Antimutagenic Activity of Terminalia chebula Fruit Extract

D. Benito Johnson*, Sai Kishore P., M. Adarsh Verma and Tamil Selvan. A.

R.V.S. College of Pharmaceutical Sciences, Coimbatore, Tamil Nadu, India.

ABSTRACT:

Conventional drugs available for the treatment of cancer exhibit cytotoxicity as well as mutations in normal healthy body cells. The fruit extract of Terminalia chebula has been shown to have anticarcinogenic activity along with antimicrobial activities etc. To obtain experimental evidence on the therapeutic efficacy of the fruit extract, we examined its effect on chromosomal aberration (CA) and micronucleus (MN) formation in C57BL hybrid mice, to assess the anti mutagenic activity. In MN formation test, single application of Terminalia chebula methanolic fruit extract at different doses of 50, 100 and 150 mg/kg dry weight 24 hours prior to i.p administration of cyclophosphamide (CP) at the dose of 50mg/kg significantly reduced the frequency of MNCPE and at the same time significantly increased PCE/NCE ratio compared to CP alone. Concerning CA test, fruit extract at all different doses significantly reduced the % CA and at the same time increased the % degree of protection in dose dependent manner in bone marrow cells of mice as compared to CP alone treated group. However Terminalia chebula fruit extract alone did not show any chromosomal aberration and/or micronucleus formation. These results therefore, indicate the antimutagenic activity of fruit extract. The anti mutagenic activity observed in this study can be attributed to the presence of flavonoids and polyphenols. Thus it could be a better choice to treat cancer without inducing mutations in healthy body cells.

KEYWORDS: Terminalia chebula, Chromosomal aberration, Micronucleus formation

INTRODUCTION:

The mainstays of cancer treatment are radiotherapyand chemotherapy, both of which are genotoxic and mutagenic.The survivors of therapy can experience the late adverse effects,including the development of secondary, treatment-related tumors¹. Another major adverse effect of radiotherapy and chemotherapyis mutagenesis, which can cause heritable genetic damage anddiseases in the offspring of survivors. It should be stressedthat the majority of chemotherapy treatments compromise normalsexual function and often lead to prolonged infertility².The long-term genetic effects of parental exposure to anticancerdrugs therefore represent a major concern to genetic toxicology.

Cyclophosphamide (CP) is an alkylating agent and is used in chemotherapy to treat cancer of ovaries, breast, blood, lymph system and nerves, retinoblastoma, multiple myeloma and mycosis fungoides³.

CP has been tested extensively for genetic effects in a wide variety of tests in-vivo and in-vitro. In drosophila it induced aneuploidy, heritable translocations and somatic and sex linked recessive mutations. In fungi, CP induced aneuploidy, recombination, gene conversion and DNA damage and induced dominent lethal mutations, Chromosomal Aberration (CA), micronuclei formation (MNF), Sister Chromatid Exchanges (SCE), mutation and DNA damage in rodent’s invivo⁴. BL sowjanya et al described that CA test using somatic cells of mice is one of the sensitive methods to predict environmental mutagens and/or carcinogens⁵. The micronucleus test invivo is based on the principle that in anaphase acentric chromatid and chromosome fragments lag behind when the centric elements move towards the spindle poles. After telophase the undamaged chromosome as well as the centric fragments, give rise to regular daughter nucleus. The lagging elements are included in the daughter cells too, but a considerable proportion is transformed into one or several secondary nuclei which are much smaller than the principal nucleus and therefore called micronuclei.

Terminalia chebula is a plant species belonging to the genus Terminalia, family Combretaceae. It is a flowering evergreen tree called as “The Black Myrobalan” in English. Terminalia chebula is rich in tannin⁶. Besides this, it contains fructose, amino acids, succinic acid, betasitosterol, resin and purgative principle of anthroquinone and also possesses sennoside nature. Flavonol glycosides, triterpenoids, coumarin conjugated with gallic acids called chebulin as well as other phenolic compounds were also isolated. The fruit of this tree has been used as traditional medicine for household remedy against various human ailments, since antiquity. Terminalia chebula has been extensively used in Ayurveda, Unani and Homoeopathic medicine and has become a cynosure of modern medicine⁷. In this study by using CA and MNF method, we have analyzed the anti mutagenic activity exerted by the Terminalia chebula fruit extract against the CP induced mutations in the Swiss Albino mice.

