Cytotoxicity and genotoxicity of Cassia angustifolia decoct

Abstract

Cassia angustifolia is one of the most commonly used medicinal plants globally. Although many studies evaluated its biological activities, their results led to opposing results, particularly when it comes to the toxicity of this species. Thus, this study aimed to further evaluate the biological properties of Sennae folium decoct especially its cytotoxicity and genotoxicity. HPLC analysis revealed the presence of 8-glucosyl rhein, sennoside A, sennoside B, sennoside C, sennoside D and rhein. DPPH test showed low antioxidant activity. RBCs hemolytic assay, Allium cepa test and lipid peroxidation evaluation confirmed the high cytotoxicity of the tested decoct when used in concentrations of 1500 μg/ml, similar to the one observed in positive controls, cells treated with H₂O₂. However, the genotoxicity assessment showed that the genotoxic properties of even the high tested concentrations of the Sennae folium decoct were much lower than the ones seen in the positive controls (p < 0.001). It can be concluded that the toxicity of the higher concentrations of this Cassia species decoct is mostly caused by its actions on the cell membrane level.

Keywords:
Cassia angustifolia; Decoct; Cyto-toxicology; Geno-toxicology.

INTRODUCTION

Cassia angustifolia Vahl., Fabaceae leaves (Sennae folium) are well known for numerous pharmacological properties, such as anti-inflammatory, anti-bacterial, anti-fungal, anti-viral, hepatoprotective, hypoglycemic, neurodegenerative, anti-obesity, and, most importantly, laxative activities. Used as a spice, it offers a unique flavor to add richness and depth to various traditional Eastern dishes. Because of that, it has been used for more than 2000 years for treating a variety of diseases, and, most importantly, as a constipation remedy (Le et al., 2021; Zibaee et al., 2023).

Having in mind its ethnopharmacological value, the fact that even 16% of adults and 33% of adults older than 60 years suffer from constipation (Bharucha et al., 2013), and the fact that these numbers are much higher in people with the most frequent health issues, such as diabetes and cancer, where constipation is occurring in 60 - 90% of patients overall (Phillips et al., 2006; Wickham, 2017), wide use of this plant species globally is not of the surprise. However, scientific evaluation of its potential benefits and its safety showed contradictory results. For instance, previous studies showed that it could be effective in treating colorectal carcinoma (Yan et al., 2019), that there is no relation between long-term use of its extract (Morales et al., 2009), and that it also may stimulate the occurrence of melanosis coli and carcinogenesis of colon cancer (Le et al., 2021). Evaluation of C. angustifolia cytotoxicity/ genotoxicity also showed opposing results - from the absence of toxicity, through mild toxicity, up to the high levels of toxicity (Heidemann, Miltenburger, Mengs, 1993; Brusick, Mengs, 1997; Silva et al., 2004; Zibaee et al., 2023).

Thus, the aim of this research was to further elucidate the biological properties of C. angustifolia decoct through evaluation of its phytochemical composition, antioxidant capabilities as well as the levels of its cytotoxicity and genotoxicity.

MATERIAL AND METHODS

Plant material

C. angustifolia leaves (Sennae folium) were obtained from the Institute for the Study of Medicinal Plants Dr. Josif Pančić, Belgrade, Serbia. The extracts were prepared according to the traditional use, in the form of decoct. 1 g of dry plant material was boiled in 100 ml of distilled water until half of the liquid evaporated. The extract was then filtered and used as a stock solution. Both stock solution and dilutions were made directly before use. Six concentrations of C. angustifolia decoct, i.e., 250, 500, 750, 1000, 1250 and 1500 μg of dry herbal weight/ml were evaluated using in vitro (DPPH radical scavenging activity and RBCs hemolytic activity) and in vivo methods (Allium cepa test, lipid peroxidation assay).

