Call Us @ +91-7020626059

Anti-Clastogenic effect of Trigonella foenum graecum in mouse bone marrow cells

HTML Full text

Anti-Clastogenic effect of Trigonella foenum graecum in mouse bone marrow cells.

Nitin Nema *1, SP Jain1, Shripad M Bairagi2, Ritu Shrivastava3, RT Lohiya4

1Rajarshi Shahu College of Pharmacy, Buldhana, M.S., India, 
2Department of Pharmaceutical Sciences, Dr. H.S. Gour Central University, Sagar, M.P., India.
3Sagar Institute of Pharmaceutical Sciences, Sagar, M.P. India.
4SKB College of pharmacy Kamptee, Nagpur, (MH) India.


ARTICLE INFO

Date of Submission: April 10, 2018
Date of Revision: April 25, 2018
Date of Acceptance: May 30, 2018

*Corresponding Author:
Email ID: drnitinnema85@gmail.com

KEYWORD
Cyclophosphamide, Methanolic extract of Trigonella foenum graecum,
Chromosomal aberration, Clastogenecity, antioxidants.

ABSTRACT

Objective: Noticeable chromosomal damage or recombination is generally considered important aspect bringing change in heritable or character which involved in multi-step process of malignancy, where genetic changes plays significant role.
Methods: Inhibitory effects of Trigonella foenum graecum was evaluated on the induction of clastogenicity on bone marrow cells induced by Cyclophosphamide in mice, using Bone Marrow Chromosomal assay.
Results: Pre-treatment with methanolic and aqueous extract of Leaves of Trigonella foenum graecum (100 & 200mg/kg b.wt, p.o) reduced structural chromosomal aberrations scored significantly along with restoration of enzymatic antioxidants like Catalase, reduced glutathione and malonaldehyde content.
Conclusion: In conclusion, it was found that Trigonella foenum graecum reduces genotoxicity induced by Cyclophosphamide and thus may decrease the risk of development of secondary tumor.


1. INTRODUCTION

Clastogenicity describes the process by which certain substances cause one or more types of structural changes in chromosomes of cells. The substances called clastogens; which are agents that cause breaks in chromosomes that result in the gain, loss, or rearrangement of chromosomal segments. The genetic damage that results in chromosomal breaks, structurally abnormal chromosomes, or spindle abnormalities leads to micronucleus formation which is a ultimate source of mutagenicity.1
”Oxidative stress” is an important mechanism of indirect genotoxicity that is triggered by exposure to exogenous factors such as UV, ionising radiation, anoxia and hyperoxia.ROS (Reactive Oxygen Species) production cause damage to DNA modifying the base and altering DNA strands, and can contribute to genotoxicity.2
Antioxidants are capable of stabilizing, or deactivating, free radicals3. Free radicals are produced in cells by cellular metabolism and by exogenous agents. These species react with biomolecules in cells and one of the important targets is DNA4.
Trigonella foenum-graecum (Linn.) belonging to the family Papilionaceae commonly known as Fenugreek. It contains active beneficial chemical constituents including steroidal sapogenins5, dietary fiber6, galactomannans7, antioxidants, and amino acids such as 4-hydroxyisoleucine, histidine, lysine, arginine and fixed oil (5-8%)8.
Also plant contains Vitamins, especially A, B1, and C; minerals (especially calcium and iron); volatile components (more than 50), which include n-alkanes, sesqui-terpenes, coumarins and other constituents.9,10 like neurin, biotin, trimethylamine11.
It possess anti-diabetic, hypocholesterolemic and hypoglycemic properties which have potential for use in the treatment of antipyretic12, antinociceptive, antifertility activity, cure leprosy galactogogue13, obesity, diabetes and cancer14, CNS stimulant15. In Ayurvedic and Unani systems of medicine, fenugreek is used for the treatment of epilepsy, paralysis, gout, dropsy, chronic cough and piles.
The present study designed to investigate the protective role of Trigonella foenum graecum Linn. in chromosomal damage induced by CYP in bone marrow cells of Swiss albino mice.

