1735-2657/05/42-100-104

IRANIAN JOURNAL OF PHARMACOLOGY & THERAPEUTICS

Copyright © 2005 by Razi Institute for Drug Research (RIDR)
IJPT 4:100-104, 2005

Prevention of Acetaminophen-Induced
Mitodepression with Myrobalan (Fruit of Terminalia
chebula) in Allium cepa Model

HEMANT SINGH RATHORE and POOJA CHOUBEY

Cell Biology Unit, School of Studies in Zoology, Vikram University, Ujjain, India.
Received April 24, 2005; Revised July 28, 2005; Accepted July 30, 2005

This paper is available online at http://ijpt.iums.ac.ir

D
ABSTRACT
Allium cepa bulbs were grown in pure tap water (group I), in seven concentrations of acetaminophen
(7.81, 15.62, 31.25, 62.50, 125, 250, 500, and 1000 ppm) in the presence (group II) and absence of my-

SI
robalan (fruit of Terminalia chebula) at a fix concentration of 0.10 mg/mL. Parameters of study were mean
root length, mitotic index, abnormal mitosis and chromosomal aberrations and morphology of root. Aceta-
minophen at all concentrations except 1000 ppm where roots did not grow at all, significantly inhibited
root growth and declined mitotic index, effect appeared concentration dependent (group II). In the pres-
ence of myrobalan (group III) acetaminophen-induced mitodepression could be checked significantly. No
morphological i.e. shape and color changes, abnormal mitosis and any type of chromosomal aberrations
of
could be detected in any group. Probable protective role of myrobalan is discussed.

Keywords: Allium cepa, Mitodepression, Acetaminophen, Myrobalan, Terminalia chebula

ive

Acetaminophen (paracetamol) has earned a promi- Test Herbal Drug

nent place as a common house hold analgesic which is Myrobalan, dried young nuts of Terminalia chebula
available without prescription in several parts of the
ch

were procured from local herbal medicine shop, and

world [1]. Genotoxicity of acetaminophen is also on were gently backed for few minutes and cooled. Swol-
record and it is suggested that use of paracetamol may len nuts were ground to a fine powder. A recent study
contribute to an increase in the total burden of genotoxic (Sharma, 2003) revealed lack of mitostatic effect of
damage in man [2]. Ayurved (ancient Indian system of myrobalan on mitosis in Allium test at 0.10 mg/mL [6];
Ar

therefore this concentration was selected for the present

herbal medicine) recommends life time use of myro-
study.
balan alone or in Trifla (meaning three fruits: T. che-
bula, T. belirica, Emblica officinalis) to maintain gen- Test Chemical
eral good health of human beings because it is safe and Acetaminophen (paracetamol) under trade name
possesses antioxidant and antimutagenic properties [3- 'medimol' dispersible 500 mg B.P. tablets made by
5]. Myrobalan alone does not exert any ill effects in synchem laboratories, Baroda (Gujrat) was used. Each
Allium test [6]; however, acetaminophen does so [7-8]. tablets was dissolved in known amount of luke warm
Aim of this study was to find out whether myrobalan tap water to prepare suspensions of known concentra-
tions.
could reduce, nullify or intensify cytotoxic effect of
acetaminophen in Allium root tip cells when adminis- Experimental Design
tered simultaneously. Experiments were planned as per standard protocol
for Allium test [9]. Onions were descaled and placed on,
twelve test tubes filled with pure tap water, (Group I,
MATERIALS AND METHODS
controls). Second series of 12 test tubes bearing onions
Allium cepa were filled with suspension of each concentration of
acetaminophen (Group 2, acetaminophen exposed).
Dry healthy onion bulbs 1.5 to 2.0 cm in diameter Third series of test tubes bearing onions were also filled
were obtained from local market. with different concentrations of acetaminophen suspen-

