Anticoagulant, antioxidant and cytotoxic potential of cinnamic acid and derivatives

Potencial anticoagulante, antioxidante e citotóxico do ácido cinâmico e derivados sintéticos

Abstract

Introduction  Since antiquity, natural products have been widely used to relieve symptoms and prevent numerous diseases. Early treatments for vascular disorders, including thrombosis, relied heavily on plant-derived substances, which later served as prototypes for the development of platelet aggregation inhibitors and coagulation-modulating agents. Among these bioactive constituents, phenolic compounds—abundantly found in various botanical sources—have gained prominence due to their well-established antioxidant properties and reported anticoagulant effects. Recent studies highlight that the structural diversity of phenolic acids, particularly cinnamic acid (CA) and its derivatives, offers a valuable chemical scaffold for developing new therapeutic agents targeting thrombogenesis and oxidative imbalance.

Objective  This study aimed to evaluate, in vitro, the pharmacological (anticoagulant and antioxidant) and toxicological (oxidant and cytotoxic) activities of cinnamic acid (CA) and five synthetic derivatives—4HDCN, 4MTCN, 3,4DHCN, 3,4DMCN, and 3H4MCN.

Methods  Anticoagulant activity was assessed using a semi-automated coagulometer by measuring clotting time after exposure of human plasma to each molecule. Antioxidant, oxidant, and cytotoxic (hemolytic) activities were determined using human erythrocytes from blood groups A, B, and O. Oxidative and antioxidant responses were quantified by spectrophotometry following exposure to phenylhydrazine as a pro-oxidant inducer. Hemolysis was evaluated at concentrations ranging from 0 to 400 mg/dL.

Results  In the anticoagulant assays, the derivative 3,4DHCN exhibited the strongest activity, prolonging clotting time to 176.6 s, slightly surpassing 3,4MTCN (165.6 s). All molecules demonstrated low or negligible oxidizing activity (<7.1%). Regarding antioxidant potential, 4MTCN showed the most effective protection against phenylhydrazine-induced oxidation, reducing oxidative damage to 8.7%. None of the tested compounds induced moderate or high hemolysis at concentrations up to 200 mg/dL. However, at 400 mg/dL, CA and 4HDCN produced high hemolytic rates across erythrocyte types. In type A cells, hemolysis reached 89.4% (CA) and 76.0% (4HDCN); in type B, 64.3% and 60.5%; and in type O, 90.1% and 70.7%, respectively.

Conclusion  Dihydroxylated and dimethoxylated cinnamic acid derivatives demonstrated notable anticoagulant activity, minimal oxidative effects, low antioxidant capacity, and an absence of cytotoxicity in human erythrocytes at therapeutically relevant concentrations. These findings indicate that such molecules represent promising candidates for the development of new anticoagulant agents with favorable safety profiles.

Keywords:
pharmacology; synthetic derivatives; toxicology

Resumo

Introdução  Desde a antiguidade o homem utiliza produtos naturais para aliviar sintomas e prevenir as mais variadas doenças. Inicialmente, doenças vasculares como a trombose foram tratadas com produtos naturais de fontes vegetais, os quais serviram como base para o desenvolvimento de inibidores da agregação plaquetária e/ou de fatores da coagulação. Os Compostos Fenólicos, obtidos a partir de diversos espécimes vegetais, são reconhecidos por possuírem pronunciada atividade antioxidante e efeito anticoagulante, conforme demonstrado em estudos recentes.

Objetivo  O presente estudo teve como objetivo avaliar, in vitro, as atividades farmacológicas e toxicológicas do ácido cinâmico (AC) e de cinco derivados sintéticos (4HDCN, 4MTCN, 3,4DHCN, 3,4DMCN e 3H4MCN).

Métodos  A avaliação da atividade anticoagulante foi realizada em coagulometro semiautomatizado. Os ensaios antioxidante, oxidante e citotóxico foram realizados utilizando eritrócitos humanos e mensurados em espectrofotômetro.

