Black Sorghum Phenolic Extract Modulates Platelet Activation and Platelet Microparticle Release

Abstract

Platelet hyper-activation and platelet microparticles (PMPs) play a key role in the pathogenesis of cardiovascular diseases. Dietary polyphenols are believed to mimic antiplatelet agents by blunting platelet activation receptors via its antioxidant phenomenon. However, there is limited information on the anti-platelet activity of grain-derived polyphenols. The aim of the study is to evaluate the effects of sorghum extract (Shawaya short black 1 variety), an extract previously characterised for its high antioxidant activity and reduction of oxidative stress-related endothelial dysfunction, on platelet aggregation, platelet activation and PMP release. Whole blood samples collected from 18 healthy volunteers were treated with varying non-cytotoxic concentrations of polyphenol-rich black sorghum extract (BSE). Platelet aggregation study utilised 5 µg/mL collagen to target the GPVI pathway of thrombus formation whereas adenine phosphate (ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation assessed by flow cytometry. Procaspase-activating compound 1 (PAC-1) and P-selectin/CD62P were used to evaluate platelet activation- related conformational changes and degranulation respectively. PMPs were isolated from unstimulated platelets and quantified by size distribution and binding to CD42b. BSE treatment significantly reduced both collagen-induced platelet aggregation and circulatory PMP release at 40 µg/mL (p < 0.001) when compared to control. However, there was no significant impact of BSE on ADP-induced activation-dependent conformational change and degranulation of platelets. Results of this study suggest that phenolic rich BSE may confer cardio-protection by modulating specific signalling pathways involved in platelet activation and PMP release.

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nutrients
Article
Black Sorghum Phenolic Extract Modulates Platelet
Activation and Platelet Microparticle Release
Borkwei Ed Nignpense 1, Kenneth A Chinkwo 1,2 , Christopher L Blanchard 1,2
and Abishek B Santhakumar 1, 2, *
1
School of Biomedical Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia;
bednignpense@csu.edu.au (B.E.D.); kchinkwo@csu.edu.au (K.A.C.); CBlanchard@csu.edu.au (C.L.B.)
2
Australian Research Council (ARC) Industrial Transformation Training Centre (ITTC) for Functional Grains,
Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, NSW 2650, Australia
*Correspondence: asanthakumar@csu.edu.au; Tel.: +61-2-6933-2678
Received: 20 May 2020; Accepted: 10 June 2020; Published: 12 June 2020


Abstract:
Platelet hyper-activation and platelet microparticles (PMPs) play a key role in the
pathogenesis of cardiovascular diseases. Dietary polyphenols are believed to mimic antiplatelet
agents by blunting platelet activation receptors via its antioxidant phenomenon. However, there
is limited information on the anti-platelet activity of grain-derived polyphenols. The aim of the
study is to evaluate the effects of sorghum extract (Shawaya short black 1 variety), an extract
previously characterised for its high antioxidant activity and reduction of oxidative stress-related
endothelial dysfunction, on platelet aggregation, platelet activation and PMP release. Whole
blood samples collected from 18 healthy volunteers were treated with varying non-cytotoxic
concentrations of polyphenol-rich black sorghum extract (BSE). Platelet aggregation study utilised
5
µ
g/mL collagen to target the GPVI pathway of thrombus formation whereas adenine phosphate
(ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation assessed by flow cytometry.
Procaspase-activating compound 1 (PAC-1) and P-selectin/CD62P were used to evaluate platelet
activation- related conformational changes and degranulation respectively. PMPs were isolated from
unstimulated platelets and quantified by size distribution and binding to CD42b. BSE treatment
significantly reduced both collagen-induced platelet aggregation and circulatory PMP release at
40
µ
g/mL (p<0.001) when compared to control. However, there was no significant impact of BSE on
ADP-induced activation-dependent conformational change and degranulation of platelets. Results
of this study suggest that phenolic rich BSE may confer cardio-protection by modulating specific
signalling pathways involved in platelet activation and PMP release.