Phytochemical analysis

HPLC analysis

HPLC with UV detection was used to identify individual compounds of the tested extract, as previously described (Madić et al., 2021), using Agilent 1200 Series with the C18 column (Zorbax Eclipse XDBC18, 5 μm, 4.6 × 150 mm), Diode Array Detector (DAD), quaternary pump, vacuum degasser, autosampler and AgilentChemStation software (Agilent Technologies, US). Elution of the samples was performed in gradient mode, by varying the volume ratios (v/v) of eluent A (0.27 M formic acid solution) and eluent B (methanol): initially 70% A (0-5 min), 70-30% A (5-20 min), 30-10% A (20-25 min). The column temperature was 25ºC, the injected sample volume was 5 μl, the wavelength range was 190-400 nm, and the detection wavelength was 280 nm. Identification of the extract components was done by comparing retention times and UV absorption spectra of constituents with commercially available standards.

In vitro study

DPPH radical scavenging activity

The antioxidant activity of the tested extract was evaluated using the DPPH radical scavenging method as previously described by Madić et al. (2021). Tested concentrations were prepared by diluting the stock solution with methanol. The positive control was 0.3 ml of methanol mixed with the DPPH radical methanol solution, while butylated hydroxytoluene (BHT) was used as a standard. Finally, the absorbance was measured at 517 nm. The radical scavenging activity (DPPHsa) was calculated using the formula:

$$\text{DPPHsa}(\mathrm{\%})=\frac{\text{Absorbance of the positive control}}{\text{Apsorbance of the tested extract}}\times 100$$

RBCs hemolytic activity

The animals were used by the principles of the Care and Use of Laboratory Animals and approved by the Ethical Committee of the Faculty of Medicine, University of Niš (No. 323-07-05754/2023-05). Red blood cells (RBCs) of male Wistar rats were prepared as previously described by Madić et al. (2019).

The hemolytic assay was done according to the slightly modified protocol of Yang, Sun and Fang (2005). Tested concentrations were prepared by diluting the stock solution with PBS. 0.5 ml of RBCs suspension was mixed with 0.5 ml of the tested extract, and the mixture was incubated at 37ºC for 2h. The samples were afterwards centrifugated at 2000 RPM for 5 minutes at 4ºC. 0.2 ml of the obtained supernatant was mixed with 3 ml of Drabkin’s reagent, incubated for 10 minutes at the room temperature, and, in the end, absorbance was measured spectrophotometrically at 540 nm. The negative control (NC) was RBCs in PBS, and the positive control (PC) was RBCs in 6% H₂O₂. Hemolytic activity (H.A.) was calculated as follows:

$$H.A.(\mathrm{\%})=\frac{\text{absorbance of the tested extract}-\text{absorbance of the}NC}{\text{absorbance of the}PC-\text{absorbance of the}NC}\times 100$$

In vivo study

Allium cepa test

The Allium cepa test was done according to the modified method of Fiskesjo (1985) as previously described by Madić et al. (2019). Briefly, the bulbs of the common onion (A. cepa) were grown in distilled water for 24 hours. Six low concentrations of C. angustifolia decoct were prepared by diluting the stock solution with dH₂O₂. The bulbs with satisfactory root lengths were exposed to the tested extracts for 48 h. The negative controls were bulbs grown in distilled water and the positive controls were bulbs exposed to 6% H₂O₂. Analysis of meristematic cells was done using a Leica microscope (Leica DM2500, Germany) at 400× and 1000×. For every tested concentration 2500 cells in total were analyzed.

Cytotoxicity and genotoxicity of tested extract were evaluated using following parameters: mitotic index (M.I.), total percent of cells with chromosomal aberrations (Chr. Ab.), total percent of dividing cells in individual phases of mitotic cycle (P.I.), i.e., prophase (Pro.), metaphase (Met.), anaphase (Ana.), telophase (Telo.), the percentage of the cells with normal chromosomes in individual phases of mitosis (N.Ph., i.e., N.Pro., N.Met., N.Ana., and N.Telo.), the percentage of chromosomal aberrations in individual phases of the mitosis (Ab.Ph., i.e., Ab.Pro., Ab.Meta., Ab.Ana., and Ab.Telo.), as well as frequency of specific chromosomal aberrations (SCA), i.e., C-mitosis, C-shaped metaphase, stickiness, bridges in anaphase and telophase, multipolar mitosis, fragmented chromosomes (chromosomal breaks).

