Cytotoxic Effects of Scutellaria discolor Colebr. Aqueous Extract in Allium cepa Root Tip Cells

Abstract

Scutellaria discolor Colebr. is a traditionally used annual herb that occurs in the Himalayan regions of India. The present study aimed to analyze the cytotoxic effects of S. discolor aqueous extract on Allium cepa root tip cells. The equal-sized roots, 48 h aged, were treated with the aqueous extract of S. discolor (AESD) (0.5–4 mg mL⁻¹) and colchicine (0.4 mg mL⁻¹) for 4 h continuously and then recovered for 16 h (4+16 h R) in distilled water. The AESD induced colchicine-like mitotic abnormalities; sticky chromosome, vagrant and laggard chromosomes, chromosomal fragments, anaphase bridges, stellate anaphase, micronucleus, and polyploidy, indicating it may have like colchicine prospective therapeutic and cytotoxic applications.

Scutellaria discolor Colebr. (Family: Lamiaceae, common name bicolor or official skullcap) is an annual herb that occurs in hilly regions of Assam, Himachal Pradesh, Manipur, Meghalaya, Mizoram, Uttarakhand, etc. It grows in hill slopes and blooms in rainy seasons. The lower side of the leaf is purple but appears green where sunlight reaches. The plant is used as a natural healthcare remedy in traditional and folklore medicinal systems at all the above places. The whole dry herb is used in China folklore medicinal system as a therapy for colds, tympanites, gastroenteritis like an anti-inflammatory, antipyretic, and antidote medicine (Tomimori et al. 1985). Application of the juice of fresh leaves for the healing wounds is common in local tribal communities of Assam (Das et al. 2008). In Nepal, leaf paste of this species is used to treat common colds and to heal insect stings and the whole plant juice also is a health remedy for the complications of rheumatism, headache, indigestion, and fever (Kunwar et al. 2010). At Manipur, juice of S. discolor is used together with that of the other plants and applied as formulations against different disorders in the Chakpa community. In the Bishnupur district of Manipur, a decoction of leaves is used in indigestion, constipation, flatulence, rheumatic and muscular complications. Article on the antimicrobial activities of a leaf extract from successive solvent fractions of S. discolor reported the antibacterial and antifungal activity at high concentrations (Neshwari Devi and Singh 2014). Cytotoxicity assays on cervical cancer cell lines are reported in the chrysin-rich bioactive fraction of S. discolor acetone extract arresting the cell division via caspase-dependent apoptosis (Laishram et al. 2015). Studies on this plant species reveal its phytochemical components such as flavonoids, neo-clerodane diterpenoids, saponins, steroids, essential oils, alkaloids, etc. (Tomimori et al. 1986, 1988, Ohno et al. 1996).

Colchicine is a spindle poison that arrests the cell cycle at metaphase by disrupting tubulin polymerization. As colchicine induced microtubule destabilization is reversible, it leads the chromatids to separate abnormally without cytoplasmic division and the cell gets more than one copy of genome i.e., becomes polyploid (Caperta et al. 2006), a condition where the tissue growth is arrested as the volume of a single cell is increased but new cell formation is ceased (Levan 1938). The cell progresses with an array of abnormalities in the course of the cycle. Vagrant chromosome appears when a chromosome moves ahead towards a pole from its group of chromosomes and separates abnormally mainly due to spindle failure (Fiskesjo and Levan 1993). Lack of synchronization in mitotic events and stellate anaphase (Tajo and Thoppil 1998) arises from the complete disturbances in the spindle (Sheela and Thoppil 2017). In addition, spindle dysfunction may lead to improper tilt in the spindle pole causing polar deviation in mitotic stages (Deena and Thoppil 2000). Chromosome fragmentation is indicative of the clastogenic effect of applied chemicals (Burke 2000, Permjit and Grover 1985). Micronucleus can be formed either from chromatid breakage or by performing abnormal separation (Elhajouji et al. 1998).