Keywords: black sorghum; polyphenols; platelets; platelet microparticles; atherosclerosis
1. Introduction
According to a World Health Organisation report, cardiovascular diseases accounted for an
estimated 31% of deaths globally with majority being a result of stroke or heart attack [
1
,
2
]. In clinical
settings, treatment involves blunting the activity of platelets using antiplatelet drugs. These drugs
interfere with the thrombotic pathophysiology—wherein a rupture of an atherosclerotic plaque triggers
platelet hyper-activation resulting in unwanted clot formation and occlusion of the blood vessel.
Macrovesicles referred to as platelet microparticles (PMPs) are released following platelet activation
and can contribute to the thrombotic situation [3,4].
The several signalling pathways involved in platelet activation and thrombus formation
include receptor-agonist pathways such as P2Y1/P2Y12-ADP, GPVI-collagen, PAR1-thrombin and the
COX-1-thromboxane [
5
]. An agonist such as collagen when exposed by atherosclerotic plaque may
Nutrients 2020,12, 1760; doi:10.3390/nu12061760 www.mdpi.com/journal/nutrients

Nutrients 2020,12, 1760 2 of 11
activate nearby platelets by binding to their GPVI receptor resulting in complex intracellular signalling
that produce a conformational change (indicated by GPIIb/IIIa receptor expression), degranulation
(indicated by P-selectin secretion) and subsequent formation of platelet aggregates [
5
]. In addition, PMP
released upon activation possess adhesive and pro-coagulant platelet-derived receptors that further
enhance thrombus formation, thereby acting as biomarkers of platelet activation [
3
]. The common
antiplatelet agents, clopidogrel and aspirin, used in clinical treatments inhibit platelet activation
and its circulating biomarkers by selectively targeting P2Y1/P2Y12-ADP and COX-1-thromboxane
respectively [
6
]. Unfortunately, because of the unresponsiveness and side effects associated
with administration there have been considerable research in dietary bioactive agents known as
polyphenols [7].
One such example of a polyphenol-rich functional food is sorghum whole grain. Although mainly
used as animal feed, studies have demonstrated that it possesses anti-inflammatory, anti-cancer and
antioxidant properties which add value to it as a food for human consumption [
8
]. Sorghum of
different types exist that are classified based on the pigmentation of the pericarp and vary in their
phenolic content [
9
]. The polyphenols found in sorghum that contribute to its bioactivity include
flavonoids, hydroxybenzoic acids and hydroxycinnamic acid [
8
]. Furthermore Francis et al. [
10
] recently
demonstrated that black sorghum rich in catechins and their derivatives may confer cardioprotective
properties. The treatment of human umbilical vein cells with flavonoid-rich extract was found to
prevent oxidative stress-related endothelial dysfunction through the modulation of gene expression.
Furthermore, these cardio-protective benefits of polyphenols apply in the context of platelet
function. Several studies have demonstrated that polyphenols may inhibit platelet activation,
adhesion, degranulation and aggregation by targeting specific thrombogenic pathways for example
P2Y
1
/P2Y
12
-ADP, GPVI-collagen, PAR1-thrombin and the COX-1-thromboxane. As reviewed by Ed
Nignpense et al. [
11
] many of the studies that investigate the polyphenol impact on platelet function
and PMP generation utilise aggregometry and flow cytometry. However there is limited research on
sorghum-derived polyphenols in modulating biomarkers of platelet activation. This study aims to
investigate the impact of black sorghum derived polyphenol extracts on collagen-induced platelet
aggregation, ADP-induced platelet activation and PMP generation.
2. Materials and Methods
2.1. Research Ethics
The study protocol was approved by the Charles Sturt University Human Research Ethics
Committee (HR17012) and the Institutional Biosafety Committee (19HB02). The study was performed
in compliance with relevant laws and institutional guidelines.
2.2. Volunteer Recruitment
Eighteen healthy volunteers between 18–65 years of age (9 males and 9 females) were recruited
from Charles Sturt University and the local community. Informed consent was obtained from all
participants prior to commencement of the study. The criteria for recruitment involved normal health
status with no history of conditions such as cardiovascular, metabolic, liver or lung disease. Other
parameters that could affect the integrity of the analysis such as alcohol consumption, smoking,
pregnancy, allergies or venepuncture difficulty were considered during the recruitment process.