M.I. was calculated as the percent ratio between the number of mitotic cells and the total number of observed cells, and Chr.Ab. as the percent ratio between the number of aberrant mitotic cells in relation to the number of observed cells. P.I. was calculated as a percent ratio between cells in a specific phase of the mitotic cycle and the dividing cells (Fiskesjo, 1985), and N.Ph. and Ab.Ph. as a percent ratio between normal/aberrated cells and total dividing cells in a specific phase (Madić et al., 2019). SCA was determined as the percentage ratio between the number of cells with specific aberrations and total number of aberrant cells (Bouzekri et al., 2023).

Lipid peroxidation assay

The level of lipid peroxidation was done using the slightly modified method described by Debnath and associates (2020). Briefly, 0.1 g of treated A. cepa roots were homogenized in 10 ml of 0.1% TCA and centrifugated at 4000 rpm for 10 minutes at 4°C. 1 ml of obtained supernatant was separately mixed with 4 ml of 0.5% TBA dissolved in 20% TCA, incubated at 100°C for 25 minutes, cooled, and centrifugated at 3000 rpm at 4°C. The absorbance was measured at 532 and 600 nm, while the concentration of MDA was calculated using the equation

$$MDA=\frac{\text{Absorbance at}532\mathrm{nm}-\text{Absorbance at}600\mathrm{nm}}{155}\times 1000$$

Statistical analysis

Statistical analysis was done using GraphPad Prism 5 (GraphPad Software, La Jolla California USA). In vitro experiments were done in triplicate, in vivo experiments in pentaplicate, and data were expressed as the mean ± standard deviation. The differences between the controls and the individual dosage groups of the tested extract were analyzed by the one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test. Statistical significance was accepted if p was less than 0.05.

RESULTS

Phytochemical analysis

HPLC-UV analysis used for the identification of laxative compounds, specially sennosides and anthraquinones, revealed the presence of six laxative compounds in the decoct of this Cassia species. By comparing the retention times (tR) and UV spectra (200-800 nm) of tested extract and authentic substances’ ones, we confirmed the presence of 8-glucosyl rhein (tR = 14.710 ± 0.10 min), sennoside A (tR = 15.86 ± 0.20 min), sennoside B (tR = 16.60 ± 0.10 min), sennoside C (tR = 17.70 ± 0.10 min), sennoside D (tR = 18.193 ± 0.10) and rhein (tR = 21.973 ± 0.20 min) in the tested extract (Figure 1).

FIGURE 1
HPLC chromatogram recorded at 280 nm of compounds with laxative activity.

In vitro study

DPPH radical scavenging activity

As shown in Figure 2, the antioxidant activity of the tested extract was low. Namely, the radical scavenging activities of all the tested concentrations of C. angustifolia decoct were lower (p < 0.001) than BHT, standard antioxidant, with IC₅₀ of tested extract 3.8 mg/ml and IC₅₀ of BHT 0.86 mg/kg.

FIGURE 2
Antioxidant activity of the C. angustifolia decoct. DPPHsa: Percent of the DPPH radical scavenging activity; BHT: butylated hydroxytoluene used as standard; 250-1500: tested concentrations of C. angustifolia decoct in µg of dry herbal weight/ml. Data were expressed as the mean ± standard deviation, n = 3. ***p < 0.001 compared to the standard.

RBCs hemolytic activity

As shown in Figure 3, the tested extract showed the concentration-depended hemolytic activity, i.e., 14.82 ± 3.21, 27.78 ± 5.55, 29.63 ± 3.2, 35.18 ± 3.21, 49.99 ± 5.55, and 62.96 ± 3.21% when treated with 250, 500, 750, 1000, 1250 and 1500 μg of dry herbal weight/ml.

