COMPARISON OF ANTI-MITOTIC AND CYTOTOXIC POTENTIAL OF PIPER CHABA (CHAVAK) AGAINST VINCRISTINE BY USING GERMINATION INHIBITION AND ALLIUM CEPA ROOT TIP ASSAY.

Mayur P. Parmar and Dipak V. Parmar. Department of Biochemistry, Shri Manibhai Virani and Smt. Navalben Virani Science College, Rajkot, Gujarat, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 06 August 2019 Final Accepted: 08 September 2019 Published: October 2019

In the present study, we used Vigna radiata germination inhibition and Allium cepa root tip assay to demonstrate the Anti-mitotic and cytotoxic potential of Piper chaba (Chavak) aqueous extract. We compared the effectiveness of the extract with known anti-cancer drugs, vincristine.
We observed nearly complete germination inhibition by the plant extract in the Vigna radiata assay. Furthermore, mitotic indices were significantly reduced compared to control. Our study revealed very effective antimitotic as well as cytotoxic activity of the Piper chaba aqueous extract like the vincristine. By 2020, about nine hundred thousand Indians are expected to affect by the disease. Not only these in some nations, cancer have also crossed the number of heart disease and became the most common cause of death (Smith, Andrews et al. 2017).
It is also unlikely that there will ever be a single cure for cancer, as "cancer" refers to a class of diseases and a variety of etiological factors are associated with it. There are many types of cancer treatments available depending upon the type of cancer and its advancement. Mostly a combination of treatments is chosen in the combat of cancer such as surgery with chemotherapy or radiotherapy. Stem cell transplant, laser treatment, and photodynamic therapy are some of the most advanced methods applied to treat cancer. Apart from these, we also face the problem of the side effects caused by synthetic drugs and resistance against chemotherapeutic agents our general preference is now diverted towards the herbal medicine, with over more than a half of the world population using medicinal plants for the basic medical treatments (Cordell 1995).
One of the major groups of medicinal importance to treat cancer till date is Anti-mitotic agents. Primarily Antimitotic drugs like Vinca alkaloids and Taxol family target microtubules and show cytotoxic effects lead to great success in the cancer therapeutics. (McGrogan, Gilmartin et al. 2008). In general, Anti-mitotic agents affect the cell division of neoplastic cells and also the normal cells leading to more complicated clinical conditions like myelosuppression (Rowinsky, Eisenhauer et al. 1993).

ISSN: 2320-5407
Int. J. Adv. Res. 7(10), 251-255 252 Despite major scientific and technological progress in combinatorial chemistry and structure-based design, natural products remain an important source of information on how to create compounds that can modulate new biological targets (Farnsworth 1988), (Cragg, Newman et al. 1997, Shoeb 2006. Green plants, in general, are the primary source of naturally occurring toxins and functioning against mutagenic agents (Gichner, Velemínský et al. 1993) and can be screened for antimitotic activity of the plant by using Vigna radiata (V. radiata) seed germination assay (Murthy, Francis et al. 2011). Allium cepa root tip meristematic cells has proved to be a reliable and cost-effective system for the rapid detection of cytotoxic and mutagenic properties ( Antiproliferative and cytotoxic activity of Piper chaba (P. chaba or commonly; Chavak) is evaluated in the present work, using V. radiata seed germination inhibition assay and Allium cepa root tip assay. P. chaba is found throughout India, and other warmer regions of Asian countries which include Malaysia, Sri Lanka, and Indonesia. It is a creeper plant that spreads on the ground. It may also grow around large trees. It is commonly used as a spice and an important constituent of certain recipes. Roots of this plant is mainly used for its strong aroma. In this study, we evaluated the antimitotic and cytotoxic potential of its aqueous extract using V. radiata and Allium root tip assay.