Resultados  Nos ensaios in vitro, 3,4DHCN apresentou o melhor efeito anticoagulante (176,6 segundos). que foi discretamente superior ao efeito do derivado 3,4DMCN (165,6 segundos). As moléculas estudadas possuem baixa ou ausente atividade oxidante (<7,1%). Na avaliação da atividade antioxidante, 4MTCN apresentou melhor efeito antioxidante contra a ação da fenilhidrazina (reduzindo a oxidação para 8,7%). Nenhuma das moléculas estudas causou hemólise moderada ou alta até a concentração de 200mg/dL. No entanto, na concentração de 400mg/dL de AC e de 4HDCN causaram alta taxa de hemólise em eritrócitos tipo A: 89,4% e 76,0%, respectivamente, em eritrócitos tipo B: 64,3% 60,5% e em eritrócitos tipo O: 90,1% e 70,7%, respectivamente.

Conclusão  As moléculas dihidroxiladas e dimetoxiladas apresentaram atividade anticoagulante com reduzido efeito oxidante, baixa capacidade antioxidante, sem efeito citotóxico sobre eritrócitos humanos, apresentando-se como promissoras para o desenvolvimento de novos fármacos anticoagulantes.

Palavras-chave:
farmacologia; toxicologia; derivados sintéticos

1. Introduction

In recent decades, life expectancy has been increasing worldwide. In addition, the mortality and debilitating disease profile has changed, resulting in an increase in the incidence of cardiovascular disease worldwide (Calazans and Queiroz, 2020). Thrombosis is the most common cause of cardiovascular disease, and its main consequences are acute coronary syndromes, venous thromboembolism, and stroke (Liberato, 2021).

The literature reports that plants of various species produce secondary metabolites with biological activity that can affect hemostasis, among these molecules, polyphenols stand out as common constituents of plants, as well as in their extracts. These constituents, in turn, have significant antioxidant and anti-inflammatory effects, in addition to being able to affect blood clotting (Lamponi, 2021).

Studies by Bijak and colleagues (2014) demonstrated that flavonoids (a molecule subgroup of the polyphenol class) are capable of inhibiting the proteolytic and amidolytic activity of thrombi-inhibition mediated by antithrombin-III. Furthermore, several studies have demonstrated the antioxidant potential of chemical compounds obtained from plant sources, including flavonoids, phenolic acids, and coumarins (Chen et al., 2020; Kostova et al., 2011).

Natural products with antioxidant activity are important allies in preventing oxidative stress. Chemical reactions occurring in organisms naturally produce Reactive Oxygen Species (ROS). However, there is a balance between the production and elimination of these oxidizing agents, when it is imbalanced, oxidative stress occurs due to the accumulation of ROS. Excess ROS, is linked to fundamental cellular signaling pathways such as apoptosis and gene expression, as well as the development of various diseases such as diabetes, cardiovascular and neurodegenerative diseases such as Parkinson's and Alzheimer's, and even cancer (Rani et al., 2018).

Thus, given the biological effects previously attributed to phenolic compounds, cinnamic acid and its synthetic derivatives emerge as phenolic compounds with potential pharmacological activity. This necessitates investigation of their pharmacological effects - especially their potential anticoagulant effects—and their toxicity assessment using in silico and in vitro methods. Thus, the objective of this study is to evaluate the pharmacological and toxicological activities of cinnamic acid and its synthetic derivatives.

2. Material and Methods

Cinnamic acid (AC) and it synthetic derivatives named: i) 4-hydroxycinnamic acid (4HDCN), ii) 4-methoxycinnamic acid (4MTCN), iii) 3,4-dihydroxycyamic acid (3,4DHCN), iv) 3,4-dimethoxycyamic acid (3,4DMCN) and v) 3-hydroxy-4-methoxycyamic acid (3H4MCN) were commercially purchased from the manufacturer Sigma-Aldrich ^®.

2.1. Ethical aspects of research

All experiments involving human biological samples were submitted to analysis and authorized by the Research Ethics Committee of the Hospital Universitário da Universidade Federal do Vale do São Francisco, obtaining ethical authorization according to protocol 7.669.047. Biological samples obtained from the University Hospital of the Federal University of Vale do São Francisco were used, and after laboratory analysis, they would be discarded.

2.2. Anticoagulant activity

Human blood samples collected in tubes containing 3.8% sodium citrate were centrifuged at 3,000 rpm for 15 minutes, according to the reagent kit recommendations. Plasma samples within the reference time for PT and aPTT formed a plasma pool, which was used in the experiment.