2. Material and Methods:

2.1 Drugs and Chemicals:

Cyclophosphamide was purchased from Sigma Chemical Co. was dissolved in sterile distilled water (50mg/Kg b.wt.) was administered orally. Colchicine was purchased by AB Enterprises, Mumbai, India was dissolved in sterile distilled water (4 mg/kg b.wt) was administered intraperitoneally 2h before killing the animals and other chemicals were procured locally.

2.2. Preparation of plant extracts:

Fresh leaves of Trigonella foenum graecum. Linn were collected from local regions of Sagar (M.P). The plant was authenticated from Department of Botany, Dr. H.S Gour Central University Sagar (M.P) India havinf voucher specimen number Bot./Her/C/1419. The leaves were shade dried and was powdered. Extraction was done by Methanol and water, obtained extract was dried in water bath and then kept in tightly closed container for further use.

2.3 Animals:

Swiss Albino mice 4-6 weeks old, weighing 23±5g were used during the study. The animals were housed in polypropylene cages and provided standard pellet diet and water ad libitum and maintained under controlled conditions of temperature and humidity, with a 12 h light/dark cycle.
The use of animal is as per Institutional Animal Ethics Committee (IAEC), Dr. Hari Singh Gour Vishwavidyalaya, Sagar and Pinnacle Biomedical Research Institute (Register number SIPS/EC/2014/48 and PBRI/13-14/IAEC/PN-367).

2.4 Experimental design:

  • The animals were randomly divided into four groups (n=6). Mice in control group were treated with (0.2ml) normal saline orally.
  • Mice in group II were received CYP-50mg/kg B.W intraperitoneally and sacrificed after 24 hours by cervical decapitation.
  • Mice in group III and IV were received 100/200 mg/kg of METFG for five days respectively. On completion of dosing schedule of five days, mice were given CYP (50mg/kg B.W, intreperitoneally) after 2 hours of extract of METFG and sacrificed after 24 hours by cervical decapitation.
    Before sacrificing, Colchicine-4mg/kg B.W was given intraperitoneally 2 hrs before.

2.5 Assessment of oxidative stress:

In-vitro assessment on AETFG and METFG was performed by DPPH assay.16,17,18 Assessment of oxidative stress in liver content such as lipid peroxidation (LPO)17,19 reduced glutathione (GSH) assay,20,17 superoxide dismutase (SOD) assay17 was done.

2.6 Bone Marrow Chromosomal Assay:

The animals were sacrificed by cervical dislocation, 24 h after the treatment. Colchicine (4 mg/kg b.wt) was administered intraperitoneally 2 h before killing the animals. The slides were prepared essentially as per modified method.26,27 Femur bones were excised and the bone marrow extracted in 0.56% KCl. In brief the harvested cells were incubated at 37°C for 20 min and then centrifuged for 10 min at 1000 rpm. Cells were fixed in Cornoy’s fixative (methanol:acetic acid, 3:1) and burst opened on a clean slide to release chromosomes. The slides stained with 5% Giemsa solution for 15 min and then put in xylene and mounted with DPX. A total of 100 well spread metaphase plates were scored for chromosomal aberration at a magnification of 1000×(100×10×) for each group. Chromatid breaks, deletions, fragmentation, ring, deletion and acrocentric association, etc., were scored to assess the chromosomal aberrations. The data were expressed as percentage chromosomal abberations 21,22

2.7 Statistical Interpretation:

Data were presented as mean ± SEM. To assess the relationship among the groups, one-way ANOVA followed by Dunnett’s test, was performed using Graph Pad Prism, Version 4.0 (Graph Pad Software, San Diego, CA, USA).