Materials and Methods:-Collection and preparation of aqueous extract for Vigna radiata assay:
Lyophilized powder of root part of the plant was purchased from a certified dealer. 100 mg / mL plant material was used extract was prepared in water and then centrifuged at 10 K for 15 minutes. A clear supernatant was used for the study.
The Vigna radiata germination assay: The Vigna radiata seeds (n = 10) of almost equal size and dry weight were selected for the assay. The seeds were pre-treated with water (washed and soaked) and were brought up to a stage where they are about to germinate. Such seeds were then imbibed in the control solutions or plant extracts. A set of 10 seeds were kept for each control and plant extracts. Seeds were incubated in microtiter plate with one seed in each well having 0.5 ml of control solutions and plant extract. The seeds were allowed to incubate for 72 hrs. Sprout length was recorded as a parameter to check the cytotoxic effect of the extract.

Collection and preparation of aqueous extract for Allium test:
Lyophilized powder of root part of the plant was purchased from a certified dealer and 20 mg/ml and 40 mg/ml aqueous extracts were prepared and centrifuged at 10000 rpm for 15 minutes. The clear supernatant was used for the assay.

Allium cepa root tip assay:
The test was performed according to the method described by Fiskesjo, 1985. Onions (Allium cepa) were grown in water under laboratory conditions (light, 24ºC). Onions having root length of approximately 5cm were incubated in 20 mg/ml and 40 mg/ml aqueous extracts. We have used four to six root tips for each concentration from analogous onions then fixed using Carnoy's Fixative (3:1 v/v mixture of Ethanol and Glacial acetic acid), macerated using HCl and stained with Acetocarmine stain.

Mounting and slide observation:
Root-tips were taken on a clean glass slide, squashed in a drop of glacial acetic acid, covered with a coverslip and observed under the "Motic" microscope (40X Magnification) having a camera and software attachment (Motic Live Imaging Module). Photographs of several fields were captured. Chromosome morphology and their changes were observed and the mitotic index was calculated.

Statistical analysis:-
The data represented were analyzed by student t-test using Sigma Stat 2.0 statistical analysis software. The normality of data was tested by the Shapiro-Wilk test prior to the Student t-test. p values *p≤0.05 was considered as statistically significant.

Results:-
Results of our study have clearly pointed out that the Piper chaba aqueous extract treated seeds were unable to germinate while healthy sprout were observed in the water control group. The cytotoxic effect of the extract was found as significant as vincristine (*p≤0.05) ( fig.-1.1 and 1.2). These results were further confirmed by using Allium test. Plant aqueous extract in two variable concentrations; 20 mg/ml and 40 mg/ml was able to reduce the mitotic index of root tips in the Allium test. Cells were found arrested in the prophase and chromosomes of the cells were condensed which indicated Anti-mitotic and cytotoxic activity of the extract. This significant reduction in the mitotic index compared to the control group was the same as the vincristine treated group indicated the strong Antimitotic and cytotoxic potential of the Piper chaba extract ( fig.-2.1 and 2.2).

Discussion:-
We used plant-based model systems to check the Antimitotic and cytotoxic effect of Piper chaba aqueous extract in vitro by Vigna radiata germination inhibition assay and Allium cepa root tip assay. Our findings were compared with both positive and negative control groups. We used water as a negative control group in which normal growth of Vigna radiata seeds as well as normal cell division in meristematic cells of Allium bulbs was observed. Potent Antimitotic agent which is also a cancer chemotherapeutic agent vincristine was used as positive control and its effect was compared to the effect of Piper chaba extract. Vincristine has significantly reduced the germination of the seeds and Mitotic index compared to the negative control group. The aqueous extract treated seeds had shown the cytotoxic effect and resulted into the same inhibition of germination as that of vincristine. Allium bulbs treated with extracts have shown prophase arrest. This antimitotic effect may be due to compound/s present in the extract affecting chromosomal morphology or maybe to the spindle orientation. Our study indicated that the Piper chaba extracts possess cytotoxic activity and may contain anticancer metabolites which can be further evaluated by various advanced methods.

Conflict of Interest:
We declare no conflict of interest