A health screening questionnaire was used to assess the already mentioned parameters. A dietary
questionnaire (adapted from WINTEC and NZ academy of sport) was used to assess the usual dietary
intake of volunteers and to avoid recruitment of participants on a high antioxidant diet. The cut-off
figure for each type of food listed in the questionnaire was based on nutrient reference ranges for
Australia and New Zealand—recommended daily nutrient intake values.

Nutrients 2020,12, 1760 3 of 11
2.3. Blood Collection and Processing
After fasting for at least 8 h, whole blood was collected from each participant by a trained
phlebotomist into a tri-potassium ethylene diamine tetra-acetic acid (EDTA-1.8 mg/mL concentration)
anticoagulant tube (Vacuette Greiner Bio-one, Interpath Services, Heidelberg West, VIC, Australia) and
a tri-sodium citrate (28.12 g/L concentration) anticoagulant tube (Becton, Dickson and Company, North
Ryde, NSW, Australia). A 20-mL syringe (Becton, Dickson and Company, North Ryde, NSW, Australia)
and 21-gauge 1.5-inch needle (Terumo Medical Corporation, Macquarie Park, Australia) were used to
draw blood from the median cubital vein. The purpose of choosing a larger gauge was to avoid the
activation of platelets while drawing or dispensing blood. Utmost care was taken to ensure samples
were not obtained through a traumatic collection and that none contained obvious clots. In addition,
care was taken to ensure minimal specimen handling and agitation in order to prevent artefactual
platelet activation. The first 2 mL of blood was discarded before drawing into the tri-sodium citrate
tubes in order to avoid the risk of collecting platelets activated by venepuncture. Tri-sodium tubes
were used for aggregometry and flow cytometry assays whereas the EDTA was used to perform full
blood examinations.
2.4. Full Blood Examination
Using an Abbott CELL-DYN Emerald 22 Haematology Analyser (Abbott Diagnostics, Illinois, USA),
a full blood examination (FBE) was performed on all samples. The FBE results of volunteers indicated
that the blood cell parameters were within normal reference ranges as determined by the Royal College
of pathologists of Australia. Individuals with cell counts, especially platelet counts, outside of the
reference range were excluded from the study. Quality control validation and maintenance were all
performed according to the Abbott CELL-DYN Emerald 22 Haematology Analyser manual.
2.5. Extraction of Black Sorghum Polyphenols
Sorghum (Sorghum bicolor) samples of six different pericarp varieties were obtained from glasshouse
trials conducted by Curtin University, Perth, Western Australia. Six pigmented varieties of sorghum were
cultivated under the same conditions, grown in a glasshouse equipped with Lumisol Clear AF cover
(200
µ
m thick, at a transparency of ca UV-A 94%, UV-B 84% and photosynthetic active radiation (PAR,
400-700 nm) 93% [
8
]. Extraction and analysis of phenolic composition and antioxidant activity were
performed previously using methods described by Rao et al. [
8
]. Among the different sorghum varieties,
the black pericarp variety (Shawaya short black 1) was selected for this study because of its relatively high
antioxidant activity when analysed with ferric reducing antioxidant power (FRAP; 20.19
±
2.69 mg/g TE)
and 2,2-dipheny-1-picrylhydrazyl (DPPH; 18.04
±
3.53 mg/g TE) antioxidant assays (Supplementary Figure
S1, Tables S1 and S2). The highest level of polyphenols found in the BSE included catechin derivatives,
catechins and pentahydroxyflavanone-(3->4)-catechin-7-O- glucoside (Supplementary Table S2). Stock
concentrations of BSE (20 mg/mL in 50% DMSO) were diluted in phosphate buffered saline (PBS) to
achieve desired concentrations (5
µ
g/mL, 20
µ
g/mL and 40
µ
g/mL) inwhole blood. Desired concentrations
were selected based on viability studies done by Francis et al. [10].