100-104 | IJPT | July 2005 | vol. 4 | no. 2

www.SID.ir
Prevention of Acetaminophen-Induced Mitodepression with Myrobalan ijpt.iums.ac.ir | 101

Table 1. Mean root length (MRL) of Allium cepa when exposed to acetaminophen alone or in combination with myrobalan (mean±SEM, n = 10)
Group of ONION bulbs
S.
Concentration GR II acetaminophen Gr. III acetaminophen % Inhibition Gr % Inhibition Gr Difference Gr II
No. Gr I Control
experimental + myrobalan I vs. Gr II I vs. Gr III vs. Gr III
1 Control 0.0 56.91±0.65
2 15.62 ppm 51.08±0.59a 54.69±0.21bc 10.40% 3% 7.40%
3 31.25 ppm 48.81±0.27a 51.22±0.44bc 14.30% 9.98% 4.32%
4 62.50 ppm 44.11±1.71a 49.08±0.37bc 22.91% 13.58% 9.33%
5 125 ppm 37.98±0.50a 42.36±1.42bc 33.63% 25.66% 7.97%
6 250 ppm 32.83±1.00a 38.02±1.02bc 42.12% 33.92% 8.20%
7 500 ppm 27.51±0.66a 32.65±0.89bc 51.60% 42.62% 8.98%
8 1000 ppm - - - - -
Statically significant based on 't' test at 5% level of significance. MRL = in mm.
a
Control vs. Gr I (acetaminophen exposed)
b
Control vs. Gr II (acetaminophen + myrabalan exposed)
c
Gr II vs. Gr III
(-) No root growth at all

sion but each contained myrobalan powder at 0.10 ploidy etc.

D
mg/mL level (Group III, acetaminophen plus myrobalan
Statistics
exposed). All solutions were changed every 24 hrs. Af-
ter 48 hr two onions out of twelve in each series with Experiments were repeated five times. Students’t’
most poorly growing roots were removed. Same day i.e. test was applied at 5% level of significance.
after 48 hours distal 2 mm of five roots were cut off
from five individual bulbs from each series, and fixed in
acetic acid - ethyl alcohol (1:3 v/v) for chromosomal
study. Every time fixation was done at a fix time 11:00
a.m.
SI Mean Root Length
RESULTS

All tested concentrations of acetaminophen (except

of
After 72 hrs total lengh of the 05 root bundles in at 1000 ppm where roots did not grow at all) caused
each series of each onion was measured to record mean significant inhibition in the growth of roots (Gr. II) in
root length. comparison to control (Gr. I). A comparison between
Squashing and Observations of Slides Gr. II and Gr. III (acetaminophen + myrobalan) revealed
that myrobalan could check acetaminophen induced root
ive

Root tips were squashed using N-HCl and 2% aceto- growth inhibition at all concentrations (Table 1).
carmine stain. Four fields from each slide were ob-
Morphology, Color and Shape of Root Tips
served to cover 50 cells in each i.e. total 200 cells per
slide, 3000-4000 cells were observed for each group of Morphology i.e. color and shape of Allium cepa root
onion. Mitotic index was calculated as percentage of tips cultivated in all test concentrations of acetamino-
dividing cells. Slides were also observed to find out phen alone (Gr. II) or acetaminophen plus myrobalan
ch

mitotic arrest, chromosomal aberrations, fragments, (Gr. III) did not reveal any change from controls (Gr. I)
abnormal orientation, lagging chromosomes and poly- (Table 2).

