Regular Article
Cytotoxic and Micronuclei Inducing Effects of Petroleum Ether Fraction of Leaf Aqueous Extract of Clerodendrum viscosum Vent. in Allium cepa Root Tip Cells
2021 年 86 巻 3 号 p. 261-266
詳細
2021 年 86 巻 3 号 p. 261-266
Clerodendrum viscosum is a traditionally used medicinal plant. The present study aimed to analyze a detailed cytotoxic effect of the non-polar petroleum ether fraction (AQPEF) of leaf aqueous extract of C. viscosum Vent. (LAECV) in Allium cepa root tip cells. The LAECV was fractionated with petroleum ether and tested for A. cepa toxicity. Micronuclei, polyploidy, and mitotic abnormalities were analyzed after AQPEF treatment. The AQPEF induced concentration-dependent increased mitotic abnormalities, micronuclei, and polyploid cell frequency in A. cepa root tip cells and that were comparable with the colchicine effects. Thus, the present study explores that petroleum ether is a suitable solvent for extraction of the active phytochemicals of LAECV; AQPEF having colchicine like micronuclei, polyploidy and mitotic abnormality inducing potentials in A. cepa root tip cells, indicating its perspective use in karyotyping, plant breeding programs, and cytotoxicity studies.
Clerodendrum viscosum (common name: bhant, ghentu; family Lamiaceae) is traditionally used medicinal plant. It has gained a reputation in traditional Ayurvedic, Unani, and Homeopathic medicine (Nandi and Lyndem 2015). In the Indian Homeopathic system, it is prescribed to treat diarrhea, postnatal complications, and fresh wounds (Hamilton 1997, Nadkarni and Nadkarni 2002). In Unani medicine, this plant is well known for its effectiveness against rheumatism (Singh et al. 1997). To treat worm infection, cough, asthma, itching, leprosy, scorpion sting, bronchitis, fever, etc., the whole plant decoction is used (Kirtikar and Basu 1991, Bhattacharjee et al. 2011). The Indian Tribals of Chotanagpur plateau uses this plant parts’ extracts as a remedy for malaria, cataract, diseases of the skin, blood, and lung (Kirtikar and Basu 1991). The leaf methanolic extract exerts blood glucose level restoring effects after streptozotocin treatment (Arvind et al. 2002, Das et al. 2011). The leaf extract showed peripheral analgesic activity at a dose of 200 mg kg−1 body and a dose 500 mg kg−1 body weight reduced blood glucose level in the blood from 130 to 36 mg dL−1 in 2 h (Chandrashekar and Rao 2012, Hossain et al. 2014). Allelochemicals present in the leaf aqueous extract have harmed the growth and germination of weeds in the agro-ecosystem (Qasem and Foy 2001, Devi et al. 2013). Leaf and stem aqueous extracts of C. viscosum have resulted in significant insecticidal activity against tea pests Helopeltis theivora and Oligonychus coffeae when compared to acaricide and Azadirachta indica induced cytotoxic lethality (Azad et al. 2013, Islam et al. 2013). A promising positive correlation was established between the plant's parts and their insect repellent and insecticidal activity (Muh et al. 2014).
The crude leaf extracts of C. viscosum contain phenolics viz. fumaric acids, acetoside, methyl esters of caffeic acids, flavonoids such as apigenin, acacetin, scutellarein, quercetin, cabrubin, hispidulin, terpenoids like clerodin, steroids such as clerodone, clerodol, clerodolone, clerosterol and some fix oils containing linolenic acid, stearic acid, oleic acids, and lignoceric acid (Saroj 2016). Acute toxicity test revealed that these plant parts are safe up to 2,000 mg kg−1 body weight (Gupta and Singh 2012).
