Mitotic Abnormality Inducing Effects of Leaf Aqueous Extract of Clerodendrum inerme Gaertn. on Allium cepa Root Apical Meristem Cells

Abstract

Clerodendrum inerme is a traditionally used medicinal plant in tropical and subtropical regions of the world. The present study aimed to analyze the mitotic abnormality-inducing potentials of the leaf aqueous extract of C. inerme (LAECI) on Allium cepa root tip cells. The 48-h aged A. cepa roots were treated with LAECI (2, 4, and 8 mg mL⁻¹) for 2 and 4 h and also allowed to recover up to 16 h and compared with the standard colchicine (0.4 mg mL⁻¹) actions. The cytological study revealed that both the LAECI and colchicine treatments induced mitotic abnormalities like sticky and vagrant chromosomes, anaphase bridge, multipolar anaphase-telophase, etc. The present study indicates that the LAECI contains bioactive substances having colchicine-like mitotic abnormality-inducing effects on A. cepa root tip cells.

Clerodendrum inerme (L.) Gaertn. (family Lamiaceae) is a perennial shrub distributed abundantly near the coastal regions of southern China, the Pacific island, Australia, Southeast Asia, and India (Wiart 2006) and it has been used in the traditional medicinal system of South Asian countries for the treatment of rheumatic pain, arthritis, fever, cough and skin diseases (Shrivastava and Patel 2007, Chethana et al. 2013). The ground leaf juice of C. inerme was used to relieve motor tics in a patient with intractable chronic motor tic disorder (Fan et al. 2009). Studies have shown that the different extracts of C. inerme leaves significantly improved methamphetamine-induced hyperlocomotion in mice (Chen et al. 2012), and shown to have antidiabetic (Rajeev et al. 2012), analgesic, and anti-inflammatory (Amirtharaj et al. 2010, Khanam et al. 2014), antipyretic (Thirumal et al. 2013), hepatoprotective (Chakraborthy et al. 2013), mosquito larvicidal (Kalyanasundaram and Das 1985), antifungal (Sharma and Verma 1991), antimicrobial (George and Pandalai 1949, Anandhi and Ushadevi 2013), and antiviral (Mehdi et al. 1997) activities. The various biological potentials of the ethnomedicinal plant extract mainly due to the presence of secondary metabolites (Mouri et al. 2020).

The phytochemical investigation of C. inerme revealed the presence of flavonoids, sterols, saponins, iridoids, diterpenes, triterpenes, tannins, carbohydrates, fixed oils, phenolics (Al-Snafi 2016). A triterpenic glycoside, lup-1,5,20(29)-trien-3-O-β-D-glucopyranoside along with friedelin, n-octacosane, and β-amyrin were isolated from leaves of C. inerme (Parveen et al. 2010). Several known flavonoids, like hispidulin, apigenin, acacetin, and diosmetin have been isolated from the aerial parts of C. inerme (Nan et al. 2005a, b, Chen et al. 2012, Huang et al. 2015, Srisook et al. 2015). Among them, hispidulin was found to be one of the active chemical constituents having various pharmacological activities including neuropsychiatric disorders (Huang et al. 2015, Liao et al. 2016, Chiou et al. 2018, Mouri et al. 2020), anti-inflammatory activity (Srisook et al. 2015), anticancer activity (Gao et al. 2014), and anti-osteoporotic_activity (Zhou et al. 2014). Several glycosides, such as 2-(3-methoxy-4-hydroxylphenyl) ethyl-O-2″,3″-diacetyl-α-L-rhamnopyranosyl-(1→3)-4-O-(E) feruloyl-β-D glucopyranoside, melittoside, inerminoside, isoverbascoside, monomelittoside, in addition to stigmasta 5,22,25-trien-3-β-ol, betulinic acid, B-friedoolean-5-ene-3-β-ol, and β-sitosterol were isolated from the aerial part of the C. inerme (Nan et al. 2005a, b, Ibrahim et al. 2014).