FIGURE 3
Hemolytic activity of C. angustifolia decoct. 250-1500: tested concentrations of C. angustifolia decoct in µg of dry herbal weight/ml. Data were expressed as the mean ± standard deviation, n = 3. a: p < 0.001 when compared to the PC group.

In vivo study

Allium cepa test

In the A. cepa test, 48h treatment with C. angustifolia decoct induced a number of different chromosomal aberrations as shown in Figure 4.

FIGURE 4
Stages of mitotic division in cells of A. cepa treated with different concentrations of C. angustifolia decoct. 1(a): interphase; 1(b): normal prophase; 2(a): sticky metaphase; 3(a): c-mitosis; 4(a): sticky metaphase; 5(a): sticky telophase with fragmented and lagging chromosomes; 5(b): vagrant chromosome in prometaphase; 6(a): lagging chromosomes in anaphase; 7(a): normal anaphase; 8(a): normal metaphase; 9(a): telophase with lagging chromosome; 10(a): star telophase; 10(b): fragmented metaphase; 10(c): c-shaped metaphase; 11(a): sticky and c-shaped metaphase; 11(b): normal prophase; 12(a): normal telophase; 13(a): sticky metaphase; 14(a): star anaphase; 14(b): fragmented prophase; 15(a): normal anaphase; 15(b): normal late anaphase; 16(a): bridge in anaphase; 16(b): sticky early metaphase; 17(a): bridges in anaphase; 17(b) lagging chromosomes in anaphase; 17(c): fragmented late metaphase; 18(a): lagging chromosome in telophase; 19(a): sticky early metaphase; 19(b): normal telophase; 19(c): normal metaphase; 20(a): bridges in anaphase. Scale bar 25 μm. Magnification 1000×.

The detailed cytogenetic analysis showed that the mitotic index in the negative control and the positive control was 22.44 ± 0.68 and 8.92 ± 1.19%, respectively. Compared to the negative control, treatment with C. angustifolia decoct caused dose-dependent decrease of M.I., i.e.,¬¬ 19.95 ± 1.15, 35.83 ± 3.18, 44.72 ± 2.59, 47.34 ± 8.57, 54.32 ± 3.84, and 56.25 ± 4.61 in bulbs treated with 250, 500, 750, 1000, 1250 and 1500 μg of dry herbal weight/ml, respectively (Figure 5 (a)).

FIGURE 5
Mitotic index (a) and chromosomal changes (b) in meristematic cells of A. cepa after 48 h exposure to C. angustifolia decoct. NC (negative control): distilled water; PC (positive control): 6% H₂O₂; M.I.: mitotic index; Chr.Ab: total percent of cells with chromosomal aberrations; 250-1500: tested concentrations of C. angustifolia decoct in µg of dry herbal weight/ml. Data were expressed as the mean ± standard deviation. The results refer to the analysis of 2500 cells per treatment. Data were expressed as the mean ± standard deviation, n = 5. a: p < 0.05 compared to the NC group. b: p < 0.05 compared to the PC group.

At the same time, the increase of the tested extract’s concentrations caused a dose-dependent increase of chromosomal aberrations in A. cepa meristematic cells in mitosis. The lowest percent of Chr.Ab. was observed in the negative control (1.77 ± 0.58%) and the highest one in the positive control (70.41 ± 9.56%). 48h of exposure of A. cepa meristematic cells to the concentrations of 250, 500, 750, 1000, 1250 and 1500 μg/ml of C. angustifolia decoct resulted in 9.56 ± 1.57, 14.68 ± 3.57, 26.65 ± 6.12, 28.76 ± 5.73, 37.16 ± 2.65, and 42.84 ± 1.13% of Chr. Ab., respectively (Figure 5 (b)).