The anticoagulant activity assessment was performed according to the methodology previously described by Sousa et al., (2019), with modifications. The experiment was performed using a 150µL volume of citrated human plasma, to which 20µL of cinnamic acid or a synthetic derivative solubilized in PBS (10x, pH 7.4) was added. The negative control for the experiment was 20µL PBS without any additives.

The plasma was then incubated in a 37°C water bath for 5 minutes. Then, aliquots of plasma (50 µL) containing the cinnamic acid derivatives or the control were transferred to cuvettes containing a metal sphere, and the PT was obtained using a semi-automated Coagumaster 2.0 Wama® equipment, which measures the variation in the movement of metal spheres in citrated plasma, allowing the calculation of plasma clotting time (Sousa et al., 2019).

2.3. Evaluation of oxidizing and antioxidant activity

After obtaining the blood samples, erythrocytes were washed by adding 0.9% saline solution (150mM NaCI), then this suspension was centrifuged at 3,000 rpm for 5 minutes, this process was repeated three times. To carry out the experiments, an erythrocyte solution composed of 30% erythrocytes, 69.4% PBS (PBS pH 7.4), 0.6% glucose at 10% was used. Triton X-100 was used to lyse the red blood cells and expose the hemoglobin, as described by Souza-Melo et al. (2021).

2.3.1. Oxidizing activity

Concentrations of 5, 50, 100, 200, and 400µg of each molecule under study were evaluated, following the methodology proposed by Souza-Melo et al. (2021), with modifications: amounts of the evaluated molecule were added to the red blood cell solution, then incubated for 1 hour at 37°C with shaking at 100 rpm and subsequently centrifuged at 3,000 rpm for 1 minute to obtain the supernatant. From the supernatant obtained in the previous step, 25µL was removed and resuspended in 750µL of phosphate buffer (PB), to be read in Nandrop® equipment, using a wavelength of 630nm.

Then, 75µL of the supernatant obtained was removed and resuspended in 750uL of phosphate buffer (PB), which were read in Nandrop® equipment, using a wavelength of 540nm, as described by Souza-Melo et al., (2021). PB was used as the equipment blank, and the erythrocyte solution without any additive was used as a negative control (Souza-Melo et al., 2021). Finally, the methemoglobin formation rate (mHb) was calculated using the Formula 1:

$\%mHb=\left(D.O.630nm\times 100\right)/\left(D.O.630nm+D.O.540nm\times 10\right)$ (1)

2.3.2. Antioxidant activity

Tests were performed at concentrations of 5, 50, 100, 200, and 400µg of each of the molecules under study following the methodology proposed by Souza-Melo et al., (2021) with modifications: the cinnamic acid derivative was added to 1000µL of erythrocyte solution, which was incubated for 1 hour at 37°C with shaking at 100 rpm, then it was centrifuged at 3,000 rpm for 1 minute and the supernatant was obtained. To the supernatant obtained, 50µL of 1M phenylhydrazine was added, they were incubated for 20 minutes at 37°C with shaking at 100 rpm, then the tubes were centrifuged at 3,000 rpm for 5 minutes. From the supernatant obtained, 25µL were removed and resuspended in 750µL of PB, which were read in Nandrop^® equipment, using a wavelength of 630nm, as described by Souza-Melo et al., (2021).

Then, 75µL of the supernatant obtained was removed and resuspended in 750µL of PB, which were read in Nanodrop^® equipment, using a wavelength of 540nm (Souza-Melo et al., 2021) . PB was used as the equipment blank, as a positive control, the erythrocyte solution added with 50µL of 1M phenylhydrazine was used without the addition of any cinnamic derivative, as described by Souza-Melo et al., (2021). Finally, the methemoglobin formation rate (mHb) was calculated using the Formula 2:

$\text{\%}mHb=\left(D.O.630nm\times 100\right)/(D.O.630nm+\left(D.O.540nm\times 10\right)$ (2)

2.4. Assessment of hemolytic activity

The direct hemolytic potential of each cinnamic derivative was evaluated on human erythrocytes of blood groups A, B and O and the tests were performed according to the methodology described by Lima et al., (2024) with some adaptations.

After obtaining the blood samples, erythrocytes were washed by adding 0.9% saline solution (150mM of NaCI), then this suspension was centrifuged at 3,000 rpm for 5 minutes, this process was repeated three times (Lima et al., 2024).

