An investigation on the cytotoxic effect of Aloe vera and Plukenetia volubilis evaluated by

Allium cepa test

Research Question

To what extent does the Aloe vera and Plukenetia volubilis extracts affect the mitotic index of

Allium cepa?

Introduction

Plants are widely used for their medicinal and therapeutic potential. 20% of the plants

known and classified have been tested for pharmacological purposes (Sahu, Giri, Singh,

Pandey, Gupta, Shrivastava, Kumar & Pandey, 2013, p.599) 1. The most common medical

applications they are given are as analgesics, antibacterials, antifungals, antivirals, cathartics,

depuratives and anti-inflammatories.

Moreover, the metabolites of some plants have proven to affect cell proliferation,

which can be used in the treatment of neoplastic diseases 2 (Cano, Gonzales-Figueroa & Peña,

2002, p.21). Research on Aloe vera and Plukenetia volubilis indicates their properties can

inhibit the uncontrolled growth of cells (Giani, L., Lissoni, P., Rovelli, F., Trabattoni, P., &

Zerbini, S., 1998). These potential medical treatments can be evaluated by identifying the

cytotoxic effects of Aloe vera and Plukenetia volubilis extracts on Aliium ceoa (most

commonly known as onions), following the procedure of A. cepa test (Guerrero-Pepinosa &

Muñoz-Solarte, 2013, p.83). This bioassay involves the examination of bulbs of A. cepa after

its exposure to the specific studied substance. The mitotic indexes of the meristematic cells

before and after are compared.

1
According to the OMS, 80% of the world population commonly administers medicinal plants as components
of pharmaceutical remedies or as natural extracts (Bermúdez, A. & Oliveira-Miranda, M. & Velásquez, D.,
2005, p. 345).
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Abnormalities in the mitotic cycle that lead to uncontrolled cell division and formation of tissue such as
tumors.
Variables

Independent. -

 The quantities of Aloe vera and Plukenetia volubilis extract, and distilled water

where the A. cepa bulbs will be introduced into. Each beaker will be filled with

100ml of the predetermined solution.

Dependent. -

 The mitotic index (MI) and the percentage of each phase evaluated by Allium Test

will depend on the cytotoxicity of A. vera and P. volubilis. This will be calculated

by the following equations:

Number of cells undergoing mitosis

mitotix index=
Total number of cells

Number of cells undergoing the phase

Phase percentage= X 100
Totalnumber of cells

Controlled. -

 The quantity of stain used in the preparation of the samples for its view under the

microscope will be measured with droplets (0.5ml).

 The time of rest of the bulbs in the extracts will be measured with a chronometer

(±0.05s).

Uncontrolled. -

 The environmental factors in the laboratory such as room temperature and the time

of exposure to sunlight and its intensity (as the samples will rest by a window)

will not be measured nor controlled.

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Materials

TABLE 1. List of materials

Material Quantity

Allium Cepa bulb 9

Aloe vera extract 300ml

Plukenetia volubilis extract 300ml

Distilled water 300ml

Beakers 8

Aceto-Orcein stain 200ml

Light microscope 1

Scalpel set 1

Watch glass 1

Wooden clamp 1

Bunsen burner 1

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Biosafety

In order to perform the experimental procedure of the investigation it is necessary to

wear a lab coat and a pair of gloves to protect clothing and the skin from the contamination of

Aceto-Orcein stain. Moreover, equipment such as the scalpel set and the Bunsen burner must

be used carefully as they can cut or burn the skin and damage other elements in the

laboratory. For flaming the A. cepa samples, the gas pump faucet will be opened strictly

when needed and the watch glass will be approached to the fire using wooden clamps.

Finally, all solutions3 used for the experiment will be gathered separately instead of pouring

them down the drainage to prevent water pollution.

Procedure

Allium cepa stain

1. Assign three A. cepa bulbs to three different beakers with 100ml of distilled water

each one. Introduce the other six bulbs to other three beakers with 100ml of Aloe vera

extract and three beakers with 100ml of Plukenetia volubilis extract.

2. After 72 hours, take the Allium Cepa out of the beakers, cut off about one or two

centimeters of the root and place the sample on a watch glass. Repeat with all Allium

Cepa bulbs.

3. Cover the root tip with two to three droplets of Orcein A.

4. Using the wooden clamp, hold the watch glass and flame it in the Bunsen burner for

about 30 seconds.

5. Take the watch glass from the flame and place the sample on a slide.

3
The extracts, distilled water and Aceto Orcein stained samples.

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 6. Cover it with two droplets of Orcein B and place a coverslip over it. Press it firmly

but gently under a paper towel in order to crush the sample and take the stain excess

away.

7. Repeat the procedure with all the bulbs in distilled water and Aloe vera extract.

Allium cepa mitosis observation under microscope

1. Place the microscope slide under the microscope and locate de sample in the center.

2. Using 4x, scan and focus on the root tip. Proceed to the same under 10x and 40x.

3. Set the image and identify the processes of mitosis in the given cells.

4. Operate the mitotic index equation (interphase does not count as part of mitotic

division) and the percentage of cells under each mitotic phase4.

5. Repeat the procedure with all the Allium cepa bulb samples. Record all data, organize

it in a chart and graph, and compare the results.

Data Collection

TABLE 2. Count of A. cepa merismatic cells under mitosis after 72 hours submerged in

distilled water, Aloe vera extract or Plukenetia volubilis extract.