In our previous study, we have reported cytotoxic effects of LAECV on root apical meristem cells of wheat and onion (Ray et al. 2012). The metaphase arrest and cell cycle delay-inducing effects were somewhat comparable to colchicine’s actions (Roy and Ray 2017, Kundu and Ray 2016, Ray et al. 2013). In another comparative study, treatment of colchicine and LAECV on Allium cepa root tip cells revealed their similar cytotoxic effects including an increased frequency in mitotic abnormalities and micronucleus (Kundu and Ray 2016). Recently, Roy and Roy (2019) assessed the cytotoxic effects of aqueous and methanolic leaf extracts of C. viscosum treatment on A. cepa roots after 5 days, however, cytotoxic activities were not studied at the early hours of treatment, and also the used concentrations of the extracts were not conclusive. In continuation of the previous reports, here non-polar solvent petroleum ether was used to extract the non-polar fraction of LAECV and analyzed its cytotoxic effects at early hours, 2 to 4 h, after the AQPEF treatment (50–200 µg mL−1) in A. cepa root apical meristem cells and at 16 h recovery. Additionally, colchicine-induced cytotoxic effects were compared with the AQPEF induced cytotoxic effects on A. cepa root apical meristem cells.
The collected plant was taxonomically identified as C. viscosum Vent. by Prof. A. Mukherjee, Department of Botany, The University of Burdwan. For future reference, a voucher specimen (No. BUTBSR011) is maintained in the Department of Zoology, B.U.
Fresh leaves of C. viscosum were collected from Burdwan University campus, washed in tap water, dried in shade, ground by Philips Mixer Grinder HL1605, and for further use, the ground leaf was stored in a sealed container. Ground leaf (100 g) was extracted in 2.5 L of boiling distilled water for 2–3 h and the extract (LAECV) was filtered with filter paper. With the help of a magnetic stirrer, the LAECV was fractionated with petroleum ether and the resulting yellow-colored petroleum ether (AQPEF) solution was concentrated by a rotary vacuum evaporator and stored in a glass bottle.
Cytotoxic effects of AQPEF in A. cepa root apical meristem cellsMitotic abnormalities, micronuclei, and polyploid cell frequencies were analyzed for the elucidation of AQPEF induced cytotoxic effects on A. cepa root-tip cells. The similar size A. cepa bulbs were surface sterilized by 1% sodium hypochlorite and allowed for root sprouting. The onion bulbs were placed in 6-well plates containing distilled water and kept in dark, within an environmental test chamber at 25–27°C. The similar-sized (2–3 cm), 48 h aged, A. cepa roots were treated with AQPEF (0, 50, 100, 150, and 200 µg mL−1) and colchicine (150 µg mL−1) for 2 and 4 h. After the treatment hours, 8–10 roots were fixed and processed for squash preparation following the standard procedure (Chaudhuri and Ray 2015). The remaining roots were allowed to grow further for another 16 h in distilled water and subsequently, root tips were fixed. The treated and untreated root tips were fixed in aceto-methanol (3 parts methanol : 1 part glacial acetic acid) for 24 h and then hydrolyzed for 10 min in 1 M HCl at 60°C. The roots were stained with 2% aceto-orcein and finally squashed in 45% acetic acid (Sharma and Sharma 1999, Ray et al. 2013). The well-spread areas of squashed roots were focused under the bright field light microscope for observation and scoring the mitotic abnormalities.
Scoring and statistical analysisIn the case of squash preparation of A. cepa root apical meristem cells, at least three randomly coded slides were observed under the light microscope. Calculation of the mitotic index was done by counting the number of dividing cells per total cells scored for each concentration. Aberrant cell frequencies were calculated by counting the number of abnormal cells scored per slide for each concentration (Bakare et al. 2000). The mitotic abnormalities were analyzed by the 2×2 contingency χ2-test.
The different extracts of C. viscosum have various pharmacological activities (Yusuf et al. 1994, Warrier et al. 1996, Jirovetz et al. 1999, Gouthamchandra et al. 2010, Aley Kutty et al. 2011, Ray et al. 2012, 2013, Kundu and Ray 2016). The present investigation analyzed a detailed cytotoxic effect of the AQPEF, a non-polar fraction of LAECV, in A. cepa root apical meristem cells at early hours (2 and 4 h) of treatment and also at 16 h recovery.
