Potential antithrombotic effect of two new phenylpropanoid sucrose esters and other secondary metabolites of Canna indica L. rhizome

Abstract Canna indica L. has been traditionally used to treat various diseases. Based on previously reported antithrombotic effect for this plant, two new phenylpropanoid sucrose esters (canindicoside A (1) and canindicoside B (2)) and seven known compounds: nepetoidin B (3), caffeic acid (4), ferulic acid (5), (R)-(+)-rosmarinic acid (6), isorinic acid (7), (S)-(-)-rosmarinic acid (8) and (S)-(-)-rosmarinic acid methyl ester (9) were isolated from the ethyl acetate extract. Compounds were elucidated by NMR and MS spectroscopic methods. The antiplatelet effect was evaluated using turbidimetric method. Anticoagulant activity was examined by measuring activated partial thromboplastine time (APTT), prothrombin time, and thrombine time (TT). It was shown for the first time that both new phenylpropanoid sucrose esters 1 and 2, 7 and 9 displayed dose-dependent antiplatelet effects. 2 and 9 had the highest inhibitory activity on both adenosine diphosphate (ADP)- and collagen-induced platelet aggregation. Moreover, 1, 7 and 9 also exhibited anticoagulant activity. At 0.4 mg/mL, both 1 and 7 prolonged APTT compared to the negative control (p < 0.05), suggesting the possible inhibitory impact on the intrinsic coagulation pathway. Moreover, 9 at 0.4 mg/mL exerted higher TT values than the negative control (p < 0.05). C. indica and its bioactive phytochemicals are potential candidates for development of anti-thrombosis therapy. Graphical Abstract

Cardiovascular diseases (CVDs) are among the leading causes of fatality in the world, representing 38% of chronic disease-related deaths (WHO 2021).One of the most feared complications of CVDs is thrombosis, which leads to other life-threatening events such as coronary artery disease (CAD), venous thromboembolism (VTE), myocardial infarction (MI) or ischaemic stroke.The mainstay of current treatment in CVDs and cerebrovascular disorders is the use of antiplatelet and anticoagulant agents.However, conventional antithrombotic agents may induce serious adverse events including internal bleeding and gastrointestinal side effects (Chan and Weitz 2019).Therefore, there is an urge for development of safer and effective antithrombotic medications.Natural resources have been a field of interest for drug development.
Recently, we described the potent antiplatelet and anticoagulant activity of some species from the genus Canna and isolated bioactive compounds possessing these activities such as epimedokoreanone A, nepetoidin B, indole-3-carboxylic acid and p-coumaric acid (Le et al. 2022, Nguyen et al. 2020).Although C. indica has been used to support treatment of heart diseases (Vo 2012), its scientific evidence is still scarce and the mechanistic basis of its functioning is yet to be studied.We previously demonstrated that the ethyl acetate fraction of C. indica rhizome had potential antiplatelet and anticogulant effect (Le et al. 2020).In this study, two new phenylpropanoid sucrose esters (canindicoside A (1) and canindicoside B (2)) together with seven known compounds (3 -9) were isolated for the first time from the ethyl acetate extract of C. indica rhizome.The antiplatelet aggregation and anticoagulant activity on human blood of isolated compounds were evaluated.

Structural elucidation of two new compounds
Compound 1 was isolated as colourless amorphous powder, [α]
Compound 2 was obtained as colourless amorphous powder, The IR and UV spectra of 2 showed absorption bands for the hydroxyl and α, β-unsaturated aromatic ester groups.The NMR spectra of 2 resembled to those of 1 except for the appearance of signals for feruloyl groups instead of p-coumaroyl groups, which indicated that 2 is phenylpropanoid sucrose containing three feruloyl groups and an acetyl group (Table S1).All the esterification and acylation positions were specified by HMBC experiment and comparing the NMR spectra data of 2 with corresponding values of 1.In the HMBC spectrum, correlations between H-3 (δ H 5.59) and C-9″′ (δ C 168.4), from H-6 (δ H 4.49/4.52)to C-9″″ (δ C 168.6), and from H-2′ (δ H 4.71) to C-9″ (δ C 168.8) confirmed the linkages of feruloyl groups at C-3, C-6 and C-2′ of sucrose moiety.The HMBC correlation from H-6′ (δ H 4.21/4.68)to carbonyl carbon of acetyl group (δ C 172.8) indicated that sucrose moiety was acetylated at C-6′ (Figure S2).Consequently, the structure of 2 was characterised as 6′-O-acetyl-2′,3,6-O-triferuloyl sucrose, a new compound named canindicoside B (Figure 1).

