equivalence (QE),
Trolox equivalent antioxidant capacity (TEAC) and vitamin C equivalent
antioxidant (VCEAC)
Antiplatelet activity of pomelo juice
PRP sample was pre-incubated with pomelo juice and then stimulated with
8 M ADP. In control, without pomelo juice, ADP-induced platelet aggregation
was about 87-94% as shown in Figure 1. The pomelo juice affected to ADPinduced platelet aggregation depending on incubation time and concentration.
Figure 1a and 1b showed that the lower concentration of pomelo juice
(50%) had slightly inhibitory effect on the value of maximum aggregation.
However, all of them altered the aggregation curve into biphasic shape. KY
marginally decreased the value of maximum aggregation to 77.8% after 5 minutes
of pre-incubation while TD and KT marginally decreased the value of maximum
aggregation to 76.1% and 82.5%, respectively after 30 minutes of pre-incubation.
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(a) 50% pomelo juice, 5 minutes
(b) 50% pomelo juice, 30 minutes
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Figure 1.
(d) 100% pomelo juice, 30 minutes
Platelet aggregations after incubation with pomelo juice for 5 and 30
minutes (TD; Thong Dee, KT; Khao Tang Kwa and KY; Khao Yai)
Platelet samples were pre-incubated with 100% pomelo juice for 5 minutes
as shown in Figure 1c. The results shown KY greatly decreased the value of
maximum aggregation (29.5%) followed by KT (60.4%) and TD (85.8%). For 30
minutes of incubation time (Figure 1d), TD decreased the value of maximum
aggregation to 51.6%, while KT and KY shown similarly response curve.
The maximum aggregation and %inhibition, which was calculated
comparing to control of each group, were shown in Table 2.
Table 2.
The maximum aggregation of pomelo juice pre-incubated platelet
and percentage inhibition of maximum aggregation of pomelo juice
Incubation
time
5 minutes
30 minutes
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(c) 100% pomelo juice, 5 minutes
Sample
Name
TD
KT
KY
TD
KT
KY
Maximum aggregation
(%)
50% juice 100% juice
91.5
85.8
86.8
60.4
77.8
29.5
76.1
51.6
82.5
63.4
88.5
64.7
% Inhibition
50% juice
2.0
12.2
12.6
5.3
-
100% juice
6.7
34.3
67.9
45.1
32.6
31.2
Discussion
Pomelo juice from TD, KT and KY contained high polyphenol content as
shown in gallic acid equivalence. The phenolic content was in millimorlar level
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which was corresponded to levels of antioxidant activities as demonstrated by
values obtained from DPPH and FRAP assay. On the other hand, the value of
flavonoids content was in micromolar. The value may underestimate because of
the limitation of technique of flavonoids assay that due to high content of water in
pomelo juice may enhance dissociation between flavonoids and aluminum (III)12.
Therefore, flavonoids content will be further determined by high performance
liquid chromatography (HPLC) technique.
The results demonstrated that pomelo juice had inhibitory effect on platelet
function. Although the low concentration and shorter pre-incubation time had not
significant effect on maximum platelet aggregation, the biphasic shape of the curve
may indicate that pomelo juice altered the activation and delay aggregation process.
Several lines of evidences have indicated the roles of flavonoids as antiplatelet
functions thru several mechanisms including inhibition of ADP receptors (P2Y1
and P2Y12)6-7. Activation of platelet induced the production of free radicals that
involve in the platelet aggregation pathway13-15. Therefore, mechanism related to
antioxidants should be considers. In addition, three cultivars of pomelo juice (TD,
KT and KY) have nitrite content in range 3-6 μM. We also suspected the effect of
nitrate which nitrite is product of nitric oxide (NO). NO is chemical compound that
potentially inhibit platelet aggregation16-17. The mechanism of antiplatelet activity
of pomelo juice will be investigated.
Conclusion
Pomelo juice had high content of antioxidants and exhibited antiplatelet
activity. The potency of antiplatelet activity depended on concentration, incubation
time and cultivars. TD showed the highest potency among cultivars. The extract
mechanism of antiplatelet activity will be future investigated.
Acknowledgements
This study was supported by Thailand Research Fund (RDG5820013) and
Development and Promotion of Science and Technology Talents Project (DPST).
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