3 Results and Discussion:

DPPH (2, 2-diphenyl-1-picryl-hydrazyl-hydrate) free radical method is an antioxidant assay based on electron-transfer that produces a violet solution in methanol. This free radical, stable at room temperature, is reduced in the presence of an antioxidant molecule, giving rise to colorless solution23.
DPPH assay of METFG and AETFG was estimated and compared with the standard curve of ascorbic acid and IC50 value was calculated as shown in (Table 1). IC50 value of Ascorbic, METFG and AETFG was 55.76 µg/ml, 38.55 µg/ml and 163.84 µg/ml respectively (Fig 1).
Superoxide dismutase catalyses the dismutation of the highly reactive superoxide anion to O2 and to the less reactive species i.e H2O2.24. The level of SOD was decreased in case of CYP treated group 1.164±0.155 when compared with control group 1.885±0.160. Amongst the test group METFG (1.772±0.210) is more effective as compared to AETFG (1.48±0.078) i.e P AETFG.
Lipid peroxidation is increased in the damaged tissue which was reflected by increased level of MDA (malonidaldehyde) measure spectrophotometrically as compared with the control 0.263±0.015. In the intercomparison study between METFG and AETFG, it was found that METFG 0.285±0.148 is nearly twice more potent than AETFG 0.535±0.049. Thus potency ratio was METFG (methanolic extract of Trigonella foenum graecum) >AETFG (Aqueous extract of Trigonella foenum graecum with respect to lipid per-oxidation. Glutathione is important water phase antioxidant enzyme and essential cofactor. Its high electron donating capacity combined with its high intracellular concentration endows GSH with great reducing power. The level of GSH was also decreased in Cyclophosphamide treated group 43.38±11.02 as compared with control subject 703.00±16.65. METFG and AETFG when compared it was found that METFG 673.40±32.70 is more potent than AETFG 495.06±23.82 and the results were found to be significant P

Table 1 Effect of extracts of Trigonella foenum graecum against DPPH radical scavenging assay.

S. No

Extract

Line of regression

R2

IC-50

1.

 Ascorbic acid

 Y= 0.241x + 36.37

0.946

55.76

2.

 METFG

 Y= 0.076x + 47.07

0.973

38.55

3.

AETFG

Y= 0.065x + 39.35

0.95

 163.84

Table 2 Effect of extracts of Trigonella foenum graecum against in-vivo antioxidants enzymes.

Treatment groups

SOD U/mg

GSH nMol/mg

LPO nM/mg

Control

1.885±0.160

703.000±16.652

0.263±0.015

CYP Control

1.164±0.155###

434.380±11.026###

0.715±0.020###

Control

1.772±0.210***

673.400±32.708***

0.285±0.148***

AETFG

4.000±0.182***

495.060±23.828***

0.535±0.049***

Fig1. Standard curve of Ascorbic acid and Trigonella foenum graecum extracts against DPPH radical scavenging assay.
Fig 2 Effect of extracts of Trigonella foenum graecum against Cyclophosphamide induced total aberration.

Fig 3 Effect of extracts of Trigonella foenum graecum against Cyclophosphamide induced fragments.

Fig 4: Effect of extracts of Trigonella foenum graecum against Cyclophosphamide induced breaks.

Fig 5. Effect of extracts of Trigonella foenum graecum against CYP induced deletions.

Photograph 1: Control group (Normal Metaphase)
CommentMice bone marrow cells revealed healthy metaphase index, suggesting of normal Chromosomal count (2n=40) and no abnormality is observed.

Photograph 2: Cyclophosphamide (CYP) Control group
Comment- Mice bone marrow cells revealed very poor metaphase index, suggesting undefined and abnormal uncountable Chromosomal structure and premature centromeric division. Following varied types of abnormality observed likewise.
  • Pulverization • Gap                   • Break                   • Deletion              • Fragmentation

Photograph 3: Test group A (METFG 100mg/kg)

Photograph 4: Test group A (METFG 100mg/kg)
Comment- Mice bone marrow cells after treatment with METFG 100/kg BW revealed good metaphasic index as compared to CYP control group and chromosomes are countable. However few aberrations like fragments, deletion still persist.
Photograph 5: Test control group B (METFG 200 mg/kg)

Photograph 6: Test control group B (METFG 200 mg/kg)
Comment– Mice bone marrow cells at dose level of METFG 200/kg BW revealed. However polycentromeric division (PCD) was observed.

