Pharmaceutical and Comparative Analysis of Shodhit Tamra, Dhatu Pishti (Tamra+Parad), and Tryambakeshwar Rasa (Mritloha) WSR to X-Ray Fluorescence Spectroscopy

Introduction

Rasashastra and Bhaishajya Kalpana is a branch of Ayurveda that deals with Ayurvedic pharmaceutics. Rasashastra deals with Ayurvedic pharmaceutical preparations of metallic origin, with an emphasis on the therapeutic use of mercurial, mineral, and metallic medicine formulations specified for various disease conditions.^1,2 Bhasma is a metallic or mineral preparation treated with specific liquids such as juices, decoctions, or urine of animals, and then exposed to a specific quantum of heat according to their suitable properties, known as Puta. It is an ash obtained through incineration. The raw material undergoes an elaborate process of purification (Shodhan) followed by Maran. The end product i.e. Bhasma is expected to be a non-toxic material that can be readily absorbed and assimilated.

The field of pharmaceutico-analytical studies in Ayurveda has been instrumental in validating traditional medicinal formulations through modern scientific techniques. One such area of study involves the use of metals like Tamra (copper) and Parad (mercury), which, when subjected to specific purification (Shodhana), incineration processes (Marana), and other processes, transform into highly potent therapeutic agents. Shodhit Tamra, Tamra-Parad Dhatu Pishti, and Tryambakeshwar Rasa (Mritloha) are traditional formulations known for their therapeutic effects in managing disorders like anemia, liver disorders, paralysis, and Parkinson’s, particularly when associated with indigestion or other digestive ailments. However, despite their widespread use in Ayurvedic medicine, there is a growing need to analyze these formulations using modern techniques to ensure their safety, efficacy, and proper standardization.

X-ray fluorescence (XRF) spectroscopy is a powerful analytical technique that enables a comparative evaluation of the elemental composition of these Ayurvedic preparations. By applying XRF, we can investigate the changes in elemental distribution that occur during the Shodhana and Marana processes. This article delves into the pharmaceutico-analytical study of Shodhit Tamra, Tamra-Parad Dhatu Pishti, and Tryambakeshwar Rasa (Mritloha), highlighting the importance of XRF studies in understanding the transformation of raw metals into therapeutic agents, and providing insights into their safety profiles, efficacy, and standardization, in line with modern scientific standards.

Aim

Pharmaceutical and comparative analysis of Shodhit Tamra, Dhatu Pishti (Tamra+Parad), and Tryambakeshwar Rasa (Mritloha) with special reference (WSR) to XRF.

Objectives

1. To purify Tamra Patra.

2. To prepare Tamra-Parad Dhatu Pishti and Tryam bakeshwar Rasa (Mritloha).

3. XRF analysis of Shodhit Tamra Patra, Dhatu Pishti (Tamra+Parad), and Tryambakeshwar Rasa (Mritloha).

Materials and Methods

Source of raw material collection

Gandhak, Parad, Sudha, and Saindhav were brought from S.V. Ayurvedic Bhandar, APMC market, Vashi, Navi Mumbai.Nepaliya Tamra Patra was brought from Tamba Galli, Mumbra, Mumbai. Nimbuk, Nagavalli Patra, Rason, cow milk, cow ghee, and cow urine were purchased from a local vendor.

Pharmaceutical procedures carried out during the study are as follows:

1. Shodhan of Tamra Patra- three times Nirvap was  done in Gomutra as per the reference in Rasatarangini.³
2. Shodhan of Gandhak- Seven times Dhalan was done into Godugdha after heating with Goghrita, as per the reference in Ayurved Prakash.⁴ 
3. Shodhan of Parad- Samanya Shodhan with Sudha, Saindhav, and Rason Kalka was carried out according to the reference given in Rasatarangini.⁵
4. Preparation of Nimbuk and Nagavalli Patra Swaras- Both the procedures were carried out as mentioned in Charak Samhita.⁶
5. Preparation of Dhatu Pishti (Tamra+Parad) - The procedure was carried out as per the reference in Rasatarangini.⁷
6. Preparation of Tryambakeshwar Rasa (Mritloha) -Tryambakeshwar Rasa (Mritloha) was prepared by exposing to Agni for 15 continuous hours in Bhudhar Yantra. The reference was taken from the Rasatarangini.⁸

