XRD, SEM- EDAX, AAS Analysis of Kukkutandatwak BhasmaAmavata, Rheumatoid Arthritis, Deepana-Pachana, Shleshmadara Kala

INTRODUCTION

Kukkutanda is a Jangama Dravya. It is included under Sudhavarga as it contains Calcium compound. In initial texts Shuklavarga was the name used to include Calcium containing drugs like Sudha, Kurmaprishta, Varatika, etc. Later texts especially the books of 19th and 20th century have used the term like Sudhavarga to include Calcium containing drugs. The number and name of drugs included in Sudhavarga varies from text-to-text and later books have included many additional drugs like Ajasthi, Kukkutandatwak, etc. In the earlier classical texts like Rasaratnasamuchaya, Rasarnava, Rasahridayatantra and Rasendra chudamani group like Sudhavarga does not appear. But in later text namely Rasamritam has mentioned Sudhavarga including Kukkudandatwak.¹

These drugs are used in Ayurveda pharmaceutics after subjecting it to the process of shodhana (purification process) and marana (incineration) to bring it into a suitable therapeutic dosage form. Various herbal media are utilized for the process of shodhana and marana, the criteria for selection being the action that is desired in therapeutics.A literary review of earlier research works on Kukkutandatwak Bhasma reveals two different Bhavana Dravya i.e., Changeri and Ghritakumari in Swarasa form being utilized for the purpose of its Marana.²

With the textual reference, Kukkutandatwak bhasma sample 1 and sample 2 was prepared and analysed by using instruments for evaluating the differences in their Characterization by X-ray diffraction (XRD), Scanning Electron Microscope (SEM) with the ED X-ray detector (SEM- EDAX) and Atomic Absorption Spectra (AAS).

MATERIALS AND METHOD

1. Preparation of both the Bhasma samples

2. Characterization by XRD

3. Characterization by SEM- EDAX

4. Characterization by AAS

1. Preparation of Changeri Bhavita KukkutandatwakBhasma (KTB sample 1) and GhritakumariBhavita Kukkutandatwak Bhasma (KTB sample 2)

Shodhana³ : 520g of raw Kukkutandatwak was taken, was soaked in 770ml of Ushnodaka for a day. Next day it was taken out and the sticky sheath or thin membranous layer was removed and washed with hot water then dried under shade.

Marana⁴ : KTB sample 1 Marana: 250g of Shuddha Kukkutandatwak was taken and Changeri Swarasa (382ml) was added and done Bhavana till Subhavita Lakshana was obtained. Then Chakrikas were prepared and dried under shade. Kept in Sharava and Sandhibandhana 3 layers were done then subjected to Puta with 4.8kg (44) cow dung cakes. The procedure was repeated till it attained Bhasma Pareeksha. Totally five Putas was required.

KTB sample 2 Marana: 250g of Shuddha Kukkutandatwak was taken and Ghritakumari Swarasa (405ml)was added and done Bhavana till Subhavita Lakshana was obtained. Then Chakrikas were prepared and dried under shade. Kept in Sharava and Sandhibandhana 3 layers were done then subjected to Puta with 4.8kg (44) cow dung cakes. The procedure was repeated till it attained Bhasma Pareeksha. Totally five Putas was required.

2.Characterization by X- Ray Diffraction⁵ X-ray diffraction (XRD) is a rapid analytical technique primarily used for phase identification of a crystalline material and can provide information on unit cell dimensions. X-ray diffraction is most widely used for the identification of unknown crystalline materials (e.g. minerals, inorganic compounds). Determination of unknown solids is critical to various geological applications.

Materials: X ray diffraction, samples

Procedure: 200mg of sample was taken. Sample was grinded to fine powder. Powder less than ~10 μm (or 200-mesh) in size is preferred. The powder was mounted on sample holder and subjected for reading.

3.Characterization by SEM- EDAX Study^6,7 A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. The electrons interact with electrons in the sample, producing various signals that can be detected and that contain information about the sample’s surface topography and composition.

Materials: Scanning Electron Microscope (SEM) with the ED X-ray detector along with beam source, pulse processor and analyzer.

Procedure: 0.5mg of the samples were taken on silver coin on that silver sputtering was done and placed over the slide, specially designed for SEM. This slide was kept in SEM analyzer and scanning of the elements was done. The image areas found in display were taken for the sample. In one image three different areas have been selected and mass percentage of elements presents in each area analyzed and mean of the percentage of these three values were taken out as total percentage of the respective element present in the sample for accuracy.