Table 2. Morphology of Allium cepa root tips following 72 hrs exposure to acetaminophen alone or in combination with myrobalan.
Ar

Morphology i.e. shapes of root tips Color of root tips

S. Abnormal Normal Normal Abnormal
Groups
No. Crochet Broken Dark brown /
Bulb Straight White Pale
hooks tips black
Gr - I control No No No Yes Yes No No
Gr. II Only acetaminophen solution
1 15.62 ppm No No No Yes Yes No No
2 31.25ppm No No No Yes Yes No No
3 62.50 ppm No No No Yes Yes No No
4 125 ppm No No No Yes Yes No No
5 250 ppm No No No Yes Yes No No
6 500 ppm No No No Yes Yes No No
7 1000 ppm - - - - - - -
Gr III Exp acetaminophen + myrobalan
0.1 mg/mL
1 15.62 ppm No No No Yes Yes No No
2 31.25 ppm No No No Yes Yes No No
3 62.50 ppm No No No Yes Yes No No
4 125 ppm No No No Yes Yes No No
5 250 ppm No No No Yes Yes No No
6 500 ppm No No No Yes Yes No No
7 1000 ppm - - - - - - -
(-) No growth at all

www.SID.ir
102 | IJPT | July 2005 | vol. 4 | no. 2 Rathore et al.

Table 3. Mitotic index (MI) of Allium cepa root tip cells when exposed to acetaminophen alone or in combination with myrobalan (mean±SEM,
n=10)
Group of ONION bulbs
S.
Concentration Gr I GR II acetaminophen Gr. III acetamino- % Inhibition Gr % Inhibition Gr DifferenceGr II
No.
Control experimental phen + myrobalan I vs. Gr II I vs. Gr III vs. Gr III
1 Control 0.0 46.17±1.29
2 15.62 ppm 39.64±1.25a 44.12 ± 1.52bc 14.12% 4.44% 9.68%
3 31.25 ppm 30.31±0.62a 38.19 ± 1.40bc 34.35% 17.28% 17.07%
4 62.50 ppm 24.81±0.60a 32.95 ± 0.66bc 46.26% 28.63% 17.63%
5 125 ppm 20.22±0.80a 29.70 ± 0.70bc 56.20% 35.67% 20.53%
6 250 ppm 17.38±0.67a 24.67 ± 0.54bc 62.35% 46.56% 15.79%
7 500 ppm 12.61±0.54a 19.25 ± 0.71bc 72.68% 58.30% 14.38%
8 1000 ppm - - - - -
Statically significant based on 't' test at 5% level of significance.
a
Control vs. Gr I (acetaminophen exposed)
b
Control vs. Gr II (acetaminophen + myrabalan exposed)
c
Gr II vs. Gr III
(-) No root growth at all

Mitotic Index turbing effect and chromosomal effects of acetamino-

D
In comparison to controls (Gr. I), all test concentra- phen (paracetamol) in animal, human and plant cells
[10-19]. Results of the present study reveal cytotoxicity
tions of acetaminophen (Gr. II) significantly lowered
mitotic index i.e. percentage of dividing cells. This mi- i.e. mitodepression mitostatic (low mitotic index to no
todepression effect appears dose dependent. Even in the
presence of myrobalan (Gr. III) acetaminophen exterted
its inhibitory action on mitosis but effect was found
significantly less pronounced indicating preventive ac-
tion of myrobalan against acetaminophen (Table 3).
SI
cell growth at all) but no chromosomal effect. This de-
scripency might be due to use of comparatively lower
concentrations in the present study of acetaminophen
than those used by other workers.
A careful persual of results indicate two findings for
discussion, firstly probable mechanism of action of
of
Abnormal Mitosis acetaminophen in root tip cells for lowering mitosis and
No chromosomal aberrations and abnormal mitosis secondly probable protective action of myrobalan.
could be seen in root tip cells of onions in any groups Acetaminophen can bind irreversibly to DNA, can
(Table 4). cause DNA break, can inhibit both replicative DNA
synthesis and repair DNA synthesis [1]. It can block
ive

DNA replication by inhibiting deoxyribonucleotide (n

DISCUSSION
NTP) synthesis in several mammalian cell types [18].
Earlier workers observed cytotoxicity, spindle dis- Also, acetaminophan is topoisomerase II poison [20]. It

Table 4. Cytological effects of different concentrations of acetaminophen alone or in combination with myrobalan suspension in tap water.
ch