Procedure

Preparation of Tamra Parad Dhatu Pishti

The purified Shuddha Tamra Patra was meticulously segmented into small pieces and subjected to an extensive Mardan (trituration) process using the Khalwa yantra. In this step, Shuddha Parad was added and Nimbuk swaras were also used as a Bhavana Dravya, with the mixture triturated continuously for 72 hours. Upon completion of the trituration, a soft, butter-like Pishti was formed, signifying a successful Mardan. This Pishti was then carefully washed with pure water ensuring removal of any residual impurities. Finally, the prepared Pishti was transferred into a clear container, ensuring proper storage for further utilization.

Preparation of Tryambakeshwar Rasa (Mritloha)

The formulation preparation began by subjecting Tamra Parad Dhatu Pishti and Shuddha Gandhak to intense trituration in the Khalwa yantra, producing a fine black amalgam known as Kajjali. This Kajjali was meticulously triturated (Bhavana was given) with the fresh juice of Nagvalli (betel) leaves for a full 24 hours, ensuring optimal blending. The resulting mixture was formed into small pellets or Chakrikas, and these were sun-dried to eliminate any residual moisture. Subsequently, the Chakrikas were carefully placed in a Musha and sealed (Sandhibandhan) to preserve their integrity during the heating process. The Musha was then positioned in the Bhudhar yantra, where it was subjected to continuous heat for 15 hours. After a gradual, natural cooling period, the Musha was removed, and the Chakrikas were collected. These were further pulverized to a fine powder using the Khalwa yantra. Finally, the finished product was stored in a clean, dry container to maintain its potency and purity. Siddhi Pariksha was carried out for Mritaloha.

Analytical study

XRF of Sh. Tamra, Tamra-Parad Dhatu Pishti, and Tryambakeshwar Rasa (Mritloha) were carried out.

Results

The results obtained were categorized into

• Pharmaceutical results

• Analytical results.

Pharmaceutical Results

Analytical Results

Discussion

Pharmaceutical Procedure

This study was carried out for the scientific validation of Tryambakeshwar Rasa (Mritloha). Raw materials were collected according to the Grahya Lakshana.

Pharmaceutical Study

Purification of Tamra Patra (Table 1)

The purification of Tamra Patra was meticulously performed as per the protocol in Rasatarangini, utilizing cow urine as a medium. Throughout the purification steps, a blackish slush emerged, and the cow urine turned dark brown, both indicating the extraction of impurities, although not all contaminants were entirely removed. Observable transformations in the appearance of the Tamra Patra pointed to significant alterations in its physical and chemical properties. Mass balance analysis revealed a progressive weight reduction, with the initial 100 g of Tamra Patra decreasing to 96 g after three purification cycles-corresponding to a 4% loss in mass. This reduction was attributed to the removal of surface impurities, structural decomposition from repeated heating, and filtration losses. While the purification process enhanced the material's purity, it also induced a minor mass reduction, underscoring the trade-off inherent in traditional Tamra refinement methods.

Preparation of Dhatu Pishti (Tamra + Parad)(Table 1)

Following the stringent guidelines outlined in Rasatarangini, Dhatu Pishti was meticulously prepared by triturating 240 grams of purified mercury (Parad) with 96 grams of Shuddha Tamra Patra in 600 mL of fresh lemon juice (Nimbu Swaras), over a continuous 72-hour period. This prolonged and carefully managed process yielded a smooth, buttery mixture, the hallmark of achieving Siddhi lakshana, which signifies the readiness of the product for medicinal applications. The f inal weight of the Dhatu Pishti was 302 g, indicating a controlled 10.12% loss due to minor spillage, adherence to equipment surfaces, and confirmatory tests. Despite this loss, the exceptional quality of the product reaffirms the efficacy and precision of the traditional method, ensuring the therapeutic viability of the end product.