4. CONCLUSION

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 cells25. Therefore, although CP activated metabolites have been shown to be beneficial for treating cancer, the side effects of these metabolites causes great concern26. CP generates active metabolites, 4- hydroxycyclophosphamide, phosphoramide mustard and acrolein26. Among these metabolites acrolein is highly toxic in nature and generates oxidative stress and damage DNA by inducing single strand breaks.28,29
The present study demonstrated that methanolic extract of Trigonella foenum graecum. (Linn) shows chemoprotective effect against CYP induced genotoxicity in mice bone marrow cells. Preliminary phytochemical studies indicates that Trigonella foenum graecum extract contains carbohydrates, alkaloids, tannins, phenolic, flavonoidal and antioxidant compounds30. Flavonoids have remarkable biological activities. The presence of high phenolic and flavonoid content has contributed directly to the antioxidant activity by neutralising the free radicals31.
Finally our study clearly concluded the potent anti-clastogenic effect of methanolic extract of Trigonella foenum graecum by its diverse medicinal properties elicited by attenuating the CYP induced genotoxicity in bone marrow. However, further investigations are needed to be explored for the active moiety present in methanolic extract of TF responsible for its anti-clastogenic effect. However, the anti-clastogenic effect could be due to its antioxidant potential as an adjuvant to CYP for preventing the adverse effects associated with these drugs.

5.REFERENCES:

1. Akanni E.O, Loke J.K, Mabayoje V.O , Saka G.O; (2010); Clastogenicity Potential Screening of Pleurotus pulmonarius and Pleurotus ostreatus Metabolites as Potential Anticancer and Antileukaemic Agents Using Micronucleus Assay British Journal of Pharmacology and Toxicology; 1(2): 56-61.

2. Shinde. A, Ganu. J, Naik. P; (2012); Effect of Free Radicals & Antioxidants on Oxidative Stress: A Review Journal of Dental & Allied Sciences;1(2):63-66.

3. Perciva.M; (1998); Antioxidants Nut031 1/96 Rev. 10/98 Clinical Nutrition Insights.

4. Rao.K; (2009); Free radicals induced oxidative damaged to DNA: Relation to Brain ageing and neurological disorder; Indian Journal of Biochemistry and Biophysics; 46:9-15.

5. Taylor W.G, Zaman M.S; (1997); Analysis of steroidal sapogenins from amber fenugreek (Trigonella foenum-graceum) by capillary gas chromatography and combined gas chromatography/mass spectrometry. J Agric Food Chem; 45: 753-9.

6. Naidu M.M, Shyamala B.N, Naik J.P, Sulochanamma. G, Srinivas P; (2011); Chemical composition and antioxidant activity of the husk and endosperm of fenugreek seeds. J Food Sci Technol; 44:451-6.

7. Wu. Y, Cui. W, Eskin. N, Goff H.D; (2009); An investigation of four commercial galactomannans on their emulsion and rheological properties. Food Res Int ;42: 1141-6.

8. Bhatia. K, Kaur. M, Atif. F, Ali. M, Rehman. H, Raisuddin. S; (2009);Aqueous extract of T. foenum-graecum L. ameliorates additive urotoxicity of buthionine sulfoximine and cyclophosphamide in mice. Food and Chemical Toxicology;
44: 1744—.

9. Ikeuchi. M, Yamaguchi. K, Koyama. T, Sono. Y, Yazawa. K; (2006); Effects of fenugreek seeds (Trigonella foenum greaecum) extract on endurance capacity in mice, J. Nutr. Sci. Vitaminol., Tokyo, Vol. 52.

10. Khan F.U, Durrani F.R, Sultan. A, Khan R.U, Naz. S; (2009); Effect of fenugreek (Trigonella foenum-graecum) seed extract on visceral organs of broiler chicks. ARPN. Journal of Agricultural

11. Michael. S, Kumawat. D; (2003); Legend and archeology of fenugreek, constitutions and modern applications of fenugreek seeds. International-symp, USA: 41—.

12. Bhatia. K, Kaur. M, Atif. F; (2006); Aqueous extract of Trigonella foenum-graecum L. ameliorates additive urotoxicity of buthionine sulfoximine and cyclophosphamide in mice. Food and Chem Toxicol; 44:1744-50.

