Colchicine Like Metaphase and Cell Cycle Delay Inducing Effects of Leaf Aqueous Extract of Clerodendrum inerme (L.) Gaertn. in Allium cepa Root Apical Meristem Cells

Abstract

Clerodendrum inerme is traditionally used for the treatment of various diseases including cancer. The present study aimed to analyze colchicine like metaphase (c-metaphase) and cell cycle delay-inducing effects of the leaf aqueous extract of C. inerme (LAECI) in onion root tip cells. Onion root growth retardation, mitotic index reduction, decreased anaphase, and telophase cumulative frequencies were analyzed for LAECI induced cell cycle delay. The LAECI (1–8 mg mL⁻¹) induced a concentration-dependent onion root growth retardation (p＜0.01), increased c-metaphase frequency at 4 h treatment, and 4 h treatment +16 h recovery in water. The LAECI could induce a reduction rate of transition from metaphase to anaphase, decreased percentage of anaphase-telophase cumulative frequency, root tip swelling and that was comparable with the colchicine action. In summary, LAECI contains secondary metabolites having c-metaphase and cell cycle delay-inducing effects in onion root tip cells.

Colchicine was originally extracted from Colchicum autumnale and functions effectively as a spindle poison by inhibiting microtubule polymerization and consequently results in microtubule destabilization, chromosome condensation, metaphase arrest, reduced metaphase to anaphase transition, micronuclei, polyploidy, etc. (Salmon et al. 1984, Ray et al. 2013) and now it is often prescribed to treat gout attacks (van Echteld et al. 2014). It can ease pain from gout outbreaks by inhibiting neutrophil motility leading to a net anti-inflammatory effect. It is used for the treatment of constipation-predominant irritable bowel syndrome in women (Verne et al. 2003), chondritis (Puechal et al. 2014), and long-term treatment of Behcet’s disease (Cocco et al. 2010). In the present study colchicine like metaphase and cell cycle delay inducing effects of leaf extract of Clerodendrum inerme were analyzed.

The genus Clerodendrum, which includes small trees, shrubs, and sub herbaceous perennials; having more than five hundred species, is widely distributed in tropical and subtropical regions of the world. In Ayurvedic medicine, C. inerme is used for the treatment of various diseases, such as rheumatism, skin disease, tumors, asthma, stomach pain, hepatitis, etc. (Kirtikar and Basu 1975). The leaf aqueous and methanolic extracts of C. inerme were investigated for significant antinociceptive, antioxidant, anti-inflammatory, analgesic, and antidiabetic activity (Farnsworth et al. 1985, Fenech and Crott 2002, El-Ghamery et al. 2003, Rajeev et al. 2012). Due to its large medicinal properties, extensive phytochemical constituents of C. inerme have been reported. Compounds such as flavonoids, sterols, iridoid glycosides, diterpenes, triterpenes, and neolignans have been isolated from various parts of C. inerme (Vendantham et al. 1977, Spencer and Flippen-Anderson 1981, Akihisa et al. 1990, Çalis et al. 1994, Chatterjee and Pakrashi 1995, Pandey et al. 2005).

It is a well fact that some antineoplastic and anticancer plant extracts exert effects through cell division machinery. Earlier we have reported antiproliferative, apoptosis, metaphase arrest, cell cycle delay, metabolic abnormality, and micronuclei inducing effects of leaf extracts of C. viscosum (Ray et al. 2013, Kundu and Ray 2017, Chandra and Sanjib 2017). Recently, Roy and Roy (2019) assessed cytotoxic effects of aqueous and methanolic extracts of C. viscosum and C. inerme using Allium cepa test, however, colchicine like metaphase, c-metaphase, and cell cycle delay inducing effects of C. inerme aqueous extract is not clear. C-metaphase is colchicine induced metaphase arrest, as described by Levan (1938) in A. cepa, is the toxic effect of colchicine, which blocks metaphase to anaphase transition by inactivating the spindle formation that results in condensed haphazardly arranged chromosomes, pro-metaphase like chromosomal arrangement (Fig. 1D, E), cell cycle delay and polyploidy or mitotic catastrophe. The A. cepa assay is an efficient test for the cyto-genotoxicity assessment and/or disturbances in the mitotic cycle induced by chemicals (Fiskesjö 1985, Cabrera and Rodriguez 1999). Therefore, the present study aimed to explore c-metaphase and cell cycle delay-inducing effects of leaf aqueous extract of C. inerme on A. cepa root tip cells.

Fig. 1. LAECI and colchicine induced c-metaphase and onion root tip swelling effects. A–E, mitotic phases of squashed onion root tip cells. A–C, typical mitotic phases of untreated root tip cells; A, Prometaphase; B, Metaphase; C, Anaphase; D, LAECI induced c-metaphase; E, C-metaphase; F–H, root swelling pattern. F, Untreated roots; G, LAECI (4 mg mL⁻¹) and H, colchicine (0.4 mg mL⁻¹) induced onion root tip swelling at 4 h treatment followed by 16 h recovery in distilled water. Scale bars=10 µm for A–E. Scale bars=1 cm for F–H.

