INTRODUCTION:

Siddha system of medicine is a form of medicine that has been widely in practice in the Indian state of Tamil Nadu with its origin going back to B.C 10,000 to B.C 4,000.¹

The unique of Siddha system is its continuous service to humanity for more than five thousand years in combating diseases and in maintaining its physical, mental, moral health².

There are 18 siddhars (Pathinen siddhargal) of which Agasthiyar, Bogar, Thirumoolar, Pulippani are some of the important Siddhars who served the mankind. This system of medicine is recognized under AYUSH (Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homoeopathy) system of medicine, Government of India, was created in November, 2003³. Even millennia ago, the Siddhars mastered the technique of creating metallic medicines in nano-formulations. In the traditional Siddha medicinal system, various metallic formulations such as Parpam (mineral or metal oxides), Chenduram (mineral or metal sulfides), Chunnam (caustic oxide preparation), and Pathangam (product of sublimation) are utilized. These formulations offer extended shelf life and enhanced efficacy even with small doses^4,5.

Although this medicine has been in human use for an extended period, it is now necessary to subject it to scientific testing in this modern era. This process is known as reverse pharmacology, which involves reversing the traditional laboratory-to-clinic discovery approach and adopting a clinics-to-laboratories perspective. The reverse pharmacology, is defined as the science of integrating documented clinical experiences and experiential observations into leads, through transdisciplinary exploratory studies, and further developing these into drug candidates through robust preclinical and clinical research⁶. The drug PMC has already undergone toxicological⁷ evaluation. It also possess pharmacological activities such as anti-proliferative⁸ and anti-cancer effects⁴ in Human Keratinocyte Cell lines (HaCaT) and Michigan Cancer Foundation-7 (MCF-7) cell lines, respectively. Furthermore, it has undergone Clinical study in psoriasis patients⁹.

The current study was conducted to establish the Standard Operating Procedure (SOP) for Siddha Herbo-mineral formulation Panchamuga Chendhuram (PMC) and to produce scientific evaluation through modern parameters such as physicochemical analysis, Scanned Electron Microscopy (SEM), Fourier Transformed Infrared Spectroscopy (FTIR), Inductively Coupled Plasma Optical Emission Spectroscopy (ICPOES).

MATERIALS AND METHODS:

Raw drug collection and Authentication:

The raw materials were obtained from a raw drug store in the Chennai market. Following thorough authentication by the relevant pharmacognosist at the Siddha Central Research Institute in Arumbakkam, Chennai, the preparation was conducted.

Standard Operating procedure:

Purification of Sulphur (Gandhagam):

Materials used: Gandhagam -100g, Milk-1liter. Equipments used: Iron spoon, mud pot, stove, tissue paper.

Procedure: Gandhagam is melted in an iron spoon, which is then heated until the Gandhagam liquefies and is poured over the milk. This process is repeated twice. Following that, the Gandhagam is removed, rinsed with cool water, and left to dry.

Precautions: Precautions include conducting the procedure in an open area, wearing a mask to avoid inhaling smoke, and taking care to prevent the overflow of sulfur during the melting process. Additionally, it is important to thoroughly wipe the sides of the iron spoon before placing it on the stove to prevent any potential fire hazards (Fig 1).

Purification of Arsenic Trisulphidium (Thalagam) (AS₂S₃):

Materials used: Thalagam-100g, limestone-500g, small cloth. Equipments used: Stove, mud pot, small cloth. Procedure: Thalagam is enclosed in a small cloth and secured with a tiny rope. Subsequently, it is buried in limestone, and water is poured until the limestone is fully submerged. The mixture is then heated until the water completely evaporates. The thalagam is retrieved, washed in cool water, and left to dry. A precaution to be observed is to avoid touching the thalagam while washing.

Purification of Mercuric Chloride (Veeram) (Hgcl₂):

Materials used: Veeram-100g, tender coconut-2, pepper, piper betel leaves. Equipments used : mud pot, small cloth, small stick, mortor with pestle. Procedure: A combination of pepper and betel leaves is blended with tender coconut water and placed in a mud pot. Veeram is enclosed in a cloth and suspended over the pot without making contact with the water. The pot is then heated for 30 minutes. The veeram is subsequently removed and left to dry.