13. Bhalke R.D, Anarthe S.J, Sasane K.D, et al; (2009); Antinociceptive Activity of Trigonella foenum-graecum Leaves and Seeds (Fabaceae). IJPT; 8: 57-9.

14. . Bhalke R.D, Anarthe S.J, Sasane K.D, et al; (2009); Antinociceptive Activity of Trigonella foenum-graecum Leaves and Seeds (Fabaceae). IJPT; 8: 57-9.

15. Natrajan. B, Muralidharan. A, Satish. R, Dhananjayan R; (2007); Neuropharmacologicial activity of Trigonella foenum-graecum Linn. Seeds. J. Nat. Rem. 7; 160—.

16. Philip M; (2004); the use of the stable free radical diphenylpicryhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J. Sci. Technol. 26(2): 211-212.

17. Sapakal VD, Shikalgar TS, Ghadge RV, Adnaik RS, Naikwade NS and Magdum CS; (2008); In Vivo Screening of Antioxidant Profile: A Review, Journal of herbal Medicine and Toxicology 2 (2) 1-8.

18. Paoletti F, Aldinucci D, Mocali A and Caparrini A; (1986); A Sensitive Spectrophotometric Method for the Determination of superoxide Dismutase Activity in Tissue Extracts, Analytical Biochemistry,153, 536-541

19. Ohkawa H, Ohishi N, and Yagi K; (1979); Assay for Lipid Peroxidase in Animal Tissues by Thiobarbituric Acid Reaction, Analytical Biochemistry, 95, 351-358

20. Ellman G.L; (1959); Tissue Sulfyhydryl Groups Archeives of Biochemistry and Biophysics, 82, 70-77.

21. Preston J; Dean, B. J.; Galloway, S.; Holden, H.; McFee, A. F.; Shelby, M. (1987); Mammalian in Vivo Cytogenetic Assays Analysis of Chromosome Aberrations in Bone Marrow Cells. Mutat. Res. Toxicol. 189 (2), 157—.

22. Nema. N, Kharya M.D; (2012); Impact of Triphala on kupffer Cell Regeneration: A Possible Mechanism International Journal of Pharmacology and Pharmaceutical Technology (IJPPT), ISSN;1:2277 —3436.

23. Garcia. E, Oldoni. T, Alencar. S, Reis. A, Loguercio. A, Grande. R; (2012); Antioxidant Activity by DPPH Assay of Potential Solutions to be Applied on Bleached Teeth Braz Dent J 23(1).

24. Joe M. McCord; (2000); the Evolution of Free Radicals and Oxidative Stress AmJ Med; 108:652—659

25. Tripathi D.N, Jena G.B; (2008); Astaxanthin inhibits cytotoxic and genotoxic effects of cyclophosphamide in mice germ cells. Toxicology; 248:96—.

26. Patel J.M, Block E.R, Hood C.I; (1984); Biochemical indices of cyclophosphamideinduced lung toxicity. Toxicol App Pharmacol; 76:128—.

27. Daikh D.I, Wofsy D; (2001); Cutting edge: Reversal of murine lupus nephritis with CTLA4Ig and cyclophosphamide. J Immunol; 166: 2913—.

28. Ludeman S.M; (1999); the chemistry of the metabolites of cyclophosphamide. Curr Pharm Des; 5:627—.

29. Crook T.R, Souhami R.L, McLean A.E; (1986) ; Cytotoxicity, DNA cross-linking, and single strand breaks induced by activated cyclophosphamide and acrolein in human leukemia cells. Cancer Res; 46:5029—.

30. Doshi. M, Mirza. A, Umarji. B, Karambelkar. R; (2012); Effect of Trigonella foenum-graecum (Fenugreek/ Methi) on Hemoglobin Levels in Females of Child Bearing Age Biomedical Research; 23 (1): 47-50.

31. Umamaheswari. M, Chatterjee T.K; (2008); In vitro antioxidant activities of the fractions of Coccinia grandis L. leaf extract. Afr J Tradit Complement Altern Med;5:61-73

PDF
Share:
0 comments on Anti-Clastogenic effect of Trigonella foenum graecum in mouse bone marrow cells

Post a comment

Your email address will not be published. Required fields are marked *