In our previous study, we have shown that LAECI has root growth inhibitory and swelling effects in A. cepa and causes an increased percentage of metaphase cells with haphazardly arranged condensed chromosomes. These effects were found to be similar to colchicine action (Barman et al. 2020). Colchicine acts effectively as a spindle poison by binding to tubulin dimer and results in microtubule depolymerization, chromosome condensation, and arrest of the cell cycle at metaphase. These mitotic inhibitory effects of colchicine have been of great usage for the metaphase chromosome preparation as well as karyotype studies (Salmon et al. 1984, Negi et al. 2015). Colchicine treatment in root apical meristem cells of A. cepa causes root growth inhibitory and swelling effects and causes an increased frequency of metaphase cells with haphazardly arranged condensed chromosomes by inhibiting mitotic spindle organization (Hague and Jones 1987, Ray et al. 2013, Roy et al. 2020). Colchicine induced an increased frequency of mitotic abnormalities in A. cepa and it was comparable with the leaf aqueous extract of C. viscosum (Kundu and Ray 2016), and clerodin (Roy et al. 2020). Roy and Roy (2019) assessed the cytotoxic effects of aqueous and methanolic leaf extracts of C. inerme treatment on Allium roots after 5 days, however, mitotic abnormalities were not scored from the early hours of treatment duration, and also the concentrations of the extracts were not conclusive. Therefore, detailed cytotoxic effects of LAECI were studied at 2 to 4 h after the LAECI treatment in A. cepa root apical meristem cells and also at 16 h recovery. Moreover, the cytotoxic effects of LAECI were compared with the colchicine-induced cytotoxic effects.

Materials and methods

Plant collection and extract preparation

Fresh C. inerme leaves were collected from Renaissance housing complex, Purba Burdwan, West Bengal, India in January 2018. The voucher specimen (SR/REN/BWN/2018/01) is maintained in the Zoology Department, BU. The collected leaves were washed in tap water, shade dried, crushed in an electric grinder (Philips Mixer Grinder HL1605, Kolkata, India), and stored in an airtight container for future use. For the preparation of aqueous extract, 100 g of C. inerme pulverized leaves were mixed with 1 L distilled water and boiled at 100°C for 1 h and these processes were repeated thrice. Finally, the extract was filtered (Whatman #1 filter paper), measured the concentration, and stored at 4°C until use.

Study of mitotic abnormalities

The similar-sized onion bulbs of A. cepa were purchased from the local Golapbag market of district Purba Bardhaman, India. The bulbs were properly cleaned and the outer scales were carefully peeled out without damaging the primordial of the root and were placed over the test tubes filled with distilled water for 48 h to sprout roots. During this period the bulbs were maintained in a B.O.D. incubator at 22±2°C with darkness. The newly emerged roots (about 2–3 cm in length) were treated with the different concentrations of LAECI (1, 2, 4, and 8 mg mL⁻¹) and 0.4 mg mL⁻¹ of colchicine, for 2 and 4 h. After 2 and 4 h, five roots from each LAECI and colchicine treated bulbs were collected, fixed, and processed for squash preparation following the standard procedure (Chaudhuri and Ray 2015). In the case of 4 h treatment, the remaining roots were allowed to grow further in distilled water for another 16 h and the root tips were fixed and subsequently processed for slide preparation. For the control group, the onion bulbs were maintained simultaneously in distilled water along with the treated groups. The root tips were fixed in 3 : 1 (v : v) methanol-acetic acid for 24 h and then hydrolyzed for ten minutes in 1 M HCl at 60°C, stained with 2% aceto-orcein and finally, root tips were squashed in 45% acetic acid (Ray et al. 2013, Barman et al. 2020). One root tip was squashed on each slide and five slides were prepared for each concentration and observed under a microscope at 40× objective lens for scoring mitotic abnormality.

Statistical analysis

Statistical analyses were performed using the origin lab 5.0 software package. Data obtained on the frequency of different mitotic abnormality (MA%) were analyzed by calculating, MA%=the number of aberrant cells/total number of dividing cells scored ×100. Total aberrant cell frequencies (AB%) were analyzed by calculating, AB%=the number of aberrant cells/total number of cells scored ×100. The level of significance at p≤0.05 or 0.01 or 0.001, between the control and treated values for the mitotic abnormalities, was determined through a 2×2 contingency χ²-test. All the data were expressed as Mean±SEM (standard error mean).

