Inflammation, atherosclerosis, and thrombosis are all viewed as cardiovascular manifestations demonstrated by a cluster of risk factors. The prevalence and fatality rate of cardiovascular diseases (CVDs) worldwide, for instance, stroke and coronary heart disease, urged the progression in medical research [
In biological perspective, free flow of blood is of utmost importance. Platelets, smallest type of blood cell, play essential role in regulating smooth blood circulation, vascular integrity, and hemostasis [
Antiplatelet agents (APA) including aspirin, clopidogrel, and cilostazol are often used in prescription and remained as mainstay medication in vascular thrombotic diseases. However, the various side effects of such medications, for example, internal bleeding, rash, drowsiness, and bruising, impede their wide use in clinical aspects [
Natural antiplatelet agents (APA) that modulated platelet functions attained great attention from the public in primary and secondary prevention of cardiovascular diseases (CVD). Policosanol, a generic term for a mixture of long chain aliphatic alcohols (C20–C30), is usually extracted from animal and plant waxes, for example, sugarcane, wheat varieties, cereal, yam, and beeswax [
ADP was purchased from Chronolog Incorporation (Havertown, PA, USA). Aspirin was a generous gift from Mr. Choy. All the reagents and solvents used in extraction and analysis were analytical grade purchased from Merck (Darmstadt, Germany). Rice bran was obtained from Bernas milling factory in Kuala Selangor, Selangor, Malaysia. All the consumables used in animal studies were supplied by Takrif Bistari Sdn Bhd (Selangor, Malaysia). Mazola corn oil (Unilever Malaysia), Nespray fortified milk powder (Nestle Malaysia), and Specialty feeds (TN, USA) were used in high fat diet preparation.
Policosanol was extracted using solid-liquid extraction according to the described procedures minor modifications [
HFD was formulated following the recipe by Levin et al. [
Experiments were conducted in accordance with the guidelines for animal use and ethical approval was obtained from institutional animal care and use committee (IACUC), Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia. The animals (36 adult male Sprague-Dawley rats) with body weight between 100 and 150 g were randomly divided into six experimental groups. All the rats were acclimatized for 7 days
Platelet aggregation study was done using ADP as agonist according to Vaiyapuri and Gibbins [
Full blood count was done using hematology analyzer (Medonic CA530). Fresh rat blood was withdrawn into EDTA anticoagulated blood tubes and analyzed within 4 hours. Baseline and final readings were taken before and after treatment for comparison.
Rat tail bleeding time was measured according to the described procedures with minor modifications [
Extrinsic and intrinsic pathways of coagulation were analyzed by measuring prothrombin time (PT) and activated partial thromboplastin time (aPTT). PT and aPTT were analyzed followed the standard protocol of Synergy Start 4 hemostasis analyzer (Diagnostica Stago Inc.).
Stable platelet metabolites, thromboxane B2 and 6-keto PGF1
The data were analyzed using minitab 16 (Minitab Inc., State College, Pennsylvania, United States) by one-way analysis of variance (ANOVA) and the values were presented as means and standard deviation (SD). The significance of the differences between comparisons was determined by Tukey’s range test. The significant difference was taken to be a value of
Platelet, despite being the smallest type of blood cell, exerts enormous influence on physiology. Disturbance of platelet functions is attributed to a series of ischemic events [
Percentage of
The inhibitory action of rice bran policosanol extract (RBE) on
Degree of
Figure
Platelet activation was always accompanied with p-selectin expression. Measurement of soluble p-selectin is a form of important marker in determining the risk of thrombotic disorders as activated platelet was shown to shed their membrane bound p-selectin into blood plasma [
Serum level of soluble p-selectin after 30 days of RBE extract or aspirin treatment on experimental rats. Means which shared the same letter were not significantly different at
Serum level of soluble von Willebrand factor (vWF) after 30 days of RBE extract or aspirin treatment on experimental rats. Means which shared the same letter were not significantly different at
As shown in Table
Full blood count analysis using hematology analyzer (baseline: day 0 of treatment; final reading: day 30 of treatment).