Purification of Mercury (Rasam) (Hg):

Materials used: Mercury-100g, Brick powder, Turmeric powder, Acalypha indica Leaf juice. Equipments used: stove, mud plate. Procedure: A quantity of 100 grams of mercury was ground with brick powder and turmeric powder, followed by a thorough wash with water. Subsequently, the mercury was heated in Acalypha leaf juice for approximately 30 minutes.

Purification of Mercuric Sulphide (Lingam) (Hgs):

Materials used: Lingam- 100g, lime juice-250 ml, milk-250ml, Acalypha indica leaf juice-250ml, Equipments used: Mud plate (agal), stove. Procedure: Lingam was placed in a mud plate and heated, lime juice, milk, Acalypha juice are mixed together and allowed to fuse lingam. After that lingam is washed away and dried.

Preparation:

This preparation had undergone 4 stages of processing (Fig 1). Trituration, Drying, Heating, Again Trituration.

Materials used:

Purified Rasam (Hydragyrum)-100g, Purified Gandhagam (Sulphur)-100g, Purified Thalagam (Arsenic trisulphidium)-100g, Purified Lingam (Mercury II sulfide)-100g, Purified Veeram (Mercuric chloride)-100g, Piper betel leaf juice.

Equipments used:

Little mud pot, big mud pot, cloth pieces.

Fig. 1: Panchamuga Chendhuram (PMC) Standard Operating Procedure

Procedure:

Purified Gandhagam was finely ground with vettrillaicharu (piper betel juice), and Purified Rasam was added and thoroughly blended. Purified Thalagam and Purified Lingam were introduced to that mixture, and finally, Purified Veeram was added and ground extensively for a day. Subsequently, small villais (poultries) were shaped, and these were placed inside a little mud pot. The little clay pot was capped with a small clay lid and securely sealed with seven layers of clay cloth. The larger clay pot was filled with sand, and the small clay pot was nested inside it. Subsequently, the large clay pot was topped with a sizable clay lid and securely sealed with seven layers of clay cloth. The heating process included mild heat (deepaagni) for 6 hours, moderate heat (kamalaagni) for 6 hours, and heavy heat (kaadaagni) for 9 hours. After allowing it to cool naturally, the seal was opened, and the product underwent an additional day of grinding. The end product (PMC) was preserved in a sealed container to prevent air exposure.

Precautions:

1. Wear gloves when triturating veeram and during preparation of villai.

Standardization of PMC:

Characterization with Siddha parameters:

The final Chendhuram underwent analysis for the purpose of ensuring quality according to the General guidelines for development of ASU drugs. This included assessment of colour, odour, taste, pH, loss on drying/moisture content, particle size, total ash, acid -insoluble ash, sulphated ash, water- soluble extractive, alcohol- soluble extractive, assay for element and various spectroscopic and microscopic techniques such as IR/XRF/XPS/XRD/SEM/EDX/AFM, lusterless, fine enough to enter within lines of finger, floats on water, smokeless, tasteless, irreversible, shelf life. The Chendhuram should meet all twenty one criteria outlined in the guidelines to ensure its quality and efficacy¹⁰.

Physicochemical Analysis:

Physicochemical parameters, such as description, estimation of Loss on drying, Ash content, Acid insoluble ash, Water/Alcohol soluble extractive, pH, etc., were determined by Central Council for Research in Siddha (CCRS)^11-15. The final product PMC was subjected to instrumental analysis at Sophisticated Analytical Instruments Facility (SAIF) at Indian Institute of Technology (IIT) in Chennai.

SEM with EDAX analysis:

The size of the PMC was determined utilizing a Scanning Electron Microscope (FEI Quanto FEG 200 - high resolution SEM). EDX (Energy Dispersive X-ray) analysis of these medications was performed using the same instrument to validate the elemental composition of the sample¹⁶.

Fourier transform Infrared analysis (FTIR):

The PMC underwent Infrared spectroscopic analysis using the Septrum One Perkin Elmer FT-IR Spectrometer. It was utilized to identify the functional groups and establish their correlation with the therapeutic properties of the drug. The KBr method, as outlined by Price in 1972, was employed for the sampling techniques¹⁷.

