|Year : 2013 | Volume
| Issue : 2 | Page : 184-186
Anticlastogenic activity of flavonoid rich extract of Cassia auriculata Linn. on experimental animal
Supriya S Deshpande1, Shailesh M Kewatkar2, Vivek V Paithankar1
1 Dr. Punjabrao Medical College, Amravati, India
2 Pinnacle Biomedical Research Institute, Bhopal, India
|Date of Submission||29-Jun-2012|
|Date of Decision||19-Jul-2012|
|Date of Acceptance||31-Dec-2012|
|Date of Web Publication||11-Mar-2013|
Supriya S Deshpande
Dr. Punjabrao Medical College, Amravati
Source of Support: None, Conflict of Interest: None
Objective: To determine antimutagenic activity of Cassia auriculata Linn. on chromosomal damage induced by cyclophosphamide (CP).
Material and Methods: In the present investigation, four groups of six Swiss albino mice in each group were used. Excepting for the first group all the remaining groups were treated with CP (50 mg/kg). Mice of third and fourth group were treated with ethyl acetate extract of C. auriculata Linn. at 100 mg/kg and 200 mg/kg with CP. Metaphase of bone marrow cells of all animals were analyzed for qualitative and quantitative chromosomal aberrations. Break, fragment, deletion, Polyploidy, pulverized, ring and total aberration were observed.
Results: Flavonoids rich extracts of root of C. auriculata Linn. provided significant protection (P < 0.05) against CP induced chromosomal aberration. Total chromosomal aberration was found to be 12.16 and 7.33% in 100 and 200 mg/kg of extract treated animals respectively.
Conclusion: From the present study it can was observed that ethyl acetate extract of C. auriculata Linn possess significant anti-mutagenic potential against CP induced chromosomal aberration.
Keywords: Bone marrow cells, Cassia auriculata Linn., cyclophosphamide
|How to cite this article:|
Deshpande SS, Kewatkar SM, Paithankar VV. Anticlastogenic activity of flavonoid rich extract of Cassia auriculata Linn. on experimental animal. Indian J Pharmacol 2013;45:184-6
|How to cite this URL:|
Deshpande SS, Kewatkar SM, Paithankar VV. Anticlastogenic activity of flavonoid rich extract of Cassia auriculata Linn. on experimental animal. Indian J Pharmacol [serial online] 2013 [cited 2022 Aug 17];45:184-6. Available from: https://www.ijp-online.com/text.asp?2013/45/2/184/108314
| » Introduction|| |
Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. These changes may involve a single gene or gene segment, a block of genes or chromosomes. The term clastogenicity is used for agents giving rise to structural chromosome aberrations. A clastogen can cause breaks in chromosomes that result in the loss or re-arrangements of chromosome segments.  In vitro and in vivo tests suggests that major chromosomal aberrations in metaphase cells can detect a wide spectrum of changes in chromosomal integrity. The assays that detect either chromosomal aberrations or micronuclei are appropriate for detecting clastogens. 
In somatic cells, cyclophosphamide (CP) produces gene mutations, chromosome aberrations, micronuclei and sister chromatid exchanges in a variety of cultured cells in the presence of metabolic activation as well as sister chromatid exchanges without metabolic activation. It can also produce chromosome damage and micronuclei in rats, mice and Chinese hamsters.  The present study designed to investigate the protective role of Cassia auriculata Linn. in chromosomal damage induced by CP in bone marrow cells of Swiss albino mice.
| » Material and Methods|| |
Fresh C. auriculata roots were obtained from local regions of Bhopal and authentication was been carried out by Safia College, Bhopal. The roots were dried under shade and powdered. Dried plant material was extracted with ethyl acetate using soxhlet apparatus. Obtained extract ethyl acetate extract of Cassia auriculata Linn. (EACA) was evaporated using rotary vacuum evaporator and kept in air tight container till any further use.
Swiss albino male mice (20-25 g) were collected at random from animal house of Pinnacle Biomedical Research Institute, Bhopal. Animals were kept on sterile husk in propylene cages with four animals per cage. They were housed in an ambient room temperature (25 ± 2°C) and relative humidity (50 ± 5%), maintained at 12:12 h dark-light cycle. Standard feeding pellets (Golden feeds, New Delhi) and water were available ad libitum. The experiment was conducted with prior permission of Institutional animal ethical committee of PBRI (Reg No. 1283/c/09/CPCSEA) and carried out under strict compliance with the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forests, Government of India. Animals were acclimatized to the experimental conditions for a period of 1-week before the actual experimentation.
| » Pharmacological Evaluation|| |
Determination of Acute Toxicity
Acute toxicity study was carried out as per the OECD 423 guideline. Dose level of 5 mg/kg, 50 mg/kg, 300 mg/kg and 2,000 mg/kg were used to evaluate acute oral toxicity. Signs of toxicity and mortality were observed as per guidelines.