2.6. Whole Blood Platelet Aggregometry
Platelets in whole blood were stimulated for aggregation using 5
µ
g/mL collagen exogenous platelet
agonists (DSKH Australia Pty. Ltd., Hallam, VIC, Australia) to investigate the effect of BSE treatment on the
platelet aggregation. Five hundred microliters of citrated whole blood were added to 100
µ
L of 0.1% DMSO
control (Sigma-Aldrich, Castle Hill, NSW, Australia) or BSE stock concentrations (5
µ
g/mL, 20
µ
g/mL and
40 µg/mL) and mixed with 400 µL of saline. The sample was then incubated at 37 ◦C for 20 min. Using a
Chrono-log model 700 lumi-aggregometer (DKSH Australia Pty. Ltd., Hallam, VIC, Australia) the samples
were analysed by means of electrical impedance (ohms) to determine the amount of platelet aggregation
occurring in the sample over a 6-min time period (Supplementary Figure S2).

Nutrients 2020,12, 1760 4 of 11
2.7. Flow Cytometry
2.7.1. Standardisation
Flow-check fluorospheres were run as quality control for optimal laser alignments. Antibody
capture beads (Anti-Mouse Ig, K CompBeads, BD Biosciences, North Ryde, NSW, Australia) were
used for single colour compensation controls in order to achieve optimal compensation. Megamix
beads (0.1
µ
m, 0.3
µ
m, 0.5
µ
m, 1
µ
m) from Biocytex, Marseille, France were used as per manufacturer’s
instructions to set up an appropriate gating to detecting microparticles. They were run before each
PMP analysis.
2.7.2. Measurement of Platelet Activation-Dependent Conformational Change and Degranulation
The effects of BSE on ADP-induced platelet activation were analysed and interpreted using a
Gallios flow cytometer (Beckman Coulter, Inc., Lane Cove NSW, Australia). The protocols were adopted
from the method described by Santhakumar et al. [
12
] with some modifications. Platelet activation and
thrombogenic indicators were assessed via activation-dependent platelet monoclonal antibodies (mAbs)
purchased from Becton, Dickinson and Company, North Ryde, NSW, Australia. Procaspase activating
compound-1 (PAC-1)-fluorescein isothiocyanate-fluorescein isothiocyanate was used to detect platelet
activation-related conformational change and P-selectin/CD62P-allophycocyanin highlighted activation
dependent degranulation. CD42b-phycoerythrin identified the GPIb-IX-V receptor, a common receptor
found on the surface of all platelets, activated and resting included. A decreased expression of mAb
exhibits alleviation of thrombogenesis. Within 5 min of collection tri-sodium citrated whole blood was
used for assay preparation to avoid artefactual activation of platelets. A volume of 40
µ
L of blood was
incubated with DMSO control or the various BSE concentration for 20 min at 37
◦
C in the dark. A 10-
µ
L
mixture of all three monoclonal antibodies (3.33
µ
L each of PAC-1, CD62P and CD42b) was added to
blood samples and incubated for 20 min at room temperature in the dark. To induce platelet activation,
10
µ
M ADP (Helena laboratories Pty. Ltd., Mt Waverly, VIC, Australia) was added, and samples
were incubated for a further 15 min in the dark at room temperature, after which erythrocytes were
lysed (575
µ
L of 10 % lysing solution). Samples were thoroughly vortexed to ensure homogeneity and
incubated in the dark at room temperature for a further 15 min and then analysed. In all, 10,000 platelet
events were acquired, gated based on light scatter and CD42b mAb expression and activated platelets
were articulated as mean fluorescence intensity (MFI) (Supplementary Figure S3).
2.7.3. Measurement of Circulatory PMPs
Using the microparticle gating established with Megamix beads, PMPs were identified and
quantified by size distribution and binding to CD42b (Supplementary Figure S4). The protocol for
circulatory PMP analysis was adapted from Lu et al. [
13
]. A volume of 1 mL whole blood was
added to micro-centrifuge tubes in the presence of PGE1 (120 nmol/L; Sigma-Aldrich, Castle Hill,
NSW, Australia). PGE1 was added to prevent artefactual activation during centrifugation. The blood
samples were incubated with the respective 100
µ
L BSE concentrations and DMSO control for 20 min
at 37
◦
C in the dark. Each sample was then centrifuged for 15 min at a 1000 rpm and the resultant
platelet rich plasma (PRP) was discarded. The remaining blood was spun a further 15 min at 3000 rpm.