Microscopic effects in percent

Vagrant (lagging) chro-

Number
Multipolar Anaphases
Sticky chromosomes
Normal Metaphase

Normal Anaphase

of
Polykaryotes

counted
Fragments
C-mitosis

S.
mosome

Bridges

Groups Treatments cells

MNC

No.
Ar

Metaphase
+
Anaphase

1 Gr. I Control (tap water) 1000 + + - - - - - - - -

15.75 ppm Aceta.
Gr. II 1000 + + - - - - - - - -
2 15.75 ppm Aceta.
Gr. III 1000 + + - - - - - - - -
+ myrobalan
31.25 ppm Aceta.
Gr. II 1000 + + - - - - - - - -
3 31.25 ppm Aceta.
Gr. III 1000 + + - - - - - - - -
+ myrobalan
62.50 ppm Aceta.
Gr. II 1000 + + - - - - - - - -
4 62.50 ppm Aceta. +
Gr. III 1000 + + - - - - - - - -
myrobalan
125 ppm Aceta.
Gr. II 1000 + + - - - - - - - -
5 125 ppm Aceta. +
Gr. III 1000 + + - - - - - - - -
myrobalan
250 ppm Aceta.
Gr. II 1000 + + - - - - - - - -
6 250 ppm Aceta. +
Gr. III 1000 + + - - - - - - - -
myrobalan
500 ppm Aceta.
Gr. II 1000 + + - - - - - - - -
7 500 ppm Aceta. +
Gr. III 1000 + + - - - - - - - -
myrobalan
+ = Present, - = Absent, Aceta. = Acetaminophen, MNC = Micronucleate cells

www.SID.ir
Prevention of Acetaminophen-Induced Mitodepression with Myrobalan ijpt.iums.ac.ir | 103

can increase membrane permeability, can deplete cellu- Genetics, University of Lund, Sweden for providing
lar glutathione level and can cause lipid peroxidation literature.
and can bind with proteins [10, 21]. During late G1 re-
striction point gate opens in the presence of complex REFERENCES
molecules at promotors of essential cell cycle genes and 1. Insel Paul A. The pharmacological basis of Therapeutics, 9th
unrepaired DNA does not allow cells to go beyond G1 Edition, New York: McGraw-Hill; 1996. p. 617.
stage [22]. It appears that acetaminophen might have 2. Rannug U, Holme JA, Hongslo JK et al. International com-
interfered in some complex manner at DNA and/or gene mision for protection against environmental mutagens and car-
cinogens. An evaluation of the genetic toxicity of paracetamol.
level resulting in mitodepression- mitostatic condition in Mutat Res 1995;327:179-200.
Allium root tip cells.
3. Bramhaverchasva. Jadi butyon dwara swasthya sanrakshan. 9th
Cytotoxicity of N-acetyl-p-benzoquinone imine Ed., Yug Nirmana Yojana Gayatri Tapobhumi, Mathura; 1997.
(NAPQI) a common intermediate of paracetamol in p. 73-80.
cultured rat hepatocytes could be prevented fully by the 4. Naik GH, Priyadarsini KI, Naik, DB, et al. Studies on the aque-
addition of N-acetylcysteine, GSH or ascorbate during ous extracts of Terminalia chebula as a potent antioxidant and a
the exposure period [10]. Cytotoxicity of acetamino- probable radioprotector. Phytomedicine 2004;11:530-8.
phen was prevented by curcumin, a natural constituent 5. Kaur S, Arora S, Kaur K, et al. The in vitro antimutagenic activ-
ity of triphala-an Indian herbal drug. Food Chem toxicol
of Curcuma longa in rat hepatocytes [23]. Curcumin 2002;40:527-34.