All animals were randomly divided in four groups. Group-1 was given vehicle only (p.o.) for 2 days at an interval of 24 h. Group-2 was given vehicle (p.o.) as mentioned in group 1 and after 1 h of last dose CP was administered at 50 mg/kg (i.p.). Group-3 was given extract (EACA) at 100 mg/kg (p.o.), for 2 days at an interval of 24 h and 1 h after last dose of extract CP was administered at 50 mg/kg (i.p.). Group-4 was given extract (EACA) at 200 mg/kg (p.o.), for 2 days at an interval of 24 h and 1 h after last dose of extract CP was administered at 50 mg/kg (i.p.).
Chromosomal Aberration Assay
Twenty-four hour post CP treatment, mice were sacrificed by cervical dislocation. Colchicine was given i.p. before 90 min of sacrificing the mice. Animal was dissected and femur bone was excised. Bone marrow was aspirated by flushing with normal saline in the centrifuge tube and the suspension was flushed in the tube properly to get good cell suspension and then it was centrifuged for 10 min at 1,000 rpm. Supernatant solution was discarded and pellet was treated with pre-warmed (37°C) KCl on cyclomixer. After this step the above suspension was warmed in a water bath (37°C) for 30 min. Again it was centrifuged and supernatant solution was discarded. The obtained pellet was treated with freshly prepared cornoy's fixative (methanol:acetic acid [3:1]) on cyclomixer. Once again it was centrifuged and supernatant was discarded and again it was treated with Cornoy's fixative for 3 times to get debris free white pellet. Cornoy's fixative was (quantity sufficient) added to pellet and good cell suspension was obtained. Slides were made with Air Drop Method. The Slides were stained with 5% Giemsa's solution for 15 min and slides were rinsed in distilled water blotted. A total of 100 well spread metaphase plates were scored for chromosomal aberrations at a magnification of 1,000 × (100 × 10) for each groups. Different types of chromosomal aberrations such as chromatid breaks/gaps, centromeric association and chromatid fragmentation were scored and expressed as (%) chromosomal aberrations. 
All data were analysed by one way analysis of variance followed by Bonferron's test. P < 0.05 was considered as level of significance.
| » Results|| |
Acute Toxicity Studies
Acute toxicity studies (OECD - 423 guideline) of C. auriculata Linn. revealed that there was no toxic effect up to dose of 2,000 mg/kg nor any significant variation in behavior of animal was observed.
Effects of Cassia Auriculata Linn. Against Cyclophosphamide Induced Chromosomal Aberration
As mentioned in [Table 1], it was observed that in chromosomes of bone marrow cells of animals treated with CP, break was 31.33 ± 3.01%, which was significantly higher (P < 0.05) as compared to vehicle treated animals in which it was 3.83 ± 1.72%. In vehicle treated animals the extent of fragment was 2.83 ± 1.47%, which was found to be significantly higher than that of the CP treated animals in which the extend was 24.17 ± 2.56. Prior treatment of extracts at 100 mg/kg and 200 mg/kg significantly lowered (P < 0.05) the presence of Break up to 9.5 ± 2.16% and 6.33 ± 2.16%, respectively [Figure 1]. Fragment was also significantly less (P < 0.05) in extract treated animals with 100 mg/kg and 200 mg/kg. In vehicle treated animals none of the metaphase were found to be having polyploidy, pulverized or, ring type of aberration and on the other hand in animals treated with CP, these aberration increased significantly (P < 0.05). Treatment of animals with extract at 100 mg/kg and 200 mg/kg provided significant protection against CP induced polyploidy, pulverized or ring type of chromosomal aberration. Total aberration in CP treated animals was 42.33 ± 3.50%, which was found to be 12.16 ± 4.16% in 100 mg/kg treated animals and 7.33 ± 1.63 in 200 mg/kg treated animals, which were significantly less (P < 0.05) as compared to vehicle treated animals.