Antiplatelet aggregation activity
Among 9 pure compounds isolated from the ethyl acetate fraction of C. indica roots, 5 compounds were previously evaluated for antiplatelet effect (Chen et al. 2022, Hong et al. 2016, Lee et al. 2014, Nguyen et al. 2020).Therefore, in this study, the antiplatelet aggregation activity was tested for 4 compounds 1, 2, 7 and 9.
All four tested compounds exhibited positive correlations between percentage inhibition of platelet aggregation and the tested dose (Pearson correlation coefficient, r > 0.8, p < 0.05).The new compound 2 and compound 9 had the highest inhibitory activity on both adenosine diphosphate (ADP)-and collagen-induced platelet aggregation (at 0.4 mg/mL, mean percentage inhibition (%I) for 2 and 9, 67.5% and 70.9% in the case of ADP, respectively; 82.7% and 87.6% in the case of collagen, respectively).In addition, %I of both 2 and 9 was not significantly diferent from that of the positive control when platelet aggregation was induced by ADP (p > 0.05), but it was lower than the positive control when platelet aggregation was induced by collagen (p < 0.05).The new compound 1 also showed inhibitory effects on both ADP-and collagen-induced platelet agggregation (at 0.4 mg/mL, mean %I, 29.7% and 31.4% for ADP and collagen, respectively), but the effect was significantly lower than the positive controls (p < 0.05).%I of 7 was significantly higher than that of 1 for ADP (p < 0.05) but significantly lower than that of 1 for collagen (p < 0.05) (Table S2).
The two parameters, AUC and slope, were negatively dependent on the tested concentration (Pearson correlation coefficient, r < −0.9, p < 0.05).In addition, all tested compounds at 0.4 mg/mL showed significantly lower AUC and slope values than the negative control for both ADP-and collagen-induced platelet aggregation (p < 0.05).This means that these compounds significantly inhibited overall platelet aggregation and decreased aggregation velocity.Both 2 and 9 at all tested concentrations significantly reduced AUC and slope compared to the negative control (p < 0.05).In addition, AUC and slope of 2 and 9 at 0.4 mg/mL were similar to the positive control in the case of ADP (p > 0.05), indicating their effects on the overall aggregation process when induced by ADP.For collagen, all compounds exerted significantly higher slope and AUC compared to the positive control (p < 0.05) (Table S2).
Phenylpropanoid sucrose esters are naturally plant-derrived compounds in many plant species.They were thought to be the main bioactive components in various plant species of different families Brassicaceae, Polygonaceae, Arecaceae, Rosaceae….whose extracts have been used in folk medicines to treat many conditions such as inflammation, cancer, viral diseases, hair loss.They possessed a wide spectrum of biological activities like antioxidant, antitumor, antiinflammatory, antiviral, antibacterial, neuro-protective and glycosidase inhibitory effects (Panda et al. 2011).However, very modest work on mechanisms of action, structure activity relationship and therapeutic potential of these compounds have been done.A recent study on the rhizome of Sparganium stoloniferum, which is traditionally used to cure the syndrome of blood stasis in China, isolated four new ferulic acid sucrose esters and four known phenypropanoids from the ethanol extract and reported inhibitory effects on ADP-induced platelet aggregation of these isolated compounds (Deng et al. 2022).Yoshikawa et al. (2002) found that the methanolic extract of the fresh flowers of Prunus mume inhibited rabit platelet aggregation induced by thrombin.Further isolation on the methanolic extract of P. mume led to the identification of two new flavonol oligoglycosides, 2′'-O-acetylrutin and 2′'-O-acetyl-3′'-O-methylrutin and two new phenylpropanoid sucrose esters, prunose I and prunose II.Notably, these two new phenylpropanoid sucrose esters were shown to inhibit thrombin-induced platelet aggregation (Yoshikawa et al. 2002).In the present study, it was noted that both new phenylpropanoid sucrose esters expressed significant antiaggregatory effect, suppressed overall platelet aggregation and inhibited aggregation velocity stimulated by either collagen or ADP (p < 0.05).However, canindicoside B had significantly higher inhibitory effect than canindicoside A for both ADP and collagen (p < 0.05).The new detected results indicate that two new compounds might have different modes of action on platelet aggregation.

Anticoagulant activity of isolated pure compounds
All tested compounds displayed significantly lower PT, APTT and TT values than the positive control (p < 0.05).However, at the highest tested concentration of 0.4 mg/mL, both 1 and 7 prolonged APTT significantly compared to the negative control (p < 0.05), suggesting the possible inhibitory impact on coagulation factors in the intrinsic pathway.Moreover, 9 at 0.4 mg/mL exerted significantly higher TT values than the negative control (p < 0.05).This indicates that 9 might affect the transformation of fibrinogen into fibrin in the final stages of blood coagulation (Koch and Biber 2007).However, no compound showed any significant difference in PT values in comparison with the negative control (Table S3).

Experimental
See supplemental material.The study was approved by the Research Ethics Committee, School of Medicine and Pharmacy, Vietnam National University, Hanoi, Vietnam (document number 02/2020/CN-HĐĐĐ).

Conclusion
This is the first study to investigate the antithrombotic activity of secondary metabolites from C. indica rhizome.Among nine compounds isolated for the first time from the ethyl acetate fraction, two novel phenylpropanoid sucrose esters 1 and 2, and two known compounds 7 and 9 were all proven for the first time to possess antiplatelet activity.Moreover, 1, 7 and 9 showed weak anticoagulant effect.Therefore, C. indica rhizome can be a promissing source for development of functional food or searching bioactive compounds to treat and prevent CVDs.Bioactive constituents from C. indica rhizome are potential candidates to develop antithrombotic agents.Further in-vitro and in-vivo studies on this valuable plant will be helpful to elucidate the mechanisms for antiplatelet and anticoagulant effect and may lead to the discovery of novel therapeutic agents for treatment of thrombosis-associated diseases.