|Year : 2010 | Volume
| Issue : 1 | Page : 22-29
In Vivo rodent micronucleus assay of Gmelina Arborea Roxb (Gambhari) extract
Rohit Sahu, Goli Divakar, Kalyani Divakar
Acharya & B.M. Reddy College of Pharmacy, Bangalore, India
|Date of Submission||18-Jan-2010|
|Date of Decision||22-Feb-2010|
|Date of Acceptance||10-Mar-2010|
|Date of Web Publication||2-Nov-2010|
Acharya & B.M. Reddy College of Pharmacy, Bangalore
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Gmelina arborea Roxb (family Verbenaceae) commonly known as 'Gambhari' tree, the various parts of the plants are widely used in diarrhoea, anti-pyretic, thirst, anemia, leprosy, ulcers, consumption, strangury, vaginal discharges. We tested the genotoxic potential of G. arborea in bone marrow cells obtained from Swiss albino mice using micronuclei formation as the toxicological endpoints. Aqueous extract of G. arborea (AEGA) was tested at the dose of 286 & 667 mg/kg body weight (b. w.). Cyclophosphamide (CPZ) 25 mg/kg b. w. was used as positive control in micronucleus test. The AEGA significantly increased the % micronucleated polychrometics at doses of 286mg/kg and 667mg/kg, after 24, 48 72h time interval. And also decreased the PCE/NCE ratio after 24, 48 and 72 h as compared to solvent control group. In this study, we investigated the effect of G. arborea on mammalian bone marrow cells using micronuclei formation to assess the genotoxicity of the herb.
Keywords: Micronucleus test, Cyclophosphamide, AEGA.
|How to cite this article:|
Sahu R, Divakar G, Divakar K. In Vivo rodent micronucleus assay of Gmelina Arborea Roxb (Gambhari) extract. J Adv Pharm Technol Res 2010;1:22-9
|How to cite this URL:|
Sahu R, Divakar G, Divakar K. In Vivo rodent micronucleus assay of Gmelina Arborea Roxb (Gambhari) extract. J Adv Pharm Technol Res [serial online] 2010 [cited 2021 Oct 18];1:22-9. Available from: https://www.japtr.org/text.asp?2010/1/1/22/70516
| Introduction|| |
Gmelina arborea Roxb (Family Verbenaceae), an important commercial timber species has been used in Ayurveda Since ancient times. A large proportion of medicines used by the locals in India are still derived from plants or their extracts and little is known about the safety and efficacy of such alternative treatments  . In vivo and in vitro studies have shown that some natural constituents of plants parts (fruits, leaves, roots) play a modulating role on xenobiotic effects  . While some herbs might be pharmacologically or clinically effective, they are not necessarily free of toxicity and side effects. Therefore, investigation into the traditionally used medicinal plants is valuable as a source for potential chemotherapeutic drugs and as a safety measure for the continued use of medicinal plants  .
It was proposed by Schmid and Heddle that a simpler approach to assess chromosome damages in vivo was to measure the micronucleus. Micronuclei (MN) are small chromatin bodies that appear in the cytoplasm by the condensation of acentric chromosome fragments or by whole chromosomes. Often induced by clastogenic substances or spindlepoison in dividing cells such as bone marrow. Micronuclei frequencies have been considered to be a reliable index for detecting chromosome breakages and loss ,, .
Mutagenicity testing assumes importance since the pollutants can cause deleterious somatic and heritable changes without showing any immediate toxic effects. Many a times these defects occur not only due to presence of genotoxic agent but also due to the lack of antimutagenic/ anticarcinogenic agent in our diet, the best way of minimize the effect is by identifying the anti mutagens and anticarcinogens in our diet and increasing their use  .