Results

LAECI and colchicine induced mitotic abnormality in A. cepa root tip cells

The different concentrations of LAECI (1–8 mg mL⁻¹) and colchicine (0.4 mg mL⁻¹) induced statistically significant increased frequencies of stickiness, anaphase bridges, vagrant chromosome, multipolar anaphase, and telophase in A. cepa root tip cells (Table 1, Fig. 1).

Table 1. Effects of LAECI and colchicine on the frequency of mitotic abnormalities in A. cepa root apical meristem cells.

Hours	LAECI Conc. (mg mL−1)	TC	TDC	Aberrant cell (%)	Ana-bridge (%)	Vagrant (%)	Cr Sti (%)	Lagging Cr (%)	Polar deviation (%)	Multipolar Ana-Telo (%)
2	0	2872	157	0.23±0.01	0	0.62±0.36	0.41±0.20	0.63±0.00	0	0
	1	4455	177	0.86±0.12***	7.93±0.56***	1.10±0.28	2.95±0.53	1.13±0.06	0	0
	2	3614	188	1.43±0.10***	2.81±0.38	7.58±0.79**	6.03±0.17*	3.55±0.48	2.64±0.46	0
	4	3885	209	1.53±0.08***	4.38±0.90	6.19±0.44**	5.99±0.92*	2.88±0.34	2.52±0.53	0
	8	3757	157	1.24±0.11***	5.54±0.44	3.10±0.78	4.21±0.22	5.44±0.41	2.53±0.32	0
	0.4@	3762	201	3.82±0.10***	2.29±0.34	4.41±0.48	3.86±0.97	0.47±0.25	0	0.67±0.19
4	0	3307	193	0.22±0.04	0.33±0.16	0.69±0.18	0.34±0.17	0.66±0.42	0	0
	1	3661	114	1.04±0.07***	2.75±1.01	3.71±0.45	4.28±0.70	3.28±0.73	3.79±0.73	0
	2	3203	185	3.11±0.06***	3.39±0.58	4.66±0.14	6.14±0.63**	2.68±0.20	1.05±0.27	0
	4	4795	175	2.74±0.12***	7.56±0.71***	7.79±0.25***	15.89±1.27***	1.1±0.55	1.30±0.47	0
	8	4671	152	1.76±0.02***	6.56±0.43**	5.47±0.46	11.81±0.4***	4.58±0.73	4.59±0.03	0
	0.4@	4109	420	8.1±0.12***	1.34±0.43	4.52±0.57*	10.71±0.25***	0.23±0	0	0.46±0.22
4+16	0	4698	285	0.14±0.02	0.34±0.00	0.34±0.00	0.46±0.11	0±0	0	0
	1	4224	271	0.44±0.03**	1.58±0.29	0.97±0.23	0.84±0.43	2.20±0.04	0±0	0
	2	3978	275	6.99±0.13***	7.37±0.18***	8.6±0.39***	5.04±0.61***	3.02±0.04	1.08±0.22	7.36±0.29***
	4	3563	525	17.54±0.08***	7.42±0.16***	9.76±0.49***	5.64±0.19***	1.58±0.23	0.56±0.10	8.12±0.37***
	8	3808	300	7.09±0.11***	7.32±0.55***	8.87±0.35***	4.67±0.75**	3.54±0.05	1.44±0.23	7.42±0.12***
	0.4@	3427	133	12.41±0.16***	2.21±0.28	0.98±0.18	1.23±0.19	0±0	0	2.21±0.28

^@Colchicine, *Significant at p＜0.05. **Significant at p＜0.01. ***Significant at p＜0.001 as compared to their respective control with 2×2 contingency χ² test with respective df=1. TC: total cells; TDC: total dividing cells; AC: aberrant cells; AB: abnormality; Meta: metaphase; Ana: anaphase; Telo: telophase; Cr: Chromosome; Sti: Stickiness.