The cytotoxic potentials of the various phytochemical substances can be deciphered by studying the mitotic abnormalities (Caritá and Marin-Morales 2008). Concentration-dependent increased aberrant cell percentages were observed at 2 and 4 h in AQPEF (100, 150, and 200 µg mL−1) treated samples. The highest aberrant cell percentage (10.45±0.46%) was scored from 150 µg mL−1 followed by 100 µg mL−1 (8.75±0.26%) concentration at 4 h treated samples. However, in 16 h recovery samples, the AQPEF induced aberrant cell frequency was considerably decreased compared to 2 and 4 h treated samples (Table 1). Colchicine (150 µg mL−1) induced 10.55±0.83, 8.12±0.37, and 0.50±0.17% aberrant cells respectively in 2, 4, and 4 h treatment followed by 16 h recovery samples (4 h T+16 h RS). The AQPEF induced a similar aberrant cells percentage at a much lower concentration than LAECV at 2 and 4 h treated A. cepa root apical meristem cells. In the case of 4 h T+16 h RS, the aberrant cell frequencies are reduced in comparison to 2 and 4 h treated cells but still aberrant cells persist more in number than the untreated ones. Our earlier reports indicated that LAECV has colchicine-like metaphase arrest and mitotic abnormalities inducing potentials (Ray et al. 2012, Kundu and Ray 2016) and this study explored that AQPEF of LAECV contains the active principles having colchicine like aberrant cell inducing potentials.
| H | Conc. (µg mL−1) | TC | TDC | Ac | Sti | Bri | Pd | Vag | Lag | MN | POL |
|---|---|---|---|---|---|---|---|---|---|---|---|
| % (Mean±SEM) | |||||||||||
| 2 | 0 | 1959 | 111 | 0.30±0.09 | 1.78±0.89 | 1.83±1.83 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 |
| 50 | 2181 | 144 | 2.58±0.24a | 6.23±1.24 | 7.0±1.5 | 2.06±0.03 | 0.00±0.00 | 1.03±0.60 | 0.00±0.00 | 0.00±0.00 | |
| 100 | 2445 | 183 | 4.59±0.33a | 5.49±1.36 | 5.62±1.08 | 5.32±1.08 | 2.67±0.35 | 2.29±0.80 | 0.00±0.00 | 0.00±0.00 | |
| 150 | 2404 | 234 | 7.13±0.31a | 3.84±0.69 | 7.24±0.63 | 3.42±1.48 | 2.50±0.63 | 0.40±0.40 | 0.00±0.00 | 0.00±0.00 | |
| 200 | 2389 | 251 | 6.65±0.10a | 6.42±0.52 | 7.23±0.51 | 3.54±1.24 | 0.84±0.42 | 0.81±0.41 | 0.00±0.00 | 0.00±0.00 | |
| 150© | 1987 | 248 | 10.55±0.83a | 2.67±0.80 | 2.55±1.81 | 0.00±0.00 | 1.50±0.75 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | |
| 4 | 0 | 2914 | 207 | 0.20±0.05 | 0.00±0.00 | 3.09±1.09 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 |
| 50 | 3423 | 352 | 6.28±0.59a | 1.76±1.31 | 5.63±0.41 | 1.43±0.58 | 2.17±0.84 | 0.60±0.30 | 0.00±0.00 | 0.00±0.00 | |
| 100 | 2162 | 222 | 8.75±0.26a | 0.00±0.00 | 3.60±0.47 | 0.00±0.00 | 1.35±0.05 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | |
| 150 | 3035 | 403 | 10.45±0.46a | 1.59±0.80 | 5.30±2.20 | 0.21±0.21 | 1.16±0.52 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | |
| 200 | 3525 | 352 | 7.61±0.76a | 1.66±0.76 | 5.50±0.86 | 0.35±0.35 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | |
| 150© | 3286 | 301 | 8.12±0.37a | 0.93±0.40 | 3.45±1.07 | 1.00±0.07 | 0.35±0.35 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | |
| 4+16 | 0 | 2449 | 241 | 0.25±0.03 | 0.00±0.00 | 2.49±0.10 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 |
| 50 | 3946 | 276 | 1.05±0.9 | 0.00±0.00 | 2.17±0.06 | 0.36±0.36 | 0.00±0.00 | 0.00±0.00 | 4.31±0.33a | 11.96±0.48a | |
| 100 | 2130 | 177 | 4.37±0.86a | 1.16±0.63 | 3.23±1.64 | 0.59±0.59 | 3.69±0.91b | 0.00±0.00 | 5.08±0.13a | 20.14±0.68a | |
| 150 | 2068 | 136 | 3.84±0.59a | 1.04±1.04 | 5.83±1.72c | 0.00±0.00 | 5.16±0.68a | 0.00±0.00 | 5.05±0.22a | 18.43±0.21a | |
| 200 | 2162 | 161 | 3.13±0.52a | 4.33±1.51b | 7.38±0.71c | 0.00±0.00 | 5.04±0.83b | 0.00±0.00 | 3.05±0.37a | 12.42±0.81a | |
| 150© | 1922 | 69 | 0.50±0.17 | 0.00±0.00 | 2.70±1.60 | 0.85±0.85 | 0.85±0.85 | 0.00±0.00 | 6.11±0.43a | 16.03±0.20a | |
a significant at p＜0.0001 and b significant at p＜0.001, c significant at p＜0.05 as compared with their respective control by 2×2 Contingency χ2-test with respective df=1. H; Hours, Comp; Compound, Conc; Concentration, Tc; Total number of cells, TDC; Total dividing cells, Ac; Aberrant cells, Sti; Stickiness, Bri; Bridge, Pd; Polar deviation, Vag; Vagrant chromosome, Lag; Laggard chromosome, MN; Micronucleus, POL; Polyploidy, ©; Colchicine.
