|Year : 2013 | Volume
| Issue : 2 | Page : 204-208
Analytical study of raw Swarna Makshika (Chalcopyrite) and its Bhasma through TEM and EDAX
Sudhaldev Mohapatra1, Chandra Bhushan Jha2
1 Lecturer, Department of Rasa Shastra, A and U Tibbia College, Karol Bagh, New Delhi, India
2 Professor, Department of Rasa Shastra, Banaras Hindu University, Varanasi, Uttar Pradesh, India
|Date of Web Publication||10-Oct-2013|
Department of Rasa Shastra, A and U Tibbia College (Govt. of NCT), Karol Bagh, New Delhi - 05
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Ayurveda, the Indian System of Medicine deals with a number of metals and minerals. Swarna Makshika (SM), a chalcopyrite one such minerals has iron (Fe), copper (Cu), and sulphur (S) as major ingredients along with other trace elements of therapeutic importance. Studies related with characterization of SM are very few. In the current study SM and SM Bhasma were analysed through Transmission Electron Microscope (TEM) and Energy Dispersive X-Ray Analysis (EDAX). Analysis reveals the presence of iron, copper, and sulphur in SM. In addition to these elements, SM Bhasma found to contain Potassium, Magnesium, Aluminum, and Silicon in trace amount. TEM study reveals that, grain size of the SM (5-10 microns) is significantly reduced in SM Bhasma to 50-200 nm.
Keywords: Bhasma , chalcopyrite, energy dispersive X-ray analysis, standardization, Swarna Makshika, transmission electron microscope
|How to cite this article:|
Mohapatra S, Jha CB. Analytical study of raw Swarna Makshika (Chalcopyrite) and its Bhasma through TEM and EDAX. AYU 2013;34:204-8
| Introduction|| |
Analytical study is one of the imperative parts for drug standardization in Ayurveda, Siddha, and Unani (ASU) systems of medicine. In today's era of science and information technology, it is essential that the details about the medicine consumed by the consumer must be explored to the scientific community and regulatory authority for its validation, authentication, and future development when necessary, for the greater benefit of the particular system and society as well. Though the Bhasmas are in clinical practice since hundreds of years, the need of the hour is to scientifically validate these by using modern tools and techniques.
Quality control methods of Bhasmas were well developed by seers based on the parameters prevailing in those days. Though the parameters have their own significance, they have certain limitations and are not sufficient to characterize the Bhasma with respect to their physicochemical nature. Therefore use of sensitive modern tools and techniques are necessary to characterize the Bhasma. Considering this, an effort has been made to analyze the raw material (SM), and finished product (SM Bhasma) through TEM and EDAX. Prior to subjecting the material to TEM or EDAX, attempts were made to examine the Bhasma through classical parameters of analyses.
| Pharmaceutical Process|| |
The pharmaceutical process included Shodhana (purification) and Marana (incineration) of Swarna Makshika (SM) to prepare genuine Bhasma. For Shodhana, the initial material was roasted at a temperatureof about 850-900°C with frequent addition of lemon juice in a charcoal furnace till it appeared red in color, and it took three days for achieving the desired features of Shodhana. , Then the material was subjected to Marana process. For the Marana, lemon juice was used as Bhavanadravya (material for wet trituration). In conventional Puta (heat source) system of heating, 11 Putas were given to achieve desired quality of Bhasma and for each Puta, 4 kg cow dung cakes were used. 
After completion of the pharmaceutical process, the Bhasma was subjected to classical quality control parameters followed by analyses through Transmission Electron Microscope (TEM) and Energy Dispersive X-Ray Analysis (EDAX) analysis.
| Materials and Methods|| |
The raw material (SM) was collected from Ayurvedic Pharmacy, Banaras Hindu University and authenticated by studying its characters as explained in classical texts. , Shodhita SM and SM Bhasma are prepared in the Department of Rasa Shastra, Institute of Medical Sciences, Banaras Hindu University.
For TEM and EDAX: Properly calibrated EDAX attached TEM (Model-Technai-20G2, Voltage-200 KV, FEI, USA), mortar and pestle, mounting stub, copper grid, sample holder were taken and maintained as required.
Ancient analytical methods
Rekhapurnatvam : A pinch of prepared SM Bhasma should be taken in between the thumb and index finger and rubbed. Properly prepared Bhasma should enter into the creases of the fingers and should not easily cleansed out. This test confirms the fineness of Bhasma.
Varitaratavam: Small amount of the prepared Bhasma is to be sprinkled over the stagnant water ina beaker. An ideal Bhasma should float over the surface of the water. 
Nisvadutvam: Genuine Bhasma should be tasteless when a small amount was kept over tongue.
AmlaPariksha: When a pinch Bhasma is mixed with little amount of Dadhi (curd) in a clean petridish, kept for 24 h, no color change should occur. Similar observations should occur when lemon juice is added.
Dantagrekachkachabhava: No grittiness should be observed, when a small amount of Bhasma is placed between the teeth.
Avami: Properly manufactured Bhasma should not produce any nausea or vomiting.
Analysis using modern parameters
The raw SM, intermediary product (Shodhita SM) and final product (SM Bhasma) were subjected to TEM and EDAX at Department of Physics, BHU.
Principle of imaging technique in TEM
In TEM, a crystalline material interacts with the electron beam mostly by diffraction rather than absorption. The intensity of the diffraction depends on the orientation of the planes of atoms in a crystal relative to the electron beam. A high contrast image is formed by blocking electrons deflected away from the optical axis of the microscope by placing the aperture to allow only unscattered electrons to pass through. This produces a variation in the electron intensity that reveals information on the crystal structures, and can be viewed on a fluorescent screen, or recorded on photographic film or captured electronically. 
