T Sathya, B Murthy, N Vardhini
ayurveda, bhasma, comet, dna damage, genotoxicity, micronucleus
T Sathya, B Murthy, N Vardhini. Genotoxicity evaluation of certain Bhasmas using Micronucleus and Comet assays. The Internet Journal of Alternative Medicine. 2008 Volume 7 Number 1.
Bhasmas, herbal preparations of ayurvedic origin, contain heavy metals in traces. Very little/ no published information is available on the preclinical toxicity or mutagenicity of these Bhasmas. Considering the recent controversy over the risk of toxic heavy metals in ayurvedic herbo-mineral preparations, we studied the genotoxic potential of few such preparations. Most popular Bhasmas were investigated - Ras Manikya Ras, Lauha Bhasma, Tamra Bhasma and Kajjali Bhasma. A single dose (200mg/kg b.w) was administered orally to Wistar rats. Peripheral blood leukocytes and bone marrow samples were collected. Micronucleus assay and the comet assay were employed to study the endpoint of chromosomal damage and single / double-strand DNA breaks. The results revealed lack of induction of Micronuclei or DNA damage as evidenced by the Comet assay, despite the presence of traces of transformed toxic heavy metals.
Rasa shastra (Vedic chemistry) , a branch of Ayurveda, describes the use of metals, gems, minerals and even poisons for manufacturing special formulations to combat chronic and difficult diseases. Several metallic preparations with organic macromolecules termed “Bhasmas”, in Ayurvedic literature, are employed in the treatment of a variety of disorders 6. Bhasma preparations involve the conversion of the metal into its mixed oxides, during which, the zero valent metal state is converted to a higher oxidation state. The significance of this “Bhasmikarana” is that the toxic nature of the resulting metal oxide is completely destroyed while introducing the medicinal properties into it 18.
Traditionally, many Indians have been using the Ayurvedic herbo-mineral preparations (Bhasmas) for the treatment of chronic ailments. Although no systematic pre-clinical and clinical studies on the efficacy and toxicity of these preparations are published, they are considered to be safe in view of clinical experience as recorded in the ancient Indian documents 7. Recently, Saper
For sometime now, we have been involved in gathering preclinical and genotoxicity information on some commonly used Bhasmas. As a part of this, we report here our findings of genotoxicity with respect to four Bhasmas - Ras Manikya Ras, Lauha Bhasma, Tamra Bhasma and Kajjali Bhasma. We selected these Bhasmas for our studies due to their extensive clinical use in India. Further, we thought that these Bhasmas would be more pertinent for toxicity and genotoxicity investigations since they contain metal ions of arsenic, mercury etc which are intentionally incorporated during their preparation and since some of these metals are known genotoxic agents.
Materials and methods
The Bhasmas - Ras manikya ras, Lauha Bhasma, Tamra Bhasma and Kajjali Bhasma were obtained commercially.
Fetal Bovine Serum (Gibco), Cyclophosphamide (Sigma), Low melting agarose (Sigma), Normal melting agarose (Sigma), Ethidium Bromide (Sigma), Tris base (Hi Media), Triton X 100 (Hi Media), DMSO (Sigma).
Six to eight week old Wistar rats weighing 100-150gm were obtained from the animal house – breeding facility, International Institute of Biotechnology and Toxicology (IIBAT), where the study was conducted. Animals were acclimatized for seven days and maintained on a standard feed and water
Animals were divided into 6 groups of 10 animals each (5 males and 5 females). Group 1 served as vehicle control receiving the vehicle alone (Corn oil) orally, Group 2 animals were dosed with the positive control chemical, Cyclophosphamide at the concentration of 25mg/kg b.w intraperitoneally. Group 3, 4, 5 and 6 received the Bhasmas- Ras Manikya Ras, Lauha Bhasma, Tamra Bhasma and Kajjali Bhasma, respectively, at the dose of 2000mg/kg b.w. suspended in corn oil by oral intubation. (Dose selected based on pilot study, data not presented here).
After 24 hrs of dosing, the animals were sacrificed by cervical dislocation. The femurs were exposed, cut just above the knee and the bone marrow was aspirated into tubes containing Fetal Bovine Serum (FBS). The cells were centrifuged and the supernatant was drawn off. The cells were re-suspended in minimal volume of FBS. The bone marrow suspension was spread onto a clean glass slide. The slides were fixed in Methanol, stained with Giemsa and mounted. With the oil-immersion objective, 2000 immature Poly Chromatic Erythrocytes were scored for the presence of “Micronucleus”, which are defined as round, darkly staining nuclear fragment indicating chromosome damage. To evaluate any cytotoxicity induced by the administered substances at the dose selected, the proportion of Polychromatic Erythrocytes to Normo Chromatic Erythrocytes was determined in 200 cells for each animal in all treatment groups.
The comet assay was performed under alkaline conditions essentially following the procedure of Singh
The objects were observed at 400x magnification in a Carl Zeiss Axiophot fluorescence microscope (Carl Zeiss, Germany). For DNA damage analysis, 100 cells were scored per slide (Singh and Stephens, 1997). Cells were assigned to various damage degrees, visually, based on their tail intensities. The percentage of damaged cells was calculated manually. An arbitrary unit (AU) was used to express the extent of DNA damage.
Where ni is the number of cells with damage degree
Table 1 shows that the ratio of Poly Chromatic Erythrocytes and the Normo Chromatic Erythrocytes
The DNA damage percentage in the different groups
Metallic herbal preparations offer advantages over plant drugs by virtue of their stability over a period, lower dosage, easy storability and sustained availability and contain minerals and metals as integral part of the formulations 2.They are being used with an intention to give therapeutic efficacy to the product of the designated illness. Use of metals in medicine is often associated with toxicity 1. It is to remove those toxic qualities of the metals that the preparations undergo various processes of purification (Shodhana, Marana, and Samskara). The metals and minerals are mixed with herbs because they are considered non-living and by treating them with herbs they are converted to a living state thereby becoming bio-compatible. The same metal processed with different herbs acts on different organs in the human body.
The results reveal that the four Bhasmas investigated are not genotoxic in the Micronucleus and the Comet assays. The micronucleus assay used in this investigation is a powerful technique that detects chromatid / whole chromosome loss. The comet assay is presently used world wide in pharmaceutical industries for preliminary screening of drugs and genotoxicity assessment of heavy metal contaminations in various test species. This assay requires fewer samples and is cost-effective. It detects DNA damages in the form of Single and double-strand breaks and alkali-labile sites (ALS).
The results reiterate the fact, Bhasmas, despite their trace heavy metal content, are safe, when appropriately manufactured and consumed as per directed instructions. It also re-emphasizes that the mere presence of a chemical compound of metallic origin does not contribute to the toxicity of the finished product as the standard manufacturing process inflicts intense changes and components of herbal origin after sequential reactions with diverse components of processing is responsible for the therapeutic action.
We wish to thank the Management of IIBAT for the support and encouragement.