Antimitotic Activity of Fruits of Momordica dioica by using Allium cepa Root Tip Assay

 

N. S. Patil*, K. B. Patil, M. R. Patil, R. A. Ahirrao

Department of Pharmaceutical Analysis, P. S. G. V. P. M’s College of Pharmacy, Shahada-425409, Dist- Nandurbar. (M.S) India.

*Corresponding Author E-mail: nayanapatil804@gmail.com

 

ABSTRACT:

Objective: Cancer is a dreadful disease and combating this disease is of great importance to public health. Phytochemical examination has been making rapid progress and herbal products are becoming popular as sources of plausible anticancer compounds. In the present study we have utilized the Allium cepa root tip meristem model to evaluate antimitotic activities of fruits of Momordica dioica commonly known as kakrol, spiny gourd or teasle gourd belongs to family cucurbitaceae. Methods: Preliminary antimitotic screening was done using Allium cepa root tip assay. The herbal powder obtained from plant part-dry fruits of Momordica dioica were extracted with aqueous solvents by maceration methods. The antimitotic activity was analysed using Allium cepa root and methotrexate was used as a standard drugs. Result and discussion: In Allium assay, aqueous extract of fruits of Momordica dioica (10 mg/ml) and methotrexate showed significant concentration dependant inhibitory influence against the dividing cells of Allium roots and decreased root growth and mitotic index as compared to control distilled water.  The results indicated the cytotoxic potential of extract due to antimitotic effects. The effect may be attributed to the presence of flavonoids, alkaloids and phenolic compounds in Momordica dioica fruits extract. Conclusion: The present work revealed that fruits of Momordica dioica contain some important chemical constituents extracted using aqueous as solvent that can be used further in the management of cancer treatment.

 

KEYWORDS: Allium cepa, Momordica dioica, cucurbitaceae, maceration, flavanoids, alkaloids and phenolic compounds.

 

 

1. INTRODUCTION:

Cancer is one of the most dangerous diseases in humans and presently there is a considerable scientific discovery of new anticancer agents from natural products1. Natural product based medicines, particularly, herbal- based drugs represented about 60 to 80 percent of all drugs in use by 19902,3.

 

The plant derived compounds have always been an important source of medicines for various diseases, and have received considerable attention in recent years due to their diverse pharmacological properties including cytotoxic and cancer chemo reventive effects4. Plants have a special place in the treatment of cancer. It is estimated that plant derived compounds one or the other way constitute more than 50% of anticancer agents5,6.

 

Momordica dioica is a perennial, dioecious climber included in the Cucurbitaceae family, which is commonly known as spiny gourd, teasel gourd or small bitter gourd worldwide whereas in India it is known as Kankro, Kartoli, Kantola, Kantroli, Ban karola or Janglee karela. Momordica dioica has been known to have many medicinal properties namely anti tumorogenic, analgesic, anti diabetic, anti inflammatory and anti allergic activity7,8,9,10.

 

There are five active constituents isolated from the dichloromethane extract of Momordica dioica roots which were found to possess anticancer activity in pharmacologic testing on cancer cell (L1210). The growth inhibitory index (%) was shown to be 50%, at the dose of 4 mg/mL11. Based on the literature survey, it is evident that no work has been carried out on the evaluation of antimitotic property of 10 mg/ ml concentration of aqueous extracts of fruits of Momordica dioica. Hence in this present study, the antimitotic potential of aqueous extract of fruits of Momordica dioica in 10 mg/ml concentration was assessed by using Allium cepa root tip assay.

 

2. MATERIALS AND METHODS:

2.1 Plant material and reagents:

Momordica dioica fruits were collected from the local area of Nandurbar (Maharashtra) and authenticated by Dr. Santosh K. Tayade, Head, Dept. of Botany, Art’s, Science and Commerce College, Lonkheda, Shahada, Dist-Nandurbar (MS). All the reagents and chemicals were purchased from Rajesh Chemicals, Mumbai.

 

2.2 Preparation of aqueous extract of fruits:

Collected fruits were washed with distilled water, shade dried and crushed to a coarse powder and extracted with water by maceration method. Extract was dried over anhydrous sodium sulphate and solvent was removed in vacuum at 40°C by sing rotary evaporator (Rotavapour Buchii, Switzerland). The aqueous extract fruits of Momordica dioica were subjected to preliminary phytochemical testing for the presence of different chemical classes of compounds12, 13.

 

2.3 Determination of Mitotic Index14, 15:

The red variety of Allium cepa (onions) were procured from the local market and stored for the entire study. The bulbs of Allium cepa were sprouted in tap water for 48 hours at room temperature. The roots thus developed were treated by dipping these in the aqueous solutions of fruits of Momordica dioica (10mg/ml) for 2 hours. Treatment of the roots with the distilled water and methotrexate (GSK, India 2.5 mg) at concentration 0.1mg/ml served as negative and positive control respectively. The roots thus treated with the above solutions were then cut to separate root tips and the root tips were transferred to the fixing solution {acetic acid (45% v/v) ethanol (95% v/v) in ratio of 1:3 v/v} for 10-15 hrs. After 10-15 hours, these were treated with 1 N hydrochloric acid and warmed in an oven at 50°C for 15 min. These root tips were then washed with distilled water and were stained with 2-3 drops of Carmine stain (LR Central Drug House Pvt. Ltd). The slide was then squashed and observed under microscope. The numbers of cells in each stage of cell division were counted in four fields for each group.

