PHYSICOCHEMICAL CHARACTERISTICS OF RASAMANIKYA-AN AYURVEDIC ARSENICAL FORMULATION

Objective: The objective of this study was standardization and Chemical characterization of rasamanikya prepared as per standard operating procedures (SOP) mentioned in the classical text. 
Methods: Rasamanikya was prepared by putting churnodaka shodhita haratala (Orpiment-As2S3) between two abhraka (white mica) sheets which are heated for a while to obtain a red colored finished product. The Ayurvedic specifications for the analysis of rasamanikya were performed through qualitative and quantitative analysis. Physicochemical analysis, assay of elements by atomic absorption spectrometer (AAS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES) were carried out and some other tests such as x-ray diffraction (XRD), x-ray photoelectron spectroscopy (XPS) and energy dispersive x-ray analyzer (EDAX) were also performed to ensure the quality of the drug. 
Results: In the finished drug Arsenic and Sulphur are present in the form of As4S4, As2S3, As2S5. On the basis of XPS survey scans, scanning electron microscopy-energy dispersive x-ray analyzer (SEM-EDAX) and carbon, hydrogen, nitrogen, sulphur (CHNS) analysis the Arsenic to Sulphur (As to S) ratio is thus standardized as 39-47: 53-61. In addition to this powder, XRD shows a major conversion into an amorphous phase. 
Conclusion: The results could be used to lay down a new set of pharmacopoeial standards for the preparation of rasamanikya for getting optimal efficacy of medicine. Therefore, the information will help the Scientists and Researchers to build comprehensive standards, to screen the compounds responsible for different bioactivities, and to elucidate the molecular mechanism of action.


INTRODUCTION
Rasamanikya is a well known ayurvedic arsenical preparation, which is prepared out of shuddha haratala. The formulation has been first described in Rasendra Chintamani in the 13th century A. D. as Rasam manikya prabham [1]. The same product has been described in Siddha Bheshaja Manimala as kumuda rasa [2]. Tala manikya is one of the synonyms of rasamani kya.
Haratala (As2S3), the only ingredient is described as dhatuvisha and phenasma in samhitas [5,6]. Rasamanikya is one among the numerous preparations made out of haratala. Being an arsenical preparation and, considering the importance of rasamanikya in clinical use; SOP for its preparation and chemical characterization, safety/toxicity studies becomes the mandate for its acceptance. In this current study, an attempt has been made to evaluate preliminary physicochemical characteristics of rasamanikya.

Pharmaceutical processing
Rasamanikya was prepared by following standard methods mentioned in the Ayurvedic Formulary of India [7]. The whole process of preparation is divided into the following steps.

Preparation of churnodaka
Lime powder (250 mg) and water (60 ml) were mixed thoroughly with the help of a stirrer. The solution thus obtained was kept aside undisturbed for 12 h. Next day morning, the whole contents were filtered through a clean cloth. The filtrate thus obtained was collected and preserved in a clean glass container [8].

Haratala shodhana
Small pieces of haratala were bundled in a muslin cloth (pottali) immersed in dolayantra containing churnodaka and subjected to moderate heat for three hours [9]. Utmost care was taken to immerse pottali completely in churnodaka throughout the procedure. At the end of three hours, haratala was taken out from the pottali. It was then washed in potable hot water, dried in open air and shodhita haratala [10] was collected.

Preparation of rasamanikya
Powdered shodhita haratala was spread in between two mica sheets and the joints were closed by using 'U' pins. This was held with the help of tongs and heated over the LPG stove. After the melting is completed, it was withdrawn from heating and ruby colored rasamanikya was collected [11].

Reagents and standards
All chemicals, reagents and solvents were used analytical grade and obtained from authentic suppliers.

X-ray diffraction
Powder x-ray diffraction (XRD) analysis of Rasamanikya was carried out using Rigaku Ultima-IV X-ray diffractometer with CuKα radiation (λ = 1.54A°) operating at 40 kV and 30mA. Pattern was recorded for angle (2θ) ranging from 10-100° at a scanning rate of 1°/second and scan step of 0.1°. Sample identification was done by matching dspacing with the standard database.

