STUDY OF EFFECT OF AYURVEDIC PHARMACEUTICAL PROCESSING NAMED SHODHANA AND MARANA ON THE CHEMICAL COMPOSITION AND CRYSTALLINE STRUCTURE OF METAL IRON AND IRON-CONTAINING MINERALS USING XRD AND XRF ANALYSIS

Objective : This study was designed to evaluate the effect of Ayurvedic pharmaceutical procedures Shodhana and Marana on the chemical composition of the raw material. Methods: Iron and four iron-containing minerals were subjected to Shodhana and Marana . For Shodhana, Loha (Iron), Suvarnamakshika (Copper pyrite) and Mandura (iron slag,) were repeatedly quenched sequentially in sesamin oil, buttermilk, cow’s urine, natural vinegar and herbal decoctions. Kasisa (green vitriol) was grinded in the juice of Eclipta Alba . For Marana , these materials were first grinded in prescribed liquids and then incinerated in closed earthenware caskets in measured pits. Powdered Gairika (red ochre) was roasted in Cow’s ghee for its Shodhana. Samples of Loha (iron)-L1, Samanya Shodhita Loha Vishesha Shodhita Loha Loha bahsma Mandura (iron slag)–M1, Shodhita Mandura Mandura Suvarnamakshika pyrite)-S1, and -G2; were studied using XRF and XRD techniques. Results : XRD findings suggested that the Chemical nature, elemental composition and Crystaline lattice structure of each substance were altered after extensive processings. XRF studies confirmed the multi-elemental nature of the final products. Reduction in particle size and other morphological changes were observed in intermediate and finished products during each procedure. Conclusion : The study indicates that the composition of material is altered as a result of Ayurvedic pharmaceutical processing, ’ Shodhana and Marana .


INTRODUCTION
A branch of Ayurvedic medicine that principally deals with metallic and mineral drug preparations is known as Rasa Shastra. The drugs of Metallic and mineral origin are processed with an objective to make them edible, therapeutically effective and completely safe in therapeutic doses. Generally, a raw metallic or mineral drug substance undergoes through the following pharmaceutical procedures.
The raw material fulfilling the required criteria of selection for its use in drug preparation is usually collected from the market. The collected raw material is first made suitable for further processing like preparation of thin metal sheets or grinding the raw material to produce a powder of suitable particle size. The thin metal sheets or the metal powder as the case may be, are subjected to a pharmaceutical procedure named Shodhana [1] in terms of Ayurveda. The literal meaning of Shodhana is purification. But translation of the term Shodhana as purification produces a misperception about the objective and the real effect of the Shodhana procedure. Hence such translation should never be used. Purification in the process of Shodhana is restricted only to physical purification. It nowhere implies or achieves chemical purity of the material being processed. In addition to physical purification, the procedure softens the processed material to some extent and makes it ready for further processing. In some cases, particularly in case of some minerals which are soft in consistency like red ochre used in the present study, the softening of the material is sufficient to put the material directly to therapeutic use. In such cases the material also becomes safe for internal use in therapeutic doses following Shodhana. Metals are usually subjected to two types of Shodhana: Samanya (common for all metals) and Vishesh (specific to a metal to be used with a specific therapeutic objective). All metals and minerals which are hard in consistency need to undergo one more pharmaceutical procedure named as Marana [2] following Shodhana.
The pharmaceutical process of Shodhana includes procedures like heating the material to red heat and then quenching it in prescribed liquids till the material under process achieves the required quality. The pharmaceutical process of Marana to be followed after Shodhana includes incineration of the material obtained as an end product of the process of Shodhana. The material obtained from Shodhana is first wet ground with prescribed medicinal substances either liquid or solid or both, depending on the prescribed method. The grinding is continued till the material is converted into a dough. The dough so produced is used to prepare small cakes of suitable size. The cakes so produced are dried and put in a closed circular earthenware container known as casket (Sharava Samputa in terms of Ayurveda) and then subjected to incineration in a measured pit using dried cow dung cakes as fuel. The process of incineration is repeated till a product of desired quality is obtained. The product obtained after completion of incineration is known as Bhasma. Both these processes named Shodhana and Marana bring about significant change in the constitution of the processed material. Naredran N. et al. (2012) have shown the reducing effect of Gallic acid from Horse gram on Loha Bhasma during Marana [3]. In the present study the metal Loha (metal Iron) and minerals Gairika (red ochre), Kasisa (ferrous sulphate), Suvarnamakshika(Copper pyrite) and Mandur (iron slag, ferric oxide) were subjected to their respective Shodhana and Marana to obtain their therapeutically useful drug forms named as Bhasma. These Bhasmas and their intermediates obtained as end products following their Shodhana and Marana were subjected to XRD and XRF analysis to study the effect of these procedures on the constitution of the processed raw metals and minerals.

