Antimitotic and cytotoxic effects of Clerodendrum indicum leaf aqueous extract in Allium cepa root tip cells

Abstract

Clerodendrum indicum (L.) Kuntze. (Family Lamiaceae, common name: bamunhati) is a medicinal plant traditionally used for various therapeutic purposes. This study aimed to evaluate the antimitotic and cytotoxic effects of leaf aqueous extract of C. indicum (LAECi) in Allium cepa root tip cells. Root tips of A. cepa were treated with varying concentrations up to 8 mg/mL of LAECi for 2 to 48 h and root growth retardation, mitotic index (MI) reduction, and the occurrence of cellular abnormalities such as hyperchromasia, nuclear lesion, nuclear erosion, and giant cells were analyzed. The results showed that LAECi induced a concentration- and time-dependent root growth retardation, MI reduction, and the formation of diverse cellular abnormalities. Thus, this study explores the antimitotic and cytotoxic effects of LAECi on A. cepa root tip cells.

Cerodendrum is a genus of angiosperms belonging to the family Lamiaceae (Harley et al. 2004). It is widely distributed in tropical and subtropical regions and includes small trees, shrubs, and herbaceous plants. The genus was first described by Linnaeus in 1753 (Munir 1985). Taxonomically, Clerodendrum is characterized by oppositely arranged, entire or toothed leaves, terete stems, terminal or axillary cymose inflorescences, hypogynous bisexual flowers, a persistent calyx, a cylindrical corolla tube with a 5-lobed spreading top, exerted stamens, a short bifid stigma, an imperfectly 4-celled ovary, exalbuminous seeds, and an endocarp that separates into four stony pyrenes (Kirtikar and Basu 1999).

Recent studies have reported the cytotoxic and micronuclei-inducing effects, cell cycle delay, pro-metaphase arrest, and c-metaphase induction effects of other species within the Clerodendrum genus, such as C. viscosum in A. cepa root tip cells (Roy et al. 2021, 2022). Similarly, colchicine-like metaphase arrest and cell cycle delays were observed following treatment with the aqueous leaf extract of C. inerme, along with the cytogenotoxic effects of 3-epicaryoptin on A. cepa root apical meristem cells (Barman et al. 2020; Barman and Ray 2023).

One notable species, C. indicum, also known as the bowing lady or tube flower, is an annual shrub that can grow up to 2–3 m tall. Its lanceolate leaves can reach up to 23 cm in length (Manandhar and Manandhar 2002). The plant’s large inflorescences bear tubular white or yellow flowers, while its fruits are round, pulpy, and transition from green to blue-black or reddish-black upon maturity. Traditionally, the leaves and roots of C. indicum have been used in the treatment of various ailments, including gastric cancer, malaria, asthma, rheumatism, hysteria, bronchitis, fever, intestinal worms, arthritis, epilepsy, febrile convulsions, and a range of gastrointestinal disorders (DeFilipps and Krupnick 2018). Additionally, the plant has shown potential as an anti-HIV-1 agent, with promise for treating acquired immunodeficiency syndrome (Bunluepuech and Tewtrakul 2009).

Clerodendrum indicum contains a variety of bioactive compounds, including hispidulin, which exhibits anticancer properties (Subramanian and Nair 1973). Furthermore, three triterpenoids—lupeol, oleanolic acid 3-acetate, and betulinic acid—are noted for their cytotoxic activities (Somwong and Suttisri 2018). Another important compound, p-coumaric acid, derived from C. indicum, has demonstrated phytotoxic effects (Kyaw et al. 2021).

Despite its therapeutic potential, there are few studies examining the antiproliferative and cytotoxic activities of C. indicum. Given the importance of evaluating its bioactivity, this study aims to investigate the antimitotic and cytotoxic effects of the leaf aqueous extract of C. indicum (LAECi) on A. cepa root tip cells in detail. The A. cepa test system, endorsed by the United Nations Environment Programme, the International Programme on Chemical Safety, and the World Health Organization, is a widely accepted model for studying mitotic abnormalities due to its rapid cell proliferation, large chromosome size, and low chromosome number (2n=16) (Cabrera and Rodriguez 1999; Gomes et al. 2013). This method is highly sensitive, cost-effective, and suitable for assessing the cytotoxicity of plant extracts (Fiskesjö 1985; Andrade et al. 2008; Bakare et al. 2012; Dal-Souto Frescura et al. 2013; Ray et al. 2013a, 2013b; Barman et al. 2020; Chakraborty et al. 2021; Das et al. 2021; Barman and Ray 2022).