The mitotic abnormalities like anaphase bridges, stickiness, polar deviation, vagrant chromosome, laggard chromosome, micronuclei, polyploidy, etc. were induced by both AQPEF and colchicine treatments in A. cepa root tip cells. The AQPEF treatment induced an increased percentage of anaphase bridges in A. cepa root apical meristem cells. The anaphase bridge percentages were scored as 7.0±1.5, 5.62±1.08, 7.24±0.63, and 7.23±0.51 at 2 h respectively at concentrations of 50, 100, 150, and 200 µg mL−1 and correspondingly, these were decreased to 5.63±0.41, 3.60±0.47, 5.30±2.20, and 5.50±0.86% at 4 h and 2.17±0.06, 3.23±1.64, 5.83±1.72 and 7.38±0.71% in 4 h T+16 h RS, except for the concentration of 200 µg mL−1, which showed the highest (7.38±0.71%) anaphase bridge percentage (Table 1, Fig. 1). Colchicine (150 µg mL−1) induced 2.55±1.81, 3.45±1.07 and 2.70±1.60% anaphase bridge respectively in 2, 4, and 4 h T+16 h RS. The LAECV has anaphase bridge inducing capabilities in 4 h and 4 h treatment followed by 16 h recovery A. cepa root tip cells (Kundu and Ray 2016). The AQPEF at a lower concentration has produced similar types of mitotic abnormalities, as induced in LAECV, in all the treated hours. It may be due to the accumulation of active principles in the petroleum ether fraction of LAECV.
Like anaphase bridge, an increased frequency of chromosomal stickiness was also observed in both the AQPEF and colchicine-treated onion root tip cells at 2 h and that was successively decreased at 4 h and 4 h T+16 h RS. The AQPEF (200 µg mL−1) induced the highest frequency of chromosomal stickiness (6.42±0.52%) at 2 h in onion root tip cells. In the case of 16 h recovery samples, the percentages of sticky chromosomes containing cells were reduced (Table 1, Fig. 1). Similarly, colchicine (150 µg mL−1) induced 2.67±0.80% and 0.93±0.40% chromosomal stickiness respectively in 2 and 4 h treated samples. The basis for the onset of the sticky chromosome may be due to sub chromatid association among chromosomes or dissolution of nucleoprotein or de-condensation of DNA (Ford and Correll 1992, Babich et al. 1997). The AQPEF induced anaphase bridges may be due to the fission and fusion of the chromatids and chromosomes. The AQPEF induced multipolar anaphase in condensed and decondensed form and such multipolar anaphases might be formed from the chromosomal bridge and sticky chromosome.
The percentage of polar deviation increased in A. cepa root apical meristem cells that were treated with AQPEF, at 2 h but decreased at 4 h treatment and 4 h T+16 h RS. At 2 h, AQPEF induced polar deviations were scored as 2.06±0.03, 5.32±1.08, 3.42±1.48, and 3.54±1.24% respectively for concentrations 50, 100, 150, and 200 µg mL−1. At 4 h AQPEF treatment and 4 h T+16 h RS, the polar deviation frequency consequently decreased as compared to 2 h treated samples (Table 1, Fig. 1). Colchicine (150 µg mL−1) induced 1.00±0.07 and 0.85±0.85% polar deviation respectively at 4 h treatment and 4 h T+16 h RS. The present investigation shows that AQPEF induced increased polar deviation frequencies at 2 and 4 h treatments but it was reduced in 4 h T+16 h RS indicating its reversible effects like colchicine. The polar deviation is a type of mitotic abnormality that was reported earlier with LAECV and colchicine in A. cepa root apical meristem cells (Kundu and Ray 2016). The polar deviation becomes evident in microtubule destabilizing drugs i.e., colchicine. Thus, the preliminary mode of action of AQPEF is almost certainly reminiscent of colchicine.
