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PHARMACOLOGICAL STUDY
Year : 2012  |  Volume : 33  |  Issue : 3  |  Page : 418-422  

Evaluation of subchronic genotoxic potential of Swarna Makshika Bhasma


1 Reader, Department of Rasa Shastra and Bhaishajya Kalpana, Ashwini Ayurvedic Medical College and Research Centre, Tumkur, Karnataka, India
2 Associate Professor, Department of Rasa Shastra and Bhaishjya Kalpana including Drug Research, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
3 Lecturer, Department of Pharmacology, SSR College of Pharmacy, Silvasa, UT of D and NH, India
4 Director, Research and Developement, SDM College of Ayurveda, Kuthpady, Udupi, Karnataka, India
5 PhD Scholar, Pharmacology Laboratory, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India

Date of Web Publication16-Mar-2013

Correspondence Address:
Pavan B Savalgi
Department of RS and BK, Ashwini Ayurvedic Medical College and Research Centre, Tumkur - 572 105, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-8520.108858

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   Abstract 

Extremely diminutive published information is available on the mutagenic activity of Ayurvedic Bhasmas. Genotoxicity of few Bhasmas were reported on single maximum dose, but no reference is available on the sub-chronic level. Hence the present study was carried to generate and evaluate genotoxic potentials of Swarna Makshika Bhasma (mineral preparation) administered at therapeutic dose for 14 days. Chromosomal aberrations and abnormal sperm assay parameters were taken in this study. Cyclophosphamide (CP) was taken as positive group and results were compared. The results revealed a lack of generation of structural deformity in above parameters by tested drugs compared to CP treated group. Observed data indicate that the Bhasmas tested were non-genotoxic under the experimental conditions.

Keywords: Abnormal sperm assay, chromosomal aberrations, cyclophosphamide, genotoxicity, Swarna Makshika Bhasma


How to cite this article:
Savalgi PB, Patgiri B, Thakkar JH, Ravishankar B, Gupta VB. Evaluation of subchronic genotoxic potential of Swarna Makshika Bhasma. AYU 2012;33:418-22

How to cite this URL:
Savalgi PB, Patgiri B, Thakkar JH, Ravishankar B, Gupta VB. Evaluation of subchronic genotoxic potential of Swarna Makshika Bhasma. AYU [serial online] 2012 [cited 2020 Feb 27];33:418-22. Available from: http://www.ayujournal.org/text.asp?2012/33/3/418/108858


   Introduction Top


Ayurveda is known and carried forward as an ancient Indian heritage. It is a traditional medical system used by a majority of Indian population. [1] The drugs known as "Bhasmas0" are well-known in the traditional Indian Ayurveda and these are chemically mixed oxides of one or more metals. [2] Their traditional preparation involves conversion of a pure metal into its oxide form following a typical procedure, available in the ancient literature of Ayurveda. [3] Recently, doubts have been raised about the safety of Ayurvedic preparations using Bhasma and concerns were expressed regarding the metal toxicity of traditional preparations containing Bhasmas. Ayurveda fraternity claims that these medicines, if properly prepared and administered are safe and therapeutic. [4] According to them toxicity can arise only from a metal in its free form, and that a Bhasma prepared according to the classical methods never contains a metal in free form. Despite these theories claiming Bhasmas are non-toxic, documented case reports of poisoning was noted. [5],[6] Hence the current study of Swarna Makshika Bhasma (SMB) would serve as a database of baseline information for genotoxicity, since there is apparently no literature on this aspect of mineral preparations.


   Materials and Methods Top


Test drugs

Three different samples of Swarna Makshika (SM) were collected from different mines across India. Bhasmas were prepared according to classical reference. [7] Coded as mentioned below:

  1. SMB prepared from samples collected from Khetri Mine, Rajasthan (SMBKR).
  2. SMB prepared from samples collected from Hatti Gold Mine, Karnataka (SMBHK).
  3. SMB prepared from Malharjkhand Mine, Madhya Pradesh (SMBMM).
Chemicals

Colchicine was obtained from Hi-media, Mumbai. Cyclophosphamide (CP) procured as Cyphos vial from Intas Pharmaceuticals, Mumbai. Potassium chloride, methanol, acetic acid, giemsa stains were obtained from Sissco Research Laboratory, Mumbai, India.

