Antimutagenic effects of Tribulus terristris fruit extract on Micronucleus

Pushpa Prasad¹*, R.C. Agrawal², Amit Roy¹ and Nirmala Gupta²

¹Columbia Institute of Pharmacy, Tekari, Raipur (C.G.)-493111, India.

²Jawaharlal Nehru Cancer Hospital and Research Centre, Idgah Hills, Bhopal (MP), India

ABSTRACT:

In this study, the protective effect of hydromethanolic fruit extract of Tribulus terristris is reported against Cyclophosphamide (CP) induced micronuclei (MN) formation in bone marrow cells of mice. Swiss albino mice were treated with a single i.p. dose of 500, 1000 and 1500 mg/kg b.w. with Tribulus terristris fruit extract prior to CP administration. A significant inhibition was noticed in the micronuclei frequencies, as it 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.

KEYWORDS: Micronucleus, Mutagenic, Tribulusterristris, hydromethanolic.

INTRODUCTION:

Medicinal plants have created the foundation of health care system throughout the world since the initial stage of humanity and still plant products are the major source drug/formulation in treatment of various diseases. These Herbal drug and their phytoconstituents resembles safety and efficacy, they produces less/no side effect when compare to synthetic drugs. Thus, these natural products are the main stay of treatment of various disorders in human beings from ancient times (Sen et al, 2011, Lamba et al, 2011). Tribulus terrestris is a natural herb used for treating many diseases like hypertension. It is a member of the Zygophyllaceae family, and an annual herb found in many tropical and moderate areas of the world, including the U.S. and Mexico, the Mediterranean region, and throughout Asia (Hussain et al, 2009). Tribulus terrestris, is also known as Puncture Vine and it contains steroidal saponins which act as a natural testosterone enhancer (Gauthaman et al, 2003). It also possesses radiomodulatory activity against Gamma radiations and also useful in the treatment of Urolithiasis (Manish et al, 2009, Arasaratnam et al, 2010).

MATERIALS AND METHODS:

1. Preparation of Tribulus terristris fruit extract:

The fruit was 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 50-60⁰C and the powder of the drug were obtained.

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 tribulus terristris 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

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 (Polychromatic erythrocytes) and NCE (Normochromatic erythrocytes) cells were counted and number of micronucleated cells was also scored. Pink coloured cells are PCE and blue coloured are NCE.

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.

RESULTS AND DISCUSSION:

This study evaluated the protective effect of Tribulus terristris fruit extract against cyclophosphamide induced micronucleus in bone marrow cells of Swiss albino mice. The results showed that when CP was given at a single dose of 50 mg/kg. b.w., (Group. VI) cause a high incidence of micronucleus formation 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 4.4 ± 0.24, which was significantly higher (t-test, p < 0.05) when compared with the experimental groups. The MNPCE frequency of the group treated with Tribulus terristris alone at concentration of 500 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 Tribulus terristris fruit extract at doses 500, 1000, and 1500 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. Thus, from the result it was clear that Tribulus terristris fruit extract alone had not induced micronucleus formation, reiterating the fact that the extract has no pro-mutagenic components.

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). Tribulus terrestris possesses tonic properties, antibacterial, anti-inflammatory, smooth muscle relaxation and diuretic actions, which are useful in genitourinary infections, painful micturation, hematuria, dysuria, benign prostatic hyperplasia, urethritis and prostatitis (Tomova and Tibestan, 1987, Singh et al, 1991, Sivarajan et al, 1994 and Arora et al, 2003). It has been reported that Tribulus terrestris contains saponins, quercetin, kaempferol, rutin, furostanol, gigenin, hecogenin, ruscogenin, gitogenin, tigonenin, terrestrinins A(1) and B(2), which are known to have antioxidant and anticancer properties (Ross, 2001, Huang et al, 2003 and Combarieu et al, 2003). The result from the present study suggests that the bone marrow cells can be protected by CP induced toxicity by Tribulus terrestris fruit extract, which was evident by the reduction in micronuclei frequency.

Graph.1 Showing the protective effect of Tribulus terristris fruit extract (Group II, III, IV) in micronucleus formation induced by CP (Group VI).

Micronucleus

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

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

Group	Description	Treatment	MNPCE Mean±SE	PCE/NCE Mean±SE
I	Treatment group (Tribulus terristris alone)	500 mg/kg	0.5±0.09	0.86±0.04
II	Experimental group (Tribulus terristris +CP)	500+CP	2.0±0.31*	0.69±0.40
III		1000+CP	1.6±0.22*	0.48±0.04
IV		1500+CP	1.2±0.18*	0.38±0.06
V	Control	Negative (Solvent alone)	0.16±0.16	0.67±0.05
VI	Control	Positive (CP alone)	4.4±0.24	0.51±0.03

Data presented as the mean and standard error (SE) among mice (n = 6). (*) Denotes statistically significant value at p<0.05

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 09.06.2012

Modified on 15.06.2012

Accepted on 25.06.2012

Research J. Science and Tech. 4(3): May-June 2012: 140-142