　

Fig. 1. LAECI induced different types of mitotic abnormalities in A. cepa root apical meristem cells. (A–D) are normal mitotic phase: (A) Prophase, (B) Metaphase, (C) Anaphase, and (D) Telophase; (E–L) LAECI induced mitotic abnormalities: (E) Sticky chromosome, (F) Polar deviation, (G) Anaphase bridge, (H) Vagrant chromosome, (I) Lagging chromosome, (J) Ring chromosome, (K) Multipolar anaphase, and (L) Multipolar telophase; (M–T) Colchicine induced mitotic abnormalities: (M) Sticky chromosome, (N) polar deviation, (O) Anaphase bridge, (P) Vagrant chromosome, (Q) Lagging chromosome, (R) Ring chromosome, (S) Multipolar anaphase, and (T) Multipolar telophase. Scale bars=10 µm.

Aberrant cell frequency

Both LAECI and colchicine treatment showed a significant (p＜0.001) increase in the total aberrant (AB) cell percentage in A. cepa root apical meristem cells. The percentages of AB cells induced by LAECI were 3.11±0.06, 2.74±0.12, and 1.76±0.02% respectively at 2, 4, and 8 mg mL⁻¹. Colchicine (0.4 mg mL⁻¹) induced 8.1±0.12% of AB cell at 4 h treatment. However, the highest AB cell frequency (17.54±0.08%) was observed with LAECI (4 mg mL⁻¹) treated sample at 16 h recovery when compared with standard (0.4 mg mL⁻¹) colchicine (12.41±0.16%) and untreated control (0.14±0.02%) (Table 1).

Anaphase bridges

Significant frequency of anaphase bridges was observed with LAECI treated onion root tip cells. The LAECI treatment for 4 h exhibited 7.56±0.71% (p＜0.001) and 6.56±0.43% (p＜0.001) of cells with anaphase bridge respectively at concentrations of 4 and 8 mg mL⁻¹. Colchicine (0.4 mg mL⁻¹) caused 1.34±0.43% and the untreated control showed 0.33±0.16% anaphase bridge. In the recovery phase, the higher frequency of anaphase bridge was maintained in LAECI treated samples and showed 7.37±0.18%, 7.42±0.16%, and 7.32±0.55% respectively for 2, 4, and 8 mg mL⁻¹, whereas colchicine (0.4 mg mL⁻¹ concentration) caused 2.21±0.28% cells with anaphase bridge (Table 1, Fig. 1).

Vagrant chromosomes

Treatment of LAECI (1, 2, 8 mg mL⁻¹) at 4 h did not show any significantly increased frequency of vagrant chromosomes except for the concentration of 4 mg mL⁻¹ of LAECI (7.79±0.25%, p＜0.001) and 0.4 mg mL⁻¹ colchicine (4.52±0.57%, p＜0.05) treatment. In the case of 16 h recovery treatment, the vagrant chromosomes frequencies were significantly (p＜0.001) increased (8.6%, 9.76%, and 8.87%) with the respective increased concentrations of LAECI (2, 4, and 8 mg mL⁻¹) treated samples (Table 1, Fig. 1).

Chromosomal stickiness

An increased percentage of chromosomal stickiness was found in both LAECI (4 and 8 mg mL⁻¹) and colchicine (0.4 mg mL⁻¹) treated roots. However, the frequency was higher in the case of LAECI treated (2, 4 h) and in 16 h recovery samples. The highest stickiness frequency (15.89±1.27%, p＜0.001) was observed in the case of 4 mg mL⁻¹ LAECI treatment after 4 h. Colchicine (0.4 mg mL⁻¹) treatment also induced a significantly increased frequency (10.71±0.25%) of sticky chromosomes at 4 h. In the case of 16 h recovery samples, the frequency was decreased to 5.64±0.19% and 4.67±0.75% respectively in 4 and 8 mg mL⁻¹ of LAECI treated samples and it was 1.23±0.19% in the case of colchicine treated sample (Table 1, Fig. 1).