Materials and methods

Plant leaf collection and preparation of aqueous extract

The plant C. indicum was collected from Joypur Forest, Bankura, West Bengal, India. The plant’s authenticity was confirmed by the Botanical Survey of India, Howrah, West Bengal, by referencing the deposited specimen (No. BU/200/01). Freshly collected leaves were thoroughly washed under running tap water, shade-dried at room temperature, and ground into a fine powder using a Philips Mixture Grinder (Model HL 1605, Kolkata, India). The powdered leaves were then stored in an airtight container for future use (Dutta and Ray 2015).

For the preparation of the crude aqueous leaf extract, 40 g of powdered leaves were mixed with 1000 mL of double-distilled water and heated slowly for 1.5 h using a regulated heater set at 80°C three times. The resulting extract was filtered through Whatman No.1 filter paper and was designated as the leaf aqueous extract of C. indicum (LAECi).

Root growth retardation assay

The A. cepa bulbs were surface sterilized using a 1% sodium hypochlorite (NaOCl) solution and placed in 6-well plates containing distilled water for root germination at room temperature. Roots of similar size (0.5–1 cm) were then treated with various concentrations of LAECi (0, 0.5, 1, 2, 4, and 8 mg mL⁻¹). The experiment was conducted in triplicate, and root lengths were measured at 24-hour intervals over a 48-hour period following treatment.

Antimitotic and cytotoxic effects

The antimitotic and cytotoxic actions of LAECi were confirmed by calculating the mitotic index (MI) and by scoring different nuclear abnormalities such as hyperchromasia, nuclear lesions, nuclear erosions, and giant cells following the standard procedure described by Ray et al. (2013b) and Prajitha and Thoppil (2016a) respectively with minor modifications. In this process, healthy onion bulbs were treated with LAECi at concentrations of 0.5, 1, 2, 4, and 6 mg mL⁻¹, while bulbs maintained in distilled water served as the negative control. Root tips were collected at 2, 4, and 6 h after treatment and fixed in freshly prepared aceto–methanol (1 : 3) for 24 h. Following fixation, the root tips were hydrolyzed in 1 M HCl for 10 min, stained with 2% aceto-orcein, and squashed in 45% acetic acid. For each concentration, at least three slides were prepared and examined under a Magnus MLX microscope, and images were captured using the Future Win Joe software (Version 1.6.5.1207, Future Optics).

Counting and statistical analysis

In the squash preparations, total cells were counted to study cytological and chromosomal abnormalities. The mitotic index (MI) was calculated by determining the ratio of dividing cells to the total number of cells scored at each concentration. The percentage of aberrant mitotic cells was calculated by dividing the number of cells exhibiting specific abnormalities by the total number of dividing cells, multiplied by 100. Similarly, the percentage of aberrant interphase cells was determined by dividing the number of abnormal interphase cells by the total number of interphase cells, multiplied by 100 (Bakare et al. 2009). Cytotoxic abnormalities were assessed using a 2×2 contingency Chi-square test, while root growth retardation was analyzed using Student’s t-test in Origin 8.5 software. All data (percentages) are presented as mean±SEM.

Results and discussion

In this study, the antimitotic and cytotoxic effects of LAECi were evaluated using a root length retardation assay and an analysis of various cell division phases and interphase aberrations in A. cepa root tip cells. According to Dal-Souto Frescura et al. (2012), the cytotoxic and antiproliferative effects of plant extracts and biochemical components can be effectively studied using A. cepa root apical meristem cells. Following this approach, we assessed the root growth inhibitory effect of LAECi at concentrations ranging from 0.5 to 8 mg mL⁻¹ over 24 and 48 h. The highest root growth inhibition (94.54±1.48% at 24 h and 93.06±0.72% at 48 h) was observed at 8 mg mL⁻¹. Even at the lowest concentration (0.5 mg mL⁻¹), significant inhibition (27.45±1.22% at 24 h and 38.07±1.01% at 48 h) was recorded (p＜0.0001). The IC₅₀ values for root growth retardation were calculated as 1.85 mg mL⁻¹ at 24 h and 1.16 mg mL⁻¹ at 48 h (Table 1).

Table 1. LAECi induced root growth retardation of A. cepa.

LAECi Concentration (mg mL−1)	Root growth (cm)/[Retardation %] (mean±SEM)
	24 h	48 h
0	1.09±0.01/[00±00]	2.15±0.02/[00±00]
0.5	0.79±0.01/[27.45±1.22]	1.33±0.01/[38.07±1.01]
1	0.6±0.01/[45.00±1.06]	1.05±0.01/[51.12±0.86]
2	0.5±0.01/[54.09±1.43]	0.90±0.01/[58.09±0.79]
4	0.21±0.02/[80.72±2.12]	0.52±0.02/[75.87±0.97]
8	0.06±0.01/[94.54±1.48]	0.15±0.01/[93.06±0.72]
IC50	1.86 mg mL−1	1.16 mg mL−1

The values of the treated groups are statistically significant compared to their respective controls (p＜0.0001), as determined by a Student’s t-test for two populations.