Collection of raw materials
The raw material i.e. Loha (metal Iron), Gairika (red ochre), Kasisa(green vitriol: ferrous sulfate), Suvarnamakshika (Copper pyrite), and Mandura (iron slag: ferric oxide) fulfilling the expected quality as described in the classical texts [4] were collected from the local market.
The raw materials so collected were subjected to their respective Shodhana and Marana procedures. The end products obtained following completion of both the procedures were Loha Bhasma, Mandura Bhasma, Suvarnamakshika Bhasma, Kasisa Bhasma, and Shuddha Swarna Gairika.

Pharmaceutical processing
Loha (metal Iron) was subjected to the following pharmaceutical processing to obtain Loha Bhasma.

Shodhana and marana of loha
Loha Bhasma preparation includes the following steps.

After Following
Samanya Shodhana, Vishesh Shodhana was performed. Loha choorna was heated till it gets got red hot and quenched seven times in the decoction of Triphala prepared in Gomutra (cow's urine). By this method, Shuddha Loha Choorna was obtained [6].

Loha marana
After Following Loha Shodhana, Shuddha Loha was subjected to Loha Marana. It was done in three steps.

Loha bhanupaka
Shuddha Loha choorna was soaked in specially prepared Triphala decoction to make it produce its slurry. The mixture was kept in an open earthen plate. This plate was exposed to blazing sunlight till all the liquid got evaporated. The process was repeated 7 times [7].

Loha sthalipaka
Loha choorna processed in Bhanupaka was washed with water. It was then mixed in specially prepared Triphala decoction (q. s.) to produce a slurry. The mixture was then subjected to heating on a high flame. This was continued till all the moisture got evaporated. The process is known as Loha Sthalipaka [8].

Loha putpaka
Loha Choorna was obtained as an end product of Bhaupaka followed by Sthalipaka, was washed with water and then ground well with specially prepared Triphala decoction to produce a soft dough. Thin flat pellets from this dough were prepared and dried. They were arranged and placed in a closed circular earthenware container known as Sharava samputa (casket) and then subjected to incineration in a measured pit named Gajaputa, using dried cow dung cakes as fuel. The process was repeated till the desired quality Bhasma was obtained [9].

Shodhana of mandura
Raw Mandura (iron slag) lumps were procured from the market. They were heated till they got red hot. Thereafter, n they were quenched in the decoction of Triphala 21 times. This decoction was prepared by boiling Triphala in cow urine instead of water. This process yielded small fragile pieces of Shuddha Mandura [10].

Marana of mandura
Shuddha Mandura obtained as an end product of the Shodhana process was subjected to wet grinding with Aloe juice (Aloe barbadensis) to produce a soft dough. Thin flat pallets from this dough were prepared and dried. They were arranged and placed in a closed circular earthenware container known as Sharava samputa (casket) and then subjected to incineration in a measured pit named Gajaputa, using dried cow dung cakes as fuel. The process was repeated till the desired quality Bhasma was obtained [11].
Suvarnamakshika (copper pyrite) was subjected to the following procedures to obtain Suvarnamakshika Bhasma.
Preparation of Suvarna makshik Bhasma includes the following steps.
The lumps were subjected to the Shodhana process by quenching them in Triphala decoction 7 times. This process yielded Shuddha Suvarnamakshika in the form of brittle pieces [12].