4.Characterization byAtomic Absorption Spectra (AAS)⁸ Atomic absorption spectroscopy (AAS) is a spectroanalytical procedure for the quantitative determination of chemical elements using the absorption of optical radiation (light) by free atoms in the gaseous state

Procedure: After choosing the proper hollow cathode lamp for the analysis, the lamp was allowed to warm up for a minimum of 15 minutes unless operated in a double beam mode. During this period, the instrument was aligned, the monochromator was positioned at the correct wavelength, the proper monochromator slit width was selected, and the hollow cathode current was adjusted according to the manufacturer’s recommendation. Subsequently, the flame was lit and the flow of fuel and oxidant was regulated, the burner and nebulizer flow rate was adjusted for maximum percent absorption and stability, and the photometer was balanced. A series of standards of the element under analysis were run and a calibration curve was constructed by plotting the concentrations of the standards against the absorbance. For those instruments which read directly in concentration set the curve corrector to read out the proper concentration. The samples were aspirated to determine the concentrations either directly or from the calibration curve. Standards were run each time a sample or series of samples are run.

OBSERVATIONS AND RESULTS

X-Ray Diffraction

In XRD the 2-theta value and intensity of peal (counts) are represented on X and Y-axis respectively. Higher the value of counts represents higher the crystallanity of the phase. For identification of each phase, peaks were chosen and compared with standard Joint Powder Diffraction File (JCPDF). The standard JCPDF number, identified phase and composition are also given.

DISCUSSION

X-Ray Diffraction

Standard XRD d-spacing data file contains thousands of standard compounds, but they lack in unique compounds like Rasaushadhis which are the mixtures of metal, minerals, etc. Hence the comparison of the sample with the standard can be done, but precise identification cannot be done. XRD data of Kukkutandatwak Bhasma sample 1has 22 peaks and sample 2 has 21 peaks showing the (2 θ), heights, d-spacing and FWHM values. Among these spectral peaks, major peaks are selected and the (2 θ) values of the peaks are connected with JCPDF standards and the substances are identified as Calcium carbonate. The peak of calcium was found more in sample 1, as the calcium component from Changeri must have been added during the process of Bhavana9.

Energy Dispersive X-Ray Spectroscopy (EDAX):

EDAX of Kukkutandatwak Bhasma sample 1 is Ca- 49.35%, S- 1.60% and C- 49.05% EDAX of Kukkutandatwak Bhasma sample 2 is Ca- 31.12%, S- 1% and C- 67.88%

The Ca component is more in sample 1, this is because of the calcium present in Changeri Swarasa that has been added during the process of Bhavana.

Scanning Electron Microscope (SEM):

The Scanning electron microscopic view of both samples of Kukkutandatwak Bhasma was viewed in 4 different magnifications from 500X to 3000X. The size of the sample given is 100µm, 50µm, 20µm and 10µm respectively. The particles are agglomerated in magnification of 500X but there is no such observation in other magnification. This indicates the smaller particle size of the Bhasma i.e., in micro meters.

Atomic Absorption Spectroscopy (AAS):

AAS method is used to detect any elements in trace amount. Kukkutandatwak Bhasma sample 1and 2 showed 3920ppm and 4185ppm of Calcium respectively.

CONCLUSION

Shodhana of Kukkutandatwak is by immersing in Ushnodaka to remove the physical impurities and the membranous sheath of the shell.

Kukkutandatwak Bhasma sample 1 was prepared by subjecting to 5 Puta with 44 cowdung cakes for every Puta with Bhavana Dravya as Changeri Swarasa and for sample 2 with Bhavana Dravya as Ghritakumari Swarasa.

The Bhasma of Kukkutandatwak Bhasma sample 1 was greyish in colour and that of sample 2 was grey indicating the different phytoconstituents added to the Bhasma when subjecting to levigation.

XRD data of Kukkutandatwak Bhasma sample 1has 22 peaks and sample 2 has 21 peaks. The peak of calcium was found more in sample 1 as the calcium component from Changeri must have been added during the process of Bhavana.

The Scanning electron microscopic view of both samples of Kukkutandatwak Bhasma was viewed in 4 different magnifications.

The weight percentage of elements in Kukkutandatwak Bhasma sample 1 is Calcium (49.35%), Sulphur (1.60%) and Carbon (49.05%) and of sample 2 is Calcium (31.12%), Sulphur (1%) and Carbon (67.88%).

Kukkutandatwak Bhasma sample 1 shows a higher percentage of calcium and sulphur, lesser amount of carbon when compared with Kukkutandatwak Bhasma sample 2, thus indicating the addition of components during the process of bhavana by the media adopted for bhavana.

Thus the extra number of peaks, moreweight percentage of elements in changeri bhavitaKukkutandatwak Bhasma throwslight on the addition of constituents by the process of bhavana indicating that bhavana not just facilitates the process of marana but also enhances the property of the drug by incorporating the constituents in it.

Scanning Electron Microscopy (SEM):

The SEM analysis of the sample Kukkutandatwak Bhasma sample 1 and 2 revealed particle of the sample given is 100µm, 50µm, 20µm and 10µm respectively