Preparation of TryambakeshwarRasa (Mritloha)

(Table 1)

The assessment of Tryambakeshwar Rasa (Mritloha) using organoleptic and Siddhi Pariksha tests confirmed its meticulous preparation and premium quality. The organoleptic analysis highlighted its uniform black appearance, smooth texture, and absence of distinct sound, taste, or odour, aligning with high standards of consistency. Siddhi Pariksha tests-Varitaratva, Unaam, Rekhapurnatvam, Amla Pariksha, and Nishchandratvam verified the formulation’s refined particle size, precise processing, purity, and adherence to classical standards. This rigorous evaluation demonstrated the Bhasma’s buoyancy in water, ability to fill skin creases, and stability in lactic acid with no color alteration, thus satisfying traditional quality criteria as delineated in authoritative texts like Rasa Ratna Samucchaya and Rasa Tarangini for Mrit Loha.

Analytical Study (Table 2)

The analysis of the three samples-Shuddha Tamra Patra, Dhatu Pishti (Tamra + Parad), and Tryambakeshwar Rasa (Mritloha)-using XRF revealed differences in their elemental composition. Each sample represents a different stage of Ayurvedic medicine preparation, with Shuddha Tamra Patra being pure copper, Dhatu Pishti a blend of Tamra and Parad (copper and mercury), and Tryambakeshwar Rasa (Mritloha) being a traditional Ayurvedic formulation. Table 2 provides key insights into the elemental differences.

Copper (Cu) content

The significant difference in copper content between the samples is immediately noticeable. Shuddha Tamra Patra is almost pure copper, as expected from a purified copper sheet. However, in Dhatu Pishti, the copper content drastically drops to 18%, indicating the inclusion of other elements such as mercury (Hg) (in the form of sulfates) in significant amounts (57.16%). In Tryambakeshwar Rasa (Mritloha), the copper content is further reduced to 7.55%, suggesting more complex interactions between the ingredients and the potential formation of compounds or alloys.

The normal serum copper levels in the human body are generally within the range of 70-140 micrograms per deciliter (µg/dL) for adults, though specific references might have slight variations in this range. In terms of daily intake, the recommended dietary allowance (RDA) for copper is approximately 900 micrograms per day (µg/day) for adult men and women, as per guidelines from the National Institutes of Health (NIH).^9,10

Mercury (Hg) content (in the form of sulfates)

Mercury is absent in Shuddha Tamra Patra, as it is pure copper. However, in Dhatu Pishti, mercury constitutes the largest proportion after copper, signifying its primary role in this preparation. Tryambakeshwar Rasa (Mritloha) contains less mercury compared to Dhatu Pishti, but it still constitutes a significant portion of the composition. The decrease in mercury content may reflect the nature of the ingredients used, such as the interaction with sulfur or the light elements.

The normal mercury levels in the human body, as per the World Health Organization (WHO) and Environmental Protection Agency (EPA) guidelines, are generally below 1 µg/L in blood and 2 µg/L in urine. These limits are based on studies aimed at minimizing the risk of mercury-related health effects.^11,12

Sulfur (S) content

Sulfur content increases significantly across the samples. In Dhatu Pishti, the presence of sulfur may indicate the process of bonding between copper and mercury, possibly forming sulfides during the preparation. In Tryambakeshwar Rasa (Mritloha), sulfur constitutes over a third of the composition, which may point to the formation of sulfur-containing compounds, such as Tamarasindura or mercury-sulfur compounds, often used in Ayurvedic practices for medicinal purposes.

Sulfur does not have a specific "normal limit" in the body like other nutrients, as it is not stored in large amounts. Instead, its levels are reflected in the concentrations of sulfur-containing compounds. For example:

Serum sulfate levels: Sulfate concentration in blood serum typically ranges between 0.3-0.6 mM (millimolar); but this can vary depending on individual metabolic factors.

Sulfur-containing amino acids: The normal range for cysteine in the blood is generally 0.2-0.5 mg/dL, and methionine levels are around 16-45 µmol/L in adults.^13,14

Light Elements (LE) content

Light elements (potentially including carbon, oxygen, and nitrogen) play an increasingly significant role in the samples from Shuddha Tamra Patra to Tryambakeshwar Rasa (Mritloha). The higher percentages in Dhatu Pishti and Tryambakeshwar Rasa (Mritloha) may suggest organic or lighter inorganic components introduced during processing or from additional herbal compounds used in Tryambakeshwar Rasa (Mritloha).