In this study, we evaluated the cytotoxic effect of LAECi on the root apical meristem cells of A. cepa. A significant difference in MI values was found between the untreated control group and the LAECi-treated samples (0.5–4 mg mL⁻¹). The maximum MI reduction, recorded at the highest concentration of LAECi, was 4.47±0.17%, 2.54±0.04%, and 0.27±0.01% at 2, 4, and 6 h, respectively. Even the lowest concentration (0.5 mg mL⁻¹) showed a reduction in MI (7.01±0.01%, 6.82±0.05%, and 4.29±0.25%) with increasing treatment durations (2, 4, and 6 h) (Table 2). Recently, Roy et al. (2021) and Das et al. (2021) reported the frequency of mitotic phases and cellular abnormalities in the root apical meristem cells of A. cepa treated with plant extracts. A reduction in the MI and the observed cytostatic effects suggest potential cytotoxicity. The significantly reduced MI values observed with LAECi treatment suggest its ability to inhibit cell growth, indicating its promise as a candidate for anticancer research. These findings demonstrate both a concentration- and duration-dependent cytotoxic effect of LAECi. These results align with previous studies, which showed that several medicinal plant extracts inhibit mitotic cell division in A. cepa, demonstrating antimitotic properties (Ray et al. 2013a, 2013b). According to multiple authors, MI reduction suggests cytotoxicity, potentially caused by a blockage during the G2 phase, insufficient ATP synthesis affecting spindle fiber formation, or disruptions in DNA synthesis that prevent cells from entering mitosis (Sreeranjini and Siril 2011; Majewska et al. 2003; Sudhakar et al. 2001). Earlier studies have also proposed that plant extracts that reduce MI can be considered cytogenotoxic (Dal-Souto Frescura et al. 2013).

Table 2. LAECi induced cytological abnormalities in A. cepa. root apical meristem cells.

Time (h)	Concentration (mg mL−1)	TCC	TDC	TIC	MI	Hyperchromasia	Nuclear erosion	Nuclear lesions	Giant cell
					% (mean±SEM)
2	0	7457	204	7253	7.24±0.10	0.01±0.01	0.13±0.03	0.00±0.00	0.00±0.00
	0.5	7581	170	7411	7.01±0.01	0.01±0.01	0.05±0.05	0.00±0.00	0.00±0.00
	1	7430	147	7283	6.13±0.08	0.03±0.03	0.15±0.03	0.09±0.06	0.00±0.00
	2	8423	162	8261	6.22±0.07	0.02±0.01	0.17±0.04	0.19±0.08b	0.00±0.00
	4	8771	151	8620	4.47±0.17c	0.23±0.02b	0.15±0.02	0.34±0.04c	0.00±0.00
4	0	10059	243	9816	7.18±0.16	0.01±0.01	0.13±0.07	0.00±0.00	0.00±0.00
	0.5	7193	155	7038	6.82±0.05	0.00±0.00	9.79±1.25c	0.00±0.00	0.00±0.00
	1	8848	167	8681	5.47±0.10c	0.03±0.02	32.60±2.72c	0.12±0.03	0.02±0.02
	2	7932	130	7802	4.04±0.07c	0.37±0.00c	38.82±4.15c	0.88±0.02c	0.13±0.02
	4	9164	75	9089	2.54±0.04c	1.26±0.02c	43.53±0.81c	1.01±0.02c	0.14±0.00b
6	0	10256	744	9512	7.23±0.08	0.07±0.00	0.08±0.05	0.01±0.01	0.00±0.00
	0.5	7594	317	7277	4.29±0.25c	0.66±0.05c	23.36±0.86c	1.06±0.11c	0.08±0.00
	1	6543	63	6480	0.97±0.03c	1.77±0.05c	73.93±5.17c	1.79±0.24c	0.66±0.03c
	2	7984	52	7932	0.65±0.01c	0.64±0.07c	90.91±4.77c	0.47±0.05c	1.20±0.07c
	4	2033	6	2027	0.27±0.01c	3.02±0.42c	97.44±1.15c	0.90±0.17c	0.30±0.06c

^a Significant at p＜0.05, ^b at p＜0.001, and ^c p＜0.0001 as compared to their respective control analyzed with 2×2 contingency χ²-test with respective d.f.=1. TCC: Total cell count, TDC: Total dividing cell, TIC: Total interphase cell, MI: Mitotic index.

Various interphase cell abnormalities, including hyperchromasia, nuclear lesions, nuclear erosion, and giant cells (Fig. 1) were observed to increase progressively with higher concentrations of LAECi and longer treatment durations, compared to the control (Table 2).

