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International Journal of Pharmacology and Pharmaceutical Sciences 2016; Vol: 3, Issue: 3, 32-38.
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Research Article
ISSN: 2394-613X
ANTI-PROLIFERATIVE ACTIVITY OF SOME MEDICINAL PLANTS
Paras Jain*, Pallavi Singh and H.P. Sharma
Laboratory of Plant Physiology and Biotechnology, University Department of Botany, Ranchi University, Jharkhand (India)
*Corresponding Author
ABSTRACT
Cancer is one of the most dreaded diseases of the 20 th century and spreading further with continuance and increasing incidence
even in 21stcentury. Unfortunately, in recent past few years the several new cancer cases has been identified. Cancer is occurs
when cells are continuously dividing or apoptosis process is disturbed. Medicinal plants are being in practice for treating several
diseases from Ancient time. Medicinal plants containing several secondary metabolites which can directly or indirectly decrease
the rate of cell division. If any compounds inhibit the rate of cell proliferation, they may be a source of anticancer drugs. In the
present investigation four medicinal plants namely Plumbago zeylanica, Bacopa monnieri, Semecarpus anacardium, Phyllanthus
amarus were selected for scientific authentication and validation of ethnomedicinal knowledge collected from local people of
Jharkhand by the Allium Assay. The result of present investigation is reveal that the all the four selected plant containing the
compounds which inhibit the cell division.
KEY WORDS: Allium assay, Ethno-medicinal Plants, Cancer, Anti-mitotic activity, Cell division.
INTRODUCTION
Cell is a basic structural and functional unit of life. And this basic unit of life shows self – reproduction which is perhaps the most
fundamental characteristic of cell. All cells reproduce by dividing in two, with each parental cell giving rise to two daughter cells
on completion of each cycle of cell division. Cell cycle is a genetically controlled series of changes that occur in a newly formed
cell by which it duplicates its content, undergoes growth and division to form two daughters. The division cycle of most cells
consists of four coordinated processes: cell growth, DNA replication, distribution of the duplicated chromosomes to daughter
cells, and cell division. In bacteria, cell growth and DNA replication take place throughout most of the cell cycle, and duplicated
chromosomes are distributed to daughter cells in association with the plasma membrane. In eukaryotes the cell cycle is more
complex and consists of four discrete phases (Hartwell et al, 1989).
Mitosis is a part of the cell cycle process by which chromosomes in a cell nucleus are separated into two identical sets of
chromosomes, each in its own nucleus. In general, mitosis (division of the nucleus) is often followed by cytokinesis, which
divides the cytoplasm, organelles and cell membrane into two new cells containing roughly equal shares of these cellular
components (Carter., et al,2014) Mitosis and cytokinesis together define the mitotic (M) phase of an animal cell cycle—the
division of the mother cell into two daughter cells, genetically identical to each other and to their parent cell.
The process of mitosis is divided into stages corresponding to the completion of one set of activities and the start of the next.
These stages are prophase, prometaphase, metaphase, anaphase, and telophase. During mitosis, the chromosomes, which have
already duplicated, condense and attach to fibers that pull one copy of each chromosome to opposite sides of the cell. The result is
two genetically identical daughter nuclei. The cell may then divide by cytokinesis to produce two daughter cells (Maton, et al.,
1997). Errors during mitosis can induce apoptosis (programmed cell death) or cause mutations. Certain types of cancer can arise
from such mutations (Kops, et al, 2005). German zoologist Otto Bütschli was one of the first researchers who might have claimed
Paras Jain. et al..; Int. J. Pharmacol. Pharm. Sci. (2016) 3:23; 32-38
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the discovery of the process presently known as "mitosis", (Fokin, 2013) a term coined by Walther Flemming in 1882 (Sharp,
1921).
When mitosis begins, the chromosomes condense and become visible. In some eukaryotes, for example animals, the nuclear
envelope, which segregates the DNA from the cytoplasm, disintegrates into small vesicles. The nucleolus, which makes ribosomes
in the cell, also disappears. Microtubules project from opposite ends of the cell, attach to the centromeres, and align the
chromosomes centrally within the cell. The microtubules then contract to pull the sister chromatids of each chromosome apart
(Zhou,et al, 2002). Sister chromatids at this point are called daughterchromosomes. As the cell elongates, corresponding daughter
chromosomes are pulled toward opposite ends of the cell. A new nuclear envelope forms around the separated daughter
chromosomes.
As mitosis concludes, the cell may begin cytokinesis. In animal cells, a cell membrane pinches inward between the two
developing nuclei to produce two new cells. In plant cells, a cell plate forms between the two nuclei. Cytokinesis does not always
occur; coenocytic (a type of multinucleate condition) cells undergo mitosis without cytokinesis.
Plants have a long history of use in the prevention and treatment of cancer. In Present study Four Medicinal plants namely Bacopa
monnier, Phyllanthus amarus, Plumbago zeylanica, and Semecarpus anacardium. have been selected to check their anti-mitotic
acivity.