The microtubule destabilization induced by colchicine is reversible in nature and microtubule destabilization results in an array of abnormalities in dividing cells (Levan 1938, Ray et al. 2013, Barman et al. 2021). In our earlier study, we have reported the effect of AESD on root growth inhibition, delay in cell cycle due to increase in metaphase as well as c-metaphase cells frequency with a decrease in anaphase cells frequency in A. cepa and found a similar action with that of colchicine (Chakraborty et al. 2021). The present study aimed to compare the AESD induced mitotic abnormalities with that of the colchicine in root meristematic cells of A. cepa.

Materials and methods

Plant collection, identification, and extract preparation

Both sides green S. discolor whole plants were collected from Langol Reserve Forest (24.8137°N, 93.8869°E), Imphal West, Manipur, India and was identified by Dr. Athokpam Pinokiyo, Assistant Professor, P.G. Department of Botany, D. M. College of Science, Imphal West, Manipur. For further references, a voucher specimen (DMC/BOT/IDENTIFICATION/4) is preserved at the Department of Zoology, The University of Burdwan. The whole plant was washed with tap water, shed-dried, and ground in powder by Philips Mixer Grinder HL1605. For aqueous extract preparation, this powder (25 g) was boiled in 2 L distilled water continuously for 1 h while stirred intermittently and after filtration through No. 1 Whatman filter paper, the obtained extract was named AESD.

Mitotic abnormality analysis

A. cepa bulbs with similar-sized roots were subjected to treatment with AESD (0.5, 1, 2, and 4 mg mL⁻¹ concentrations). Distilled water and colchicine (0.4 mg mL⁻¹) were used as negative and positive control respectively. The treatment was continued for 4 h continuously after which, for recovery, the bulbs were maintained in distilled water for 16 h (4+16 h R). Roots from control, colchicine, and AESD treated bulbs were collected (at 2, 4, and 4+16 h R) and were fixed in aceto-methanol (3 parts methanol and a part acetic acid) for 24 h. The fixed root tips were hydrolyzed in 1 M HCl for about 10 min and stained in 1% aceto-orcein following standard protocols (Sharma and Sharma 1999, Ray et al. 2013). For each concentration, at least three slides were prepared to score the mitotic abnormalities. From the well-spread region of squashed root tip cells, the mitotic abnormalities were visualized and scored with a bright-field optical microscope, and digital images were captured using the software Future Win Joe, Future Optics (Version 1.6.5.1207).

Statistical analysis

The frequencies of mitotic abnormalities were statistically analyzed by a 2×2 contingency chi-square test in GraphPad Prism 8.4.3. The percentages of abnormal mitotic cells were obtained by dividing the total number of cells of a particular abnormality by total diving cells multiplied by 100. Micronucleus (MN), polyploidy (PC), and Giant cells (GC) percentage were calculated by the respective number of cells/total cells multiplied by 100. The differences between the control and treatment groups were represented as significant at the level of p≤0.05 as ^c, p≤0.01 as ^b, and p≤0.001 as ^a. All of the data are denoted as mean±SEM (standard error of the mean from at least three separate experiments).