X-ray photoelectron spectroscopy (XPS)
XPS measurements of rasamanikya were obtained on a KRATOS AXIS 165 instrument equipped with dual aluminum-magnesium anodes using Al Kα radiation. The x-ray power supply was run at 15 kV and 5 mA. The pressure of the analysis chamber during the scan was 10 -9 Torr. The peak positions were based on calibration with respect to the C 1s peak at 284.6 eV. The obtained XPS spectra were fitted using a nonlinear square method with the convolution of Lorentzian and Gaussian functions after the polynomial background subtraction from the raw data ( fig. 3).

RESULTS AND DISCUSSION
The Organoleptic observation shows that the prepared rasamanikya is in the form of a yellowish fine powder having no characteristic odour and taste. The qualitative analysis shows the positive test for the presence of arsenic and sulphur. The chemical analysis revealed that it contains 60.0% of Arsenic and 39.5% of Sulphur on average together with minor elements viz. boran, iron, magnesium, chromium, aluminum, lead, and calcium. Moisture content 0.20% was found when the determined loss on drying at 105 °C. Total ash content (approx 2.75%) is left after burning of volatile matter (96.75%). The observations show that watersoluble (1.75%) and alcohol soluble (2.0%) matter are also present in this formulation. (Details are mentioned in table 2). Particles size is about 300 nm, and is homogeneously distributed which showed the presence of the microfine particle. The three rasamanikya (3 batches) samples have near identical XRD patterns with two small but prominent peaks at 26.7 and 27.9 degrees respectively; only one sample shows a peak at 67.8 degrees to the samples were compared with earlier reported XRD patterns [19]. This shows the XRD of the 'haratala' (arsenic trisulphide/Orpiment) used to make these samples by heating haratala at high temperatures while holding the samples between mica sheets. The XRD of rasamanikya samples show a complete transformation into an amorphous state, barring two samples showing a peak due to a crystal phase (some fraction of hartala might have remained).
The present set of samples too, show a major conversion into an amorphous phase but the peaks seen at 26.7, 27.9 and 67.8 degrees could not be specifically assigned to any crystalline phase but the XRD patterns quite resemble the amorphous patterns of samples as mentioned therein [19]. The XRD pattern is shown in fig. 1 and fig. 2. The observed binding energy peaks for both As 3d and S 2p clearly suggest the coexistence of these two metals in multivalent states. Based on the literature, the various oxidation states of As and S and the corresponding binding energies are given in table 3and4. In addition to As in metallic form, it is present in the form of As4S4, As2S3, As2S5 with characteristic binding energy peaks at ~ 42.6 eV, 44.5 ev and 45.9 eV respectively. From the area under the curves the approximate percentage of the various forms, namely As (0), As4S4, As2S3, As2S5 are 32.1%, 36.2%, 20.7%, 11.0%, respectively.   XPS survey scans do not show any remarkable changes for all the three batches. However, the atomic concentration quantification report for the elements As and S for batch II shows a ratio of 39:61 (As: S) whereas in batches I and III they are in the ratio 47:53 and 43:57 respectively. As: S ratio was also determined by SEM-EDAX for all batches and were found to be 45. 6:54.4, 49.9:50.1 and 49.8:50.2, respectively. Sulphur content in batches I, II, and III as determined by elemental analysis by CHNS showed 36.30, 39.05, and 37.73 % respectively. On the basis of XPS survey scans, SEM-EDAX and CHNS analysis the Arsenic to Sulphur (As to S) ratio is thus standardized as 39-47: 53-61.

CONCLUSION
The preliminary profiles of rasamanikya evaluated in this attempt could be used to lay down a new set of pharmacopoeial standards. This information will help the scientists and researchers to build comprehensive standards, to screen the compounds responsible for different bioactivities, and to elucidate the molecular mechanism of action.