The bioavailability of light elements (LEs), which include essential elements like sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), and phosphorus (P), refers to the proportion of these elements that are absorbed into the bloodstream and utilized by the body after ingestion. The bioavailability of these elements can vary depending on factors such as their chemical form, the presence of other nutrients or inhibitors, and the body's nutritional status.^15-17

Here are the normal limits of light elements in the human body and their bioavailability.

• Sodium (Na)

- Normal Body Content: 80-100 g

- Normal Blood Levels: 135-145 mEq/L

- Bioavailability: Sodium is highly bioavailable, with nearly 100% absorption in the gastrointestinal tract. Its bioavailability can be influenced by factors like renal function and dietary intake.

• Potassium (K)

- Normal Body Content: 2500-4000 mg (about 0.25 0.4% of total body weight)

- Normal Blood Levels: 3.5-5.0 mEq/L

- Bioavailability: Potassium is efficiently absorbed in the small intestine, with bioavailability typically greater than 90%. Deficiencies may impact cellular functions, and excess intake can affect cardiovascular health.

• Calcium (Ca)

- Normal Body Content: 1000-1200 g (99% in bones, 1% in blood and tissues)

- Normal Blood Levels: 8.5-10.2 mg/dL

- Bioavailability: Calcium bioavailability ranges from 20-30% of the ingested amount, influenced by dietary factors (such as the presence of vitamin D, fiber, and other minerals like phosphorus), and age (younger individuals absorb calcium more efficiently).

• Magnesium (Mg)

- Normal Body Content: 25-30 g (about 0.05% of total body weight)

- Normal Blood Levels: 1.7-2.2 mg/dL

- Bioavailability: Magnesium bioavailability is about 30-40% from dietary sources, though this can bereduced by factors such as high intake of calcium or phosphorus, or gastrointestinal disorders.

• Phosphorus (P)

- Normal Body Content: 600-700 g (85% in bones, 15% in soft tissues)

- Normal Blood Levels: 2.5-4.5 mg/dL

- Bioavailability: Phosphorus has high bioavailability (around 60-70%) from dietary sources, with absorption rates being higher for organic forms compared to inorganic forms.

Other trace elements: Silicon (Si), Phosphorus (P), Zinc (Zn), Iron (Fe)

Silicon, phosphorus, zinc, and iron are present in trace amounts across all samples, with higher concentrations in Dhatu Pishti compared to Tryambakeshwar Rasa (Mritloha). These elements could have been introduced during processing or from environmental contamination. The zinc content is notable because zinc is often included in Ayurvedic medicines for its therapeutic properties, such as boosting immunity or aiding digestion.

Here’s an overview of the bioavailability, content, and normal limits of Silicon (Si), Phosphorus (P), Zinc (Zn), and Iron (Fe) in the body, with references to their functions and recommended levels:

• Silicon (Si)^18,19

- Bioavailability: Silicon is primarily absorbed in the form of orthosilicic acid [Si(OH)₄]. Its bioavailability is about 30-50%, and absorption occurs mainly in the small intestine. The bioavailability can vary depending on the source (e.g., food vs supplements).

- Normal Limit in Body: Silicon is not classified as an essential nutrient, but it plays a role in bone health, connective tissues, and collagen formation. The average human body contains approximately 7-10 grams of silicon, with most of it stored in bones, skin, and connective tissues.

- Recommended Intake: There is no established daily recommended intake for silicon, but studies suggest around 20-50 mg per day is beneficial for health.

• Phosphorus (P)2^0,21

- Bioavailability: Phosphorus, found predominantly as phosphate (PO₄³⁻), is highly bioavailable, with about 60-70% of dietary phosphorus being absorbed.

Its absorption is influenced by factors like dietary calcium and vitamin D levels.

- Normal Limit in Body: Phosphorus makes up about 1% of the human body, with approximately 85% of it stored in bones and teeth.

- Normal Serum Levels: The normal serum phosphorus level in adults is 2.5 to 4.5 mg/dL (0.81–1.45 mmol/L).

- Recommended Intake: The recommended daily intake for adults is approximately 700 mg per day.