Results and discussion

Here, a comparative account of the cyto-genotoxic potential of the AESD and colchicine was assessed. Like colchicine, the AESD induced a variety of mitotic abnormalities like sticky chromosomes, laggard and vagrant chromosomes, chromosome bridges, polar deviations, fragmented chromosomes, stellate chromosomes, micronucleus, and polyploid cells in A. cepa root apical meristem cells. The percentages of mitotic abnormal cells (MAC) were increased significantly (p＜0.001) in all the used concentrations of AESD and colchicine treated samples than the control. In 4 mg mL⁻¹ AESD, it reaches 50.78±1.41% at 2 h and 80.88±0.29% at 4 h and that decreases to 46.97±2.38% at 4+16 h R. In colchicine, it was 49.82±1.19, 83.39±0.43 and 24.33±1.08% respectively at 2, 4 and 4+16 h R (Table 1, Fig. 1). Earlier we have reported that the most frequent abnormality induced by AESD is c-Metaphase (cM) (Chakraborty et al. 2021). Owing to the persisting metaphase signal in cM condition, chromosomes get highly condensed producing sticky chromosomes (SC), and delay in metaphase to anaphase transition is observed (Ade and Rai 2010, Chakraborty et al. 2021). The percentage of SC increases significantly in both the AESD and colchicine at 2 and 4 h than the control. It reaches up to 17.51±0.59% (p＜0.001) at 4 h of AESD treatment (4 mg mL⁻¹). At 4+16 h, the percentage decreases but maintained concentration-dependent increasing patterns and AESD (4 mg mL⁻¹) shows a similar value (11.90±1.46%; p＜0.001) like colchicine (11.50±0.70%; p＜0.001) (Table 1). The AESD induced increased frequency of Vagrant Chromosome (VC) over untreated samples but it shows no significant difference (Table 1). The increased SC percentages strongly correlate with that of c-metaphase frequencies. The VC appears when a chromosome moves ahead towards a pole from its group of chromosomes and separates abnormally mainly due to spindle failure (Fiskesjo and Levan 1993). Stellate anaphase (SA) frequency is 6.55±0.25 (p＜0.001), 1.68±0.22 (p＜0.05) and 1.18±0.12% in 2 mg mL⁻¹ of AESD at 2, 4 and 4+16 h R respectively and 1.34±0.04 (p＜0.05), 0.38±0.20 and 1.04±0.17% (p＜0.05) in colchicine (Table 1). Lack of synchronization in mitotic events and SA, observed in AESD as well as in colchicine (Fig. 1) and that may arise from the complete disturbances in the mitotic spindle (Tajo and Thoppil 1998, Sheela and Thoppil 2017).

Table 1. AESD induced mitotic abnormalities in A. cepa root tip cells.

h	Conc. (mg mL−1)	TC	TDC	MA %	Different types of MA %
					Sticky chromosome %	Laggard chromosome %	Vagrant chromosome %	Anaphase bridge %	Stellate anaphase %	Polar deviation %	Fragmented chromosome %
2	0	5645	350	4.07±0.45	0.56±0.33	0.42±0.18	0.18±0.42	0.63±0.63	00±00	0.98±0.22	0.27±0.27
	0.5	6765	433	23.19±0.22a	2.23±0.62	1.35±0.16	0.59±0.30	1.33±0.14	1.23±0.16c	3.16±0.08b	0.59±0.31
	1	5362	370	28.38±1.17a	3.66±0.32c	2.49±0.49	0.66±0.33	1.57±0.28	4.66±0.20a	2.83±0.20b	1.17±0.17
	2	4907	373	55.36±1.19a	5.40±0.12b	3.47±0.40	0.67±0.34	1.82±0.21	6.55±0.25a	3.63±0.42	1.65±0.23
	4	3365	276	50.78±1.41a	7.26±0.10b	4.24±0.66	0.86±0.54	1.36±0.25	3.63±0.05a	3.63±0.05	1.50±0.20
	0.4C	5478	378	49.81±1.19a	5.78±0.38b	5.06±0.38	0.48±0.29	2.71±0.11	1.34±0.04c	0.14±0.14	0.43±0.43
4	0	5375	344	4.08±0.65	0.84±0.42	0.53±0.30	0.20±0.20	0.51±0.51	00±00	0.53±0.29	0.29±0.29
	0.5	6354	394	34.02±0.32a	4.01±0.47b	2.77±0.31	0.78±0.40	1.77±0.15	1.77±0.15c	4.01±0.47	0.63±0.32
	1	5768	473	50.52±0.07a	7.03±0.31a	3.20±0.19b	1.61±0.21	1.38±0.19	2.99±0.03b	3.54±0.28	1.38±0.19
	2	4070	232	66.11±0.88a	12.98±0.33a	5.65±0.23b	1.33±0.14	1.83±0.16	1.68±0.22c	3.47±0.38c	2.18±0.47
	4	7431	327	80.87±0.29a	17.51±0.59a	6.16±0.79a	0.75±0.37	2.13±0.15	0.51±0.29	2.22±0.73c	2.50±0.52c
	0.4C	4625	444	83.40±0.43a	12.21±0.4a	6.05±1.00a	0.50±0.29	2.07±0.12	0.38±0.19	0.38±0.19	0.82±0.33
4+16 h R	0	5354	332	3.95±0.55	0.68±0.34	0.25±0.34	0.32±0.25	0.37±0.32	00±00	0.93±0.37	0.48±0.10
	0.5	2961	255	15.75±1.60a	2.85±0.41	0.67±0.34	0.91±0.52	1.86±0.85	0.65±0.32	4.79±0.31c	1.27±0.26
	1	5009	217	24.09±0.94a	3.80±0.41c	1.82±0.17	1.89±0.17	1.82±0.17	1.89±0.17c	5.12±0.37b	3.30±0.28b
	2	5070	260	31.75±1.34a	7.64±0.41a	2.35±0.23	1.53±0.32	2.70±0.32	1.17±0.12	4.70±0.47c	4.35±0.72b
	4	3693	236	46.96±2.38a	11.90±1.46a	3.44±0.18b	0.96±0.52	2.09±0.15	2.09±0.15b	5.19±0.54c	8.58±0.34a
	0.4C	10230	399	24.32±1.08a	11.50±0.7a	5.51±0.54a	0.18±0.18	1.04±0.17	1.04±0.17c	0.86±0.23	0.27±0.27