Treatment with C. angustifolia decoct only slightly affected the phase index compared to the positive control, in terms of the fact that higher tested doses increased the percent of the cells in anaphase and the telophase of the mitotic cycle (Figure 6 (g, j)). At the same time, as shown in Figure 6 (f, i, l), treatment doses higher than 750 μg/ ml increased the percent of chromosomal aberrations in metaphase, anaphase and telophase compared to the negative control (p < 0.001), while the highest tested dose (1500 μg/ml) caused the accumulation of chromosomal aberrations in prophase as well (Figure 6 (c)) ((p < 0.05)).

FIGURE 6
Phase index in meristematic cells of A. cepa after 48 h exposure to C. angustifolia decoct. NC (negative control): distilled water; PC (positive control): 6% H₂O₂; 250-1500: tested concentrations of C. angustifolia decoct in µg of dry herbal weight/ml. n = 5. a: p < 0.05 compared to the NC group. b: p < 0.05 compared to the PC group.

The lowest percent of chromosomal aberrations was observed in negative control (1.28 ± 1.31, 3.27 ± 4.94, 2.36 ± 3.24, and 1.18 ± 2.63% in prophase, metaphase, anaphase, and telophase, respectively) (Figure 6 (c, f, i, l)). The highest percent of chromosomal aberrations in prophase and telophase was observed in the positive control (78.7 ± 11.56 and 67 ± 22.25%, respectively) (Figure 6 (c, l)), while the highest percent of aberrations in metaphase and anaphase was seen in meristematic cells treated with highest tested concentration of tested extract, where treatment with 1500 μg/ml caused 69.86 ± 10.26% of Ab.Meta. and 83.8 ± 7.79% Ab.Ana (Figure 6 (f, i)).

Yet, none of the tested concentrations caused such a high accumulation of chromosomal aberration in prophase as observed in positive controls. (Figure 6 (c)) (p < 0.001).

Moreover, as shown in Figure 7, treatment with tested decoct caused a variety of chromosomal aberrations in A. cepa meristematic cells. The lowest number Chr.Ab. was observed in negative control (1.76 ± 0.58%), where the occasional mutations included C-mitosis (50 ± 50%) and multipolar mitosis (26.67 ± 25.27%) (Figure 7 (a, e)). The highest Chr.Ab. was recorded in the positive control (70.41 ± 9.56%), where the most prominent aberration was fragmented chromosomes (93.22 ± 4.17%). Stickiness of the chromosomes in the cells treated with C. angustifolia decoct was similar to the ones treated with 6% H₂O₂, while bridges in anaphase and telophase were more frequent in cells treated with tested decoct than in the positive control group (p < 0.01), however, the dose-dependence was not observed.

FIGURE 7
Types of chromosomal aberrations in A. cepa meristematic cells induced by 48h exposure to C. angustifolia decoct. NC (negative control): distilled water; PC (positive control): 6% H₂O₂; 250-1500: tested concentrations of C. angustifolia decoct in µg of dry herbal weight/ ml. Data were expressed as the mean ± standard deviation, n = 5. a: p < 0.05 compared to the NC group. ^b: p < 0.05 compared to the PC group.

Lipid peroxidation assay

The results of the lipid peroxidation test showed that 48h of the treatment with C. angustifolia decoct increased the level of the MDA in the A. cepa roots in a concentration-dependent manner. As shown in Figure 8, the lowest tested concentration of the extract (250 μg/ml) did not increase the level of MDA compared to the negative controls (0.05 ± 0.003 and 0.05 ± 0.009 mmol/g, respectively). Yet, the level of MDA in the group treated with the highest tested concentration of the extract (1500 μg/ml) was almost as high as in the positive control group, treated with H₂O₂ (0.12 ± 0.02 and 0.14 ± 0.007 mmol/g, respectively).

FIGURE 8
Level of MDA in A. cepa roots after 48h exposure to C. angustifolia decoct. NC (negative control): distilled water; PC (positive control): 6% H₂O₂; 250-1500: tested concentrations of C. angustifolia decoct in µg of dry herbal weight/ml; Data were expressed as the mean ± standard deviation, n = 5. a: p < 0.05 compared to the NC group. b: p < 0.05 compared to the PC group.

