Aegle marmelos Fruit Extract Prevents Cyclophosphamide Induced Micronucleus Formation in Mice

 

Nirmala Gupta¹*, R.C. Agrawal¹, Vinoy Shrivastava², Amit Roy³ and Pushpa Prasad³

¹Jawaharlal Nehru Cancer Hospital and Research Centre, Idgah hills, Bhopal (MP), India

²Dept. of Biosciences, Barkatullah University, Bhopal (MP), India

³Columbia Institute of Pharmacy, Raipur

*Corresponding Author E-mail: niru84_biotec@rediffmail.com

ABSTRACT:

The aim of the present study was to investigate the protective efficacy of hydromethanolic fruit extract of Aegle marmelos on Cyclophosphamide (CP) induced micronuclei (MN) formation in bone marrow cells of mice. Swiss albino mice were treated with a single i.p. dose of 450, 675 and 900 mg/kg b.w. with Aegle marmelos fruit extract prior to CP administration. The result demonstrated that the micronuclei frequencies gradually decreased with increase in concentration of the extract suggesting a protective effect and the extract alone had not induced MN formation, reiterating the fact that the extract has no pro-mutagenic components.

 

 

 

1. INTRODUCTION:

Now-a-days beneficial aspects of dietary constituents of plant origin have been extensively studied (Shukla et. al., 2002). Traditionally, several members of the family Rutaceae are being used for its various ethno medicinal properties (Siddique et. al., 2010). One of the members of this family, Aegle marmelos Correa commonly known as Bael has been largely used in indigenous medicine due to its various medicinal properties (Brijesh et. al., 2009). The pharmacological functions of Bael including antioxidant (Siddique et. al., 2010), antidiabetic (Sabu and Ramadasan, 2004) and hepatoprotective (Kalaivani et. al., 2009) effects have been described. Many reports have indicated that it prevents carcinogenesis and act as anticarcinogenic agent during promotion/progression stages of different cancer (Lampronti, 2003; Veras et. al., 2005). It also exhibits antigenotoxic effects against doxorubicin induced micronucleus (Venkatesh et. al., 2007). Thus, the present study was carried to investigate in vivo antimutagenic effect of hydromethanolic fruit extract of Aegle marmelos on CP induced micronuclei formation in mouse bone marrow cells.

 

2.       MATERIALS AND METHODS:

2.1.  Preparation of Aegle marmelos fruit extract: The fruit pulp was carefully taken out from the hard shell, shade dried and powdered. About 100 gm of plant material was kept in petroleum ether to defat the extract for 1 hour. The crude extract was defatted to remove the lipid present in plant material and it was then subjected to separating funnel extraction using 50% methanolic solvent by refluxing for 36 hrs. at 50-6ºC and the powder of the drug were obtained.

 

2.2.  Mice treatment: Both sexes of Swiss albino mice of 6-7 weeks old and weighing 15-20 gm were obtained from the animal colony of our Research Centre. Animals were housed in plastic cages and provided standard pellet diet and water ad libitum. The requisite dose of Aegle marmelos fruit extract was dissolved in ddw and administered as single i.p. dose of 0.2 ml/mouse 24 hr prior to the CP administration, to 6 animals. Control mice were injected with an equal volume of vehicle alone. The positive control group also received a single i.p. injection of 50mg/kg CP in 0.9% saline.

 

2.3.  Extraction of Bone marrow: The animals were killed 24 hr after the CP administration by cervical dislocation, and slides of bone marrow were prepared essentially as described by Schmid (1975) and modified by Aron et. al. (1989). After staining with May-Gruenwald and Giemsa, a total of 1000 cells were scored at a magnification of ×1000 (100×10x) for each animal. About 1000 PCE and NCE cells were counted and number of micronucleated cells was also scored.