To carry out the experiments, 2,000µL of a 0.5% erythrocyte solution in 0.9% NaCI was used. Tests were performed at concentrations of 5, 50, 100, 200, and 400µg of the molecules under study, which were added to the erythrocyte solution and incubated for 1 hour at 37°C with agitation at 100 rpm. Subsequently, it was centrifuged at 3,000 rpm for 1 minute, and the supernatant was obtained (Lima et al., 2024).

The supernatant obtained was read in Nanodrop^® equipment using a wavelength of 540nm. DMSO/PBS was used as a negative control, while the positive control was Triton X-100 (Lima et al., 2024). The percentage of hemolysis was calculated according to Hubert et al. (1997), using the Equation 3:

$\%hemolysis=100\times \frac{\left(Absdaamostra-Abscontrolenegativo\right)}{\left(Abscontrolepositivo-Abscontrolenegativo\right)}$ (3)

2.5. Statistical analysis

All experiments were performed in triplicate, the results were expressed as mean ± standard error of the mean (SEM) and statistically analyzed using the One-way ANOVA test, followed by the Dunnetts multiple comparisons test. Values of p<0.05 were considered significant. All results were evaluated using GraphPad Prism software, version 8.0 (GraphPad Software Inc., San Diego, CA, USA).

3. Results

3.1. Evaluation of anticoagulant activity

By analyzing the anticoagulant potential of the molecules under study, it was possible to verify that cinnamic acid caused a delay in clotting time when compared to the control clotting time. However, it was possible to observe that the presence of a hydroxyl group substituent at the 4-position of the aromatic ring of 4HDCN abolished the anticoagulant effect identified for cinnamic acid. The presence of two hydroxyls, one at the 3-position and the other at the 4-position of 3,4DHCN, further potentiated the anticoagulant effect when compared to the effect observed for cinnamic acid (Figure 1).

Figure 1
Evaluation of the anticoagulant effect caused by cinnamic acid and its synthetic derivatives on human citrated plasma. AC: Cinnamic acid, 4MTNC: 4-methoxycinnamic acid, 3H4MCN: 3-hydroxy-4methoxycinnamic acid, 4HDCN: 4-hydroxycinnamic acid, 3,4-DMNC: 3,4-dimethoxycinnamic acid, 3,4-DHCN: 3,4-dihydroxycinnamic acid. Results expressed as mean ± standard error of the mean (SEM) (n=3), where ns: not significant for p-value >0.05; ****: p-value <0.00001.

When the hydroxyl group in position 4 of the aromatic ring is replaced by a methoxy group (3-hydroxy-4-methoxycinnamic), there is a strong reduction in the anticoagulant effect when compared with the compound 3,4DHCN. However, if there are two methoxy groups, one in position 3 and the other in position 4 of the aromatic ring, the anticoagulant effect is reestablished (Figure 1).

These results point to three important characteristics of these chemical groups: i) substitution by a methoxy group in position 4 of the aromatic ring improves the anticoagulant effect in relation to cinnamic acid; ii) substitution in positions 3 and 4 with different chemical groups (hydroxy or methoxy) also improves the anticoagulant effect in relation to cinnamic acid; iii) if the substitution in positions 3 and 4 are with the same radicals, an even greater improvement is observed in relation to the effect observed for cinnamic acid.

3.2. Evaluation of oxidizing activity

In the evaluation of the oxidizing potential of cinnamic acid and its chemical derivatives, it was possible to observe that cinnamic acid did not cause a statistically significant increase in methemoglobin production when compared to the control (Figure 2a). The acids 4MTCN and 3H4MCN did not cause hemoglobin oxidation greater than 4% at any of the concentrations evaluated (Figure 2b and 2c).

Figure 2
Evaluation of the oxidizing effect of cinnamic acid and its synthetic derivatives against human hemoglobin. C-: negative control AC: Cinnamic acid, 4MTNC: 4-methoxycinnamic acid, 3H4MCN: 3-hydroxy-4methoxycinnamic acid, 4HDCN: 4-hydroxycinnamic acid, 3,4-DMNC: 3,4-dimethoxycinnamic acid, 3,4-DHCN: 3,4-dihydroxycinnamic acid. Results expressed as mean ± standard error of the mean (SEM, n=3), where ns: not significant for p-value>0.05; *: p-value<0.01; **: p-value<0.001; ***: p-value<0.0001 and ****: p-value<0.00001.