Materials and methods

Chemicals

Glacial acetic acid, orcein, and methanol were purchased from Merck Ltd. Mumbai, India. Colchicine was obtained from Himedia Laboratories Pvt. Ltd. Mumbai, India. The other chemicals were purchased from reputed manufacturers.

Plant collection and extraction

The leaves of C. inerme were collected from Renaissance housing complex, Purba Burdwan, West Bengal, India in January 2018 and taxonomically authenticated by Prof. A. Mukherjee, Department of Botany, University of Burdwan. The voucher specimen (SR/REN/BWN/2018/01) is maintained in Department of Zoology, The University of Burdwan. The freshly collected leaves were washed in running tap water and dried in shade for 2–3 days. The dried leaves were then crushed in a mixture grinder. Fifty gram of C. inerme leaf powder was extracted in 600 mL distilled water for 3 h each at 100°C. The extract was then filtered using Whatman #1 filter paper. Finally, the extract was coded as LAECI, leaf aqueous extract of C. inerme, and stored in a refrigerator (−20°C).

Allium cepa root growth retardation and root tip swelling assay

The similar-sized onions were allowed for root sprouting in distilled water at 25±2°C and the 48 h aged roots (1.5–2 cm) were treated with LAECI (1, 2, 4, and 8 mg mL⁻¹) and colchicine (0.4 mg mL⁻¹). The root length was measured at 24, 48, and 72 h, and the root tip swelling patterns were analyzed at 4+16 h (4 h in treatment and then 16 h recovery in distilled water).

C-metaphase and cell cycle delay analysis

The onion roots (about 1.5–2 cm) were exposed with LAECI and colchicine for 2, 4 and 4+16 h (4 h treatment followed by 16 h recovery in water). In the case of control groups, the onion roots were maintained in distilled water. For slide preparation, the control and treated onion root tips were collected at each 2, 4, and 4+16 h recovery and were fixed in aceto–methanol 1(acetic acid): 3(methanol) for 24 h, then hydrolyzed in 1 N HCl at 60°C for 10 min, stained with aceto-orcein (2%) and finally squashed in 45% acetic acid (Sharma and Sharma 1999, Ray et al. 2012). The squashed root tips were observed in a microscope, digital images were taken with Future Win Joe, Future Optics (Version 1.6.5.1207), and the c-metaphase, prophase, metaphase, anaphase, telophase cell percentage as well as the mitotic index (number of dividing cells/total number of cells ×100) were scored from the micrographs.

Statistical analysis

Data obtained on the mitotic index, c-metaphase frequency, and cell frequencies at different phases were analysed by using 2×2 contingency χ² test while root length retardation effect was analysed by Student’s t-test. The difference between the control and treated groups were considered significant at p≤0.05 or 0.01 or 0.001. All the data were expressed as mean±SEM (standard error of the mean).

Results and discussion

The LAECI (1–8 mg mL⁻¹) induced root growth retardation (RGR) and root tip swelling (RTS) effects were analyzed and compared with colchicine (0.4 mg mL⁻¹). A concentration-dependent growth retardation effect was observed in LAECI treated A. cepa roots. The maximum RGR (90.81±2.11%) effect was observed at 72 h of LAECI (8 mg mL⁻¹) treatment and a comparable RGR (94.00±2.28%) and RTS (45.4%) effects were also observed with colchicine (0.4 mg mL⁻¹). The IC₅₀ value LAECI for RGR was determined as 1.65 mg mL⁻¹ at 48 h (Table 1). In others and our previous studies have shown that the plant extract mediated root growth inhibition increases with the increasing extract concentrations (Ray et al. 2013, Murthy et al. 2011). Also, the comparable pattern of root swelling by colchicine and LAECI treatment (Fig. 1) indicates that the LAECI contains active principles that may act like colchicine, which interacts with the mitotic system, and result in the cell cycle arrest at metaphase, delay in the cell cycle, followed by cytotoxic effects/polyploidy induction and swollen root tips (Lodhi 1976, Hague and Jones 1987, Ray et al. 2013).

Table 1. Root growth retardation and root tip swelling effects of LAECI and colchicine on A. cepa.