Study design

Animals


Adult Swiss albino mice (weighing 25 ± 5 g) procured from an institutional animal house attached to SSR College of Pharmacy, Silvasa, where the study was conducted. Animals were maintained with controlled temperature (23 ± 2°C), relative humidity of 50 ± 10% and 12 h light/dark photo-period. The animals were acclimatized for 7 days prior to the experiment and provided with standard mice feed and distilled water ad libitum. Study was carried out after obtaining approval from Institutional Animal Ethics Committee (IAEC/2011/01).

Animals were randomly divided into five groups (5 mice per group) after an acclimatization period. Group I served as positive control and challenged with CP single-dose of 25 mg/kg body weight intra-peritoneally 24 h prior to termination. Group II served as vehicle control. Vehicle was prepared in combination of honey and deionized water (with a ratio of 1:1.5) and administered in the dose of 0.5 ml/kg body weight as per CCRAS/NIN guidelines. Group III, IV, and V received SMBKR, SMBHK and SMBMM at therapeutic dose 4.5 mg/kg body weight respectively along with vehicle for 14 consecutive days and sacrificed on the 15 th day. The doses of test drugs were calculated as per the reference of Paget and Barnes (1969).

Body weight

Animals were examined throughout the experimental period for signs of gross toxicity. Body weight was recorded initially and at the time of sacrificed on the 15 th day.

Experimental procedure

Chromosomal aberration assay


Animals were injected colchicine intra-peritoneally at the dose of 4 mg/kg body weight, on the 15 th day in order to arrest dividing cells in metaphase [8] and sacrificed by cervical dislocation, 90 min after the colchicine treatment. Bone marrow cells from both femurs were extracted, subjected to hypotonic shock treatment (KCl 0.075 M), for about 30 min, at room temperature and then centrifuged at 1000 rpm for 10 min. The cells were fixed 5 times using freshly prepared methanol-acetic acid (3:1). The cells were spread on clean glass slides that were dried on a hot plate at 40°C. One more drop of fixative was added on slides to see more reliable pictures of chromosomes and then the slides were air dried at room temperature and finally stained with a 5% dilution of Giemsa reagent in a phosphate buffer (pH 6.8) for 15 min. The chromosomes of 1000 cells in metaphase abnormalities were analysed with a ×100 oil immersion objective, using a Trinocular Research Carl Zeiss Microscope (Germany). Metaphases with chromosomes and chromatid breaks, gaps, rings, stickiness, centric fusion, and deletion were recorded. [9]

Sperm abnormality assay

The method of Wyrobek and Bruce [10] was used for investigating sperm morphology abnormality assay. The test preparations were administered for 14 days, to correlate the results with positive control group. On 15 th day, the overnight fasted animals were sacrificed by cervical dislocation and dissected out. Both the cauda epididymus were removed and placed in a watch glass containing 1 ml phosphate buffered saline (pH 7.2). Then minced and teased carefully well with fine scissors and forceps to release the spermatozoa. After gentle pipetting, the suspension was separated from the tissue fragments and filtered through double layers of muslin cloth to remove the tissue debris. A drop of Eosin Y solution (10:1) was added to this suspension and kept for 30 min. Air dried smears were prepared on clean, grease-free glass slides and a uniform smear was made. About 1000 sperms per animal were examined at ×400 magnifications from each treatment and control groups for the presence of sperm morphological abnormalities.

Statistical analysis

Statistical methods were carried out only to assess change in body weight by applying paired t-test. Statistics were not applied in CA assay and sperm abnormality assay. Different kinds of morphological changes were observed in CAs and sperm abnormal aberrations.