Groups | RBC (1012/L) | MCV | RDW | HCT | PLT (109/L) | MPV (fl) | HGB (g/L) | MCH (pg) | MCHC (g/L) |
---|---|---|---|---|---|---|---|---|---|
(fl) | (%) | (%) | |||||||
NP (before treatment) | 8.005 ± 0.04 |
53.95 ± 0.35 |
10.7 ± 2.12 |
40.85 ± 3.75 |
712 ± 97.58 |
6.7 ± 0.14 |
148.5 ± 9.19 |
19.75 ± 0.64 |
367 ± 14.14 |
HFD (before treatment) | 7.49 ± 0.68 |
48.58 ± 1.36 |
14.35 ± 1.32 |
36.21 ± 3.23 |
844.25 ± 157.46 |
6.93 ± 0.12 |
133.78 ± 10.45 |
17.91 ± 0.78 |
366.3 ± 6.70 |
NP (after treatment) | 7.21 ± 1.01 |
52.97 ± 1.89 |
13.83 ± 2.46 |
38.1 ± 4.42 |
730 ± 97.58 |
7.27 ± 0.45 |
138.67 ± 20.50 |
19.27 ± 0.72 |
363.67 ± 13.65 |
HFD-NC (after treatment) | 7.8 ± 0.71 |
49.47 ± 1.29 |
13.53 ± 0.67 |
38.53 ± 2.51 |
1155.33 ± 160.55 |
6.77 ± 0.06 |
143.33 ± 8.08 |
18.37 ± 0.67 |
372 ± 3.61 |
500 mg/kg | 7.66 ± 1.04 |
47.27 ± 1.88 |
15.53 ± 0.67 |
36.3 ± 6.16 |
756 ± 86.27 |
7.53 ± 0.91 |
131.67 ± 23.44 |
17.13 ± 1.03 |
362.33 ± 8.74 |
250 mg/kg | 8.29 ± 0.25 |
47.37 ± 1.25 |
14.87 ± 0.84 |
39.3 ± 1.31 |
961 ± 147.89 |
7.13 ± 0.40 |
143.33 ± 4.62 |
17.27 ± 0.55 |
365 ± 2 |
100 mg/kg | 7.86 ± 0.31 |
48.23 ± 1.32 |
15.23 ± 1.67 |
37.9 ± 1.4 |
772 ± 288.5 |
7.13 ± 0.61 |
138 ± 6.08 |
17.57 ± 0.40 |
364 ± 3.46 |
Aspirin-PC | 8.31 ± 0.23 |
46.53 ± 0.81 |
15.3 ± 1.42 |
38.67 ± 0.64 |
902 ± 106.07 |
7 ± 0.7 |
140.33 ± 3.51 |
16.87 ± 0.23 |
363 ± 3.61 |
NC: negative control; PC: positive control; NP: rats which consume normal pellet; HFD: rats which consume high fat diet throughout 3 months study period,
Bleeding time, the time taken for bleeding to stop, was an important test in platelet function assessment [
Tail bleeding time (BT), activated partial prothrombin time (aPTT), and prothrombin time (PT) in secs of various experimental groups after oral administration of RBE or aspirin for one month,
Groups | BT (secs) | aPTT (secs) | PT (secs) |
---|---|---|---|
HFD-negative control | 484 ± 45.36 |
19.7 ± 1.8 |
19.8 ± 0.8 |
500 mg/kg | 573.6 ± 39.11 |
16.8 ± 3.1 |
17.7 ± 1.5 |
250 mg/kg | 540.8 ± 93.59 |
17.5 ± 1.8 |
18.8 ± 0.2 |
100 mg/kg | 521.2 ± 97.16 |
16.3 ± 0.3 |
17.7 ± 1.6 |
Aspirin-PC | 597 ± 96.77 |
15.1 ± 0.1 |
18.4 ± 0.8 |
The presented value was mean ± standard deviation. Means that shared the same letter were not significantly different at
Practically, platelets were activated by numerous physiological factors through a multitude of mechanism, for instance, interaction with platelet receptors or glycoproteins, modulation of intracellular platelet messengers, and regulation of platelet signaling products. Despite different pathway of platelet activation, platelet responded in the same series initiated with shape change, secretion, liberation including prostaglandins, or lipooxygenase products, followed by aggregation [
Serum level of thromboxane B2 after 30 days of aspirin/RBE treatment,
Serum level stable metabolite of prostaglandin I2 (6-keto PGF1
From the result obtained, we can boldly hypothesize that RBE might serve as antagonists of receptor GPIIb/IIIa (fibrinogen receptor), P2Y1 and P2Y12, thus preventing the ADP-induced
Additionally, antioxidant ability of RBE was a potential factor in reducing platelet aggregation. Inhibition power of RBE on endogenous and exogenous free radicals from attacking platelets must not be discarded [
Organs weight (heart, liver, spleen, and kidney) of sacrificed rats from various experimental groups. The presented values were mean ± standard deviation and means denoted with same letters were not significantly different at
Increasing trend of the body weight (in grams) of rats from various experimental groups (negative control group, policosanol-treated groups, and aspirin-treated group),
The present study demonstrated that rice bran policosanol extract (RBE) potentiates antiplatelet effect using
The authors of this study report no competing interests in the work.
Maznah Ismail and Eusni Rahayu Mohd Tohit provided the ideas for the paper. Wai-Teng Wong and Yi-Da Zhang designed the experiment and conducted the laboratory works. Wai-Teng Wong participated in analyzing the data and drafting the manuscript. Rasedee Abdullah helped in data analysis and discussion. All authors read, revised, and approved the final manuscript. Maznah Ismail gave the final approval of the version to be published.
The financial support from Ministry of Education Malaysia (Knowledge Transfer Program, Project no. 6228130) is gratefully acknowledged. The authors thank Padiberas Nasional Berhad (Selangor, Malaysia) for providing rice milling by-product and IBS staffs for the kind assistance. The authors would like to acknowledge Mustapha Umar Imam who participated in analyzing the data and drafting the manuscript.