Different mitotic abnormalities induced by AESD (0, 0.5, 1, 2 and 4 mg mL⁻¹) and colchicine (0.4 mg mL⁻¹) on A cepa root tip cells at 2, 4 and 4+16 h; Conc.: Concentration; 0.4^C represents colchicine (0.4 mg mL⁻¹). Significant at the level of p≤0.05 as ^c, p≤0.01 as ^b, and p≤0.001 as ^a as compared to their respective control with 2×2 contingency χ² test with respective df=1. Total cell (TC), Total diving cell (TDC), Mitotic abnormalities (MA).

Fig. 1. The AESD and colchicine induced mitotic abnormalities in A. cepa root tip cells. U, C, and A represent respectively untreated, colchicine, and AESD induced different mitotic abnormalities. In control: Prophase (U1), Metaphase (U2), Anaphase (U3), Telophase (U4), Interphase (U5). In colchicine and AESD: LC (C1 and A1), VC (C2 and A2), asynchronous cell (A3, C7, and A11), SC (C3 and A4), AB (A6, C5, and A7), SA (C6 and A8), fC and cM (C8 and A10), PD (C4 and A6), Telophase Bridge (A7), MN (C9 and A14), PC (C9 and A13) and GC (C10 and A15). Scale bars=10 µm.