DISCUSSION

This study aimed to evaluate the ethnopharmacological value C. angustifolia decoct through assessment of its phytochemical composition, antioxidant activity, and, most importantly, potential in vitro and in vivo toxic effects.

Phytochemical analysis revealed the presence of six laxative compounds in the tested C. angustifolia decoct, i.e., 8-glucosyl rhein, sennoside A, sennoside B, sennoside C, sennoside D and rhein (Figure 1). Interestingly, tested decoct showed very low antioxidant activity, lower than BHT (Figure 2), contrary to the study of Vargas and associates (2004), but similar to the research of Towanou and colleagues (2023). Namely, in Vargas and associates’ study, rhein, one of the bioactive substances of the tested decoct, was more effective as an antioxidant than even vitamin C. However, Towanou and colleagues’ study (2023) showed that other species rich in laxative compounds had lower antioxidant capacity than standard commercial antioxidants. This is not of a surprise because biological properties of pure compounds have different biological properties than crude medicinal plant extracts such as decocts, due to the complexity of their phytochemicals that can work synergistically, additively or antagonistically (Prajitha, Thoppil, 2016).

Both in vitro (Figure 4) and in vivo cytotoxicity evaluation (Figure5 (a)), Figure 6 (a, d, g, j)) confirmed that exposure of the cells to the C. angustifolia decoct had a dose-dependent cytotoxic effect.

Having in mind that when comparing the percent of cytotoxic activity to the negative control, where cytotoxicity of the plant extracts can be classified into four groups: (a) neutral, when the cytotoxic activity of the tested extract is similar to the negative control; (b) weak, with cytotoxicity < 20% of the control; (c) potent, when cytotoxic activity was higher than 20% of the control, and (d) high, when cytotoxic activity is > 50% of the control (Antosiewicz, 1990; Khalili, Ebrahimzadeh, Safdari, 2014), it can be observed that only the lowest tested concentration of C. angustifolia decoct (250 μg of dry herbal weight/ml) had minimal cytotoxic activity, while higher tested doses had medium and high cytotoxic activity. The observed hemolytic activity can be explained by the downregulation of aquaporins (Cao et al., 2018) as well as by the increase of Ca²⁺ induced hyperpolarization of the membrane, cell membrane scrambling and eryptosis (Lang, Lang, 2015; Mischitelli et al., 2016).

In the RBCs hemolytic testing, even the highest tested concentration (1500 µg/ml) was not as cytotoxic as 6% H₂O₂ used as positive control (p < 0.001) (Figure 4). However, contrary to the RBC’s hemolytic test, in the A. cepa cytotoxicity evaluation, the cytotoxicity of the highest tested concentration of decoct was similar to the cytotoxicity of H₂O₂ (Figure 5 (a)).

Interestingly, lower tested doses of C. angustifolia decoct (250 and 500 µg/ml) increased the total percent of the cells in the prophase (Figure 6 (a)) but had no significant effect on the other phases of the treated meristematic cells’ mitotic cycle. At the same time, higher tested concentrations of the extract (1250 and 1500 µg/ml) showed a tendency to increase the percent of the cells in the latter stages of mitosis (Figure 6 (d, g, j)), confirming the neutral/low cytotoxicity of low concentrations of tested decoct, and high cytotoxicity of the extract when used in high concentrations (Fiskesjo, 1985).

Similarly to the evaluation of cytotoxicity, the genotoxicity assessment showed that the lowest tested concentration of the extract (250 µg/ml) was only mildly genotoxic. However, contrary to the results of the cytotoxicity assessment, where the highest tested concentration of the extract (1500 µg/ml) was highly cytotoxic, i.e., of similar toxicity as the positive control, the genotoxicity analysis revealed that its genotoxicity is much lower. To wit, the treatment with the highest tested concentration of the decoct induced much fewer chromosomal aberrations than the treatment with H₂O₂ (p < 0.001) (Figure 5 (b)).

