Chemopreventive potential of Aegle marmelos fruit extract against 7,12-Dimethylbenz (a)anthracene-Induced Skin Papillomagenesis 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

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

3 Columbia Institute of Pharmacy, Raipur.

ABSTRACT:

The present investigation was undertaken to explore the chemopreventive action of Aegle marmelos fruit extract on 2-stage carcinogenesis, induced by a single topical application of 7, 12-Dimethylbenz(a)anthracene (DMBA) (104µg/100µl acetone), and one week later, promoted by repeated application of croton oil (1% in acetone/twice in a week) till the end of the experiment (16 weeks) in Swiss albino mice. Single topical application of Aegle marmelos fruit extract at a dose of 900 mg/kg b.w. along with DMBA+Croton oil was found to be effective in decreasing the cumulative number of papillomas, tumor incidence, tumor yield and tumor burden as compared to control group (p<0.05). The depleted levels of glutathione were also restored in Aegle marmelos fruit extract treated group. Thus, the present study revealed the chemopreventive role of Aegle marmelos fruit extracts against DMBA-induced skin carcinogenesis in mice

 

KEYWORDS: Chemopreventive, Aegle marmelos, DMBA, Papilloma, Skin carcinogenesis, Glutathione..

 

INTRODUCTION:

Aegle marmelos commonly known as Bael, belonging to the family Rutaceae has been widely used in indigenous systems of Indian medicine due to its various medicinal properties. This plant grows widely in some areas of the Southeast and South Asia countries such as India, Sri Lanka, Indonesia, Malaysia and Vietnam (Nugroho et. al., 2010; Brijesh et. al., 2009). Aegle marmelos Correa has several pharmacological activities such as anti inflammatory, antipyretic, analgesic (Arul et. al., 2005), antioxidant (Sabu et. al., 2004), and antidiabetes (Upadhya et. al., 2004). Moreover, several compounds of this plant have been isolated and evaluated for their pharmacological effects such as aegeline and skimmiarepin possessing hypoglycemic and insecticidal activities, respectively (Narender et. al., 2007; Samarasekera et. al., 2004). It is necessary to focus and develop these compounds to be effective drugs. All parts of this tree, viz. root, leaf, trunk, fruit and seed are useful in several ailments. In fact, as per Charaka (1500 B.C) no drug has been longer or better known or appreciated by the inhabitants of India than the Bael (Singanan et. al., 2007; Brijesh et. al., 2009). The roots are useful for treating diarrhoea, dysentery, and dyspepsia.

The leaf is used for opthalmia, diabetes, and asthmatic complaints. Unripe fruit is useful for treating diarrhea and dysentery (Arumugam et. al., 2008

There are several studies reported for its use in the treatment of several cell lines of cancer (Jagetia et. al., 2005; Lampronti et. al., 2003). Therefore, we have planned to carry out this study to see the chemopreventive effects in experimental animals.

MATERIALS AND METHODS:

Animals:

The study was conducted on random bred, male Swiss albino mice (6-7 weeks old: Body weight 25±2). These animals were housed in polypropylene cages in the animal house under controlled conditions of temperature (25±1oc) and light (12 light: 12 dark). All animal studies were conducted according to the guidelines of the Committee for the purpose of Control and Supervision of Experiments on Animals, Government of India. The animals were fed on standard mice feed procured from Golden feeds, New Delhi, and water ad libitum. The hairs on the dorsal skin of the animals in the interscapular area were shaved one day before the commencement of the experiment.

 

Chemicals:

The known skin initiator DMBA and Croton oil (used as promoter) were obtained from Sigma Chemicals Co. (St. Louis, USA). DMBA was prepared in acetone at a concentration of 104µg/100µl. Croton oil was diluted in acetone to give a solution of 1% dilution.

 

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 50-600c and the powder of the drug were obtained. The required dose for treatment was prepared by dissolving the pellets in double distilled water at a dose of 900 mg/kg body weight.