The acids 4HDCN and 3,4DMCN, caused hemoglobin oxidation by more than 4% at some of the concentrations evaluated; however, this result was not statistically significant (Figure 2d and 2e). 3,4DHCN caused a slight increase in methemoglobin production, ranging from 4.6 to 7.1%, at concentrations of 50µg/mL and 400µg/mL, respectively (Figure 2f).

The results obtained point to molecules with no oxidation-inducing effect on human hemoglobin (cinnamic acid, 4-hydroxycinnamic, 4-methoxycinnamic, 3-hydroxy-4-methoxycinnamic, 3,4-dimethoxycinnamic) or low oxidizing effect on human hemoglobin (3,4-dihydroxycinnamic). Furthermore, it is possible to infer that: i) the low oxidizing effect observed in cinnamic acid is maintained in chemical modifications in which there is a chemical group in position 4 of the aromatic ring (4-hydroxycinnamic or 4-methoxycinnamic); ii) the low oxidizing effect is also related to the presence of a chemical group in position 3 and another in position 4, provided that the chemical group in position 4 is a methyl group; iii) in the presence of a hydroxyl group in position 4 of the aromatic ring, a slight increase in the oxidizing effect is observed compared to the negative control.

3.3. Evaluation of anti-oxidant activity

When evaluating the antioxidant potential of the molecules under study, it was possible to verify that AC, 4HDCN and 3,4DHCN did not cause a reduction in hemoglobin oxidation induced by phenylhydrazine at any of the concentrations evaluated (Figure 3a-c). The derivated 3H4MCN was unable to prevent the oxidizing effect caused by phenylhydrazine, acting as an oxidation potentiator at all concentrations (>46%, control= 32%) evaluated (Figure 3d).

Figure 3
Evaluation of the antioxidant effect of cinnamic acid and its synthetic derivatives against human hemoglobin.. Results expressed as mean ± standard error of the mean (SEM, n=3), where ns: not significant for p-value>0.05; *: p-value<0.01; **: p-value<0.001; ***: p-value<0.0001.

3,4DMCN acid caused a slight reduction in hemoglobin oxidation at all concentrations evaluated; however, only at concentrations of 50 and 100µg/mL was the oxidation reduction statistically significant (17.7% and 15.2%, respectively) (Figure 3e). 4MTCN acid, on the other hand, showed a good antioxidant effect, reducing the oxidation caused by phenylhydrazine from a concentration of 100µg/mL (≤15.5%) (Figure 3f).

3.4. Hemolytic activity

The hemolytic effect of cinnamic acid and its derivatives was evaluated on human blood types A, B and O, all belonging to the Rh-positive group. 3,4DHCN and 3,4DMCN did not cause hemolysis in any of the blood groups and at any of the concentrations evaluated (Figures 4, 5 and 6).

Figure 4
Evaluation of the hemolytic effect of cinnamic acid and its synthetic derivatives, against human red blood cells belonging to groups A. Results expressed as mean ± standard error of the mean (SEM) (n=3), in which p-values ns<0.05 were considered significant (****: p-value<0.00001).

Figure 5
Evaluation of the hemolytic effect of cinnamic acid and its synthetic derivatives, against human red blood cells belonging to groups B. Results expressed as mean ± standard error of the mean (SEM) (n=3), in which p-values ns<0.05 were considered significant (****: p-value<0.00001).

Figure 6
Evaluation of the hemolytic effect of cinnamic acid and its synthetic derivatives, against human red blood cells belonging to groups O. Results expressed as mean ± standard error of the mean (SEM) (n=3), in which p-values ns<0.05 were considered significant (****: p-value<0.00001).

In the evaluation of the cytotoxic potential of the other cinnamic acid derivatives in group A red blood cells, it was possible to observe that none of the molecules tested was capable of causing relevant hemolysis up to a concentration of 100µg/mL (Figure 4).

In type A erythrocytes the AC and 4HDCN caused a low rate (19.8% and 14%, respectively) of hemolysis at a concentration of 200µg/mL, while at a concentration of 400µg/mL they caused a high rate of hemolysis (88.4% and 75.9, respectively). On the other hand, the other molecules studied caused a low rate of hemolysis (Figure 4).