D
protected against paracetamol induced lipid peroxida-
6. Sharma, Anjali. Cytological effects of myrobalan (fruit of T.
tion, increased cellular GSH and lowered LDH leakage. chebula) in Allium test. M. Phil dissertation in Zoology. Vikram
Crude extract of seaweed, Sargussum polycysturn University, Ujjain India, 2003.
(brown alga) could afford protection against acetamino- 7. Fiskesjo and Levan A. Evaluation of the first ten MEIC chemi-
phan induced lipid peroxidation through their free radi-
cal scavenging property [24]. It is likely that aceta-
monophen induced peroxidative damage could result in
the decline in mitosis but if myrobalan possesses anti-
oxidant properties it can reduce toxic effects of aceta-
SI
8.

9.
cals in the Allium test. ATLA 1993; 21: 139-49.
Musinovic-Aganovic M, Todic, Becic F, et al. Genotoxic
evaluation of paracetamol applying Allium test. Med Arch
2004;58:206-9.
Fiskesjo, Geirid. Allium test. In: Methods in Molecular Biology,
Vol. 43: In vitro toxicity testing protocols. Eds, S.O.' Hare and
of
minophen. Infact myrobalan has been shown to exert Atterwill, C.K., Humana Press Inc. Totowa, N.J. USA; 1995. p.
antioxidant and free radical scavenging activities [4, 25- 119-27.
26]. 10. Holme JA, Dahlin DC, Nelson SD, et al. Cytotoxic effect of N-
Unrepaired DNA does not allow cells to go beyond acetyl-P-benzoquinone imine, a common arylating intermediate
G1 stage [22] and acetaminophen may damage DNA of paracetamol and N-hydroxyparacetamol. Biochem Pharmacol
1984;33:401-6.
ive

[2]. It is likely that acetaminophen induced DNA dam-

11. Holme JA, Soderlund E. Species differences in cytotoxic and
age could have been remedied by myrobalan because it genotoxic effects of phenocetin and paracetamol in primary
is known to exert antimutagenic activity in bacteria monolayer cultures of hepatocytes. Mutat Res 1986;164:167-75.
against direct acting mutagens sodium azide and 4- 12. Holme JA, Hongslo JK, Bjornstad C, et al. Toxic effects of
nitro-o-phenylene diamine, [27] later this property was paracetamol and related structures in V79 Chinese hamster cells.
attributed to tannins [28]. Mutagenesis 1988;3:51-6.
ch

The Allium root cells also posses certain enzymes, 13. Hongslo JK, Christensen T, Brunborg G, et al. Genotoxic effects
the mixed function oxidases like that of mammalian of paracetamol in V79 chinese hamster cells. Mutat Res
1988;204:333-41.
hepatocytes that can activate promutagens to mutagens
14. Topinka J, Sram RJ, Sirinjan G, et al. Mutagenecity studies on
[9]. In case of metal toxicity detoxification in root cells paracetamol in human volunteers II, unscheduled DNA synthesis
takes place in the cytoplasm and cell wall within 12-24
Ar

and micronucleus test. Mutat Res 1989;227:147-52.