Micronucleus assay in mice is a well established method to study the mutagens and antimutagens  . G.arborea is a moderate sized unarmed deciduous tree, reaching 18 m.high; bark grayish yellow, rather corky; branchlets and young parts clothed with fine white mealy pubescence  . Its decoction is used as a diuretic for loosening phlegm as an appetite stimulant and in the treatment of various stomach disorders, fevers, skin problems, liver disorders ,, . G.arborea. is an important ingredient of generic Ayurvedic formulations "Dashamularishta" prescribed for several gynecological disorders and used in several commercial Ayurvedic preparations  . In vitro studies on bark and fruit extract showed anti oxidant activity and protected liver slice culture cells by alleviating oxidative stress-induced damage to liver cells  . The ethanolic extract of the G. arborea (steam bark) possess significant antiinflammatory activity  . G. arborea contains substances that possess significant anti diarrhoeal activity  . Wound healing study was conducted on the alcoholic extract of the dried leaves of G. arborea. G. arborea possess significant wound healing activity  . Thus, the aim of this preliminary study was to evaluate the incidence of micronuclei/chromosome damages induced by G. arborea when administered orally to mice at different doses and harvest periods.
| Materials and Methods|| |
Plant material used in this study was G.arborea. The leaves of G. arborea were shade dried and reduced to coarse powder in a mechanical grinder. The powdered material obtained was then subjected to extraction using Aqueous solvent in a Soxhlet extractor. The extract obtained was evaporated at 100ºC to get a semisolid mass. The extract prepared at the Dept. of Pharmacology, Acharya & B.M. Reddy College of Pharmacy, Bangalore (India). The plant extract was mixed with distilled water and made solution of 286mg/kg and 667mg/kg, of the mouse body weight.
Chemicals and reagents
Cyclophosphamide (Cadila health care Ltd, Goa), Bovine Serum Albumin (BSA) (Himedia), May-Grunewald's Stain (SD Fine Chemicals, Bangalore), Geimsa stain (NICE Chem Pvt Ltd Cohine), Methanol (Karnataka Fine Chemicals, Bangalore) were used in the study. All chemicals used in this study were of analytical grade purity and all test solutions were freshly prepared before each experiment.
Swiss albino mice of either sex weighing between 22-25g were used. Institutional Animal Ethics Committee approved the experimental protocol l997/c/6/CPCSEA; animals were maintained under standard conditions in the animal house approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA). Swiss albino mice were used in this study was obtained from the IISc, Bangalore. The animals were housed in Poly propylene cages and maintained at 24 ± 2ºC under 12 h light/ dark cycle and were fed ad libitum with standard pellet diet and had free access to water. The animals were given standard diet supplied by Pranav Agro Industries Ltd. Sangli. The composition of the diet are protein 10%, Arachis oil 4%, Fibers 1%, Calcium 1%, Vitamin A 1000 IU/gm and Vitamin D 500 IU/gm.
Young adult mice of the Swiss albino strain were procured from Indian Institute of sciences (IISc), Bangalore and acclimatized for at least 7 days before being placed on study. Mice were housed in polycarbonate cages in groups of six animals for each of the vehicle control, positive control and plant extract treatment (Test control). AEGA were dissolved in distilled water, which also served as vehicle control. Distilled water was administered by single oral gavage in a volume of 20 ml/kg (control), to groups scheduled for the 24,48 and 72 h harvest time point. AEGA served as test control and was administered by single oral gavage at a Low dose (286 mg/kg body weight) and High dose (667mg/kg body weight) to groups scheduled for the 24, 48 and 72 h harvest time point. Cyclophosphamide dissolved in sterile deionized water served as positive control and was administered in a dose of 25 mg/kg, i.p. to groups for marrow harvest at the 24, 48 and 72 h time point.
Bone marrow extraction
The animal was killed by cervical dislocation or made unconscious using anesthetic ether. Femur and tibia were cut open. Upper end of femur was cut open. Small opening was visible. Needle is inserted through aperture and 0.5 ml of suspending medium was flushed through the lower epiphyseal end in a clean cavity block. Flushing was performed four times to collect about 2 ml of bone marrow in a suspending medium. It was then centrifuged at 1000 rpm for 8 min , .
Fixing and staining
Narrow film was prepared by smearing of the supernatant and dried. Smear fixing is performed in absolute methanol for 10 min. Slide was then is kept in coupling jar containing May-Grunwald's stain in phosphate buffer (1:1, pH 6.8). The slides were transferred to Geimsa stain freshly diluted to it phosphate buffer (1:6) and kept for 30 min. Slide were washed (3-4 times) with distilled water. Slide is air-dried , .
Each treatment and control group consisted of six mice. Values are expressed as mean ±SEM. Asterisk symbol indicates *p <0.0001 extremely significant, **p < 0.01 very significant, as compared to control group.