LAECI Conc. (mg mL−1)	Root length (cm) increase [in hibition %]			Roots breadth (mm)		% increase
	(Mean±SEM)			(Mean±SEM)
	24 h	48 h	72 h	4+16 h
				Non swollen portion	Swollen tip
0	0.95±0.04	1.66±0.03	1.96±0.02	0.77±0.02	—	—
	[0]	[0]	[0]
1	0.70±0.02*	1.27±0.01***	1.60±0.05**	0.75±0.03	0.96±0.02**	21.9
	[25.37±3.98]	[23.18±1.78]	[18.46±2.56]
2	0.36±0.03***	0.66±0.02***	0.79±0.03***	0.77±0.01	1.46±0.04***	47.2
	[61.96±4.07]	[60.28±1.7]	[59.45±2.05]
4	0.16±0.02***	0.21±0.04***	0.22±0.02***	0.78±0.02	1.83±0.07***	57.4
	[82.51±2.75]	[87.29±2.58]	[88.44±1.57]
8	0.14±0.05***	0.16±0.03***	0.18±0.04***	0.82±0.01	1.33±0.04***	38.3
	[84.66±6.23]	[90.17±2.24]	[90.81±2.11]
0.4©	0.08±0.02***	0.07±0.04***	0.11±0.04***	0.83±0.03	1.52±0.02***	45.4
	[91.24±2.93]	[95.80±2.26]	[94.00±2.28]
IC50		1.65 mg mL−1

^©symbol=Point for colchicine treatment. LAECI; Leaf aqueous extract of C. inerme, Conc.; concentration, SEM; Standard error of mean, 4+16 h; 4 h treatment followed by 16 h recovery in water. * Significant at p＜0.05, ** p＜0.01 and *** p＜0.001 Student’s t-test analysis compared to untreated control.

LAECI induced alteration in the mitotic index and dividing cells percentage were documented in Table 2. Data indicate that LAECI (1–8 mg mL⁻¹) treatment causes a dose-dependent decrease in MI at 2 and 4 h treated onion root tip cells. The minimum MI (1.99±0.17) was scored from the used highest concentration (8 mg mL⁻¹) of LAECI at 4 h treatment in comparison to control (5.52±0.25). The alteration in the MI directly correlates with the cell cycle kinetics. Here, LAECI treatment caused a concentration-dependent decrease in MI at both 2 and 4 h treated onion root tip cells. The highest reduction of MI was scored from the used highest concentration (8 mg mL⁻¹) of LAECI at 4 h of treatment. There are reports of mitotic index depression with the various plant extracts (Salam et al. 1993, Abdel Salam et al. 1997). In contrast to these MI reducing tendency, it showed the increased MI in case of 4+16 h recovery treatment in water. The highest MI increase was found in 4 mg mL⁻¹ (12.23±0.14%). Whereas colchicine (0.4 mg mL⁻¹) induced the highest MI increase (10.2±0.22%) was found at 4 h and again decreased (3.58±0.30%) at 16 h recovery samples. The observed increased MI might be due to the increased frequency of c-metaphase cells with both the LAECI and colchicine treated sample (Davidson et al. 1966, Ray et al. 2013).

Table 2. Effect of LAECI and colchicine on mitotic index, c-metaphase, and dividing cell percentages in A. cepa root tip cells.

Dose (mg mL−1)	Hours	Total cells scored	Cells percentage at different phases
			Mitotic	Pro	Meta	Ana	Ana-Telo	c-metaphase
			Mean±SEM
0	2	1584	5.65±0.29	37.7±1.34	28.26±0.28	18.18±0.39	34±1.48	1.12±0.06
1		3157	3.3±0.12c	34.39±0.42	34.42±0.71	22.59±0.38	31.14±1.02	10.49±0.95c
2		2159	4.11±0.29c	28.47±0.35	42.51±1.20a	20.54±0.41	28.99±1.36	13.11±0.13c
4		2449	4.26±0.22c	36.77±1.21	34.77±0.54	15.51±0.73	28.43±1.74	11.05±0.72c
8		2189	3.41±0.27c	38.06±1.06	37.67±1.44a	14.39±0.85	25.1±2.45	18.48±0.93c
0.4©		2459	5.56±0.38	35.76±1.80	56.2±2.13c	6.56±0.58c	7.28±0.73c	48.17±0.89c
0	4	1876	5.52±0.25	22.83±0.17	29.25±0.58	23.47±0.49	47.01±0.81	0.99±0.60
1		1979	2.48±0.21c	28.29±0.92	44.06±1.25a	12.01±1.23a	27.62±0.40b	24.82±1.76c
2		1853	5.4±0.21	24.25±0.22	60.54±0.92c	10.28±0.59c	15.18±1.09c	43.24±0.60c
4		3206	3.01±0.09c	23.4±0.99	62.22±0.34c	9.23±0.80c	14.34±1.13c	51.58±0.58c
8		2916	1.99±0.17c	24.54±0.29	47.64±1.47c	20.09±2.50	27.8±1.57b	35.49±0.36c
0.4©		2664	10.2±0.22c	16.17±0.87a	81.98±1.02c	1.1±0.09c	1.28±0.20c	66.91±0.64c
0	4+16	3322	6.09±0.10	29.27±0.10	29.3±0.58	20.87±0.22	41.4±0.61	0.81±0.15
1		2813	5.95±0.09	41.35±0.3b	25.23±0.29	13.91±0.14a	33.38±0.17	0.78±0.18
2		2803	5.5±0.12	18.33±0.6b	59.87±0.76c	6.87±0.55c	21.98±0.65c	40.17±0.41c
4		2144	12.2±0.14c	15.73±0.66c	67.34±0.55c	9.78±0.20c	16.89±0.88c	58.57±0.30c
8		2261	6.92±0.19a	26.77±0.47	44.51±0.62c	16.41±0.45	28.69±0.21b	24.65±0.34c
0.4©		2032	3.58±0.30c	52.05±1.86c	24.65±0.89	15.06±0.71	20.53±1.19c	4.56±0.75