 

2.4.  Statistical analysis: The data from the micronucleus assay were statistically analyzed by Student’s t-test, comparing the treated groups with positive control. The data was presented in MNPCE±SE and PCE/NCE±SE. The significance level considered was P < 0.05. The percentage of reduction in the frequency of CP-induced DNA damage was calculated according to Manoharan and Banerjee (1985), by the following formula:

% reduction= [(mean frequency of damage in A- mean frequency of damage in B)/ (mean frequency of damage in A- mean frequency of damage in C)] ×100

Where, A= positive control group treated with CP

B= group treated with extract

C= negative control group

 

3.      RESULTS:

The results revealed that CP when given at a single dose of 50 mg/kg. b.w., (Group. I) cause a high incidence of micronucleus induction in Swiss albino mice. The CP dose of 50 mg/kg caused bone marrow toxicity as evidenced by a decrease in the proportion of PCE/NCE ratio. For CP -treated groups the frequency of MNPCE was 4.5 ± 0.4, which was significantly higher (t-test, p < 0.05) when compared with the experimental groups. In the treatment group (AMF), the MNPCE frequency at concentration of 450 mg/kg b.w. was comparable to the positive control group suggesting that the extract is not genotoxic to the bone marrow cells of mice. For the experimental groups, pre-treated with the AMF extract at doses 450,675, and 900 mg/kg b.w, followed by CP administration the MN frequencies appeared to gradually decrease with increase in concentration of the extract suggesting a protective effect. The Aegle marmelos fruit extract alone group had not induced micronucleus formation, reiterating the fact that the extract has no pro-mutagenic components. On the other hand, the % of reduction in the frequency of CP-induced DNA damage was significantly increased in a dose dependent manner. AMF injected intraperitoneally at the concentration of 450, 675, 900 mg/kg, 24 hrs. prior to CP administration reduced the frequency of MN in 42%, 61%, 80%.

 

4.      DISCUSSION:

The present study evaluated the antimutagenicity of Aegle marmelos fruit extract on mice bone marrow cells. Intraperitoneal treatment was preferred over other because it maximizes the absorption and penetration of target cells (Preston et. al., 1981). Formation of micronuclei is an important cytogenetic endpoint that is routinely used in genotoxicity evaluation (Krishna et. al., 1991). Micronuclei are believed to be formed from chromosome fragments or entire chromosome lagging behind during cell division and thus, this assay can detect genotoxic compounds that cause chromosome breakage or other chromosomal aberrations (Krishna et. al., 1995). Considerable emphasis has been laid down on the use of dietary constituents preventing mutagen induced cytogenetic damage due to their relative non-toxic effects (Renner, 1990). The result of the present study clearly shows that extracts of hydromethanolic fruit extract of Aegle marmelos have a modulatory effect on CP induced micronuclei (Table-1). CP is a drug commonly used to treat cancer malignancies and as an immunosuppressive agent. But it itself is mutagenic and induces many side effects including alteration of male spermatozoa leading to sterility (Cevallos et. al., 2008).

 

Graph.4(a) Showing the protective effect of A. marmelos fruit extract (Group II, III, IV) in micronucleus formation induced by CP (Group VI).

 

Graph.4(b) Showing the ratio of PCE/NCE of different groups treated with A. marmelos fruit extract (Group II, III, IV) in comparison to CP (Group VI)

 

Photogragh.1. Showing Micronucleus (MN) in Polychromatic erythrocytes.

Table.1. Results showing the protective effect of AMF in micronucleus formation induced by CP.

Group	Description	Treatment	MNPCE Mean±SE	PCE/NCE Mean±SE	% reduction in the frequency of CP induced DNA damage
I	Treatment group (AMF alone)	450 mg/kg AMF	0.66±0.20	0.96±0.12	-
II	Experimental group (AMF+CP)	450+CP	2.6±0.31*	0.51±0.04	42%
III		675+CP	1.8±0.4*	0.71±0.08	61%
IV		900+CP	1.0±0.25*	0.98±0.08	80%
V	Control	Negative (Solvent alone)	0.16±0.16	0.67±0.05	-
VI		Positive (CP alone)	4.5±0.4	0.51±0.02	-

Data presented as the mean and standard error (SE) among mice (n = 6).

(*) Denotes statistically significant value at p<0.05

 

 

5. ACKNOWLEDGEMENT

The authors are thankful to research staff of Jawaharlal Nehru Cancer Hospital and Research Centre, Bhopal (M.P), India for their contribution in the piece of study.

 

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Received on 30.08.2011          Modified on 20.09.2011

Accepted on 03.10.2011         © RJPT All right reserved