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ISSN 2229 6840

Chemopreventive Potential of Methanol Extract of Stem Bark ofCassia Fistula L. In Mice

*Linu Mathew1, Shankar Shashidhar

2

1School of Biosciences, M.G. University, Kottayam, Kerala, INDIA

2Azeezia Medical College, Kollam, Kerala, INDIA

*Corresponding author: Linu Mathew Email address: [email protected]

IJPIs Journal of Pharmacognosy and Herbal Formulations

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ABSTRACT:

Ethnomedical survey has shown that stem bark ofCassia fistula. L is used to treat skin diseases by

tribal people of forest ranges of India. Therefore, bark extracts were tested for their chemopreventive

potential in two-stage murine skin chemical carcinogenesis model. The papilloma were produced on

mice skin by DMBA and promoted by croton oil. The methanol extract of stem bark ofCassia fistula L.

(MEC) was found to prevent the formation as well as persistence of papilloma in treated mice compared

to untreated control mice.

Keywords:Cassia fistula L., Chemical carcinogenesis, DMBA, croton oil, papilloma.

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Vol 2:8 (2012) IJPIS Journal of Pharmacognosy and Herbal Formulations

Linu Mathew et al Page 23

1. INTRODUCTION

Cassia fistula L. (Fa. Caesalpiniaceae) is a tropical evergreen tree with seasonal flowering and is a popular

avenue tree in Indian subcontinent. The common name of the plant is Indian Laburnum or golden shower. Most

parts of this tree have medicinal uses in the traditional Indian systems of medicine. The stem bark of the plant is

considered, an astringent and antihelminthic, in the Ayurveda system of medicine. The powdered bark of the plant is

used to treat several skin abnormalities by the mountain tribes of the states of West Bengal (Das and Chattopadhyay,2003) and Madhya Pradesh (Acharya and Rai, 2005). The bark of the plant is rich in flavonoids (Siddhuraju et al.,

2002). The chemical constituents of the stem bark are fully characterized (Murty et al., 1967). In the present study to

verify the ethnomedical claim, the methanolic extract of C.fistula L. (MEC) was tested for its chemopreventive

potential in mouse two stage skin chemical carcinogenesis model.

2. MATERIALS AND METHODS

2.1 Plant material:

Stem bark of C. fistula was collected from the premises of School of Biosciences, M G University, Kerala,

India and authenticated by Dr. V. T. Antony, Taxonomist, St. Berchmans College, Changanacherry, Kerala, India and

a Voucher specimen was deposited in the Regional herbarium, Botanical Survey of India (Specimen. No. 4589) in St.Berchmans College, Changanacherry, Kerala, India. The bark was dried in shade and 100g of powdered bark was

extracted with different organic solvents of increasing polarity in a soxlet apparatus. The methanol extract (MEC) was

found to possess maximum activity in pilot studies. The extract was dried with a rotary evaporator under reduced

pressure at 40-450c. The yield of the extract was 18%. The extract was dissolved in 0.1% DMSO.

2.2 Animals:

Balb/c male mice weighing 18-20g were procured from Small Animal Breeding Station, College of Veterinary

Sciences, Trichur, Kerala, India. The animals were acclimatized in the animal experimentation facility in School of

Biosciences. They were given standard commercial pellet diet (Lipton India Ltd), and water ad libitum and maintained

at 22-280C, relative humidity 60-70% and 12h darklight cycle. All the animal experiments were performed according

to the rules and regulations of the CPCSEA, Government of India.

2.3 Chemopreventive activity:

For the long-term carcinogenicity bioassay, random bred male Balb/C mice of 18-20g were selected. They

were kept in a group of ten animals per polypropylene cage under controlled environmental condition. During the

course of the experiment, hair was removed from the interscapular region over an area of 2cm3 using electric clippers

that were not lubricated with oil or grease. Animals were observed for 3 days and animals in the resting phase of hair

cycle were selected for the study. Mouse skin papilloma were induced in all groups by topical application of 7, 12,

dimethyl benz (a) anthracene (DMBA, Sigma chemical Co., St. Louis, USA) at the rate of 100 nmol in acetone in the

2nd week of experiment and promoted by 1% croton oil (Sigma chemical Co., St. Louis, USA) in acetone thrice weekly

until 15 weeks. The animals were divided into 3 groups comprising 10 animals.

Group1: Untreated control, receiving 0.1% DMSO topically thrice weekly (on two days interval)

Group2: Receiving MEC, weekly thrice @ 100mg/kg body wt by topical application on 1st and 3rd week of

experiment, and 0.1% DMSO for the rest of the duration of the experiment

Group3: Receiving MEC thrice weekly @ 100mg/kg body weight by topical application throughout the study period.

The shaven skin of the treated mice was observed daily for the eruption of papilloma, and body weight was

taken weekly. The time of first papilloma eruption (latency period), cumulative and average no. of papilloma, no. of

animals bearing papilloma on 15th week, total and average no. of papilloma per mouse at 8 th, 12th and 15th week were

observed.