 

Mitotic Index was calculated using the formula:

 

                          Number of dividing cells

Mitotic Index = --------------------------------    X 100

                              Total number of cells

 

3. RESULTS:

Table No.1 showed the antimitotic activity after treatment of Allium cepa roots with aqueous extracts of fruits of Momordica dioica (10 mg/ml) and standard drug methotrexate. The percentage of non dividing cells in water control, aqueous extract (10 mg/ml) and standard group (methotrexate) was found to be 35%, 59%, and 45% respectively.  In water (control), the percentage of dividing cells in prophase, metaphase, anaphase and telophase was found to be 48, 14, 2 and 1 respectively. In methotrexate (standard) drugs, the percentage of dividing cells in prophase, metaphase, anaphase and telophase was found to be 36, 3, 1 and 1 respectively. In 10 mg/ml concentration of aqueous extracts of Momordica dioica, the percentage of dividing cells in prophase, metaphase, anaphase and telophase was found to be 47, 5, 2 and 1 respectively. The average mitotic index in water (control), methotrexate (standard) and 10 mg/ml concentration of aqueous extracts of fruits of Momordica dioica was found to be 65, 41 and 55 respectively. From graph 1 and 2, it was observed that aqueous extracts of fruits of Momordica dioica in 10 mg/ml concentration shows better antimitotic activity as compared to water (control group).

 

Table 1: Antimitotic activity after treatment of Allium cepa roots with 10 mg/ml aqueous extracts of fruits Momordica dioica, methotrexate and control water.

Sr. No

Different

solutions used for treatment

% of Non dividing cells

% of dividing cells

Mitotic

Index

Mitotic Index

P

M

A

T

Avg.

SD

SEM

1

Water(control)

35

48

14

2

1

65

65

1.828

1.265

2

Methotrexate

(0.1 mg/ml positive control)

59

36

3

1

1

41

41

1.517

0.6782

3

Aqueous extract (10 mg/ml)

45

47

5

2

1

55

55

1.842

0.507

P-Prophase, M-Metaphase, A- Anaphase, T-Telophase

 

 

 

 

Graph No: 1 % of dividing cells in after treatment of Allium cepa roots with water (control), methotrexate and aqueous extracts of Momordica dioica (10 mg/ml).

 

 

Graph No: 2 Mitotic index of water (control), methotrexate and aqueous extracts of fruits Momordica dioica (10 mg/ml)

 

4. DISCUSSION:

The fruits of Momordica dioica shows wide spectrum of medicinal activities. The plant contains various phytochemical compounds having diverse chemical structure and nature. The major constituents of the plant are flavanoids, alkaloids, saponins, sterols, tannins and phenolic compounds. In the present study, attempts were made to investigate antimitotic activity of aqueous extracts of fruits of Momordica dioica in 10 mg/ml concentration by using Allium cepa roots tip assay. In Allium cepa root meristem model, commonly known as Allium assay, root meristematic cells are used for screening of drugs with antimitotic activity16,17. In meristematic region, the cell division is similar to cancer cell division in humans. Therefore, these meristematic cells can be evaluated for screening of drugs with potential antimitotic activity. Allium assay is considered a rapid, highly sensitive and reproducible bioassay for detecting cytotoxicity and genotoxicity. The root growth inhibition and antimitotic effects provide the indication of genotoxicity. The good genotoxic assay performance of Allium cepa as a plant system has been attributed to the easily studied karyo type of plant and the ability to correlate outcomes of assays with those of mammalian cells in the course toxic evaluations18. The Allium cepa species [common onion] being characterized by homogenous meristematic cells and very large sixteen numbers chromosomes is ideal for use in bioassays19. In Allium cepa assay, aqueous extract of fruits of Momordica dioica (10 mg/ml) was found to exhibit dose dependent antimitotic action on Allium cepa root meristematic cells as indicated by inhibition of root growth and decreased mitotic index after treatment. This suggests that the aqueous extract of fruits of Momordica dioica has fair antimitotic potential in concentration 10 mg/ml.  In Allium cepa assay, mitotic index is considered as an indicator of cell proliferation biomarkers which measure the proportion of cells in the M-phase of the cell cycle20,21. From result, it was conclude that 10 mg/ml concentration of aqueous extracts of fruits of Momordica dioica inhibits cell division in Allium cepa assays and suggests that the fruits may exhibit inhibitory influence on abnormal cell growth as like in cancer. Though the present study validates the traditional use of extract in the treatment of cancer, further studies in cancer cell lines is necessary.

5. ACKNOWLEDGEMENTS:

Authors are thankful to P. S. G. V. P. M’s College of Pharmacy, Shahada, District- Nandurbar. (M.S) for providing necessary support for research purposed.

 

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Received on 01.08.2018       Accepted on 11.10.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2018; 8(4): 221-224.

DOI: 10.5958/2231-5691.2018.00037.0