Multipolar anaphase and telophase

The LAECI (2, 4, and 8 mg mL⁻¹) treated roots for 2 and 4 h did not show any multipolar anaphase and telophase cells. In the case of 16 h recovery samples, the LAECI caused a significantly (p＜0.001) increased frequency of multipolar anaphase and telophase cells. The concentration of 2, 4, and 8 mg mL⁻¹ LAECI treatment respectively induced 7.36±0.29%, 8.12±0.37% and 7.42±0.12% of multipolar anaphase and telophase frequencies, and colchicine (0.4 mg mL⁻¹) induced relatively lower frequency (2.21±0.28%) (Table 1, Fig. 1).

Discussion

Clerodendrum inerme is a traditionally used medicinal plant in India (Kiritikar and Basu 1989). Here, in the present work, a comparative mitotic abnormality-inducing effects of colchicine and LAECI were studied in A. cepa root apical meristem cells at early hours of treatment and 16 h recovery. A. cepa is widely used as a plant test system for toxicity study and contributes to a better understanding of the harmful effects of ethno-phytomedicines on living organisms (Levan 1938, Ray et al. 2013, Kundu and Ray 2016, Barman et al. 2020, Roy et al. 2020). The LAECI has shown the ability to promote mitotic alterations in onion root tip cells. Effects of LAECI were analyzed by observing cytological abnormalities, including anaphase-bridge, vagrant chromosome, stickiness, polar deviations, lagging chromosome, multipolar anaphase-telophase. The results obtained from the cytotoxic studies revealed that the higher dose of LAECI induced an increased percentage of mitotic abnormalities and that were significantly different from the untreated controls. The study of mitotic abnormalities is considered to be a promising test to determine the cytotoxic potentials of the applied substances (Caritá and Marin-Morales 2008). Chromosome stickiness is a type of physical adhesion involving mainly the proteinaceous matrix of chromatin material (Patil and Bhat 1992). Chromosome stickiness specifies a highly irreversible toxic effect of the extract and it could happen due to the subchromatid linkage between chromosomes (Fiskesjö 1985, Ajay and Sarbhoy 1988). According to Mercykutty and Stephen (1980), stickiness may arise due to the effects of DNA depolymerization, chromatids breakage, and the stripping of the protein covering of DNA in chromosomes (Mercykutty and Stephen 1980). The chromosomal abnormalities can be classified into clastogenic and aneugenic. The most important chromosomal aberration that illustrates clastogenic effects is chromosome bridges and breaks. The LAECI induced chromosome bridges originating during anaphase and telophase were formed possibly by breakage and fusion of chromatids materials or may be due to unequal exchange of chromatids during translocation (Liman et al. 2010, Chatterjee and Ray 2019). However, the aneugenic effect indicates improper chromosomal segregation and inhibition of cytokinesis due to dysfunction of the mitotic spindle during cell division (Uhl et al. 2003). The induction of vagrant chromosomes is the result of the precocious movement of chromosomes in spindle poles which leads to an unequal number of chromosomes separation in the daughter nuclei and results in the formation of daughter cells with unequal sized interphase nuclei. The LAECI induced laggard’s chromosome indicates the delayed movement of the chromosome at poles and it is believed to be formed by inhibition of tubulin polymerization or may be due to inhibition of cytoskeletal proteins (El-Ghamery et al. 2003). The most frequent aberrations induced by LAECI were sticky chromosomes, followed by vagrant chromosomes, anaphase bridge, and multipolar anaphase-telophase. The occurrence of sticky, laggard, and multipolar movement of the chromosome was indicative of abnormal DNA condensation and chromosome coiling and inactivation of the spindles. The result obtained from this study revealed that treatment of LAECI and colchicine has shown similar aneugenic effects in A. cepa root tip cells. Thus, LAECI may contain active principles having colchicine-like aneugenic cytotoxic effects in A. cepa root tip cells and further studies are needed to isolate the bioactive substance(s) from LAECI and to explore their efficacy as well as the mode of action.

Acknowledgments

The authors acknowledge the financial support of CSIR JRF-09/025(0229)/2017-EMR-I Dated: 26.10.2017, and UGC MRP, DST-PURSE, DST-FIST, and UGC-DRS-sponsored facilities in the Department of Zoology.

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