MATERIALS AND METHODS:

Plant Material:

The Terminalia chebula fruits were collected from the local garden and dried for few days under shade. Then it was made into coarse powder with the help of grinder. 50 g of powdered material was extensively extracted with 50% methanol at room temperature. Resulting solution was filtered and concentrated at 40˚C under reduced pressure. Extract was weighed which was used for experiment.

Chemicals:

Cyclophosphamide (Sigma - CAS: 50-18-0) was purchased and diluted in distilled water and used as positive control and as damage inducing agent in the antimutagenicity tests at dose of 50 mg / kg administered via i.p, Giemsa was purchased from (Sigma, St Louis, MO), Fetal Calf Serum (Laborclin), Dibasic sodium phosphate (Na₂HPO₄12H₂O) (Merck). All other chemicals and media used were of analytical grade and purchased locally.

Acute toxicity test:

The Terminalia chebula fruit extract was screened for acute toxicity, following the standard method (OECD/OCDE). C57BL mice of either sex weighing 25g were divided into 7 groups and each consisting of 4 mice. Animals were maintained on normal diet and water prior to and during the course of experiment. The fruit extract was administered by i.p route. The acute toxicity studies were tested at the doses of 100, 200, 300, 400, 500, 1000 and 2000 mg/kg. The number of survivors in each treatment were observed in times of 5, 15, 30 minutes, 1, 2, 4, 24, 48, and 72 hours. At the end of this period, the number of survivors was counted. The LD₅₀valuewas estimated by the graphic method i.e. plotted a graph taking log dose on X-axis and Probit value of % mortality on Y-axis and LD₅₀was found to be 398.10±0.01 mg/kg or approximately 400 mg/kg.

Antimutagenicity test:

Melanoma cell lines were obtained from Cell sciences Research Institute, Pune and maintained in Dept. of Research, JN Cancer Hospital and Research Center, Bhopal, India. The C57BL hybrid mice of both sexes of the mean weight of 25 gm and 6-7 weeks old were obtained from the animal colony of the institute. They were housed in good condition in the department’s animal house and given standard mouse pellet diet and water ad Libitum. All the mice were kept at controlled light condition (light: dark, 12:12 hr) and temperature 22 ± 1º C. Animal experiments were carried out following the guidelines of the animal ethics committee of the institute.

Micronucleus (MN) assay:

The method of schmid⁸ was employed for micronucleus assay. Experimental animals were randomly divided into 4 groups and each group consists of 4 mice.

1. Group- I: CP (50 mg/kg)

2. Group- II: Extract (50 mg/kg) + CP (50 mg/kg).

3. Group- III: Extract (100 mg/kg) + CP (50 mg/kg).

4. Group- IV: Extract (150 mg/kg) + CP (50 mg/kg).

All treated mice were humanely sacrificed by cervical dislocation 24 hours after the treatment and bone marrow was collected for the micronucleus assay by washing the femurs with 1 ml of fetal calf serum in a centrifuge tube containing an additional 1 ml of serum, homogenizing the cell suspension and centrifuging it at 1000 rpm for 5 min, after which the supernatant was partially discarded to leave about 0.3 ml of fetal calf serum in which the cell pellet was re-suspended and then smeared on clean and dry slides which were dried at ambient temperature for 24 hours, fixed with absolute methanol for 10 min and stained for 8 min with 5% (v/v) Giemsa stain diluted in phosphate buffer ( Na₂HPO₄ 0.06 M and KH₂PO₄ 0.06 M, P^H 6.8 ). 1000 polychromatic erythrocytes (PCEs) were counted per animal to ascertain the frequency of MNPCE, 1000 normocromatic erythrocytes (NCE – normochromatic erythrocytes) were counted, as well as the frequency of polycromatic erythrocytes within the same microscope fields was analysed. Cells were scored blind according to the established criteria using a Nikon binocular optical microscope fitted with a 1,000 X (100 x10X) objective lens. Group and the Student t-test (p < 0.05) used to test for significance.