   Observations and Results Top


The effect of SMBs on body weight, chromosomal aberration and sperm abnormality assay are shown in [Table 1], [Table 2] and [Table 3].
Table 1: Effect of SMBs on body weight

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Table 2: Effect of SMBs on chromosomal aberration

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Table 3: Effect of SMBs on sperm abnormality assay

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   Discussion Top


In vivo CA assay is one of the most frequently used and sensitive tests for the detection of the genotoxic profiles of drugs. The test has been recommended for routine analysis and data obtained are considered highly relevant in human context. [11],[12] In the present study a 14-day sub-chronic genotoxicity of SMBs prepared by different samples are evaluated by employing in vivo CA assay and abnormal sperm assay (ASA). Although the genotoxic profile of some of Bhasmas have been evaluated in various studies, [13] Till date no reports of sub-chronic genotoxicity studies on Bhasmas and SMB are available. With this view body weight of animals also recorded after 14 days of drug administration and compared with CP group [Table 1]. All treated groups exhibited significant gain in body weight in comparison to CP. Body weight loss is an indicator of marked tissue loss in the body protein degradation. Gain of body weight is indicating that test drugs are not bearing degenerative potentials.

Colchicine is effective in causing metaphase stasis in cell dividing matrix. [14] Thus used to arrest metaphase, when chromosome structure seen noticeably. It inhibits microtubule polymerization by binding to tubulin, one of the main constituents of microtubules. Availability of tubulin is essential to mitosis, and therefore, colchicine effectively functions as a "mitotic poison" or spindle poison. Hypotonic solution (KCl) causes the cells to swell and enhances eventual separation of the chromosomes to facilitate visual analysis. CP is an anticancer drug that is widely used in anti-neoplastic therapy as well as in the treatment of some non-malignant diseases like rheumatoid arthritis. It is also used as an immunosuppressive agent prior to organ transplantation. [15] In somatic cells, CP has been shown to produce 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. The compound also produced chromosome damage and micronuclei in rats, mice and Chinese hamster. [16] Its use as a positive control chemical in genotoxicity tests has been recommended. [17] It increased the number of chromosome aberrations in the given dose with relatively high frequencies of chromosome breaks, centric fusion compared with other types of chromosome abnormalities. Gap, ring formation as well as stickiness were also frequent in CP treated group [Figure 1]a-f. This may be because almost all mouse chromosomes are acrocentric. These types of chromosomes have the exceptional facility to merge with each other. Only structural aberrations were enumerated in all treated groups [Figure 1]g-j against CP treated group, with special emphasis on chromosome and chromatid gap, breaks and centric diffusions placed in [Table 2].
Figure 1:

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Morphological abnormalities of sperms are described as two types as head and tail abnormalities. The head abnormality included amorphous shape, without hook, banana shaped and folded head. CP treated group observed maximum number of abnormalities in both head and tail as shown in [Table 3]. Amorphous shaped head, hook less head and coil-tailed abnormalities were more frequent than other abnormalities of head and tail of CP treated group [Figure 2]a-d. The test preparations observed negative results in sperm abnormality showing non-toxic to sperms [Figure 2]e-h. Wyrobek [18] reported that large reductions in sperm number or mortality or large increases in sperm with abnormal shapes are associated with reduced fertility.
Figure 2:

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   Conclusion Top


Present study revealed that SMB prepared from different samples were found to be safe after the administration for 14 days at the therapeutic doses. No abnormality was noticed in CA and sperm abnormal aberrations in all trial groups. Further, above findings provide new information that may be more imperative for the use of Bhasmas.


   Acknowledgment Top


Authors acknowledge to Chairman/Principle, SSR College of Pharmacy for his kind permission for the experimental study. Authors are also indebted to Director, Prof. M.S. Baghel, I.P.G.T. and R.A., G.A.U., Jamnagar, for financial support for the project. The authors are thankful to Prof. P. K. Prajapati, Head, and Dr. Galib, Asst. Prof., Dept of R.S. and B.K., I.P.G.T. and R.A., G.A.U., Jamnagar, for their valuable help.