In the evaluation of the cytotoxic potential of cinnamic acid and other chemical derivatives in group B red blood cells, it was possible to verify that none of the molecules tested was capable of causing relevant hemolysis up to a concentration of 200µg/mL in group B red blood cells (Figure 5), having been classified as molecules with low hemolytic potential (hemolysis rate < 40%).

At a concentration of 400µg/mL, AC and 4HDCN caused a high rate of hemolysis (64% and 60%, respectively). In this blood group, 4MTCN, 3,4DHCN, 3,4DMCN, and 3H4MCN caused hemolysis ranging from 9% to 39.3% (hemolysis rate = 4MTCN > 3,4DMCN > 3H4MCN and 3,4DHCN), unlike the effect observed in blood groups O and A, which may indicate that this blood group may be more sensitive to these compounds (Figure 5).

Furthermore, it was possible to identify that, in general, the evaluated molecules that contain one or two methoxy groups in positions 3 and/or 4 of the aromatic ring tend to cause a lower rate of hemolysis, the exception occurring when there are two substitutions by hydroxyl groups in these positions, which attributes the lowest hemolytic effect to the molecule.

In the evaluation of the cytotoxic potential of cinnamic acid and its chemical derivatives in group O red blood cells, it was identified that none of the molecules evaluated was capable of causing relevant hemolysis in concentrations less than or equal to 200µg/mL (Figure 6).

However, when the molecules were evaluated at a concentration of 400µg/mL, it was found that 4MTCN, 3,4DHCN, 3,4DMCN, and 3H4MCN caused a low rate of hemolysis (>36%). On the other hand, AC and 4HDCN caused a high rate of hemolysis, reaching 90% and 70%, respectively. Thus, AC was configured as the most toxic molecule for human erythrocytes with blood group O, among the molecules evaluated (Figure 6).

4. Discussion

There are reports in the literature of the anticoagulant effect of plant extracts containing phenolic compounds, for example, Khoo et al. (2015) evaluated the anticoagulant activity of crude extract of Melastoma malabathricum Linn and its constituents, and identified a significant prolongation of the activated partial thromboplastin time (aPTT) caused by both the crude extract and the fraction containing the constituents cinnamic acid and p-hydroxycinnamic acid (major and minor constituents, respectively).

Luo et al., (2017) studied several derivatives of caffeic, coumaric and ferulic acids and concluded that a caffeic acid derivative and a ferulic acid derivative that differed only in the chemical group in position 3 of the aromatic ring (hydroxyl and methoxyl, respectively) presented an excellent anticoagulant effect, in which the ferulic acid derivative presented a greater effect, since the presence of the methoxyl group makes the molecule larger and easier to fit into the receptors, which are coagulation factors.

Oxidative stress results from an imbalance between the body's oxidant-antioxidant mechanisms, in favor of the former (Petricevich et al., 2022). This imbalance is related to the accumulation of free radicals, which in turn cause damage to different macromolecules such as lipids, proteins, RNA and DNA (Ohiagu et al., 2025). This damage is directly linked to cellular aging, as well as the emergence of neurodegenerative and cardiovascular diseases, diabetes, carcinogenesis, among others (Halliwell and Whiteman, 2004).

Phenolic acids are known to have antiradical activity (free radical scavengers) and, in some cases, as metal chelators, which often gives them an antioxidant effect either in the initiation stage or in the propagation stage of oxidative processes (Santos et al., 2020).

According to the methodology used in this study, only methoxycinnamic acid was able to reduce the oxidizing effect of phenylhydrazine on human hemoglobin when compared to the positive control. In evaluating the antioxidant potential of phenolic compounds, the scavenging method of the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) (a known oxidizing agent) is often used; this technique can be used in over 90% of antioxidant activity studies, according to Oliveira (2015).

In this study, cinnamic acid and hydroxylated derivatives showed little or no reduction in the oxidizing effect caused by phenylhydrazine, unlike the results found by Natella et al. (1999), in the present research, cinnamic acid derivatives that contain methoxyl groups in the aromatic ring were more efficient in preventing oxidation caused by phenylhydrazine. This can initially be explained by the different methodologies for evaluating the oxidizing effect adopted between the studies, since in the study by (Natella et al., 1999) an evaluation was carried out of the inhibition of oxidation of human Low Density Lipoprotein – LDL induced by both copper chloride and 2,2′-Azobis(2-methylpropionamidine) dihydrochloride – AAPH (Schmitz et al., 2008).