Chromosomal Aberration (CA) assay:

The C57BL hybrid mice were randomly divided into 4 groups as in earlier test. Each group consists of four mice. Group-I animals were administered with CP 50 mg/kg and group II, III and IV animals were treated with CP 50 mg/kg administered after 24hours of Terminalia chebula fruit extract 50, 100 and 150mg/kg respectively i.p. The animals were sacrificed. Two hours prior to sacrificing, 0.5 ml of 0.05% colchicine was injected to all animals to inhibit spindle formation in order to get well spread metaphases. All the animals were sacrificed and the femur bones were dissected out and metaphase plates were prepared by “air drying method”⁹ i.e. in brief, the bone marrow was flushed out with 6ml of hypotonic solution (0.6% CH₃COOK) and prepared a fine cell suspension by using a sterile syringe with 26-gauge needle. The suspension was incubated at 37⁰C for 25 min and centrifuged for 10 min at 1000 rpm. The supernatant was collected and added with pre-chilled Carney’s fixative (3:1 methanol: acetic acid) to the pellet. The pellet was allowed to disperse evenly by flicking the tube constantly with the index finger and allowed to stand for 10 min. After 10min, it was centrifuged again and the supernatant was collected and fresh fixative was added to the pellet. This process was repeated 3 to 4 times and the final suspension was made in 1 ml of fresh fixative. One or two drops of final suspension were dropped on to grease free pre-chilled slides. The slides were dried, coded and stained in 4% Giemsa (4ml of Giemsa + 2 ml of phosphate buffer + 44ml of distilled water) for 30 min and mounted with DPX. For each animal, 100 well spread metaphase plates were scored at a magnification of 1,000 X (100 x10X) for the presence of various types of chromosomal aberrations such as gaps, breaks, fragments, association, and rings.

Statistical analysis:

For the MN and CA assay, the mean frequencies and the standard deviations were calculated for one thousand cells for each treatment group and the Student t-test (p< 0.05) was used to test for significance.

RESULTS:

MN assay:

In C57BL hybrid mice which were injected with CP (50 mg/kg) i.p. resulted in micronuclei formation. Single application of Terminalia chebula fruit extract at the dose of 50, 100 and 150 mg/kg dry weight, 24hours prior the i.p. administration of CP (at the dose of 50 mg/kg) significantly prevented the micronucleus formations in dose dependent manner in bone marrow cells of mice as compared to CP. Polychromatic erythrocytes and nonchromatic erythrocyte ratio (PCE/NCE) was increased upto 1.64 ± 0.369 from positive control value (0.455 ± 0.219) and Micro Nucleus polychromatic erythrocytes (MNPCE) values were decreased to 0.6 ± 0.547 in Group- IV animals from positive control value (3.36 ± 0.56). All the data was significant with P<0.05. MN formation in PCE cells was shown (Image1). However, the fruit extract alone did not induce any micronucleus formations in bone marrow cells as compared to control group. The effect of fruit extract on Micronucleus formation in Mouse bone marrow cells was shown (Table 1).

CA assay:

In C57BL hybrid mice which were administered with CP (50 mg/kg) i.p. resulted in large percentage of (68.80±3.16) chromosomal abnormalities or aberrations in their bone marrow cells. The single administration of Terminalia chebula fruit extractat the doses of 50, 100 and 150 mg/kg 24 hours prior to administration of CP (50 mg/kg) produced the dose dependent protection. Percentage of CA was decreased to 34.23±0.031 from positive control value (68.80 ± 3.16) and Percentage of degree of protection was increased to 45.42% in Group- IV animals. The percentage degree of protection was increased in the dose dependent manner (Table2).

All the data was significant with P<0.05. Ring formation and association of CA assay were shown Image (2 and 3).

However, the fruit extract alone did not show any chromosomal aberration in bone marrow cells compared to positive control group.

Table 1: Effect of Terminalia chebula fruit extract on Micronucleus formation in Mouse bone marrow cells

S No	Treatment Group	Dose (mg/kg)	MNPCE	PCE/NCE RATIO
1	Group I: Cyclophosphamide (CP)	50	3.36 ± 0.56	0.455 ± 0.219
2	Group II: CP+ Extract	50	1.4 ± 0.55*	1.38 ± 0.319*
3	Group III: CP+ Extract	100	0.8 ± 0.83*	1.33 ± 0.188*
4	Group IV: CP+ Extract	150	0.6 ± 0.547*	1.64 ± 0.369*

*denotes statistical significance as compared to cyclophosphamide at P<0.05. Data value ±SEM, CP – cyclophosphamide, MNPCE – micronucleus formation in polychromatic erythrocytes, PCE -- polychromatic erythrocytes, NCE –Non chromatic erythrocytes

Table 2: Protection against Chromosomal Aberrations by Termianlia chebula fruit extract