 
   References Top

1.Gogtay NJ, Bhatt HA, Dalvi SS, Kshirsagar NA. The use and safety of non-allopathic Indian medicines. Drug Saf 2002;25:1005-19.  Back to cited text no. 1
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2.Wadekar MP, Rode CV, Bendale YN, Patil KR, Prabhune AA. Preparation and characterization of a copper based Indian traditional drug: Tamra bhasma. J Pharm Biomed Anal 2005;39:951-5.  Back to cited text no. 2
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3.Kulkarni-Dudhgaonkar SB. In: Rasaratna Samuchyaya of Vagbhatta. Kolhapur, India: Shivaji University Publication 1970. p. 158.  Back to cited text no. 3
    
4.Ernst E. Heavy metals in traditional Indian remedies. Eur J Clin Pharmacol 2002;57:891-6.  Back to cited text no. 4
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5.Araujo J, Beelen AP, Lewis LD, Robinson GG, DeLaurier C, Carbajal M, et al. Centres for Disease Control and Prevention. Lead poisoning associated with use of Ayurvedic Medications - five states, 2000-2003. MMWR Morb Mortal Wkly Rep 2004;53:582-4.  Back to cited text no. 5
    
6.Buwa S, Patil S, Kulkarni PH, Kanase A. Hepatoprotective action of abhrak bhasma, an ayurvedic drug in albino rats against hepatitis induced by CCl4. Indian J Exp Biol 2001;39:1022-7.  Back to cited text no. 6
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7.Vagbhatta, Rasaratna Samucchaya. In: Kulkarni DA, editor. 3 rd ed., verse 2/78-80. New Delhi: Meharchandra Laxshandas Publication; 1987. p. 28.  Back to cited text no. 7
    
8.Mueller GA, Gaulden ME, Drane W. The effects of varying concentrations of colchicine on the progression of grasshopper neuroblasts into metaphase. J Cell Biol 1971;48:253-65.  Back to cited text no. 8
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9.Tjio JH, Whang J. Chromosome preparations of bone marrow cells without prior in vitro culture or in vivo colchicine administration. Stain Technol 1962;37:17-20.  Back to cited text no. 9
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10.Wyrobek AJ, Bruce WR. The induction of sperm shape abnormalities in mice and humans. In: Hallender A, de Serres FJ, editors. Chemical Mutagens. Vol. 5. NewYork: Plenum Press 1975. p. 275-85.  Back to cited text no. 10
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11.Anonymous. International commission for protection against environmental mutagen and carcinogens report of committee. Mutat Res 1983;114:120-77.  Back to cited text no. 11
    
12.World Health Organization. Guidelines to short-term tests for detecting mutagenic and carcinogenic chemicals. Environmental Health Criteria 51. Geneva: WHO; 1985. p. 100-14.  Back to cited text no. 12
    
13.Vardhini NV, Sathya TN, Murthy PB. Assessment of genotoxic potential of herbo-mineral preparations - Bhasmas (Research communications). Curr Sci 2010;99:1096-100.  Back to cited text no. 13
    
14.Fraser IEB. The use of colchicine and colcemid for metaphase stasis in the matrix cells of wool follicles. Aust J Biol Sci 1962;16:211-17.  Back to cited text no. 14
    
15.Jackson MA, Stack HF, Waters MD. Genetic activity profiles of anticancer drugs. Mutat Res 1996;355:171-208.  Back to cited text no. 15
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16.Anderson D, Bishop JB, Garner RC, Ostrosky-Wegman P, Selby PB. Cyclophosphamide: Review of its mutagenicity for an assessment of potential germ cell risks. Mutat Res 1995;330:115-81.  Back to cited text no. 16
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17.Krishna G, Petrere J, Anderson J, Theiss J. Use of cyclophosphamide as a positive control in dominant lethal and micronucleus assays. Mutat Res 1995;335:331-7.  Back to cited text no. 17
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18.Wyrobek AJ, Gordon LA, Burkhart JG, Francis MW, Kapp RW Jr, Letz G. An evaluation of the mouse sperm morphology test and other sperm tests in non-human mammals: A report of the US Environmental Protection Agency Gene Toxicology Program. Mutat Res 1983;115:1-72.  Back to cited text no. 18
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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