Total
Cells under
Solution Sample Prophase Metaphase Anaphase Telophase number of
mitosis
cells

1 42 13 6 4 65 110

Distilled
2 40 12 7 3 62 105
water

3 39 10 5 3 56 96

4
Equations are shown in the dependent variables (see page number 3).

5
 4 63 12 7 2 84 124

Aloe vera
5 67 5 4 0 76 102
extract

6 61 5 0 1 67 119

7 58 4 3 0 65 112
Plukenetia
volubilis
extract
8 52 3 2 1 58 99

9 60 6 3 2 71 114

Data Processing

TABLE 3. Mitotic index and percentage of cells in specific mitotic phases of each A. cepa

sample

A. cepa Mitotic Percentage of cells in specific phase

Solution
sample index Prophase Metaphase Anaphase Telophase

1 0.59 38.18 11.82 5.45 3.64

2 0.59 38.10 11.43 6.67 2.86

Distilled
water
3 0.58 40.63 10.42 5.21 3.13

mean 0.59 38.97 11.22 5.78 3.21

S.D. 0.00 1.44 0.72 0.78 0.40

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 4 0.68 50.81 9.68 5.65 1.61

5 0.75 65.69 4.90 3.92 0.00

Aloe vera
extract
6 0.56 51.26 4.20 0.00 0.84

mean 0.66 55.92 6.26 3.19 0.82

S.D. 0.09 8.46 2.98 2.89 0.81

7 0.58 51.79 3.57 2.68 0.00

Plukenetia 8 0.59 52.53 3.03 2.02 1.01

volubilis
extract
9 0.62 52.63 5.26 2.63 1.75

mean 0.60 52.31 3.95 2.44 0.92

S.D. 0.02 0.46 1.16 0.37 0.88

Mitotic index mean for A. cepa samples in distilled water, A. vera and P.
volubilis extract
0.68

0.66

0.64

0.62

0.6

0.58

0.56

0.54
Distilled water Aloe vera extract Plukenetia volubilis extract
G

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RAPH 1. Mitotic index mean for A. cepa samples in Distilled water, A. vera and P. volubilis

extract

Percentage of cells in each mitotic phase of A. cepa samples in distilled

water, A. vera and P. volubilis extract
60
55.92
52.31
50

40 38.97 Distilled water

Aloe vera extract
30 Plukenetia volubilis extract

20

10 11.22
5.78
6.26
3.95 2.443.19 3.21
0 0.82 0.92
Prophase Metaphase Anaphase Telophase

GRAPH 2. Percentage of cells in each mitotic phase of A. cepa samples in distilled water, A.

vera and P. volubilis extract

Conclusion

The information gathered and processed shows the mitotic index of the evaluated A.

cepa samples in distilled water (for standardization), A. vera and P. volubilis extracts. The

meristematic cells submerged in the evaluated solutions evidenced a higher mitotic index

mean, being the ones in A. vera extract more present during the mitotic process at 0.66

(followed by the samples in P. volubilis extract at 0.60).

Moreover, GRAPH 2 indicates the percentages of cells in each mitotic phase also tend

to change in the cells affected by the solutions. Whereas the standardized samples presented a

considerably lower percentage of cells under prophase, both groups of samples previously

submerged in the extracts were present in lower percentages.

From both graphs, it can be concluded that the Aloe vera and Plukenetia volubilis

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extracts accelerate the start of the mitotic process in the meristematic cells. This result does

not corroborate the hypothesis that stated these solutions inhibited the uncontrolled mitosis.

However, under the same solutions the A. cepa cells were halted from entering into the next

mitotic stages showing in the end to effectively prevent the formation of new cells (as they

cannot enter cytokinesis).

Evaluation and Future Recommendations

The main limitation of the present investigation was the scarce number of samples used

to evaluate the natural extracts. The analysis of more A. cepa bulbs could lead to more

conclusive results and show a notorious trend of the cytotoxic effect of the solutions.

Additionally, for future recommendations, it is strongly suggested to include a wider range of

variables in regards to the different extracts evaluated. This should be done by carefully

selecting the new possible natural solutions to be tested. One of the first limitations of the

project was the election of natural oils instead of extracts, for example. These had to be

changed, as the squash technique of the A. cepa test did not permit a proper visualization of

the cells under the microscope. Finally, further investigation could properly identify the

reason of the tendency of the extracts to inhibit the entering of the meristematic cells into

metaphase, anaphase, telophase and cytokinesis. This could give answers to the search for a

natural treatment to the uncontrolled cell growth and possible tumor formations.

Bibliography

Bermúdez, A. & Oliveira-Miranda, M. & Velásquez, D. (2005). La investigación

etnobotánica sobre plantas medicinales: una revisión de sus objetivos y enfoques

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 actuales. Red de revistas científicas de América latina y el Caribe, España y Portugal.

Interciencia. Caracas, Venezuela, 30(8), 453-459.

Cano, M., Gonzales-Figueroa, H. & Peña, J. (2002). Alteraciones de los índices de fases y

mitótico en meristemos de Allium cepa, inducidas por extractos de Lepidium meyenii

Walp maca. Revista de la facultad de medicina humana, 3(1), 21-22.

Guerrero-Pepinosa, N. & Muñoz-Solarte, D. (2013). Allium test para evaluar el efecto

citotóxico y genotóxico de extractos naturales en células meristeméticas de Allium

cepa. Memorias, 11(19), 83-86.

Kumar, A., Giri, D., Gupta, S., Pandey, K., Pandey, P., Sahu, P., Shrivastava, A. & Singh, R.

(2013). Therapeutic and medicinal uses of aloe vera: a review. Pharmacology &

Pharmacy, 4(08), 599-610.

Giani, L., Lissoni, P., Rovelli, F., Trabattoni, P., & Zerbini, S. (1998). Biotherapy with the

pineal immunomodulating hormone melatonin versus melatonin plus aloe vera in

untreatable advanced solid neoplasms. Natural immunity, 16(1), 27-33.

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