ISSN   0974-3618  (Print)                    www.rjptonline.org

            0974-360X (Online)

 

 

RESEARCH ARTICLE

 

Protective Efficacy of Phyllanthus amarus L against Radiation – Induced Chromosomal Damages in Onion Root Meristems

 

O.S. Vivekanandan1, Dr. R. Kavya2 and R. Radhai3

1Dept of Biotechnology/Bioinformatics, School of Life Science,

VELS University, Pallavaram Chennai – 600117. India

2Saveetha Dental College, Poonthamalle, Chennai – 600077. India

3Research Student, Dept. of Biotechnology, VELS University

*Corresponding Author E-mail: oyessvi70@rocketmail.com

 

ABSTRACT:

The protective effect of Phyllanthus amarus L in the irradiated roots of onion was evaluated. The results were indicated that the Phyllanthus extract showed a radio modifier effect by reducing the frequency of aberrant cells in the meristematic cells exposed to gamma rays.

 

KEYWORDS: Gamma rays, aberrant cells, chromosomes, gyres, antimutagens.

 

 

INTRODUCTION:

With the increasing application of diagnostic radiotheraphy in medical practice and the possible expanded use of nuclear fuel as a substitute for our dwindling fossil reserves, there is growing concern over the impact of radiation exposure on human health especially with respect to genetic hazards. Deleterious reactions of these oxyradicals with biomolecules result in the induction of a variety of structural aberrations of chromosomes including DNA strand brakes, protein oxidation and lipid peroxidation11,1. Unrepaired and misrepaired damages lead to drastic biological effects like cell death, induction of mutations, cell transformation and carcinogenesis1. Hence, it has become absolutely essential to seek a measure to minimize the radiation – induced genetic damage. A variety of pre and post- treatments have been studied with an attempt to reveal, the protective effects of vitamin C and E against gamma radiation3.  The role of various plant extracts as desmutagens and antimutagens are being increasingly recognized, as several of them are shown to have anti-mutagenic and anti-carcinogenic properties21.

 

 

 

Received on 22.01.2015       Modified on 19.02.2015

Accepted on 25.02.2015      © RJPT All right reserved

Research J. Pharm. and Tech. 8(4): April, 2015; Page 389-391

DOI: 10.5958/0974-360X.2015.00065.7

 

A large number of vegetable juices were also found to reduce chromosomal irregularities in rat bone marrow cells induced by dimethyl benz(a) anthracene12. Abraham et al., (1986), also showed the suppression of micronuclei induced by cyclophosphamide in mice feed with carrot and spinach juice2. Herbal preparations of Phyllanthus amarus L and its related species have generated a great deal of intrest in recent years for a wide range of protective effects against various chemical mutagens16,7. The herbal extracts are used extensively in indigenous systems of medicine for treating ailments such as leucorrhoea17, scurvy5. Besides, they have been recorded to have antibacterial20 and antiviral18 properties. Since these preparations are extensively consumed in various quantities, their effects in organisms exposed to physical and chemical pollutant in our environment need to be assessed8. Hence, this study involved the evaluation of modifying effects of the crude extract of Phyllanthus amarus L against gamma radiation – induced chromosomal changes in Allium cepa L root meristems.

 

 

 

 

 

 

MATERIALS AND METHODS:

The experiments were carried out using the root meristems of onion as the test system (2n=16). Irradiation was carried out in a Gamma camera having the source of Co60, and potency of 7 curie, at MERADO laboratory, CSIR complex, Taramani, Chennai, India. Aqueous extracts were prepared from the leaves of Phyllanthus amarus by grinding them in distilled water and further diluting to make 0.5 and 1% decoction. The onion root meristems were exposed to gamma rays at 1.5 and 2.00 gy for 1h. Some of the irradiated cells were fixed in 1:3 acetic acid and ethanol while the other exposed root tip cells were thoroughly washed and post-treated with the crude extract of Phyllanthus extract. Parallel distilled water controls were maintained. The treated and control samples were squashed following the Heamotoxylin staining technique 14. Mitotic indices were recorded from the treated and control root samples by examining 2000 to 5000 cells involving 6 root tips from 3 bulbs10. The types of structural changes in chromosomes were classified4. The magnitude of protection afforded by Phyllanthus during post-treatments was calculated19. Statistical significance of the difference between the control and treated groups were evaluated using students t-test. Further analysis was also carried out to compare the significance of differences, if any, between the irradiated and post-irradiated samples22.