The supernatant rich in PMPs (40
µ
L) was collected into flow tubes and incubated with 4
µ
L of CD42b
and 6
µ
L of stain buffer (Becton, Dickson and Company, North Ryde, NSW, Australia) in dark room for
15 min. Four percent formaldehyde was used to fix any activation of platelets left in the supernatant.
After a 10-min incubation period the samples were run for PMP analysis on the flow cytometer.
2.8. Statistical Analysis
A two-way ANOVA following Tukey’s post comparison test was performed using GraphPad
Prism version 8.0 for Windows (GraphPad Software, La Jolla, California, USA). A minimum sample
size of 14 participants in total was required for 80% power to detect a 5% variation in the laboratory

Nutrients 2020,12, 1760 5 of 11
parameters measured where a 3–5% standard deviation exists in the population, assuming an alpha
error of 0.05. All the data were expressed as mean
±
standard deviation (SD). Differences between the
groups were significant when p<0.05. Any significant statistical interactions were included in the
analysis where applicable.
3. Results
The baseline parameters including full blood counts for all 18 participants were within normal
reference ranges set by the Royal College of Pathologists of Australasia (Table 1) [14].
Table 1. Baseline full blood counts of participants.
Parameters Mean ±SD
Age (years) 26 ±8
WBC (×109/L) 5.5 ±1.3
Neutrophil (%) 48.6 ±9.5
Lymphocytes (%) 37.8 ±9.2
Monocytes (%) 10.6 ±2.3
Eosinophils (%) 2.8 ±1.3
Basophils (%) 0.1 ±0.1
RBC (×1012/L) 4.6 ±0.5
Haemoglobin (g/L) 147.9 ±16.1
PCV (%) 0.41 ±0.04
MCV (fL) 90.0 ±3.5
MCH (pg) 35.8 ±15.2
MCHC (g/dL) 360.1 ±6.0
RDW (%) 14.9 ±0.8
Platelet count (×109/L) 248.3 ±50.0
MPV 8.41 ±0.89
Values are represented as mean
±
Standard deviation (SD). RBC, red blood cell, PCV, packed cell volume, MCV,
mean cell volume, MCH, mean cell haemoglobin, MCHC, mean cell haemoglobin concentration, RDW, red cell
distribution width, MPV, mean platelet volume.
3.1. Effect of BSE on Whole Blood Platelet Aggregation and Platelet Activation
BSE at 40
µ
g/mL concentration significantly reduced platelet aggregation stimulated by collagen
by 19 % (p=0.0004) (Figure 1). BSE at lower concentrations did not exhibit any significant reduction
in aggregation.
It was observed that whole blood treatment with the varying concentrations of BSE did not
significantly affect ADP-induced platelet conformational change and degranulation indicated by PAC-1
and P-selectin expression respectively (Supplementary Figures S5 and S6).
3.2. Effect of BSE on Circulatory PMPs
BSE at a concentration of 40
µ
g/mL significantly reduced the amount of circulatory PMPs in whole
blood by 47% (p=0.0008). Lower concentrations of BSE did not exhibit any significant reduction to the
amount circulatory PMPs (Figure 2).

Nutrients 2020,12, 1760 6 of 11
Figure 1.
The effect of varying concentrations of BSE on collagen-induced aggregation. BSE at 40
µ
g/mL
significantly reduced platelet aggregation (5.3
±
1.3; pvalue =0.0004). BSE at 5
µ
g/mL and 20
µ
g/mL
did not reduce platelet aggregation when compared to control (pvalue >0.1). N =18 and data is
represented in aggregation (Ohms) versus BSE concentrations. *** signifies statistical significance
p<0.001 compared to control. Error bars expressed as mean ±SD.
Figure 2.
The effect of varying concentrations of BSE on circulatory PMP production
in vitro
. BSE at
40
µ
g/mL significantly reduced the amount of circulatory PMPs (<24190
±
4935, p=0.0008). BSE at
5
µ
g/mL and 20
µ
g/mL did not reduce platelet aggregation when compared to control (pvalue >0.1).