Coded slides were analysed for micronuclei and the incidence of micronuclei in polychromatic erythrocytes (PCE) was recorded in 2000 PCE in each slide. Six animals were analysed for each group. Besides, the ratio of polychromatic to normochromatic erythrocytes (NCE) was also estimated by counting the 500 erythrocytes in each slide , .
A compound is considered mutagenic in this test system if at any of the preparation intervals, a statistically significant increase in the number of micronucleated PCEs is found in comparison to the vehicle controls. Data were analyzed using ANOVA followed by Dunnet comparison test to observe any significant differences amongst the dosage sets and harvest periods to asses the genotoxicity effects at p<0.05 level of significance. All the statistical analysis was performed using INSTAT statistical programme.
| Results|| |
The purpose for administration of AEGA during proliferation of haematopoietic cells was to observe whether the test compound can cause chromosome damages or inhibition of mitotic apparatus. The chromosome fragments or whole chromosomes may lag behind during cell division and form micronuclei as seen in positive control Cyclophosphamide-treated mice. The results of the in vivo micronucleus tests are presented in [Table 1]. The % micronuclei induced after administration of AEGA at three different harvest times (24, 48 and 72 h) and PCE: NCE cells ratio were statistically analyzed. The values were comparable with the negative/vehicle control values. A compound is considered to be mutagenic in this test system if at any of the preparation dose (286 and 667 mg/kg of body weight) and harvest periods (24, 48 and 72 h), has increase significantly in the number of MNPCEs when compared to the vehicle controls (distilled water).
In the present study, a dose 667 mg/kg b.w. very significantly (p < 0.01) increased the % micronuclei, 2.26, 6.26 and 7.25% after 24, 48 and 72 h as compared to solvent control group. The 286 mg/kg b.w. dose also induced the micronuclei formation in the above time period very significantly (p < 0.01). The % micronuclei were 2.05, 2.98 and 4.81% after 24, 48 and 72 h as compared to solvent control group. Cyclophosphamide, an established mutagen and positive control at the dose of 25 mg/kg b.w. induced the number of micronucleated PCE in all the time periods. The % micronuclei were 2.16, 5.19 and 9.13% after 24, 48 and 72 h as compared to vehicle control group.
A dose of 667 mg/kg b.w. and 286 mg/kg b.w. decreased the PCE/NCE ratio after 24, 48 and 72 h as compared to solvent control group.
| Discussion|| |
The mouse bone marrow micronucleus test is one of the several available in vivo mammalian test systems for the detection of chromosomal aberrations. Genotoxicity activity is indicated by statistically significant dose-related incidence of MNPCEs in the treatment group (AEGA). Inhibition of cell proliferation in the bone marrow illustrates the cytotoxicity of AEGA. Bone marrow cell toxicity (or depression) is normally indicated by a dose related decrease in the proportion of immature erythrocytes. Young adult mice were used in this study because of their high proliferative activity and low fat content which favour good quality preparations.
The evaluation of micronucleus frequencies in vivo is one of the primary genotoxicity tests recommended internationally by regulatory agencies for product safety assessment. An increase in the frequency (percentage) of micronucleated polychromatic erythrocytes (MNPCEs) in treated rats is an indication of induced chromosome damages. Decrease in the PCE: NCE ratio due to either direct cytotoxicity or micronuclei formation and heavy DNA damages leading to cell death or apoptosis ,,, .AEGA at the tested dose 667 mg/kg and 286 mg/kg b.w. induced a very significant increase in the frequency of micronuclei and decrease in PCE/NCE ratio after acute treatment [Table 1], confirming the mutagenic potential of the AEGA extract from leaves of G. arborea.
Therefore, the AEGA extract may act on the DNA, causing breaks and/or loss of entire chromosomes, and giving rise to micronuclei in the blood cells of the treated animals. The exact mechanism of action of AEGA is still unclear. It is important to know whether micronuclei induced by this extract are the results of chromosome breakages or chromosome loss.
| Acknowledgements|| |
I would like to acknowledge Mr. B. Premnath Reddy, Chairman, Acharya & B.M. Reddy College of Pharmacy, Bangalore, for his financial support for conducting my research work.