 

Experimental protocol:

Experiment was performed as per the method reported by Berenblum (1975) and standardized by Agrawal et. al. (2009). A total of 30 animals were randomized in to control and experimental groups, and divided in to 5 groups of 6 mice in each experimental group. One day before the commencement of experiment, hair on the interscapular region of the mice were clipped. Only the mice showing no hair growth were considered for the study. Body weights of the animals were recorded weekly.

 

Treatment groups:

Group-I: Vehicle Control- received topical application of acetone (100µl/mouse) on the shaven dorsal skin, 2 times/week up to 16 weeks.

 

Group-II: DMBA alone- a single dose of 104µg DMBA in 100µl of acetone was applied topically over the shaven area of the skin of the mice.

 

Group-III: Croton oil alone- 100µl of 1% croton oil in acetone was applied two times per week until the end of the experiment.

Group-IV: Extract alone- 100µl of AMF at the dose of 900 mg/kg was applied two times per week until the end of the experiment.

 

Group-V: Carcinogen control- applied topically with a single dose of DMBA (104µg/100µl of acetone) over the shaven area of the skin of the mice. Two weeks later, croton oil (1% in acetone) was applied two times per week up to 16 weeks.

 

Group-VI: AMF Experimental- applied topically with a single dose of DMBA over the shaven area of the skin of mice. Two weeks later, they were treated with A. marmelos fruit extract (900 mg/kg b.wt.) which was given one hour before each application of 1% croton oil 2 times/week up to 16 weeks.

 

Biochemical study:

Biochemical alterations were studied in all the groups at the time of termination of the experiment (i.e. 16th week). The hepatic level of glutathione (GSH) was determined by the method of Moron et. al. (1979). The GSH content in blood was measured spectrophotometrically using 5-5 dithiobis-2-nitrobenzoic acid (DTNB) as a coloring reagent, according to the method of Beutler et. al. (1963). GSH concentration was calculated on the basis of millimolar extinction coefficient of 13.6 and a molecular weight of 307.

 

Data Analysis: The differences in the incidence of tumors among different groups were considered to be significant at 5% significance level (p<0.05) when evaluated by student’s ‘t’ test.

 

RESULTS:

The findings of the present study with the skin tumor model showed that Single topical application of DMBA followed 2 weeks later, by repeated application of croton oil (twice a week), skin papilloma appeared in all animals which started appearing from 5th week onwards. The cumulative no. of Papilloma induced during the observation period was 40. The incidence in carcinogen control group reached 100% by the termination of experiment (i.e. 16 weeks). The average no. of Papilloma per mouse (tumor yield) as well as the Papilloma per Papilloma bearing mice (tumor burden) was found to be 6.6±1.4. Mice of the treatment group-VI, given a continous treatment of Aegle marmelos fruit extract topically, additionally at the dose of 900 mg/kg 2 days/week for 16 weeks, showed a significant reduction in the tumor incidence i.e. 66.6% as compared with the control group. The cumulative no. of Papilloma during the experimental period was found to be 11, which was significantly less than control group (p<0.05). The average no. of Papilloma per mouse (tumor yield) as well as the Papilloma per Papilloma bearing mice (tumor burden) was 2.7±0.6 and 1.8±0.6. Animals of Group-I (vehicle alone), Group-II (DMBA alone), Group-III (Croton oil), Group-IV (extract alone), showed no incidence of papilloma during the entire experimental period. The results are summarized in Table1.

 

TABLE.1. Chemopreventive Effect of Aegle marmelos Fruit extract against 7, 12-Dimethylbenz (a) anthracene (DMBA)/Croton Oil–Induced Skin Papilloma

Groups

Treatment

Body weight (Mean±SE)

Cumulative no. of Papilloma

Tumor Incidence

(%)

Tumor Burden

Tumor Yield

Average Latent Period

Initial

Final

I

Vehicle alone

26.7±1.6

30.3±1.9

0

0

0

0

0

II

DMBA alone

25.0±1.3

30.3±1.3

0

0

0

0

0

III

Croton oil alone

25.6±2.2

26.8±2.8

0

0

0

0

0

IV

AMF extract alone

27.0±1.5

30.9±1.1

0

0

0

0

0

V

DMBA+ Crotonoil

20.74±1.2

26.99±6

40

100

6.6±1.4

6.6±1.4

5.1±1.5

VI

DMBA + Croton oil + AMF

27.35±0.39

28.9±0.5

11

66.6

1.8±0.6*

2.7±0.6*

7.6±1.9

*Significance level among different groups at p<0.05

 

