Abstract/Summary

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.