|Figure 1: Flavonoid reach crude extract treated (100 mg/kg and 200 mg/kg) test groups III and IV showing less percentage of chromosomal abbreviation in comparison with cyclophosphamide alone treated group II|
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|Table 1: Effect of ethyl acetate extract of roots of Cassia auriculata Linn. on cyclophosphamide induced chromosomal aberration|
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| » Discussion|| |
Effective cancer chemotherapy as well as immunosuppressive therapy with CP is severely limited due to its unwanted toxicity. The cytotoxic effect of CP is attributed to the inhibition of cell division by damaging the DNA of proliferating cancerous cells. However, at the same time it also damages the DNA of the healthy tissues with high cellular turnover such as the bone marrow, gastro-intestinal tract and germ cells.  Therefore, although CP activated metabolites have been shown to be beneficial for treating cancer, the side effects of these metabolites causes great concern.  CP generates active metabolites, 4-hydroxycyclophosphamide, phosphoramide mustard and acrolein.  Among these metabolites acrolein is highly toxic in nature and generates oxidative stress and damage DNA by inducing single strand breaks. ,
The present study demonstrated that ethyl acetate extract of C. auriculata shows chemoprotective effect against CP induced genotoxicity. Preliminary phytochemical studies indicates that C. auriculata extract contains carbohydrates, glycosides, alkaloids, tannins, phenolic and flavonoidal compounds. , Flavonoids are a group of polyphenolic compounds, which exhibits biological effects.  The presence of high phenolic and flavonoid content has contributed directly to the antioxidant activity by neutralising the free radicals. 
In conclusion, the present study indicates that flavonoid rich extract of C. auriculata pre-treatment attenuates the CP induced genotoxicity in the bone marrow. The ethyl acetate extract of C. auriculata Linn possess significant anti-mutagenic potential against CP induced chromosomal aberration. The chemoprotective potential of C. auriculata could be due to its antioxidant property. Thus, C. auriculata has the potential as an adjuvant to cyclophoshamide for preventing the adverse effects associated with these drugs.
| » References|| |
|1.||EFSA Scientific Committee. Draft Scientific Opinion on Genotoxicity Testing Strategies applicable in food and feed safety assessement. EFSA 2011 Available from: http://www.efsa.europa.eu. |
|2.||Jena GB, Kaul CL, Ramarao P. Genotoxicity testing, a regulatory requirement for drug discovery and development: impact of ICH guidelines. Indian J Pharmacol 2002;34:86-99. |
|3.||Suchitra P, Suresh J, Archana T. Therapeutic use of cyclophosphamide and its cytotoxic action: A challenge for researchers. J Pharm Res 2011;4; 2755-7. |
|4.||Agrawal RC. Induction of chromosomal aberrations by propoxur in mouse bone marrow cells. Biomed Environ Sci 1999;12:292-5. |
|5.||Tripathi DN, Jena GB. Astaxanthin inhibits cytotoxic and genotoxic effects of cyclophosphamide in mice germ cells. Toxicology 2008;248:96-103. |
|6.||Patel JM, Block ER, Hood CI. Biochemical indices of cyclophosphamide-induced lung toxicity. Toxicol Appl Pharmacol 1984;76:128-38. |
|7.||Daikh DI, Wofsy D. Cutting edge: Reversal of murine lupus nephritis with CTLA4Ig and cyclophosphamide. J Immunol 2001;166:2913-6. |
|8.||Ludeman SM. The chemistry of the metabolites of cyclophosphamide. Curr Pharm Des 1999;5;627-43. |
|9.||Crook TR, Souhami RL, McLean AE. Cytotoxicity, DNA cross-linking, and single strand breaks induced by activated cyclophosphamide and acrolein in human leukemia cells. Cancer Res 1986;46:5029-34. |
|10.||Erickson LC, Ramonas LM, Zaharko DS. Cytotoxicity and DNA cross-linking activity of 4-sulfidocyclophosphamides in mouse leukemia cells in vitro. Cancer Res 1980;40:4216-20. |
|11.||Pijush K, Subrata L. A brief resume on the genus Ailanthus: chemical and pharmacological aspects. Phytochem Rev 2010;9:379-412. |
|12.||Cao G, Sofic E, Prior RL. Antioxidant and prooxidant behavior of flavonoids: Structure-activity relationships. Free Radic Biol Med 1997;22:749-60. |
|13.||Umamaheswari M, Chatterjee TK. In vitro antioxidant activities of the fractions of Coccinia grandis L. leaf extract. Afr J Tradit Complement Altern Med 2008,5:61-73. |
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