hr. which was held responsible for the mitotic activity at 15. Kocisova J, Sram RJ. Mutagenicity studies on paracetamol in
low concentration exposures [29]. Similar action to- human volunteers, III, cytokinesis block micronucleus method.
wards acetaminophen at low concentrations in the pre- Mutat Res 1990;244:27-30.
sent case can not be ruled out. 16. Giri AK, Sivam SS, Khan KA. Sister-chromatis exchange and
chromosome aberrations induced by paracetamol in vivo in
Individual plant components like sulfhydryl and fla-
bone-marrow cells of mice. Mutat Res 1992;278:253-8.
vonoid compounds, gallic acid, ellagic acid, mucic acid,
17. Kirkland DJ, Dresp JN, Marshall RR, et al. Normal chromoso-
citric acid, reducing sugars and tannins can modulate mal aberration frequencies in peripheral lymphocytes of healthy
effects of many genotoxicants [30], myrobalan pos- human volunteers exposed to a maximum daily dose of
sesses many of such compounds [31], which can be held paracetamol in a double blind trial. Mutat Res 1992;279:181-94.
responsible for reducing cytotoxic effects of acetamino- 18. Brunborg G, Home JA, Hongslo JK. Inhibitory effects of
phen in Allium cepa root tip cells in the present case. paracetamol on DNA repair in mammalian cells. Mutat Res
1995;342:157-70.
19. Jansen KG, Poulsen HE, Doehmer J, et al. Paracetamol-induced
ACKNOWLEDGEMENTS spindle disturbances in V79 cells with and without expression of
human CYP1A2. Pharmacol Toxicol 1996;78:224-8.
Head of department gave departmental facilities. We 20. Bender RP, Lindsey RH Jr, Burden, DA, Osheroff N. N-acetyl-
thank Prof. Dr. Bernad Mechler, Department of Dev- P-benzoquinone imine, the toxic metabolite of acetaminophan, is
lopmental Genetics, Deutsches Krebsforschungzentrum, a topo isomerase II poison. Biochemistry 2004;30:3731-9.
Heidelberg, Germany and Dr. G. Fiskesjo, Institute of 21. Holme JA, Wirth PJ, Dybking E, et al. Cytotoxic effects on N-

www.SID.ir
104 | IJPT | July 2005 | vol. 4 | no. 2 Rathore et al.

hydroxy-paracetamol in suspensions of isolated rat hepatocytes. 27. Grover IS, Saroj B. Antimutagenic activity of Terminalia che-
Acta Pharmacol Toxicol (Copenh) 1982;51:87-95. bula (myrobalan) in Salmonella typhimurium. Indian J Exp Biol
22. Pollard DT, Williams, CF. The G1 phase and the regulation of 1992;30:339-41.
cell proliferation In-Cell Biology, Saunders, USA; 2002. p. 687- 28. Grover TS, Simran K. Antimitogenecity of hydrolyzable tannins
76. from Terminalia chebula in Salmonella typhimurium. Mutat Res
23. Donatus IA, Sarajoko, Vermeulen NP. Cytotoxic and cytopro- 1998;41:169-79.
tective activities of curcumin. Effects on paracetamol induced 29. Cummings JR, Taylor GJ. Mechanism of metal tolerance in
cytotoxicity, lipid peroxidation and glutathione depletion in rat Plants: Physiological adaptations for exclusion of metal ions
hepatocytes. Biochem Pharmacol 1990;39:1869-75. from the cytoplasm In: Eds R.G. Alscher, J.R. Cummings, Stress
responses in plants: adaptation and acclimation mechanism. New
24. Raghvendra HR, Sathivel A, Devki T. Protective effect of Sar-
York: Wiley-Liss; 1990. p. 329-59.
gassum polycystum (brown alga) against acetaminophan-induced
lipid peroxidation in rats. Phytother Res 2005;19:113-5. 30. Sarkar D, Sharma A. Plants extracts as modulators of genotoxic
effects. Bot Rev 1996;6:275-300.
25. Naiwu Fu, Lanping Q, Huang L, et al. Antioxidant activity of
extracts of Terminalia chebula and its preventive effect on DNA 31. The Wealth of India, Raw Materials Vol. X: S-W. New Delhi:
breakage in human white cells induced by TPA. Chinese Trad PID-CSIR; 1960; p. 171-7.
Herbal Med 1992;23:26-9.
Address correspondence to: Dr. H.S. Rathore, School of
26. Cheng Huae Yew, Lin Tachen, Yu Kuohua, et al. Antioxidant Studies in Zoology, Vikram University, Ujjain, India.
and free, radical, scavenging activities of Terminalia chebula. E-mail: hemant2005hemant@sancharnet.in
Biol Pharm Bull 2003;26:1331-5.

D
SI
of
ive
ch
Ar

www.SID.ir