Energy dispersive X-ray analysis
It is an analytical technique used for the elemental analysis or chemical characterization of a sample. Being a type of spectroscopy, it relies on the investigation of a sample through interactions between electromagnetic radiation and matter and then analyzing the X-rays emitted by the mater in response to being hit with charged particles. Its characterization is due to the fundamental principle, that each element has a unique atomic structure. ,
EDAX systems are found attached on TEM equipped with a cathode and magnetic lenses to create and focus a beam of electrons. A detector is used to convert X-ray energy into voltage signals; this information is sent to a pulse processor, which measures the signals and passes them onto an analyzer for data display and analysis. 
Preparation of the sample
Sample was grinded in mortar and pestle and was made sufficiently fine. A stub was taken; the sample to be analyzed taking in a grid, was mounted to the stub, and was subjected to scanning under TEM.
Scanning the sample under TEM
The mounted sample was placed inside the vacuum chamber of microscope through an air-tight door, and then the air was pumped out. After the air was pumped out of the chamber, a beam of high electron beam was emitted by an electron gun from the top. This beam travels downward through a series of magnetic lenses designed to focus the electrons to a very fine spot. Near the bottom, a set of scanning coils made the focused beam to move back and forth across the mounted sample, row by row. As the electron beam hits each spot on the sample, secondary electrons are back scattered from its surface. A detector counts these electrons and sends the signals to an amplifier. The final image was built up from the number of electrons emitted from each spot on the sample. 
It was found during the classical analytical parameters that, the Bhasma passed all the tests of analysis. It implies that, the finished product is genuine.
TEM reveal that, the grains in the raw material and the Shodhita sample are not uniformly arranged [Figure 1], [Figure 2], [Figure 3] and [Figure 4], while they were distributed uniformly in Bhasma [Figure 5]. More clustering of grain was observed in the final product [Figure 6], whereas in raw material [Figure 1] and in the intermediary product [Figure 3] the grains were found scattered. The size of the grain was significantly reduced in the final product. The grain in the raw material was in between 5 to 10 microns [Figure 1] and in the Bhasma it was 50 to 200 nm [Figure 6].
EDAX study reveals that raw material contains Cu-33.08%, Fe-28.60%, S-32.85%, and O-5.47% w/w [Figure 7]. Shodhita material found to contain Cu-21.45%, Fe-49.25%, S-08.38%, and O-20.92% w/w [Figure 8], while SM Bhasma contains Cu-29.40%, Fe-32.26%, S-02.45%, O-24.05%, Mg-03.05%, Al-02.23%, Si-05.28%, and K-01.23% [Figure 9]. It was observed from the analysis that the quantity and quality of trace elements are increasing with succeeding the number of Putas with decreasing of the sulfur percentage and increasing of the oxygen percentage.
|Figure 9: EDAX report of raw Swarna Makshika after 11th Puta (Swarna Makshika Bhasma)|
Click here to view
| Discussion|| |
Ancient pharmaceutical processes are known to convert metals and minerals into therapeutically potential drugs that are vogue since centuries. But need of the time is to explain the physico-chemical nature of raw material and the finished products as well. Considering importance of this aspect, the raw SM and its Bhasma were analyzed by TEM and EDAX todevelop analytical profile of both the samples.
Analysis of TEM reports of raw material, and Bhasma elucidates gradual reduction of the grain size, increase in regularity, and uniformity of the grain with increasing the number of Putas. The significant reduction of the particle size of the final product justifies the Rekhapurnata and Varitaratwa character of Bhasma claimed in the ancient texts. It highlights the ancient technology of size reduction and their applicability in biomedical system in view of the current nanotechnology.
Size of the grains of SM was 5-10 microns, whereas size of the grains in SM Bhasma was 50-200 nm. It was observed that the grain size of the Bhasma was not having a particular fixed size rather; it was in a range and the particle size was significantly less in comparison with that of the raw material. This indicates that the process generates uniformity, regularity, and gradual reduction in size. In addition, the study revealed the crystalline diffraction pattern and three dimensional observation of surface phenomenon of the sample under high resolution.
From the EDAX study, it was observed that the amount of sulphur gradually decreases and amount of oxygen increases from raw material to Bhasma and the Bhasma contains least amount of sulphur and maximum amount of oxygen. This happens due to sublimation of sulphur from raw material while heating and substitution of oxygen during the process. Thus, the Bhasma may be more suitable to the body as it contains more oxygen and least sulphur.
It was also observed that trace elements like Mg, K, Si, and Al were found in the prepared Bhasma, which might enter into finished product through the process of Bhavana (wet trituration) with lemon juice. Lemon juice is reported to contain many trace elements.  The presence of silicon may be due to using of earthen casseroles in the Puta system of heating in Marana process. The relative percentage of iron is increased in prepared Bhasma because of the loss of sulphur, and some iron may be present in the Bhavana drugs used and some may be entered from the utensils used in roasting of raw material during Shodhana. Analyzing critically, it was observed that the trace elements increase both quantitatively and qualitatively with increasing of the number of Puta.
| Conclusion|| |
This study concludes that the ancient pharmaceutical processes are very effective in converting the macro elements into therapeutically effective medicines of micro form. The size of the raw material (5-10 microns) was reduced to 50-200 nm in the Bhasma. It implies that, the classical procedures reduce the particle size to a considerable level. Increase of Fe and Cu along with other trace elements and decrease of sulphur was observed in the Bhasma in EDAX study. Findings of elemental analysis of EDAX showing quantitative analysis of the samples can be considered in future studies.
| Acknowledgment|| |
Authors would like to acknowledge Prof. O. N. Srivastava and Dr. Devanand, Department of Physics, BHU for their cordial support and cooperation during the analytical study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]