| References|| |
|1.||Hosny M, Rosazza JPN. Gmelinosides A-L, Twelve Acylated Iridoid Glycosides from Gmelina arborea. J Nat Prod. 1998:61(6):734-742. |
|2.||Roncada, T., Vicentinii V.E.P. & Mantovani, M.S. Possible modulating actions of plant extracts on the chromosome breaking activity of MMC and Ara-C in human lymphocytes in vitro. Mutation Research.2004: 18: 617-622. |
|3.||Verschaeve, L., Kestens, V., Taylor, J.L.S., Elgorashi, E.E., Maes, A., Puyvelde, L.V., Kimpe, D.N. & Staden, J.V. Investigation of the antimutagenic effects of selected South African medicinal plant extracts. Toxicology In Vitro.2004: 18: 29-35. |
|4.||Fenech, M. The in vitro micronucleus technique. Mutation Research.2000: 445: 81-95. |
|5.||Martinez, V., Creus, A., Venegas, W., Arroyo, A., Beck, J.P., Gebel, T.W., Suralles, J. & Marcos, R. Micronuclei assessment in buccal cells of people environmentally exposed to arsenic in northern Chile. Toxicology Letters.2005: 155: 319-327. |
|6.||Lajmanovich, R.C., Cabagna, M., Peltzer, P.M., Stringhini, G.A. & Attademo, A.M. Micronucleus induction in erythrocytes of the Hylapulchella tadpoles (Ampphibia: Hylidae) exposed to insecticide endosulfan. Mutation Research.2005: 587: 67-72. |
|7.||Ames B.N. science.1979: 204: 587-593. |
|8.||Rabbani S.I., Kshma Devi. Anti-mutagenic effect of citral against Mitomycin-C. J Nat Remedies. 2006; 6(1):77-82. |
|9.||Nadkarni AK, Indian Materia Medica. 1st ed. Bombay: Popular prakashan; 1976. p. 584. |
|10.||Sharma B.P., Balakrishnava N.P. Flora of India, 2, Botnical survey of Calcutta, India, 1993: 402-413. |
|11.||Sharma B.P., Yelne M.B., Yelne T.J. Database on medicinal plants used in Ayurveda, 3, Central council for research in ayurveda and siddha, Department of ISM and ministry of health and family welfare, Government of India, 2001: 217--228. |
|12.||Rastogi R.P., Mehrotra B.N. Compendium of Indian medicinal plants, 1, Central Drug Research Institute and Publication and Information Directorate, Lucknow, New Delhi, India, 1990: 203-210. |
|13.||Tewari D.N., A monograph on Gamari (Gmelina arborea Roxb). International Book Distributors. Dehradun, India, 1995: 1-84. |
|14.||Sinha S., Dixit p., Bhargava S., Ghaskadbi S. Pharma.Biol. 2006: 44: 237-243. |
|15.||Agrawal VK, Gambhir SS, Wahi AK. A preliminary study on Anti-inflammatory activity of some medicinal plants. Ind J Nat Prod 1994; 10 (1):14-15. |
|16.||Agunu A, Yusuf S, Andrew G.O., Zezi AU, Abdurahman EM. Evaluation of five medicinal plants used in dirrhoea treatment in Nigeria. J Ethanopharmacol 2005; 101(1):27-30. |
|17.||Shirwaikar A, Ghosh S, Rao PGM. Effect of Gmelina arborea Roxb. Leaves on wound healing on rats. J Nat Rem. 2003; 3(1):45-48. |
|18.||Health Effect Test Guidelines. 1998. Mamalian erythrocyte micronucleus test, OPPTS 870, 5395 United States Office of Prevention, Pesticides and Toxic Substances Environmental Protection Agency (7101). [Available from: URL:< href="http://www.epa.gov/epahome/researcnone h.htm">.http://www.epa.gov/epahome/researcnone h.htm |
|19.||Zaizuhana S, Puteri J, Noor MB, Noral AY, Hussin, MA, Bakar, Zakiah Ismail et al. The in vivo rodent micronucleus assay of Kacip Fatimah (Labisia pumila) extract. Tropical Bio med 2006; 23 (2):214-9. |
|20.||Krishna, G. & Hyashi, M. In vivo rodent micronucleus assay: protocol, conduct and data interpretation. Mutation Research.2000: 455: 155-166. |
|21.||Ouanes, Z., Abid, S., Ayed, I., Anane, Mobio, T., Creppy, E.E. & Bacha, H. Induction of micronuclei by Zearalenone in Vero monkey kidney cells and in bone marrow cells of mice: protective effect of Vitamin E. Mutation Research. 2003: 538: 63-70. |