Bacopa monnieri L. (family: Scrophulariaceae) locally known as Brahmi is a reputed drug of Ayurveda. It is widely used in
traditional medicine to treat various nervous disorders, cardio tonic, digestive aid, memory enhancer. The main principal bioactive
compound of this plant is Bacosides.
Phyllanthus amarus (Family: Euphorbiaceae) is a herb lacally known as Bhumyamala. it is an annual herb (weed) which grows in
the wild after first showers of monsoon. Maharshi Charak has considered this herb to be most effective in the treatment of
jaundice, asthma, increasing appetite, improving digestion, stimulating liver, and producing laxative effects. In the Unani System
of medicine this herb is good for sores and chronic dysentery. Its seeds are used in the treatment of ulcers, wounds, scabies and
ringworms. The main principal bioactive compound of this plant is lignin.
Plumbago zeylanica Linn (Family: Plumbaginaceae) is shrub which is locally known as Chitrak and Doctorbush. The literature
reveals its wide application in traditional system of medicines against various diseases, as anti-inflammatory, anti-malarial, antifertility, anti-microbial, anti-oxidant, blood coagulation, wound healing, memory enhancer and anti-cancer. The main principal
bioactive compound of this plant is plumbagin (Jain et al.,2014).
Semecarpus anacardium L. (family Anacardiaceae) is a tree commonly known as “Bhilawa”. It is found in moist tropical forests.
It is a plant well-known for its medicinal value in ayurvedic and siddha system of medicine. Several experiments have prooved
it’s anti-atherogenic, anti-inflammatory, antioxidant, antimicrobial, anti-reproductive, CNS stimulant, hypoglycemic,
anticarcinogenic and hair growth promoter activities. The most significant component of the S anacardium Linn. is bhilwanols
(Jain et al., 2013).
Allium has proven a rapid, reliable, and inexpensive system by which the antimitotic effects of various chemical compounds may
be monitored (Andrade et al., 2008). Characterized by rather homogenous meristematic cells, very large chromosomes and only
sixteen chromosome numbers, the Allium cepa species (common onion) is ideal for use in bioassays (Leme and Marin-Morales,
2008). It has also been widely used for detection of cytostatic, cytotoxic and mutagenic properties of different compounds,
including anticancer drugs of plant origin (Havey et al.,2002). The results obtained by Allium test could be useful in correlating
the antimitotic effect of Bacopa monnier, Phyllanthus amarus, Plumbago zeylanica, and Semecarpus anacardium. Studies and
test on the isolated compound from the above mentioned medicinal plants have proved its anti cancerous activity.
MATERIALS AND METHODS
Paras Jain. et al..; Int. J. Pharmacol. Pharm. Sci. (2016) 3:23; 32-38
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Collection of plant sample
Various plant sample were collected from different area
Bacopa monnieri
: Ranchi university campus
Phyllanthus amarus
: Ranchi University campus
Plumbago zeylinaca
: Medicinal Garden of Ranchi University
Semecarpus anacardium : Hilly area of Parasnath, Gridhi
Extract preparation
The sample collected was dried in shade which took approximately 4 week and then grinded in fine powder. The grinded powder
was weighed and incubated in methanol for 4 days on shaker. On 4th day the incubated sample was filtered using filter paper and
left at room temperature for evaporation. After evaporation of methanol the stock plant extract is ready.
Fixative Preparation
Aceto alcohol was prepared (v/v) by mixing C2H4O2and ethyl alcohol in 1:3 ratio then a pinch of FeCl3 was added.
Preservative
For preserving the root tips 70% alcohol was used.
Anti-mitotic Assay
The root of Allium cepa was grown in autoclaved water for 72hr in laboratory condition. After reaching a length of 3cm (±0.5
cm), roots were incubated in plant extract dissolved in DMSO for 3 hr. This was named as “treated”. DMSO and pure water was
kept as control. After 3hr of incubation all the roots were washed, cut and incubated in Para Dichloro Benzene for 4hr, pretreatment. For this 5-8 root tips were taken from 3 analogous onions. Then the root tips were washed and subsequently kept in
fixative for 24hr. As the incubation of 24hr was completed the tips were washed and preserved in preservative for observation.
Slide preparation
The preserved root was warmed in acetocarmine till the tips become soft. Then on the slide a drop of 45% C 2H4O2was dropped
and root tips were kept on it and covered with cover slip. This was then squash and observed under 10x and 45x. An agent that
prevents or disrupts mitosis is called as antimitotic agent. Antimitotic constituents can stop the mitosis in anywhere of the cell
cycle. Methanolic extract of Bacopa monnieri, Phyllanthus niriuri, Plumbago zeylanica and Semecarpus anacardium reduced
mitotic index significantly after 3 hour extract treatment (4 mg/ml) which was comparable with mitotic index of Allium cepa root
tip treated with DMSO and autoclaved water.