Inductively Coupled Plasma Optical Emission Spectroscopy (ICPOES):

To quantitatively analyze heavy metal content in PMC in parts per million (ppm), an Inductively Coupled Plasma Optical Emission Spectrometer (ICPOES), specifically the Perkin Elmer Optima 5300DV with a 40 MHz RF generator, was employed¹⁸.

RESULTS:

Table 3: ICPOES Anslysis of PMC

S.No	Heavy metals	Results	Specification as per AYUSH/ WHO/ FDA
1.	Lead	BDL	NMT 10 ppm
2.	Mercury	BDL	NMT 1.0 ppm
3.	Arsenic	0.09	NMT 3.0 ppm
4.	Cadmium	BDL	NMT 0.3 ppm

Table 1: Organoleptic character of PMC

S.No	Characters	Inference
1.	Floating on water	Floats on water
2.	Finger furrows test	Goes into Finger furrows
3.	Luster	Lusterless
4.	Taste	Tasteless
5.	Odour	Odourless
6.	Colour	Brick red
7.	Appearance	Fine powder

Table 2: Physiochemical Analysis of PMC

S. No	Parameters %	Results
1.	Loss on drying	0.69
2.	Water soluble extractive	1.43
3.	Loss on ignition	1.01
4.	Acid insoluble ash	0.33
5.	Alcohol soluble extractive	1.61
6.	pH (5% aqueous solution)	4.75

Fig. 2: Panchamuga Chendhuram (PMC) SEM with EDAX

Fig. 3: Panchamuga Chendhuram (PMC) SEM with EDAX

DISCUSSION:

Physicochemical Analysis:

The organoleptic examination indicates that the PMC produced is a brick-red powder with no discernible odor or taste (Table 1). When placed in water, PMC particles float, signifying that the surface potential of the formulation surpasses the weight of the particle¹⁹. The research demonstrates that a positive surface charge promoted cellular uptake, resulted in a favorable distribution in the small intestine, and substantially enhanced oral bioavailability^20. The loss on drying assessment of PMC at 105°C reveals a minimal loss of less than 0.69%. (Table 2). This minimal moisture content is crucial in preventing degradation of the medicine. Consequently, the shelf life of the drug becomes longer, as documented in Siddha literature which states that chendhuram medicines have 75 years shelf life. The lower moisture content can help inhibit the growth of bacteria, fungi, or yeast. Water soluble extractive of PMC is 1.43. Substantial ash

Fig. 4: Panchamuga Chendhuram (PMC) Fourier Transformed Infrared Spectroscopy (FTIR)

content and a lower value of water-soluble acids extract suggest potential issues such as contamination, substitution, adulteration, or negligence in the drug's preparation, processing, and storage. However, the physiochemical parameters were observed to fall comfortably within the established standards, indicating that the product has been genuinely and appropriately prepared²¹.

In recent years, the measurement of pH has become increasingly significant. Monitoring pH values has become essential in controlling and regulating chemical and biological processes²². The stability of a formulation in a water-based solution is significantly influenced by the pH level. It is crucial to ascertain the highest level of stability to ensure the quality of the preparation, and this stability must be maintained throughout the entire established period of validity and preservation³. The pH value of PMC was found to be of 4.75 which indicate the acid nature of the drug. For any medicine it is essential to maintain the pH within a specific range to prolong their preservation. The pH of medicine must be kept at a low level to prevent bacteria from degrading the medicine. The determination of pH remains a reliable quality indicator as it needs to remain consistent and reproducible.