N=14 and data are represented in number of PMP events versus BSE concentrations. *** signifies
statistical significance p<0.001 compared to control. Error bars expressed as mean ±SD.
4. Discussion
There is growing interest in understanding the therapeutic benefits of functional foods. Sorghum
for example is one of the functional foods that is showing promise in this area. With sorghum-derived
polyphenols already having demonstrated anti-inflammatory, anti-cancer and antioxidant properties,
the current study aimed to evaluate the effects of polyphenol-rich BSE on platelet function in terms of
aggregation, conformational change, degranulation and circulatory PMP production [
8
–
10
]. It was
observed that BSE significantly inhibited collagen-induced platelet aggregation and decreased the
release of circulatory PMPs but did not have a significant effect on ADP-induced platelet conformational
change or degranulation. Although these results do not reflect a typical dose-dependent inhibition,
they suggest a potential role of BSE polyphenols at optimum concentrations to interfere with pathways
in the GPVI-collagen signalling and the release of circulatory PMPs but little or no effect on P2Y
1
/
P2Y12-ADP pathway.

Nutrients 2020,12, 1760 7 of 11
To the best of our knowledge only a few studies have investigated the antiplatelet effects of
sorghum extracts. Li, Yu and Fan et al. [
15
] extracted alditols and monosaccharides from sorghum
vinegar to evaluate their anti-aggregation activity using the turbidimetric method. Results from
their study indicated a significant dose-dependent inhibition of aggregation via multiple agonists,
arachidonic acid, collagen, ADP and thrombin. Furthermore, a different study by Fan et al. [
16
]
reported
in vitro
inhibition of ADP- and thrombin- induced rabbit platelet aggregation by methanolic
extracts of aged sorghum vinegar with the half maximal inhibitory concentrations (IC
50
) of 1.7
±
0.3
and 8.9
±
1.9 mg/mL respectively. When rats were orally administered the extracts (>100 mg/kg),
both collagen- and epinephrine-induced pulmonary thrombosis were inhibited. In comparison with
the present study it is to be noted that these studies employed platelet-rich plasma rather than
whole blood hence not accounting for the possible involvement of other blood cells and extracellular
mediators involved in thrombus formation. In addition, sorghum vinegar extracts were used at higher
concentrations; milligrams compared to micrograms used in this study. This raises the question of
bioavailability and the importance of employing physiological concentrations of extracts.
Although the BSE concentration of 40
µ
g/mL at which antiplatelet effect were observed is relatively
lesser in concentration than used in the other studies, the most bioactive compounds with respect to
antioxidant activity were catechins and other flavonoids which are usually considered to have low
bioavailability [
8
]. It has been suggested that the total plasma polyphenol concentration rarely exceeds
1
µ
M and that their antiplatelet effects are only found at high non-physiological concentrations (greater
than 50
µ
M) [
17
,
18
]. However, it is likely that these plasma concentrations are underestimations
due to the ability of polyphenols to bind to the surface of red blood cells and thereby exert their
bioactivity [
19
]. Furthermore polyphenols (structurally related to catechin) and their metabolites have
been shown to inhibit platelet function
in vitro
[
20
]. This highlights the possibility of sorghum catechins
and their metabolites having antiplatelet effects
in vivo
despite bioavailability concern. Interestingly,
an
in vivo
human dietary intervention trial compared consumption of red and white wholegrain
sorghum-based pasta to a control pasta in order to investigate its acute effect on the total phenol content
and antioxidant activity in the plasma of healthy subjects [
21
]. Results showed that when compared to
the control pasta, the red sorghum pasta showed significantly increased net plasma phenolic content
and antioxidant activity post consumption (from 216.90
±
2.62 at baseline to 269.40
±
2.33 at 2 h;
p<0.001), thus demonstrating a plausible correlation between antioxidant activity and sorghum
polyphenol consumption—which in turn may contribute to antiplatelet effects.
The antiplatelet effects was observed with BSE-included inhibition of collagen-induced aggregation
and circulatory PMP production but no effect on ADP-induced platelet activation. The absence of
antiplatelet effects on the P2Y
1
/P2Y
12
-ADP activation pathway suggests that BSE polyphenols are
not mimicking the action of drugs such as clopidogrel that antagonise P2Y
12
receptor activation [
22
].