The efficiency of phenolic acids as antioxidant compounds is directly related to the number, binding site and position of hydroxyl groups in the aromatic ring (Chen et al., 2020). Oliveira (2015) reports that the use of the antioxidant evaluation method using DPPH has as one of its limitations the different adaptations that have been added to the method over time, which makes it more difficult to compare the results obtained by different research groups.

Despite this limitation, Kucukoglu and Nadaroglu (2014) concluded that 4-hydroxycinnamic, 3-hydroxy-4-methoxycinnamic and 3,4-dihydroxycinnamic acids showed a strong antioxidant effect against DPPH, an effect even greater than that obtained for BHA, quercetin and α-tocopherol, which were used as standard antioxidant molecules in the experiment. This is not in agreement with the data obtained in this research, which may suggest that the mechanism of antioxidant action identified by Kucukoglu and Nadaroglu (2014) for these molecules diverges in its mechanism of action and cannot reverse the oxidizing effect exerted by phenylhydrazine on hemoglobin (Souza-Melo et al., 2021).

A different point of view than what is usually found in the literature, Chen et al. (2020) states that starting from the same “mother” chemical structure, the more methoxyl groups there are, the greater the antioxidant activity of phenolic acids. This finding corroborates the result found in this research, in which 3,4-dimethoxycinnamic acid was the only molecule to obtain effective protective activity against the oxidizing effects of phenylhydrazine.

Studying the cytotoxic potential of different compounds and molecules, whether synthetic or derived from natural products, is important even in the early stages of a study to verify the safety of product use, therefore, assessing cytotoxicity in human erythrocytes is a key tool for verifying the protective or toxic effects of various compounds and/or molecules with promising biological/pharmacological activities (Souza-Melo et al., 2021).

Erythrocytes are an important tool for evaluating the oxidizing effect caused by compounds and molecules since they have a membrane structure similar to that of cells and are permanently exposed to damage caused by free radicals, in this case oxygen (Ravikumar et al., 2020). However, due to the absence of a nucleus and mitochondria, erythrocytes have a limited metabolic capacity to deal with oxidative stress, which can result in the progressive accumulation of Reactive Oxygen Species - ROS, which in turn contribute to the peroxidation of lipids on the surface of erythrocytes, causing hemolysis (Keshavarzi et al., 2022).

Sousa et al. (2023) defined hemolytic potential based on the percentage of hemolysis obtained, dividing it into low hemolysis when the hemolysis rate obtained is less than 40%, moderate hemolysis when the hemolysis rate obtained is between 40 and 80%, and high hemolysis when this rate exceeds 80%. According to the methodology adopted in this research, none of the molecules studied showed a significant hemolysis effect, which indicates the safety of using these molecules to proceed to more complex toxicological tests.

5. Conclusion

It was concluded that the dihydroxylated and dimethoxylated molecules exhibited excellent anticoagulant activity with low oxidative effect, reduced ability to prevent free radical-mediated hemoglobin oxidation, and no cytotoxic effect on human erythrocyte membranes, regardless of ABO antigen expression.

The dihydroxylated cinnamic derivative exhibited slightly superior anticoagulant effect to the dimethoxylated cinnamic derivative, this may occur due to a better interaction/inhibition of this molecule with coagulation factors, when compared to the other molecules studied. This biological activity profile suggests that the molecules studied, particularly 3,4-dihydroxycinnamic acid, represent promising base molecules for the development of new anticoagulant drugs.

It is necessary to further explore the anticoagulant mechanism caused by these molecules, whether through direct or indirect inhibition of coagulation factors, as well as to study their molecular docking with these enzymes.

Acknowledgements

This investigation was supported, in part, by a master grant from CAPES, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil.

Financial resources of the laboratories and researchers directly involved with the experiments described in the present study.

Data Availability Statement

The entire data set supporting the results of this study was published in the article itself.

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Edited by

• Editor:
  Marcelo A.M. Esquisatto

Publication Dates

• Publication in this collection
  29 May 2026
• Date of issue
  2026

History

• Received
  02 Dec 2025
• Accepted
  06 Mar 2026