 

RESULTS AND DISCUSSION:

Table 1, shows the effect of Co60 and aberrations compared to control samples (P<0.005) Mitotic inhibition was also observed in the meristems treated with the plant extracts of Phyllanthus and distilled water (P<0.005) (Table 1). The irradiated root meristems were post-treated with Phyllanthus extract for 1 hour to study mitogenic potential. However, these cells did not attain the normal frequency of cell division. And the extent of radiation –induced mitotic inhibition was further increased following treatment with Phyllanthus (P<0.005). Hence, the results clearly demonstrate that Phyllanthus does not exert discernible influence on the mitotic frequency and that therefore it is mitotoxic. The gamma exposure of meristems resulted in a substantial increase (P<0.005) in the induction of mitotic irregularities, and structural aberrations. The irradiated root meristems showed the occurance of many anaphasic irregularities such as anaphasic bridge, multiple fragments and lagging chromosomes. The frequency of radiation–induced alterations were progressively decreased when the exposed roots were left for recovery in distilled water. The optimal dose of Phyllanthus for eliciting maximum radioprotection appears to be 0.5%. Further, the protection afforded was greater if the irradiated cells were exposed to Phyllanthus extract for 24h (81.99%). Continuous application of radiation in medical practice remains debatable as they are harmful to some healthy cells as well. Plant extracts of a variety of Phyllanthus species have been reported to afford protection against various chemical mutagens 6,15,9.

 

In the present analysis, Phyllanthus extract significantly reduces the frequency of damaged cells at 0.05% during longer exposure following 1.5 gy of gamma radiation (Table 1). This decrease in frequency of aberrations following treatment with Phyllanthus indicated its interference with the formation of radiation induced aberrations. A reduction in the yield of X-rays induced damage by treatment with Phyllanthus amarus has been shown in mouse bone marrow cells13. A similar decrease in the frequency of micronuclei and aberrant metaphases were observed following pre and post-treatments with vitamin C and vitamin E in the gamma irradiated mouse bone marrow cells19. The results revel that the Phyllanthus extract exerts radioprotection when administered immediately after irradiation. A similar situation, where, in post-treatment with vitamin E enhanced the 30 day survival of mice treated with 8 gy of gamma radiation has been reported13. Farooqi and Kesavan (1992), also reported that caffeine post treatments afforded significant radio-protection to bone marrow cells of whole body irradiated mice. The possible mechanism of P.amarus for exerting radioprotection is due to the suppression of the formation of reactive oxygen species generated as a result of radiation18. The higher protection afforded by the plant extract observed in the study may be due to Phyllanthin an active principle of Phyllanthus amarus or to the combined action of all compounds of Phyllanthus. Hence, the findings indicate that Phyllanthin or all ingredient of Phyllanthus amarus collectively reduce the radiation induced chromosomal damage when administered immediately after the radiation raises hopes regarding its suitability as therapeutic agent in emergencies.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table: I Protective efficacy of Phyllanthus plant extract in the onion root tip cells exposed to gamma rays.

Mode of Treatment

Radiation dose gy

Plant extract Concentration

Duration of treatment Hr

No. of cells in division

Mitotic index X+SE

Percentage of Aberration X+SE

Magnitude of protection %

Control DW

-

-

1

1179

7.85±0.33

1.48±0.39

-

Gamma Radiation

1.5

-

-

149

2.86±0.44

64.20±5.12

-

-do-

2

-

-

143

2.88±2.03

73.31±6.95

-

Plant extract

-

0.5

1

111

2.20±0.41

12.80±3.23

-

-do-

-

1

1

120

2.31±0.26

51.30±5.87

-

Irradiated roots post treatment with D.W

1.5

-

-

33

1.36±0.21

35.07±8.93

45.42

Irradiated roots post treatment with plant extract

2

0.5

24

37

0.77±0.12

11.57±5.93

81.99

X- Mean; SE- Standard error;  P<0.05; P<0.01; P<0.005.; Gy; gyres; Magnitude of protection = (Irradiated-Treated sample) X 100/ Control

 

 

 

 

ACKNOWLEDGEMENT:

The first and third author is grateful to Dr. Ishari K Ganesh, Chancellor, Vels University, and other members of the Vel’s Educational Trust for providing the necessary facilities and encouragement to carry out this work in the department of Biotechnology. The second author thanks the management of Saveetha Dental College, Chennai for support and encouragement.