However, the inhibition of collagen-induced aggregation suggest that BSE polyphenols interfere
with GPVI-collagen signalling pathways by either blunting the GPVI receptor directly or by other
mechanisms [
5
]. Previous studies have demonstrated that flavonoids, specifically quercetin and
catechin, can act synergistically to inhibit collagen-induced aggregation by blunting the associated
burst of H
2
O
2
and subsequent PLC activation [
23
,
24
]. Thus, a possible mechanism of inhibition
BSE flavonoids may be a synergistic antagonism of the positive feedback activation of intracellular
signals triggered by H
2
O
2
. Moreover, it has been shown that the phosphorylation cascade initiated
by collagen can be inhibited by flavonoids [
25
,
26
]. Flavones, especially apigenin and luteolin, by
virtue of a double bond in the C2-C3 and the keto group in C4 can also inhibit collagen-induced
activation by antagonizing the TxA
2
receptor activation which is also involved in the positive feedback
loop [
27
]. Besides inhibition of the GPVI-collagen signalling, BSE polyphenols showed inhibition of
the circulatory PMP production.
To the best of our knowledge, this is the first study investigating the effect of sorghum-derived
polyphenols on PMP production. In contrast to this study, other PMP studies have employed the
use of Annexin V as well as the platelet specific antibody CD42b, to identify pro-coagulant PMPs

Nutrients 2020,12, 1760 8 of 11
by their phosphatidylserine expression and to limit background noise [
28
]. However, because of the
heterogeneity of PMPs, not all PMPs express phosphatidylserine [
29
]. Moreover, the measurement of
CD42b-positive PMPs alone is significant as its increase has been associated with an increased risk
of coronary heart disease [
30
]. From the current study, the significant inhibition of CD42b-positive
circulatory PMPs observed
in vitro
may be attributed to the antioxidant properties of BSE polyphenols.
It is believed that the inhibition of PMP generation may be the result of neutralisation of H
2
O
2
,
scavenging of other free radicals or interaction with intracellular signalling leading to PMP release.
The juxtaposition of both the present study and that of an earlier study by Francis et al. [
10
]
highlights the multifaceted role of BSE polyphenols in cardio-protection. The group investigated the
effects of BSE polyphenols on the expression of antioxidant- and inflammatory-linked genes involved
in endothelial dysfunction under oxidative stress. Results indicated that BSE polyphenols alleviate
oxidative stress–induced damage to endothelial cells. Since vascular dysfunction is a precursor to
cardiovascular diseases, the current study builds upon earlier findings by exhibiting the antiplatelet
effects of BSE. In the context of endothelial dysfunction, platelet activation and circulatory PMPs
play central roles in the pathogenesis of atherothrombosis. The disruption of the plaque exposes
collagen that binds to the GPVI receptor resulting in platelet activation and subsequent thrombus
formation [
31
]. Circulatory PMPs may contribute to thrombosis via GPIb-IX-V receptor binding and
have a pro-inflammatory effect to promote the development of the plaque [
32
]. Therefore, by reducing
collagen-induced platelet aggregation and circulatory PMP generation, BSE polyphenols may be
displaying the potential to augment thrombosis.