Marana of suvarna makshika
Shuddha suvarmnamakshika was mixed with castor oil (q. s.) to produce a slurry. This slurry was subjected to heating in an open iron pan till all the oil part was evaporated. Oil catches fire during the process.
The material obtained as an end product of the above-mentioned procedure was subjected to wet grinding with lemon J juice to produce a soft dough. Thin flat pallets from this dough were prepared and dried. They were arranged and placed in a closed circular earthenware container known as Sharava samputa (casket) and then subjected to incineration in a measured pit named Gajaputa, using dried cow dung cakes as fuel. The process was repeated till the desired quality Bhasma was obtained [13].

Kasisa(green vitriol: ferrous sulfate) was subjected to the following procedures to obtain Kasisa a Bhasma
Preparation of Kasisa Bhasma includes the following steps.

Kasisa shodhana
Kasisa Choorna was procured from the local market. It was subjected to wet grinding with a sufficient quantity of juice of Bhringraj (Eclipta Alba) in a quantity sufficient to soak and immerse the Kasisa powder in it completely. The mixture was wet ground firmly till all the powder of Kasisa gets dried by grinding. This process was repeated three times consecutively. Thus Shuddha Kasisa was obtained as an end product of this grinding in the form of very fine powder [14].

Kasisa marana
Shuddha Kasisa obtained as an end product of the Shodhana process was wet ground with a sufficient quantity of lemon juice to produce a soft dough. Thin flat pallets from this dough were prepared and dried. They were arranged and placed in a closed circular earthenware container known as Sharava samputa (casket) and then subjected to incineration in a measured pit named Gajaputa, using dried cow dung cakes as fuel. The process was repeated till a desired quality of Bhasma was obtained [15].

Gairika (red ochre) was subjected to the following procedures to obtain Shuddha Gairika
This compound is not used in Bhasma form. Gairika is used after Shodhana only. Gairika powder was mixed with Cows ghee taken in 1/8th of its measure. Further, it was roasted on mild heat, till ghee mixed in Gairika powder disappears by frying and a freeflowing powder of Gairika is obtained. This processed Gairika is used as medicine. This is called Shuddha Gairika [16].

Table 1: Schematic representation of processing's of Iron metal and other Iron containing drugs
Processing  Description of XRF studies-X-Ray fluorescence was done at Varsha Bullion Lab in Mumbai.

RESULTS
The results were recorded as follows.     S3 is more crystalline compared to S2 and S1. The peaks are also sharp and intense. The XRF studies of Swarnamakshika Bhasma (S3), showed the presence of ferric oxide 29.9%, Sio2 46.3%, CuO 3.6%. The rest of the elements are present in a minor amount.

