Ethnobotanical Leaflets 10: 272-279. 2006.
Prevention of Aluminium Chloride-Induced Mitodepression with Myrobalan (Fruit of Terminalia chebula, Retz, Combretaceae) in Allium cepa Model
H.S. Rathore*, Shazia Bi, Anjali Sharma and Mukesh Makwana
Cell Biology Unit,
*(Corresponding Author e-mail: firstname.lastname@example.org)
Allium cepa bulbs were grown in pure tap water
(Group I), in five concentrations (10-1M to 10-5M) of
aluminium chloride in the absence (Group II) and in the presence (Group
Key words: Allium cepa, mitodepression, AlCl3, tannins antioxidant, myrobalan, T. chebula).
Aluminium is a ubiquitous element found in
almost every food  and is debatable and suspected etiological factor in
neurodegenerative disorder like Alzheimer's brain as it modulates
Dry healthy onion bulbs 1.5 to 2.00 cm in diameter were obtained from local market.
Test herbal drug
Myrobalan, dried young nuts of Terminalia chebula was procured from local herbal medicine shop and were gently backed for few minutes and cooled. Swollen nuts were grinded to fine powder. A recent study  revealed lack of any adverse effect of myrobalan in Allium test at 0.10 mg/ml, therefore, this concentration is selected for the present study.
Aluminium chloride hydrated: AlCl3.6H2O
made by Sarabhai Chemicals,
Administration of Drug
Very fine powder of myrobalan was added to each solution of each concentration of aluminium chloride to prepare a suspension of 0.10 mg/ml.
Experiments were planned as per protocol of Fiskesjo  for Allium test. For each set, twelve test tubes were filled with pure tap water (Group I, controls). Another series of 12 test tubes were filled with each concentration of aluminium chloride (Group II, aluminium exposed). Third series of test tubes (12) were also filled with different concentration of aluminium chloride but each one contained myrobalan powder in it (0.10 mg/ml).
All solutions were changed every 24 hr. After 48 hr two onions out of twelve in each series with most poorly growing roots were removed. Same day i.e. after 48 hr. distal 2 mm of five roots was cut off from five individual bulbs from each series and fixed in aceto-alcohol (1:3 v/v acetic acid and absolute alcohol) for chromosomal study. Every time fixation was done at a fixed time,
After 72 hr total length of the 05 root bundle in each series of each onion was measured to record mean root length.
Squashing of root tips and observation of slides
Root tips were squashed in N-HCl and 2% acetocarmin (BDH) stain. Four fields from each slide was observed to cover 50 cells in each i.e. total 200 cells per slides and 3000-4000 cells were observed for each group of onion. Mitotic index was calculated as total number of dividing cells per 100 observed cells. Slides were also observed to find out mitotic arrest, chromosome fragments, abnormal orientation, lagging chromosomes, nucleolar disorganization, polyploidy and apoptosis etc.
Experiments were done trice. Student t-test was applied at 5% level of significance.
1. Mean Root Length (
Root did not grew
at 10-1M and very poorly at 10-2M. Root grew in 10-3M
Table 1. Mean
root length (
concentration of AlCl3 alone or in combination with myrobalan
Statistically significant based on t-test at
5% level of significance. 'a' = Control Vs Gr. II, 'b' = Control Vs Gr.
2. Morphology: colour and shape of root tips
Morphology i.e. colour and shape
of Allium cepa tips
cultivated in all test concentrations of aluminium chloride (Group II) and in
the presence of drug (Group
3. Mitotic Index (MI, Table - 2)
Significant low MI is found at 10-2M to 10-5M. Presence of drug could not check Al-induced mitodepression at 10-2M and 10-3M but drug could significantly reduce Al-induced mitodepression at only at 10-4M and at 10-5M.
Table 2. Mitotic Index (MI) of Allium cepa root tip cells following 48 hrs
cultivation in AlCl3 alone or in combination with myrobalan (mean ±
Statistics and other symbols are same as detailed below Table 1.
4. Cytological Effects
No chromosomal aberrations and any type of abnormal mitosis could be seen in the root tip cells after any treatment with AlCl3, AlCl3 + myrobalan or in controls.
5. Morphology of Nucleoli (Table - 3)
Aluminium chloride exposure at 10-2M to 10-5M caused hypertrophy of nucleoli in all the nuclei of root tip cells but disorganisation as reported by Fiskesjo  could not be observed. Drug could maintain usual means i.e. control like nucleoli at 10-4M and 10-5M.
Table 3. Nucleolar morphology in the nuclei of Allium cepa root tip cells after 48 hr cultivation in AlCl3 or AlCl3+myrobalan.
NG = No growth (n = 100 - 200)
Earlier reports have shown both
i.e. aluminium induced declined in mitosis and chromosomal aberrations in
plants [10, 14] and
A perusal of results indicate that two issues emerge out which deserve discussion, first one is to understand probable mechanism of action of aluminium chloride in root tip cells for lowering mitosis and second one is for explaining probable protective role played by myrobalan against Al-toxicity.
Probable action of AlCl3 induced mitodepression
Aluminium chloride induced inhibition of root growth and low mitosis is not unexpected findings as several earlier similar reports do exist in the literature.
Aluminium chloride induced
progressive root growth inhibition from 10-5M to
In plants, aluminium causes
increased production of reactive oxygen species i.e.
Probable protective role of myrobalan against aluminium toxicity
a) Based on antioxidant property of myrobalan
The common feature of Al-toxicity
in plants and animals/human cells is increased production of reactive oxygen
Present results show that at lower
concentrations of AlCl3 (10-4M and 10-5M)
drug could significantly counteract Al-induced mitodepression
effect. This is possible only if myrobalan
possesses antioxidant properties and indeed myrobalan
has already been shown to exert such action. Fu etal
 reported antioxidant action of T. chebula and found preventive effects on
b) Based on probability of formation of an inert Al-drug complex
In Al-accumulators, Al is usually complexed with organic acids or other organic compounds to make it non toxic. The predominant Al form is Al-catechins in the leaves of tea plant , Al-citrate in Hudrangea leaves  and Al-oxalate in buckwheat  rendering the high total tissue concentrations non-phytotoxic to the cell cytosol. In addition, the cytosol is protected by Al-accumulating predominantly in the cell wall or vacuoles . Binding of Al in the cell wall is mainly to pectic substances as shown in Melastoma malabatrichum . In black tea infusions, 10-19% of total Al was present as cations species, whereas 28-33% was present as hydrolysable polyphenol complex . Myrobalan also possesses polyphenols which can bind with Al thereby reducing toxicity. Myrobalan possesses large number of components some of which can bind with Al making it inert. Further research is needed to pin point exact role played by myrobalan against Al-toxicity in Allium cepa root tip cells.
Authors thank HOD for providing
departmental facilities and to Dr. G. Fiskesjo of
Department of Genetics,
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