title: Impact of edible plant extracts and isolated compounds on the modulation of platelet function
Creators: name: Albadawi, Dina Abdulrahman I.
Publisher: name: University of Reading
Year: 2022

description: Atherothrombosis is a systemic, progressive inflammatory disease that can affect 
coronary or peripheral arteries. It can lead to ischemic heart disease or stroke which are the 
principal causes of mortality and disability worldwide. In 2019, 85% of the 17.9 million total 
cardiovascular diseases (CVDs) deaths was due to heart disease and stroke. Platelets play 
a vital role in atherothrombosis by facilitating atherosclerosis initiation, propagation, and 
subsequently thrombosis. Anti-platelet drugs are of fundamental importance for the 
treatment and/or prevention of such diseases. However, the usefulness of anti-platelet drugs 
can be compromised by their serious adverse effects such as major haemorrhage and 
severe abdominal pain. In addition, drug-drug interactions can increase or decrease the 
therapeutic effect of anti-platelet drugs. Moreover, resistance can develop towards anti-platelet drugs. Thus, there is an urgent demand for safer and more effective novel anti-platelet drugs.
An unhealthy diet is one of the modifiable risk factors of atherothrombosis. Numerous 
research studies concluded that the consumption of a healthy and balanced diet is 
associated with better patient outcomes, particularly plant-based diets. Many edible plants 
are known to exert pharmacological effects on cardiovascular diseases (CVDs) as reported 
in traditional medicine and by various research studies. Indeed, some edible plant extracts 
and/or isolated compounds showed significant anti-platelet effects. Herein, the effects of
different Asian edible plants' (fruit, vegetables, spices and herbs) extracts on platelet
activation were probed. Dimethyl sulfoxide (DMSO) was used as extraction solvent due to 
its ability to extract different phytochemicals. Different preparations were prepared from each 
plant, i.e., fresh samples (named as F), dried samples (named as D), and dried samples that 
were subjected to heat during the extraction process (named as H). Extracts were tested in 
human platelet-rich plasma (PRP) against cross-linked collagen-related peptide (CRP-XL)
and in human whole blood against collagen using aggregometry. In PRP, cinnamon D and 
H, ajwain D, okra H, brown mustard H and broccoli H extracts exhibited the highest inhibition
effects. However, whole blood (WB) aggregation was inhibited significantly by cinnamon H, 
okra H, cocoa H, ajwain H, clove H and curry leaves H. In addition, in WB the inside-out 
signalling to integrin αIIbβ3 was inhibited mainly by red onion D, tomato H, cinnamon H, tomato 
D, okra H, black pepper H, fennel H and curry leaves H. However, curry leaves H, broccoli 
H and okra H extracts had the most inhibitory effects α-granule secretion in WB. The inside-out signalling to integrin αIIbβ3 was augmented by bird eye chili F, broccoli H and brown
mustard H extracts. On the other hand, P-selectin exposure on platelets was activated
insignificantly by ridge gourd H, drumstick H, red onion D and bird eye chili F extracts. Based 
on the results, Abelmoschus esculentus (okra) pods and Murraya koenigii (curry) leaves 
were selected for further investigation along with their isolated compounds phytol (from okra), koenigicine and mahanimbine (from curry leaves) because of their promising anti-platelet 
effects and no data is available in the literature about their anti-platelet effects. Both plants
were subjected to different extraction methods using different solvents. The extracts of both 
plants and isolated compounds were tested using different ex vivo platelet functional assays.
Ethanolic and aqueous extracts of okra pods were obtained using maceration and Soxhlet 
extraction. Liquid-liquid extraction (LLE) was also performed using n-hexane, ethyl acetate, 
chloroform, and n-butanol. Room temperature ethanolic extract of okra pods showed 
significant inhibition on CRP-XL-induced PRP activation. Most of preparative thin layer 
chromatography (PTLC) fractions significantly inhibited platelet aggregation against ADP.
However, extracts obtained from LLE did not affect platelet activation. Anti-platelet effects 
of phytol against different agonists were examined after the spectroscopic characterisation 
using Fourier transform infrared (FT-IR), proton nuclear magnetic resonance (1H NMR), 
carbon nuclear magnetic resonance (13C NMR), and gas chromatography-electron 
ionization/mass (GC-EI/MS) spectrometry. Phytol inhibited ADP and collagen human PRP
activation and collagen isolated platelet aggregation. Fibrinogen binding and P-selectin 
exposure levels were inhibited to different extends against ADP, collagen, and TRAP-6. Clot 
retraction and platelet adhesion to immobilised collagen were also significantly inhibited by 
phytol.
 Curry leaves were extracted using the same extraction techniques and solvents as 
okra pods. Alkaloid extraction was also performed due to high alkaloidal content of curry 
leaves. The crude ethanolic extract of curry leaves displayed the highest inhibitory effects on 
platelet activation against CRP-XL activated PRP. In addition, the alkaloid rich extract of 
curry leaves demonstrated significant reduction on platelet activation indued by ADP, 
collagen, and U46619. Koenigicine and mahanimbine were isolated from curry leaves
characterised using the same spectroscopic methods used for phytol. The effects of these 
compounds were examined on the modulation of human platelet function using different 
platelet functional assays. Koenigicine and mahanimbine significantly inhibited collagen, 
ADP, and CRP-XL-activated human PRP aggregation and collagen-induced isolated 
platelet aggregation. The compounds showed different inhibitory effects on fibrinogen 
binding and P-selectin exposure levels in human PRP stimulated by ADP, collagen, CRP-XL, U46619, and TRAP-6. The two compounds significantly reduced platelet adhesion 
under static conditions and clot retraction.
 Overall, these data demonstrate that these extracts and isolated compounds exert 
prominent inhibitory effects on human platelets, and thus, they may act as potential anti-platelet agents. Moreover, regular consumption of okra pods and curry leaves is likely to 
prevent unnecessary activation of platelets within the circulation.