S No	Treatment Group	Dose (mg/kg)	%CA	CB	CG	RF	AN	CF	%DP
1	Group I: Cyclophosphamide (CP)	50	68.80±3.16	18.6	16.4	19.8	11	-	-
2	Group II: CP+ Extract	50	52.72±0.17*	25	8	9	4	12	15.9
3	Group III: CP+ Extract	100	44.95±0.22*	19	7	8	6	9	28.3
4	Group IV: CP+ Extract	150	34.23±0.031*	14	6	6	3	9	45.4

*denotes statistical significance as compared to cyclophosphamide at P<0.05. Data value ±SEM CP – cyclophosphamide, CA—Chromosomal aberration, CB-Breaks, CG-Gaps, RF-Ring formation, AN-Association, CF-fragment, %DP- percentage degree of protection

DISCUSSION:

Micronuclei arise as a consequence of clastogenic or aneugenic action and this end-point is widely used to evaluate the genotoxic potential of test agents^10-11. Micronuclei (MN) separated from and in addition to the main nucleus of a cell are the results of acentric fragments or lagging chromosomes that fail to incorporate into either of the daughter nuclei during telophase of the mitotic cells. The frequency of MN in polychromatic erythrocytes (PCE) of mouse bone marrow is a very sensitive index of damage produced by ionizing radiation and by chemical mutagens^12-13. This test presents some advantages compared to other kinds of assays, in which we may mainly mention the low cost and the reliability. In addition, this assay utilizes mammalians, which present capacity of metabolization similar to humans that hardly can be reproduced in totality in "in vitro" assays¹². The intraperitoneal via (i.p.) was applied because this procedure maximizes the exposure of the bone marrow to chemical mutagens¹⁴.

The micronuclei test used in this study also detects cytotoxic effects by the PCE/NCE relationship. When normal proliferation of the bone marrow cells is affected by a toxic agent, the number of immature erythrocytes (PCE) is prejudiced in relation to mature erythrocytes (NCE). Thus, the PCE/NCE ratio may decrease¹². In our results, at any dose there is a significant increase of PCE/NCE relationship compared to CP alone indicated that the extract did not possess any cytotoxic action. Terminalia chebula extract did not present a significant increase of MNPCE frequency, instead it decreased the MNCPE frequency dose dependently (50,100 and 150 mg/kg) compared to CP alone. The Pre-treatment of lymphocytes with bark extract of Terminalia arjuna before ADR treatment resulted in a significant decline in micronuclei formation¹⁵. These results indicated that the extract exhibit the anti mutagenic effects in PCE of the bone marrow of mice.

The CA is one of the widely used parameters for testing the protective effects of natural compounds on the drug and chemical induced toxicity. The modulatory effect of natural compounds on CA induced by various kinds of chemicals and drugs is well established. In our investigation, % CA, CB, CG, RF and AN decreased as dose of extract increased linearly, at the same time % degree of protection increased dose dependently when compared to the CP treated group. In the presence of Terminalia chebula, cadmium-induced mitodepression, abnormal mitosis and aberrations could be appreciably prevented¹⁶. The Terminalia chebula fruit extract might have been either delayed the promotion phase of carcinogenesis, or down regulated the reactive oxygen species formation. The fruit extract possesses many of such compounds¹⁷ especially flavonoids which are ideal antioxidants^18. A great number of scientists reported that some flavonoids have antimutagenic and anticarcinogenic activities against a number of genotoxic agents^19-20. It is suggested that flavonoids may act as antioxidant, free radical scavengers, inhibitors of tumor cell growth, inducers of apoptosis, modulators of DNA repair or carcinogen inactivators^21-22. Hence flavonoids can be held responsible for reducing CP genotoxicity in mice. Infact the presence of some polyphenols (tannins, gallic acid and tannic acid) in fruit extract may also contribute towards the anti tumour and antimutagenic activity significantly.

In summary, our results indicate that the antimutagenic effect of methanolic extract of Terminalia chebula towards CP was strongly demonstrated and it did not exhibit any cytotoxic effect on mice. Possibly, the antimutagenic activity exhibited might contribute to an anticarcinogenic effect. However, further studies are required to better characterize the antimutagenic activity of Terminalia chebula fruit extract and to identify their active compounds and mode of action.

ACKNOWLEDGEMENT:

We are thankful to Dr. R. Venkatanarayanan, Principal and The Management of RVS College of Pharmaceutical sciences, Coimbatore, Tamilnadu for their valuable support for the completion of this work.

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Received on 25.09.2010

Accepted on 30.09.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(6): Nov. - Dec. 2010, 455-459