In addition, the effect of AESD in polar deviation (PD) frequency is much greater than colchicine. There is no significant pattern of increase or decrease, but a high frequency is maintained in AESD treated samples (Table 1). Spindle dysfunction may lead to improper tilt in the spindle pole causing polar deviation in mitotic stages (Deena and Thoppil 2000). Fragmented chromosome (fC) % in AESD is much higher than colchicine. In 4 mg mL⁻¹ concentration of AESD, it is 2.50±0.52% (p＜0.05) at 4 h and increases to 8.58±0.34% (p＜0.001) after 16 h recovery. In the case of colchicine, there was no significant change (Table 1). The AESD induced fC is detected in large numbers which define the clastogenic effect of AESD and similar effects with plant extracts are also available (Permjit and Grover 1985, Burke 2000). In 4 mg mL⁻¹ of AESD, the anaphase bridge (AB) frequencies are 2.13±0.15% (at 4 h) and 2.09±0.15% (at 4+16 h R). In colchicine, it was 2.07±0.11 and 1.04±0.17% respectively at 4 and 4+16 h R (Table 1). Chromosome bridges, fragmentation, and fusion in chromosome parts, translocation, etc. may further contribute to more aneugenic aberrations (Uhl et al. 2003, Liman et al. 2010). SC and AB display an irreversible and toxic effect that may govern cell death. Micronucleus (MN) is not found in early hours but after recovery in water, a significant amount of micronucleus was formed which increases with the increased concentrations and reaches 2.31±0.25% (p＜0.01) in 2 mg mL⁻¹ of AESD and again lowers to 1.08±0.60% (p＜0.05) in 4 mg mL⁻¹. In the case of colchicine (0.4 mg mL⁻¹), at 4+16 h R, the MN% is 8.00±0.06 (p＜0.001) (Fig. 2). MN, in AESD, possibly formed from acentric fragments or an intact chromosome, is indicative of the extract’s clastogenic and aneugenic effects. The laggard chromosome (LC) frequency also increases at 2 and 4 h followed by a subsequent decrease at recovery. At 4 h both in colchicine and 4 mg mL⁻¹ of AESD, the frequencies become 6.17±0.79 (p＜0.001) and 6.05±1.00 (p＜0.001) respectively but at 4+16 h R in 4 mg mL⁻¹ of AESD it is 3.44±0.18 (p＜0.01) whereas in colchicine it is 5.51±0.54% (p＜0.001). However, a much similar increased percentages in LC in AESD than control due to delayed migration may result from the disturbances in the mitotic spindle formation as in colchicine which leads to chromosome loss and aneugenic MN formation later in cell division (Cimini et al. 2004, Fenech et al. 2011). A clear positive correlation is observed between MN% and polyploidy cell (PC) percentages (Fig. 2) both in AESD and colchicine at 16 h recovery after 4 h continuous treatment. The PCs are not found at early hours but, after 16 h recovery, it increases as concentration increases and the value reaches 17.31±0.09% (p＜0.001) in 2 mg mL⁻¹ of AESD treatment which again decreases slightly to 14.76±0.37% (p＜0.001) in 4 mg mL⁻¹. In the case of colchicine, it shows a value of 30.39±1.76% (p＜0.001) (Fig. 2). Here, swelling in the root tip (Fig. 2) occurred but growth was arrested as the volume of a single cell was increased but new cell formation ceased deciphering the antiproliferative activity (Levan 1938). Colchicine-induced PCs form when chromosomes remain scattered for a long time due to microtubule destabilization and later during division nuclear membrane is formed around these haphazard chromosomal groups further producing more aneugenic MNs. Additionally, an unseparated chromosome in bridges induces incomplete cytokinesis and thus a cell gets more than one copy of the genome i.e., becomes polyploid (Caperta et al. 2006). Giant cells (GC) are formed after 16 h recovery in water and the percentages are 5.74±0.39, 3.78±0.54, and 5.58±0.24% respectively at 1, 2, and 4 mg mL⁻¹ concentrations of AESD and 3.13±0.88% in colchicine, which significantly differs from control (Fig. 1). GC, found in AESD treatment, maybe as a result of blocks in the interphase or divisional phase. From the Pearson correlation and simple linear regression analysis, MN% shows a strong negative correlation with MAC% (Pearson’s correlation coefficient (r)=−0.952) considering their relative changes in frequencies at 2, 4, and 4+16 h R. The same pattern was also observed in the case of polyploid cells (r=−0.952). A similar pattern of correlation was observed both in the colchicine (r=−0.823 both in MN% and PC%) and AESD. Thus, the present study explored cytotoxic potentials of AESD and compared with colchicine effects; indicating, like colchicine, its chemical constituents may have therapeutic and cytotoxic applications.

Fig. 2. Showing the root tip abnormality at 4+16 h R in control (A), colchicine (B), and AESD (C). Arrow indicates root tip swelling. Graphical representation of MN and PC frequencies at 4+16 h (D; 0.4^C represents colchicine (0.4 mg mL⁻¹). Significant at the level of p≤0.05 as ^c, p≤0.01 as ^b, and p≤0.001 as ^a as compared to their respective control with 2×2 contingency χ² test with respective df=1. Correlation studies in colchicine (COL) and AESD between MAC and MN (E), PC (F) considering their relative changes in frequencies at 2, 4, and 4+16 h R with respective Pearson’s correlation coefficient (r).

Acknowledgments

The authors acknowledge Dr. Athokpam Pinokiyo, Assistant Professor, P.G. Department of Botany, D. M. College of Science, Imphal West, Manipur for authentication of plant species and the financial support to CSIR [SRF-09/025(0267)/2018-EMR-I (Dated: 26.02.2021)] and DST-PURSE, UGC-DRS, UGC MRP, and DST-FIST sponsored facilities in the Department of Zoology, The University of Burdwan.

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