In this study, LAECi treatment significantly (p＜0.0001) increased hyperchromasia, with levels reaching 3.02±0.42% and 1.26±0.02% at the highest concentration (4 mg mL⁻¹) after 6 h and 4 h, respectively. Hyperchromasia is a morphological result related to the presence of darkly pigmented nuclei due to an abundance of genetic materials. This is a recognized abnormality and serves as a well-established apoptotic marker in A. cepa meristematic cells under stress (Andrade-Vieira et al. 2012). Stress conditions can damage cellular macromolecules, including nuclear DNA, proteins, and lipids, ultimately leading to apoptosis or necrosis (Sarsour et al. 2009). Certain phytochemicals may act as pro-oxidants, generating reactive oxygen species (ROS) and causing cellular stress (Galati and O’Brien 2004).

In this study, the highest frequencies of nuclear lesions were observed at 1 mg mL⁻¹ (1.79±0.24%) and 0.5 mg mL⁻¹ (1.06±0.11%, p＜0.0001) of LAECi after 6 h of treatment. Nuclear lesions are structural defects of the cell nucleus, such as chromatin condensation, fragmentation, or envelope irregularities. According to Boumaza et al. (2016), nuclear lesions are a common cytological abnormality during interphase, potentially caused by disruptions in the S-phase or DNA synthesis. Mercykutty and Stephen (1980) also reported nuclear lesions in A. cepa root meristem cells caused by plant-derived chemicals, suggesting that cytotoxic agents can break down nuclear material. Significant nuclear lesions, indicating total nuclear destruction, have been observed in A. cepa root meristem cells treated with leaf extracts of Corymbia (Saj and Thoppil 2006).

The extent of nuclear erosion was inversely proportional to the MI, with the highest frequency recorded at 6 h, reaching 97.44±1.15% at 4 mg mL⁻¹ LAECi treatment. Nuclear erosion is a cell abnormality in which the nuclear membrane, the boundary of the cell nucleus, gradually breaks down, resulting in a loss of structural integrity and probable disruption of cellular activities (Renjana and Thoppil 2013). Caetano-Pereira et al. (1999) proposed that stress and environmental mutagens might cause chromatin degeneration, resulting in apparent erosion zones in the nucleus. This suggests the activation of a cellular restriction checkpoint in response to DNA damage, preventing cells from entering mitosis (Xavier et al. 2021).

LAECi induced a significant (p＜0.0001) percentage of giant cells (1.20±0.07%) at a concentration of 2 mg mL⁻¹ after 6 h of treatment. Giant cells are those with an aberrant mononucleus and a nuclear volume double or more than that of a normal cell (Prajitha and Thoppil 2016b). These are formed due to the fusion of vacuoles with the plasma membrane under stress conditions (Gokbayrak et al. 2022). Furthermore, experimental research has indicated that giant cells are subjected to a variety of stresses and frequently fail to control the enormous bulk of their protoplasm, making them prone to injury (Menzel 1988). Giant cells are generally polyploid cells with a larger size than normal cells (Bonciu et al. 2018). It occurs when cells attempting to enter mitosis fail to complete cytoplasmic division. They expand in size and undergo DNA replication and nuclear division before dying (Kenne et al. 1986). According to Cross et al. (1995), Fukasawa et al. (1996), and Morgan et al. (1996), multinucleate giant cells are created when karyokinesis is blocked during replication, resulting in the creation of a tetraploid giant cell.

In summary, this study highlights the antimitotic and cytotoxic potentials of LAECi in A. cepa root tip cells, likely due to its active phytoconstituents. Therefore, further research is necessary to isolate the cytotoxic components for detailed anticancer pharmacological evaluation.

Fig. 1. LAECi induced cytotoxic effects in A. cepa root tip cells.

Panels A–E show normal cell phases: (A) Interphase, (B) Prophase, (C) Metaphase, (D) Anaphase, (E) Telophase (F) Hyperchromasia, (G) Nuclear lesions, (H) Nuclear erosion, and (I) Giant cells. Scale bar=10 µm.

Acknowledgments

The authors thankfully acknowledge Dr. Karthigeyan Kaliyamurthy, scientist-E, (BSI), Howrah, India, for authentication of the plant species and the financial support of CSIR SRF- 09/025(0283)/2019-EMR-I Dated: 30.11.2019, and UGC MRP, DST-PURSE, DST-FIST, and UGC-DRS-sponsored facilities in the Department of Zoology. The author also used ChatGPT to enhance readability and language.

Author contributions

Gobinda Dhibar: Conducted the investigation and wrote the original draft.

Sanjib Ray: Supervised the study, contributed to the methodology and formal analysis, and was responsible for validation, conceptualization, and reviewing and editing the manuscript.

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