 

REFERENCE:

1.       Abraham, S.K. maharajan, S.Kesavan, P.C (1986), inhibitory effects of dietary vegetables on the in vitro clastogenecity of cytophosphamide, Mut res. 172:514.

2.       Alldrick A.J flymm, Rowland I.R(1986), effects of plant derived flavonoids and polyphenolic acids on the activity of mutagens from cooked food, Mut.res., 172:51-54.

3.       Ames,B.N (1983) Dietary carcinogens and anticarcinogens, Science,221:1256-1264.

4.       Buckton, K.E Evans H.J (1973), methods for the analysis of human chromosome aberrations, world health organization, Geneva 1973.

5.       Chopra, R.N. Nayar, S.C. Chopra I.C (1956), In:Glossary of Indian medicinal plants, CLRI, NewDelhi:154.

6.       Dhir, H.Agarwal, Kalpana Sharma, A.K talukdar, G. (1991), modifying role of Phyllanthus embilica and ascorbic acid against Ni clastogenecity in mice, Cancer letters, 59(1):9-18.

7.       (1991), production afforded by aqueous extracts of Phyllanthussp, against cytotoxicity induced by lead and aluminium salts, Phytotheraphy res, 4(5):172-176.

8.       Farook, S.I., Kesavan, P.C. (1992), radioprotection by caffeine pre and post treatment in bone marrow cells of mice given whole body gamma radiation, Mut res., 269:225-250.

9.       Gowrishankar. B and Vivekanandan. O.S. (1994), in vitro studies of a crude extract of Phyllanthus amarus L, in modifying the genotoxicity induced in Vicia faba L, by tannery effluents, Mut res., 322:185-192.

10.     Grant, W.F, (1982), Chromosome aberrations in Allium, A report of the U.S. Environmental protection agency, Mut res., 99:273-291.

11.     Halliwell, B. Gutteridge, J.M.C (1989), In: Free radicals in biology and medicine, Clarendom Press, Oxford:20-25.

12.     Ito, Y., Meada, S., Sugiyama, T.(1986), suppression of 7,12-dimethylbenzo(a) anthracene induced chromosome aberrations in rat bone marrow cells by vegetable juices, Mut.res., 172:51-54.

13.     Maliek, M.A. Roy, R.M and Sternberg. J(1978), effects of vitamin E, on post irradiation in mice, Experentia, 34:1216-1217.

14.     Marimuthu, K.M, and Subramaniyam, M.K (1960), an iron alum heamotoxylin squash schedule for the root tips of Dolichos lablab. Cur.Sci., 29:482-483.

15.     Padma R, Vivekanandan O.S., Gowrishankar and Ramakrishnan (1993), Radio modifying effect of Phyllanthus amarus on mouse bone marrows (abstract report), International symposium on emerging frontiers in radiation biology, EMSI conference, Mumbai:148.

16.     Polya, G.M., Wang, B.H., and Foo. (1955), Inhibition of signal regulated protein kinases by plant derived hydrolysable tannins, Phytocherm., 38:307-314.

17.     Rao T.S., Kusumakumari., Netaji, B.Subhkota (1985), A pilot study of Suetapradara with amalakaguggulu, J.Res. Ayurvedic Siddha, 6:213-237.

18.     Saigopal, D.V.R. Siva Prasad, V, Sreenivasalu, P.(1986), antiviral activity in extracts of Phyllanthus frateruns, cur, sci., 55:264-295.

19.     Sarma, C., Kesavan, P.C (1993), Protective effect of vitamin C and E against gamma ray induced chromosomal damage in mouse, International J. Radiation Biol., 63:759-764.

20.     Vinayagamoorthy, (1982), antibacterial activity of some medicinal plants of Srilanka, Ceylon, J. Sci and Bio. Sci., 15-50.

21.     Yoshikawa, K., Ravi R., Kada, T (1981), desmutagenic action of vegetable extracts on the mutagenic pyrolysis products of foods., Abstracts of 3rd International conference on environmental mutagenesis., 19:3.

22.     Zar, J.H., (1974), Biostatistic Analysis, Prentice Hall., Englewood Cliff.