5. Conclusion and Future Considerations
In summary, the present study contributes to the growing body of literature on bioactivity of
sorghum polyphenols and highlights possible mechanisms of antiplatelet action that may result in
cardiovascular health benefits. Because of the ability to reduce collagen-induced platelet activation and
circulatory PMP generation, BSE polyphenols demonstrate the potential to interfere with pathological
processes involved in vascular disorders and thrombotic complications. However, a larger panel of
agonist for the flow cytometry and aggregometry studies will aid to further elucidate antiplatelet
mechanisms. Because of the bioavailability concerns, well-controlled dietary intervention trials
using larger sample sizes to evaluate the antiplatelet effects of sorghum consumption in healthy and
pro-thrombotic populations are warranted to justify our findings. Because of the varied phenolic
profiles of the different sorghum varieties, further research comparing the antiplatelet therapeutic
potential of different grains is also warranted. Furthermore, this study attest to the measurement of
circulatory PMPs as a biomarker of platelet activation to assess the bioactivity of functional foods.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2072-6643/12/6/1760/s1,
Figure S1: Characterisation of phenolic compounds in BSE. Ultra-high-performance liquid chromatography
(UHPLC) was employed to quantify the different phenolic compounds identified by the peak (on top). An online
2,2
0
-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was coupled with UHPLC to quantify the relative
antioxidant activity (peaks below) of each compound identified, Figure S2: A report derived from the collagen
induced platelet aggregation study using the Chrono-log model 700 lumi-aggregometer (DKSH Australia Pty. Ltd,
Hallam, VIC, Australia). The blue tracing represents the control (whole blood with no BSE) and the black tracing
represents the whole blood pre-treated with 5
µ
g/mL BSE. The addition of BSE reduced the maximum platelet
aggregation expressed in Ohms from 15 ohms to 11 ohms, Figure S3: A report from the ADP-induced platelet
activation analysis using Kaluza Flow Cytometry Software (Beckman Coulter, Brea, CA, USA). Results indicate
the gating of whole platelet population (CD42b positive events) and the proportion of activated platelets indicated
by PAC-1 and P-selectin expression, Figure S4: A report from the PMP analysis using Kaluza Flow Cytometry
Software (Beckman Coulter, Brea, CA, USA). Microparticle gating was established using Megamix beads of
standard sizes. PMPs were distinguished from other microparticles by size (0.5
µ
m - 0.9
µ
m) and expression of
CD42b. The number of CD42b positive events in the microparticle gate was used to quantify the PMPs, Figure S5:
The effect of varying concentrations of BSE on PAC-1 expression. BSE did not significantly reduce ADP-induced
platelet conformational change detected by PAC-1 expression (pvalues >0.1 compared to control) N=14 and data
is represented in mean fluorescence intensity (MFI) versus BSE concentrations. Error bars expressed as mean
±
SD,
Figure S6: The effect of varying concentrations of BSE on P-selectin expression. BSE did not significantly reduce
ADP-induced platelet degranulation detected by P-selectin expression (pvalues >0.1 compared to control ) N=14

Nutrients 2020,12, 1760 9 of 11
and data is represented in mean fluorescence intensity (MFI) versus BSE concentrations Error bars expressed as
mean
±
SD, Table S1: Phenolic composition and antioxidant activity of sorghum varieties on as is basis, Table S2:
List of top ten phenolic compounds identified in the black sorghum phenolic rich extracts by Q-TOF LC/MS and
quantified using UHPLC-Online ABTS system (Adapted from Rao et al., 2018).
Author Contributions:
B.E.N. conducted the experiments outlined in this study and drafted the manuscript.
K.A.C., C.L.B. and A.B.S. were involved in the designing and critical reviewing of the manuscript. All authors
have read and agreed to the published version of the manuscript.
Funding:
This study was funded by the Australian Research Council Industrial Transformations Training Centre
for Functional Grains (Project ID 100737).
Acknowledgments:
The authors would like to acknowledge Graham Centre for Agricultural Innovation for
providing funding towards open access publication of this article. Borkwei Ed Nignpense is a recipient of a
PhD Scholarship from the Australian Government Research Training Program and also a recipient of a top-up
scholarship by the Graham Centre.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
ADP Adenosine diphosphate
ATP Adenosine triphosphate
BSE Black sorghum extract
COX-1 Cyclooxygenase-1
DMSO Dimethyl sulfoxide
EDTA Ethylene diamine tetra-acetic acid
FBE Full blood examination
GAE Gallic acid equivalents
MCH Mean cell haemoglobin
MCHC Mean cell haemoglobin concentration
MCV Mean cell haemoglobin
MFI Mean fluorescence intensity
MPV Mean platelet volume
PAR Protease-activated receptor
PAC-1 Procaspase activating compound-1
PBS Phosphate buffered saline
PCV Packed cell volume
PGE1 Prostaglandin E1
PMP Platelet microparticle
RBC Red blood cell
SD Standard deviation
WBC White blood cell
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