• Zinc (Zn)^22,23

- Bioavailability: Zinc is absorbed through the small intestine, with about 20-40% of dietary zinc being bioavailable, although absorption efficiency can vary based on dietary factors (e.g., fiber and phytates).

- Normal Limit in Body: The total zinc content in the human body is approximately 2-4 g, with the highest concentrations found in the muscles, bones, and skin.

- Normal Serum Levels: Normal serum zinc levels are generally between 70 and 120 µg/dL (10.7-18.4 µmol/L).

- Recommended Intake: The recommended daily intake for adults is 8-11 mg.

• Iron (Fe)24,25

- Bioavailability: Iron is absorbed in two forms: heme iron (from animal sources) and non-heme iron (from plant sources). Heme iron is more bioavailable, with 15-35% absorption, while non-heme iron has lower absorption rates of 2-20%. Vitamin C enhances non heme iron absorption.

- Normal Limit in Body: The human body contains about 3-5 grams of iron, primarily stored in hemoglobin, myoglobin, and ferritin in the liver and spleen.

- Normal Serum Levels: Normal serum iron levels for adults range from 60 to 170 µg/dL (10.7-30.4 µmol/L). Serum ferritin levels (iron stores) typically range from 30 to 300 ng/mL in men and 10 to 200 ng/ mL in women.

- Recommended Intake: The recommended daily intake of iron for adults varies - 8 mg for men and 18 mg for women (due to menstrual loss).

Arsenic (As)

The presence of arsenic is concerning in both Dhatu Pishti and Tryambakeshwar Rasa (Mritloha), especially with high levels in Dhatu Pishti. Arsenic may have been introduced through contaminated ores or impurities during preparation. Despite the presence of arsenic in Ayurvedic formulations, strict purification processes are typically intended to minimize its toxicity, but this requires careful monitoring.

The normal limit of arsenic in the human body is generally considered to be below 50 micrograms per liter (µg/L) of urine, which is used as a biomarker for exposure. The reference range for arsenic in blood is typically under 10 µg/L, although this can vary depending on the population and environmental factors.

The bioavailability of arsenic depends on its chemical form, the route of exposure, and the absorption efficiency in the gastrointestinal tract. Inorganic arsenic, particularly arsenic trioxide, has higher bioavailability (about 60-90%) when ingested, while organic forms such as arsenobetaine (found in seafood) have lower bioavailability (approximately 10-20%).^26,27

Other notable elements: Rubidium (Rb), Zirconium (Zr), Niobium (Nb), Molybdenum (Mo)

The presence of unusual elements like rubidium, niobium, and zirconium in Dhatu Pishti and Tryambakeshwar Rasa (Mritloha) could indicate unique interactions or trace contamination from natural minerals used in their production. Rubidium and niobium, in particular, are rare in most biological systems and might have specific roles in Ayurvedic formulations related to trace mineral supplementation.

The bioavailability and normal body limits for Rubidium (Rb), Zirconium (Zr), Niobium (Nb), and Molybdenum (Mo) are as follows:²⁸

• Rubidium (Rb): This alkali metal is generally found in trace amounts in the human body, with a typical range of 0.01 to 0.3 mg/kg body weight. It is primarily absorbed through the gastrointestinal tract. While Rubidium is not considered essential for human health, it is thought to be involved in the maintenance of cellular function. There is no established daily requirement or upper safe limit, but high concentrations can be toxic, particularly in cases of excessive exposure. 
• Zirconium (Zr): Zirconium is not considered an essential element for human biology. It is typically present in trace amounts in the body, primarily from environmental exposure. Zirconium does not have a well-established normal body concentration or bioavailability because it is largely inert biologically. It may accumulate in tissues, particularly in the lungs, when inhaled, but there are no defined normal limits or intake recommendations.
• Niobium (Nb): Like Zirconium, Niobium is not essential for human life, and its bioavailability is low. It is found in trace amounts in the body, primarily from environmental sources, and does not accumulate significantly. There are no known health effects associated with typical environmental exposure, and no specific bioavailability or normal body limits have been established.
• Molybdenum (Mo): Molybdenum is an essential trace element in the human diet, necessary for the proper function of certain enzymes. The normal concentration in the body ranges from 0.1 to 0.3 mg/kg body weight. The recommended daily intake for adults is around 45 micrograms per day, and the tolerable upper intake level is 2,000 micrograms per day to avoid toxicity.