Mitotic index
The cytotoxic level of a test chemical/compound can be determined based on the increase or decrease in the mitotic index (MI),
which can be used as a parameter of cytotoxicity in studies of environmental biomonitoring (Stegner, et al., 1996). Significant
reduction in MI, noted in the present study may be due to the inhibition of DNA synthesis or the blocking in the G2 phase of the
cell cycle (Sudhakar , et al., 2001). Several other chemicals have been reported to inhibit mitosis (Turkoglu, 2007). Inhibition of
mitotic activities is used for tracing cytotoxic substances. The cytotoxic level can be determined by the decreased rate of mitotic
index. A mitotic index decrease below 22% of negative control causes lethal effects on test organism while a decrease below 50%
has sub lethal effects (Sharma, et al., 2012) and is called cytotoxic limit value (Nagpal, et al., 2012 and Panda, et al., 1985).
Paras Jain. et al..; Int. J. Pharmacol. Pharm. Sci. (2016) 3:23; 32-38
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MI =
No. of dividing cells
× 100
Total no. of cells
RESULTS AND DISCUSSION
The in vitro antimitotic activity was observed by fixing the roots of onion (Allium cepa) at the interval of 15 min starting from
11:30 am. The roots were fixed at 11:30 am, 11:45 am, 12 pm, 12:15 pm, and 12:30 pm. The best result was seen between 12:15
to 12:30 pm.
The roots treated with water (Control 1) and DMSO (Control 2) exhibit 14-18 (Prophase 6 cells, Metaphase 5 cells, Anaphase 4
cells and Telophase 2 cells) and 18-22 (Prophase 8 cells, Metaphase 6 cells, Anaphase 4 cells and Telophase 2 cells) dividing cells
respectively. Whereas the roots treated with plant extracts exhibit the less dividing cells Plumbago zeylanica 6 (Prophase 3 cells,
Metaphase 2 cells, Anaphase 1 cells and Telophase 0 cells); Semecarpus anacardium 7 (Prophase 3 cells, Metaphase 2 cells,
Anaphase 2 cells and Telophase 0 cells); Phyllanthus amarus 8 (Prophase 4 cells, Metaphase 2 cells, Anaphase 2 cells and
Telophase 0 cells); Bacopa monnieri 5 cells (Prophase 2 cells, Metaphase 2 cells, Anaphase 1 cells and Telophase 0 cells) in
dividing stage, presented in table 1 and Fig 2 .
The mitotic index were also calculated which were significantly decrease in treatment group as compare to Control1 and control 2.
The control 1 group shows the 20% mitotic index and control 2 group show 23% mitotic index. While treated group show less
Mitotic index Plumbago zeylanica (8%), Semecarpus anacardium (10%), Phyllanthus amarus (15%), Bacopa monnieri (5%)
respectively; mention in table 1, Fig. 1 and Fig. 2. The plant extract of selected medicinal plant has some bioactive (cytotoxic)
constituents, which can inhibit or cease the cell division or cell cycle.
Table 1: Effect of Plant extracts on Mitotic Index
Total number of
Number of dividing cells in each phases (Mean±SD)
cell observed
Prophase
Metaphase
(P)
(M)
Anaphase
Telophase
(A)
(T)
Mitotic Index (%)
Control 1
82±5
6±1
5±1
4±0.5
2±0.5
18-22
Control 2 (DMSO)
85±6
8±1
6±1
4±0.5
2±0.5
20-25
Plumbago zeylanica
75±4
3±0.5
2±0.0
1±0.0
-
7-9
Semecarpus anacardium
72±3
3±0.5
2±0.0
2±0.0
-
9-11
Phyllanthus amarus
51±4
4±0.5
2±0.0
2±0.0
-
14-16
Bacopa monnieri
85±4
2±0.5
2±0.0
1±0.0
-
4-6
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Fig.1. Mitotic index of contrl 1 (water), Control 2 (DMSO) and plant extract treated groups
A
C
E
B
D
F
Fig.2 Photomicrograph of control and treated onion root tip : A. Control (Water); B. Control (DMSO); C. Treated
(Bacopa monnieri); D. Treated (Semecarpus anacardium) E. Treated ( Plumbago zeylanica) F. Treated (Phyllanthus
amarus)
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CONCLUSION
The present preliminary toxicological investigation confirms the in vitro antimitotic effects of extract from Bacopa monnieri,
Phyllanthus amarus, Plumbago zeylanica and Semecarpus anacardium against Allium assay. However, more positive results are
definitely necessary in other test systems involving in vitro and in vivo animal and human cancer cell lines for qualifying
prospective anticancer activity. The present preliminary investigation provides comprehensive in vitro evidence that the plant
extract has remarkable anti – cancerous activity.
From this study, it could be suggested that selected plants are promising source of natural drug which has the ability to modify the
physiological function of cells and hence acts as anticancer drugs to arrest the proliferation of cancer cells. Therefore, it can be
concluded that the Bacopa monnieri, Phyllanthus amarus, Plumbago zeylanica and Semecarpus anacardium extracts have shown
a commendable antimitotic activity.
ACKNOWLEDGEMENTS
Authors are heartily thankful to Dr. A. K. Srivastava, Head and Dean University Department of Botany, Ranchi University,
Ranchi for providing all necessary facilities to carry out this research work. We also acknowledge UGC for providing financial
assistance under Major Research Project.
CONFLICT OF INTEREST
Authors declare that there is no conflict of interests regarding the publication of this research work.
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