An increased percentage of the vagrant chromosomes were observed in the case of 4 h T+16 h RS than 2 and 4 h of AQPEF treatments. The frequency of vagrant chromosome was scored to be 3.69±0.91, 5.16±0.68, and 5.04±0.83% respectively for the concentration of 100, 150, and 200 µg mL−1 of AQPEF in 16 h recovery samples (Table 1). The AQPEF treatment induced an increased percentage of cells with laggard chromosomes at early hours (2 h) of treatment. Here, 50, 100, 150, and 200 µg mL−1 concentration of AQPEF induced respectively 1.03±0.60, 2.29±0.80, 0.40±0.40, and 0.81±0.41% of laggard chromosome containing cells at 2 h of treatment (Table 1, Fig. 1). Mitotic abnormalities like vagrant chromosomes and laggard chromosomes can also form due to spindle poisoning. Here, in A. cepa root tip cells, AQPEF induced both types of abnormalities at 2 and 4 h of treatment. The frequencies of vagrant and laggard chromosomes were decreased in 4 h of AQPEF treatment followed by 16 h recovery, indicating the induced effects are reversible like colchicine action.
The AQPEF treatment induced a significant (p＜0.0001) increase in micronuclei frequency and were scored 4.31±0.33, 5.08±0.13, 5.05±0.22, and 3.05±0.37% respectively for the concentrations 50, 100, 150, and 200 µg mL−1. However, the incidence of micronuclei was not observed in the case of early hours (2 h and 4 h) of AQPEF treated samples (Table 1, Fig. 1). Above result correlates with the result obtained from colchicine treatment, where 150 µg mL−1 colchicine induces 6.11±0.43% micronucleus in 4 h T+16 h RS. Data indicates that the AQPEF and colchicine treatments on onion root tip cells could induce a significantly (p＜0.0001) increased polyploid cells frequency. The polyploidy was induced only in the case of 4 h treatment followed by 16 h water recovery samples in both AQPEF and colchicine treatment. The highest polyploid frequency (20.14±0.68%) was found in 100 µg mL−1 followed by 150 µg mL−1 (18.43±0.21%) and 200 µg mL−1 (12.42±0.81%) concentrations of AQPEF treatment (Table 1, Fig. 1). 150 µg mL−1 colchicine treatment induces 16.03±0.20% polyploidy. Here, 150 µg mL−1 is the most effective concentration of AQPEF to decipher its activity similar to colchicine (150 µg mL−1). Disruption of the mitotic spindle inhibits cytokinesis and such inhibition of cytokinesis and reconstruction of nuclei leads to the formation of polyploid cells (Levan 1938, Chauhan et al. 1986, Chauhan and Sundararaman 1990, Fenech and Crott 2002, Carvalho et al. 2019). In addition to polyploid cells, the microscopic analysis also revealed that AQPEF induces micronuclei in A. cepa root apical meristem cells at 4 h T+16 h RS. Reconstruction of c-metaphases, vagrant chromosomes, and chromatid breaks result in the formation of micronuclei. A positive correlation between the formation of c-metaphase, vagrant chromosome, and polyploidy was evident in A. cepa root tip cells (Levan 1938, Carvalho et al. 2019). In conclusion, petroleum ether is a suitable solvent that can extract the bioactive phytochemicals of LAECV having a cytotoxic effect on A. cepa root tip cells. The AQPEF of LAECV has shown to have effective micronuclei, polyploidy, and mitotic abnormality-inducing potentials in A. cepa root apical meristem cells.
The authors acknowledge Prof. A. Mukherjee for plant species authentication and the financial support of UGC-SRF (FC(Sc)/RS/UGC/ZOO/2018-19/129, w.e.f. 07.04.2018, dated: 04.02.2019), and the DST-PURSE, DST-FIST, and UGC-DRS and MRP sponsored facilities in the Department of Zoology.