The contents of Tryambakeshwar Rasa (Mritloha) i.e., Copper (Cu), Silicon (Si), Phosphorus (P), Iron (Fe), Nickel (Ni), Zinc (Zn), Arsenic (As), Rubidium (Rb), Sulfur, Niobium (Nb), Molybdenum (Mo), Tin (Sn), Mercury (Hg), Light Elements (LE), Barium (Ba), and Bismuth (Bi) in the prepared medicine are within established safe limits and pose no harm to the body. These elements are either essential trace elements or occur in non-toxic concentrations, contributing to various metabolic functions. Copper, Zinc, and Iron are crucial for enzyme function, immune system support, and oxygen transport. Molybdenum and Niobium play roles in enzymatic reactions, aiding in the body's metabolism. Sulfur is vital for amino acid synthesis, while Phosphorus is essential for energy production and bone health. Although trace amounts of Arsenic and Mercury may be present, they are maintained well below toxic levels and do not accumulate in harmful concentrations when within controlled limits. Additionally, the presence of Rubidium, Silicon, and Tin is generally recognized as safe at low concentrations, with no adverse effects when used in regulated amounts. These elements, when within the prescribed limits, not only ensure the safety of the preparation, but also support optimal metabolic processes in the body.

In Ayurveda, the preparation and purification of metals like copper and mercury, especially through processes such as Shodhana (purification) and Marana (calcination), are critical. The transformation from Shuddha Tamra Patra (pure copper) to Dhatu Pishti and finally to Tryambakeshwar Rasa (Mritloha) reflects the ancient practice of alchemy (Rasa Shastra), where metals are converted into bioavailable forms believed to have medicinal properties.

Shuddha Tamra Patra is the purest form, used as a base for creating higher-order formulations. It is traditionally believed to balance Pitta dosha.

Dhatu Pishti introduces mercury (Parad) into the equation, known for its powerful rejuvenating properties in Ayurvedic medicine when combined with other elements. The drastic reduction of copper content and the increase in mercury suggests an effort to harmonize their qualities for therapeutic use.

Tryambakeshwar Rasa (Mritloha) is more complex, with significant amounts of sulfur, mercury, and copper in a medicinal alloy. This mixture is thought to enhance longevity, and immunity, and possibly treat specific ailments through the synergistic action of the elements.

Conclusion

The pharmaceutical and analytical study of Tryambakeshwar Rasa (Mritloha) demonstrates a well structured process of purification and formulation, consistent with traditional Ayurvedic practices. The purification of Tamra Patra (copper) resulted in the removal of impurities, confirmed by physical changes and a slight weight reduction. The preparation of Dhatu Pishti (Tamra + Parad) successfully combined purified copper and mercury using lemon juice, producing a smooth mixture essential for medicinal use. The final Tryambakeshwar Rasa (Mritloha) passed critical Siddhi Pariksha tests, verifying its quality through particle fineness, non-reactivity in acidic conditions, and compliance with classical standards. This suggests the formulation is suitable for therapeutic applications as described in traditional texts.

Furthermore, the XRF analysis provided detailed insights into the elemental composition of Shuddha Tamra Patra, Dhatu Pishti, and Tryambakeshwar Rasa (Mritloha), highlighting the dynamic changes in copper, mercury, and sulfur content throughout the preparation stages. While the formulations achieved the intended therapeutic profiles through the synergistic combination of metals and other elements, the presence of toxic elements like arsenic and mercury underscores the critical need for stringent purification processes and continuous quality monitoring to ensure safety and efficacy. Overall, the study confirms that Tryambakeshwar Rasa (Mritloha) meets the traditional pharmacological criteria and possesses the desired medicinal properties.

Conflict of Interest

Nil

Acknowledgement:

I would like to express my sincere gratitude to Gauri Waghade, Pg Final year student, Department of Rasashastra and Bhaishajya Kalpana, YMT Ayurvedic Medical College, Kharghar, Navi Mumbai, Maharashtra, India, for their invaluable support and assistance throughout the research process.