Fourier Transformed Infrared Spectroscopy (FTIR):

FTIR analysis is conducted to discover the functional groups of biomolecules, elucidate the structure, and confirm the active molecules responsible for the therapeutic effects of the drug ²³. The study revealed that PMC contains carbon along with few organic functional groups might have been derived from the betel leaf juice used in drug preparation (Fig 4). Mercury, sulphur, arsenic was the major ingredient, but it became negligible in the finished product may be due to the catalytic activity of Betel leaf juice it also changes the particle size of the medicine. The band indicates the existence of the following groups in the PMC. The band at 1743 corresponds to C=O (Carbonyl group), band at 1609 corresponds to N-H (Nitrogen compound), band at 1111 corresponds to (Hydroxyl group) (alcohol), C-N, C=S, S=O, and band between 2924-2852 corresponds to C-H stretching (alkane). These findings indicate that PMC possesses preservative, anti-microbial, disinfectant activities. The herbal coating applied to the surface of herbo-mineral preparations in Siddha medicine resembles the contemporary process of producing metal oxide nanoparticles mediated by surfactants.²⁴

Morphology and surface characteristics:

The SEM was utilized for the morphological analysis of the PMC formulation, captured at magnifications of 20,000X, 50,000X, and 100,000X from various surface regions, verified the presence of nano-sized particles (Fig 2). At 100,000X magnification, smaller particles ranged from approximately 28nm to 42nm, while at 20,000X, larger particles were observed within the range of about 300nm to 977nm. On average, the particle size spanned from 50nm to 500nm (Fig 2). Due to the presence of nano and near-nano-sized particles, the formulation can effectively adhere to cell surfaces and easily penetrate cells, increases the absorption and bioavailability, and to expedite rapid pharmacological effects at specific locations, a minimal drug dosage is sufficient for treating diseases²⁵. The repeated heating process influenced the transformation of bulk material into mixed compounds; it has a tendency to cause drug particles to cluster together, leading to variations in particle size. This outcome underscores the impact of the preparation method on morphology, particularly in terms of prolonged trituration of metals and herbs, heating temperature and duration^26,27.

An analysis of the selected area using EDAX spectral analysis indicated the existence of C (22.35%), O (3.45%), S (16.88%), Hg (44.87%), and As (12.43%). This confirms the predominance of Mercury (Hg) as the primary component in the formulation (Fig 3).

Inductively Coupled Plasma Optical Emission Spectroscopy (ICPOES):

As per reports from the World Health Organization (WHO) and the Food and Drug Administration (FDA), the acceptable thresholds for heavy metals in safe clinical practice are arsenic (10 ppm), lead (10 ppm), cadmium (0.3 ppm), and mercury (1 ppm)²⁸. The concentrations of elements in the PMC are detailed in Table 3. The majority of analyses, including mercury, were assessed using ICP-OES. Other researchers have also employed ICP-OES for analyzing Siddha drugs or their constituents ²^9–³². The lead, mercury, and cadmium concentrations were either near or below the detection limits for emission spectrometry, thus requiring determination via GF-AAS, a technique slower but more sensitive than ICP-OES.

The complete preparation procedure for the PMC formulation mirrors the synthesis of nanomaterials. The initial step involving the grinding of raw materials corresponds to the top-down approach in assembling nanostructured materials. In the subsequent stage, the addition of herbal juices parallels the green synthesis of nanoparticles.

CONCLUSION:

The conclusions and criteria outlined in this research serve as a crucial instrument for establishing standards and ensuring quality in the production of the PMC. This information can prove beneficial for researchers, scientists, and academics, and could also be contemplated for setting the pharmacopoeial benchmarks for PMC. Additionally, the presence of various elements in different concentrations was observed, enhancing bioavailability and contributing to the therapeutic value of PMC. The alterations in size (Nanomedicine) and chemical composition observed in the formulation result from physical treatments such as raw material grinding and prolonged exposure to heat. Findings from ICP-OES, FTIR, SEM, EDAX studies can serve as reliable physicochemical fingerprints for PMC validation and offer insights into its therapeutic properties. This study demonstrates that PMC, produced through ancient classical pharmaceutical processes, has been validated by modern analytical standards, and reverse pharmacology has also been confirmed. It offers a guideline for creating the distinctive characteristics or "fingerprint" of PMC as a herbomineral formulation.

CONFLICT OF INTEREST:

The authors declare no conflict of interest

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Received on 15.02.2024 Revised on 17.06.2024

Accepted on 21.09.2024 Published on 01.07.2025

Available online from July 05, 2025

Research J. Pharmacy and Technology. 2025;18(7):3067-3072.

DOI: 10.52711/0974-360X.2025.00439

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