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2.4 Statistical analysis:

The means were expressed as Mean SEM and the results were analyzed by one-way analysis of variance

(ANOVA). The means were compared against untreated control by Dunnetts test of multiple comparison.

3. RESULTS

Average and total number of papilloma appearing in different treated groups showed statistically significantdifference compared to untreated control, at all time periods of observation namely 8

th, 12

thand 15

thweeks. Untreated

control animals had maximum number of papilloma followed by groups G2 and G3. Similarly, the time of initiation of

first papilloma (latency period) was 8 weeks for G1 and G2, but for G3 it was 12 weeks. In the case of G1 and G2 by

the end of 15th week 80% of animals developed papilloma. Nevertheless, in the case of G3 only 30% animals

developed papilloma, by the end of the period of observation (15th

week) (Table 1).

Table 1: Effect of MEC on DMBA and croton oil induced two-stage murine skin carcinogenesis

Groups Treatment

Quantal

15 week

survival

Total and Average no. of papillomas

Latency

period

Animals

with

papilloma

on week

15

8th

week 12th

week 15th

week

Total Average Total Average Total Average

G1 Untreated control 9/10 8 0.90.07 27 2.70.6 31 3.10.62 8 week 80%

G2

Receiving MEC @

100mg 1kg body wt

thrice weekly

(promotional stage)

on 1st and 3rd week of

the experiment

10/10 0 0** 9 0.70.06* 8 0.80.09** 9 week 50%

G3

Receiving MEC @

100mg 1kg body wt

throughout the study

period thrice weekly

(Both pre initiational

and promotional

stage)

10/10 0 0** 2 0.20.01* 5 0.50.17`** 12 week 30%

**significantly different from G1 at P

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groups (Table 1). The ability of the MEC treatment to increase the latency period is suggestive of its ability to reduce

clonal expansion and thereby, tumour promotion.

Antioxidant principles of many plants are reported to possess antitumour activity (Ruby et al, 1995). All plant

parts ofC. fistula L. possess in vitro antioxidant activity, and among these maximum antioxidant activity was shown by

the methanol extract of stem bark (Siddhuraju et al, 2002). This property may be attributed to the abundant

polyphenolics, anthraquinones, xanthones, proanthocyanidines, and flavonols in the stem bark of the tree (Siddhuraju

et al, 2002). These compounds are well known for their antioxidant, antimutagenic and chemopreventive potential(Jovanovic et al, 1994, Aitken et al, 1994). The presence of these compounds in the MEC may be the reason for its

chemopreventive potential. However, further investigations are essential for the isolation of the active principle of

MEC and its mechanism of action.

7. REFERENCES

(1) Acharya, D., Rai, M.K., 2005.Traditional Knowledge for Curing Various Ailments among Gonds and Bhariasof Patalcot Valley, Madhya Pradesh, India. http://www.selfgrowth.com/articles/Acharya12.html/.

(2) Aitken, R. A., Bibby, M. C., Double, A., Phillips, R. M, Sharma, S. K., 1994. Synthesis and antitumouractivity of 6 methyl derivatives of flavone -8-acetic acid (FAA). Bioorganic and Medicinal Chemistry Letters

4, 2313-2316

(3) Berenblum, I., Shubik, P., 1949. An experimental study of the initiating stage of carcinogenesis, and a re-examination of the somatic cell mutation theory of cancer. British Journal of Cancer 3, 109 118.

(4) Cerrutti, P.A., 1985. Prooxidant States and Tumour Promotion. Science 227, 375381.(5) Copeland, E.S., 1983. Free radicals in Cancer. Cancer Research 43, 56315637.(6) Das, N., Chattopadhyay, R.N., 2003. Studies on Ethnomedical Plants of Purulia Dist, West Bengal, India.

Journal of Nontimber forest products 10(3/4), 218-229.

(7) Jovanovic, S. S. V., Steen Ken, S., Tosic, M., Marjanovic, B., Simic, M.G., 1994. Flavonoids asAntioxidants. Journal of the American Chemical Society 116, 4846-4851.

(8) Klaunig, E.J., Kamendulis, M.L., 2004. The Role of Oxidative Stress in Carcinogenesis. Annual Review ofPharmacology and Toxicology 44, 239267.

(9) Murty, V.K., Rao, T.V.P., Venketeswarlu, V., 1967. Chemical Examination of Cassia fistula. Tetrahedron 23,515518.

(10)Owens, D. M., Carolyn Wei, S. J., Smart, C. R., 1999. A multihit, multistage model of chemicalcarcinogenesis. Carcinogenesis 20(9), 1837-1844.

(11)Ruby, A.J., Kuttan, G., Babu, K.D., Rajasekaran, K.N., Kuttan, R., 1995. Antitumour and AntioxidantActivity of Natural Curcuminoids. Cancer Letters 94, 783789.

(12)Siddhuraju, P., Mohan, P.S., Becker, K., 2002. Studies on the antioxidant activity of Indian Laburnum (Cassiafistula L): a preliminary assessment of crude extracts from stem bark, leaves, flowers, and fruit pulp. Food

Chemistry 79, 61 -67.

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