 

A significant fall in the level of glutathione reductase (GSH) was noticed in blood and liver of the animals which received DMBA+Croton oil as compared to Aegle marmelos fruit extracts experimental group, at the time of termination of the experiment (after 16 weeks). Treatment with Aegle marmelos fruit extracts resulted in an enhanced level of GSH (p<0.05) in such groups. The results are summarized in Table 2.

 

Table.2. Variation in the glutathione level during DMBA-induced skin carcinogenesis with/without Aegle marmelos fruit extract treatment

Treatment Group

Glutathione level

Blood (µg/ml)

Liver

(µmoles/gram)

Normal mice

5.1±.06

54.9±0.67

Carcinogencontrol (DMBA+CO

2.27±0.09

23.2±1.3

DMBA+Aegle marmelos fruit extract (900 mg/kg)+CO

3.7±0.05

24.0±2.1

*Significance level among different groups at p<0.05

 

DISCUSSION:

The Skin carcinogenesis model in experimental animals has been found useful for investigating the influence of dietary chemopreventors both mechanistically and operationally (Morse, 1985). The present study demonstrates that topical administration of Aegle marmelos fruit extract on DMBA induced skin tumorigenesis showed a significant reduction in tumor incidence, tumor burden, tumor size and cumulative no. of papillomas with a significant increase in the average latent period of tumor appearance and GSH levels. Literature suggests that one subminimal dose of carcinogen “initiates” tumorigenesis and the treatment with Croton oil “promotes” it to the visible tumor stage (Berenblum and Shubik, 1947). Our findings revealed the same with 100% tumor incidence in the control group i.e. carcinogen control. It was reported that application of promoter to the mice skin results in the rapid accumulation of inflammatory cells such as neutrophils and macrophages and an increase in the release of active oxygen species (Copeland, 1983; Cerrutti, 1985; Lewis and Adams, 1987).

 

Evidence also suggests that tumor promotion may be due to free radicals (reactive oxygen species), which play an important role in tumor initiation by enhancing or facilitating the metabolic activation and/or initiating the effects of carcinogens (Ather, 2002) and promotion of multistage carcinogenesis. Various studies exhibit the chemopreventive actions of plants such as Solanum lycopersicum fruit extract (Agrawal et. al., 2009); Aloe vera (Chaudhary et. al., 2007) and Acacia nilotica (Meena et. al., 2006) which have been tested in mammalian system and were found to be quite effective. 

 

The glutathione activity was noticed in the skin papilloma model. GSH level was measured in blood and liver of animals. Treatment with Aegle marmelos fruit extracts resulted in an enhanced level of GSH (p<0.05). During the multistage carcinogenesis process, the activity of antioxidant enzymes tend to decrease which leads to a pro-oxidant state of the cell, facilitating tumor promotion and propagation (Oberley, 1993). In the present study, it was noticed that the treatment with the initiator and promoter alone group i.e. DMBA + Croton oil lowered the glutathione (GSH) in blood and liver, while treatment with Aegle marmelos fruit extracts resulted in an elevated level of GSH in blood and liver samples. One of the mechanisms of Aegle marmelos protection against carcinogen can be due to elevation in glutathione level that is mediated through the modulation of cellular antioxidant level. GSH is a major antioxidant that is found in large amount in all cells and it has many functions in metabolism, free radical scavenging and regeneration of other antioxidants such as Vitamin E. The most important function of GSH is to provide protection against oxidative damage induced by reactive oxygen species, many of which are generated during normal cellular metabolic processes (Bystrianyk, 2005).

 

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Received on 25.08.2011

Modified on 01.09.2011

Accepted on 08.09.2011                                               

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Research J. Pharmacology and Pharmacodynamics. 4(2): March - April, 2012, 87-90