DISCUSSION
The XRD and XRF studies of 5 iron-containing drugs and their intermediates revealed interesting findings. These procedures brought a change in the elemental composition of medicinal raw material. The extensive heat treatments and other procedures on raw materials of Ayurvedic drugs change their structure and composition. The XRD data has revealed that some elements were removed, changed, or minimized after Shodhana and Marana procedures. This clearly shows the effectiveness of the adopted pharmaceutical procedures.
In some samples in the above study, although no constitutional change is observed in the processed material, the brittleness of the material was observed to increase notably at the end of Shodhana. An increase in brittleness is known to facilitate further processing of Marana in such cases.
It is believed that the use of different medicinal liquids in the form of decoctions and expressed juices for grinding the raw materials to facilitate pharmaceutical processing like Shodhana and Marana are responsible for the therapeutic action of the end product Bhasma. These Bhasmas are used with different adjuvants in medical practice by the Ayurvedic practitioners to derive different therapeutic effects. These processes make this one single end product able to be used in various conditions with different adjuvants. They make them a broad spectrum Ayurvedic drug.
XRD data has revealed that there is a change in the arrangements of the lattice structure of crystals. The amount of crystallinity has increased. This is important because the bioavailability of the drug in the body depends upon its structure. In some cases (L4 and S3), major changes in crystal structure have been observed. This could be the result of the co-crystallization of compounds. Co-crystals are multicomponent crystalline solids that can alter the Physicochemical properties of solids. They can increase or decrease the solubility of the drug compounds depending on the interaction between the solids. The observed change in XRD pattern for L4 and S3 possibly be a result of crystallization between phenolic compounds that can further be evaluated in a separate study.
Sharp intense peaks and clear smooth graphs in XRD of the final products compared to raw material and intermediates denote the uniform homogeneous phase of the final drug. This is achieved with the help of extensive Shodhana and Marana procedures. These treatments turn raw material into the final drug of acceptable quality and efficacy.
A common pattern regarding the change in the chemical constitution of all 5 drugs is observed in this study. This pattern indicates the transformation of all raw materials into their oxides. All the 5 ironcontaining raw materials were observed as being transformed into iron oxide following Marana. The oxides are known to be more acceptable in the body. Singh. N et al. have observed similar findings in their study on Loha Bhasma. According to them the ultimate phase of Loha Bhasma was observed as Iron oxide only [17].
After the cascade of these intense procedures, particle size of all these bhasma was reduced significantly. PAvani et al. have confirmed the particle size of Loha bhasma to be 28.7 nm [18].
Some part of every bhasma is in nanodomain also. There is a trend of preparation of metal nanoparticles with green synthesis method. Patil Y et al. have recently reported the antimicrobial activity of magnetic iron nanoparticles. These magnetic iron nanoparticles were prepared by green synthesis process using leaf extract of medicinal plant Tridax Procumbens. These magnetic iron nanoparticles exhibited good antibacterial activity against gramnegative (E. Coli) bacteria [20]. This is clear that these finally prepared Bhasmas have some nanomaterial part in them. In our opinion these bhasmas can exibit superior pharmacological activity over the synthetically prepared nanoparticles. The resion reason is that they have gone throuogh extensive processes during their preparation. Hence their bioavailablity and bioassimilable nature could be superior to these synthetically prepared nanoparticles. This point needs a separate study for its evaluation. Further, these bhasmas are in clinical use since ages hence using these bhasmas will save lots of energy and time of various preclinical studies as their safety is already well explored in several different studies.
Although XRD studies revealed the major elemental phase composition of these Iron-containing drugs, XRF studies showed that each Bhasma and even Shuddha Gairika are compound formations comprising of multiple trace elements. This explains the broad therapeutic activity of these drugs. With no dispute, these microelements must have entered into the end products as a result of the use of different types of medicinal plant material used in Shodhana and Marana procedures. Bhargava S. also confirms the presence of several trace elements in Loha Bhasma [21] Ashwini A et al. also confirm the multielemental nature of Ayurvedic bhasmas. However they braught brought to notice that Bhasma prepared by different pharmacies differ in their elemental composition which can be attributed to different preparation procedures adapted by them pharmacies [22].
During the entire procedure of Shodhana and Marana, a strong possibility of the formation of herbal ligands with metals is felt. Such a possibility presents a new vista of therapeutically effective herbmetallic products. It is believed that the material adopts the therapeutic activity of the other material with which it is processed. Therefore, different plant materials are used in different Shodhana and Marana procedures. In this context, it is stated that, "Tad Tad Vyadhyupyuktanam aushdhanam jale Ayasah prakshepam prah"(A. P.3/246) This means, as per the therapeutic requirement, one can use a different medium for Shodhana and Marana procedures in different diseases. This suggests that different herbal media impose a different effect on metals and minerals while processing.
The formation of different ligands from respective plants may be the reason for the difference in therapeutic activity of the drug product processed with it.

CONCLUSION
Pharmaceutical procedures such as Shodhana and Marana described in Ayurvedic classics, bring about a change in elemental composition and crystalline structure of the processed material. In the present study, such a change was studied and confirmed through XRD and XRF analysis. It is conclusively said that a change in the chemical constitution and crystalline structure is observed in the metal Loha (Iron) and iron-containing minerals Gairika (red ochre), Mandura (iron slag), Kasisa (green vitriol), and Suvarnamakshika (Copper pyrite) as a result of Shodhana and Marana. The basic iron element is transformed into its oxide. Apart from Iron oxide, the end product Bhasma is also observed to contain few trace elements like-K, Ca, P, AL, O, Ma, Zn. The presence of trace elements is deemed to be a result of the processing of the raw material with different types of plant materials.