Observed high cytotoxicity of higher tested concentrations of C. angustifolia decoct can be explained by the variety of accumulated chromosomal aberrations, such as c-mitosis, c-shaped metaphases, multipolar mitosis (including star-shaped anaphases and telophases), the stickiness of chromosomes, but most importantly - the chromosome bridges (Figure 4, Figure 7) caused by the formation of anthraquinone-G-quadruplex complex, a well-known inducer of telomere disruption (Dey et al., 2022), which can lead to several different outcomes, including chromosome breakage, cell cycle arrest and even the death of the cell (Pampalona et al., 2016).

The mechanism of the formation of observed chromosomal aberrations by laxative compounds present in the tested decoct (Figure 1) has already been characterized in the literature. Namely, detected anthraquinones and sennosides are well known for their capability to modulate a variety of cellular molecules such as cell-cycle proteins, transcription factors, enzymes, oncoproteins, and tumor suppressor proteins (Henamayee et al., 2020; Le et al., 2021). Thus, rhein can inhibit cell proliferation by inducing a cell-cycle arrest in G0/G1 and S phases through the decrease of the levels of β-catenin (Fu et al., 2004; Fang et al., 2007; Shi, Huang, Chen, 2008; Liu et al., 2018) as well as in G2/M phase through formation of the Pin1/c-Jun complex (Wang et al., 2015). Additionally, due to the high cytotoxicity owned to the inhibition of topoisomerase II, intercalation of DNA, and formation of free radicals, which further leads to DNA damage and the death of the cell, anthraquinones are already used in cancer treatment (Gamen et al., 1997; Van Gorkom et al., 2002).

Nevertheless, since none of the tested concentrations caused such a high accumulation of chromosomal aberration in prophase as observed in positive control (Figure 6 (c)) (p < 0.001), meristematic cells treated with H₂O₂, a well-known genotoxic agent (Benhusein et al., 2010), and the fact that most frequent aberration observed in positive controls, not only in this phase but in all the phases of mitotic cycle, was just the most severe chromosome aberration - fragmented chromosomes, a major form of mitotic cell death as a response to stress (Stevens et al., 2011), it could be concluded that high concentrations of decoct of Sennae folium possess much higher cytotoxic than genotoxic properties.

Finally, the results of the lipid peroxidation test confirmed the high cytotoxic properties of the tested extract. Namely, the level of MDA in roots treated with the highest tested concentration (1500 µg/ml) of the extract was almost as high as in the positive control group, treated with H₂O₂ (Figure 8). Having in mind that MDA is the final product of membrane lipid peroxidation that accumulate when cells are subjected to the elevated levels of oxidative stress (Ünyayar et al., 2006), caused by anthraquinones and sennosides present in the tested decoct (Huang, Chan, 2017) and that levels of cytotoxicity were similar in the groups treated with high concentrations (1250 and 1500 µg/ml) of the extract in both RBCs hemolytic experiment, A. cepa test and lipid peroxidation assay, it can be noticed that although the extract act as a cytotoxic agent, mainly on the cell membrane level, its overall toxicity is not that high because cell managed to reduce the genotoxic effect themselves (Łuczaj, Skrzydlewska, 2003).

It can be concluded that, despite low antioxidant activity, the tested decoct of C. angustifolia may be used for its antitumor activities. However, it cannot be neglected that treatment with all the tested concentrations, even with the low doses, of the extract caused at least some level of both cytotoxicity and genotoxicity, and that high concentrations showed even higher toxicity levels, due to the well-known phenomena called “Janus effect” (Zeiger, 2003). Thus, precautions are necessary when using the decoct of this Cassia species as an easily available herbal remedy.

ACKNOWLEDGMENTS

This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No. 451-03-65/2024-03/200124 and 451-03-66/2024-03/200124).

DATA AVAILABILITY STATEMENT

Data available from the corresponding author upon reasonable request.

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Edited by

• Associate Editor:
  Carlota Rangel Yagui

Publication Dates

• Publication in this collection
  28 Aug 2026
• Date of issue
  2026

History

• Received
  02 Sept 2024
• Accepted
  29 Jan 2025