^a Significant at p＜0.05. ^b Significant at p＜0.01. ^c Significant at p＜0.001 as compared to their respective control with 2×2 contingency χ² analysis with respective df=1. ^© symbol=Point for colchicine treatment. Pro: prophase; Meta: metaphase; Ana: anaphase; Telo: telophase; MI: mitotic index

Data indicate that the LAECI treatment caused a statistically significant increased c-metaphase frequency in A. cepa root tip cells. The mean percentages of c-metaphase induced by LAECI were 43.24±0.60, 51.58±0.58, and 35.49±0.36 at 4 h and 40.17±0.41, 58.57±0.30, and 24.65±0.34 at 4 h treatment followed by 16 h respectively at the concentrations 2, 4, and 8 mg mL⁻¹. The LAECI (4 mg mL⁻¹) treatment showed the highest c-metaphase frequency (58.57±0.30, p＜0.001) in the 16 h water recovery samples and that was also higher than the 2 and 4 h treated samples. In the contrary, colchicine (0.4 mg mL⁻¹) treatment showed the highest c-metaphase frequency (66.91±0.64) at 4 h treated sample and it was significantly decreased (4.56±0.75) at 16 h treatment recovery samples (Table 2). In case of dividing cell percentage, it was found that LAECI (2, 4 and 8 mg mL⁻¹) treatment for 4 h and 4+16 h and colchicine (0.4 mg mL⁻¹) treatment for 2 h and 4 h could significantly increase the metaphase cells frequency and decreased the prophase, anaphase, telophase cells frequency (Table 2). The LAECI induced significantly (p＜0.001) increased metaphase cells percentage, 62.22±0.34 and 67.34±0.55% with 4 mg mL⁻¹ concentration respectively at 4 h treated and 16 h recovery samples. In the case of colchicine (0.4 mg mL⁻¹) treated roots, the increase in metaphase cells percentage was observed at both 2 h (56.2±2.13%) and 4 h (81.98±1.02%) and again that decreased at 4+16 h (24.65±0.89%) sample. In the case of dividing cell percentage, it was found that LAECI (2, 4, and 8 mg mL⁻¹) treatment for 4 h and 4+16 h could induce metaphase arrest and results in a significant increase in metaphase cell percentage. The colchicine was used for comparative metaphase-arresting activity analysis (Ray et al. 2013, Levan 1938). Similarly, colchicine (0.4 mg mL⁻¹) treatment also caused an increase in the metaphase cells percentage at both 2 and 4 h, indicating that LAECI has metaphase arresting activity. With the increasing metaphase cells frequency, statistically significant decreased in the anaphase (9.23±0.80% and 9.78±0.20% respectively at 4 h and 4+16 h recovery) and telophase (5.11±0.39% and 7.11±0.71% respectively at 4 h and 4+16 h recovery) cells frequencies were also observed with the LAECI treatment in comparison to control. Like LAECI, in colchicine treated (0.4 mg mL⁻¹) samples the lower anaphase frequencies, 6.56±0.58%, 1.1±0.09% and 15.06±0.71% and telophase frequencies, 0.72±0.15%, 0.18±0.11% and 5.47±0.62% were observed respectively at 2 h, 4 h and 4+16 h (Table 2).Thus, it is evident that both LAECI and colchicine could decrease the anaphase-telophase cumulative frequencies that in turn may positively correlate with their cell cycle delay inducing effects.. The dividing cells undergo a mitotic arrest, metaphase arrest, and suppression of anaphase, producing typical c-metaphase configurations in colchicine treated samples (Davidson et al. 1966, Ray et al. 2013).

In conclusion, the present study revealed that, like colchicine, LAECI could induce onion root growth retardation, mitotic depression; metaphase arrest with haphazardly arranged condensed chromosomes in c-metaphase, cell cycle delay, etc. and that may be due to the presence of colchicine like microtubule destabilizing compounds in the C. inerme extracts. Therefore, further studies were needed to isolate the active principles.

Acknowledgments

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

References

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