The Siddha system is a prominent traditional medicinal practice in South India. It characterizes medicine as a substance that enhances physical strength normalizes bodily functions and helps reduce or eliminate diseases. The Siddha approach includes two categories of medicines: internal and external, each containing 32 distinct types. One internal medicine example is Mezhugu, which is prepared by finely grinding various ingredients with specific juices or extracts until a soft, waxy texture is achieved. This research aims to standardize the Siddha herbo-mineral formulation known as Vaalai Rasa Mezhugu (VRM), indicated for treating conditions such as tumors, throbbing pain, cervical lymphadenitis, fistulas and rheumatoid arthritis (Sambasivampillai, 1931; Thiyagarajan and Jeevam, 2013). Standardization is crucial today as it develops a detailed quality profile while ensuring safety monitoring and comprehensive quality assurance for herbal medicines. Our focus on establishing a comprehensive national regulatory framework and safety monitoring systems for Siddha medicine is the need of the hour (Abarnaet al., 2024). Currently, there is a lack of sufficient documentation regarding its standardization and investigative aspects. This study systematically standardizes Vaalai Rasa Mezhugu using PLIM guideline-based analytical methods, including evaluations of organoleptic properties, HPTLC profiling, physicochemical tests and instrumental analyses for heavy metals, microbial contamination, specific pathogens, pesticide residues and aflatoxins.

The test drug “Vaalai Rasa Mezhugu” is one of the Herbo mineral formulations for arthritis and tumor which is indicated in the Siddha literature “Siddha marunthu sei perumuraigal” written by V. Balarammaiya (Balarammaiyaet al., 1980).

The raw drug was purchased in a country drug store, in Chennai and authenticated by the “Department of Gunapadam, National Institute of Siddha, Chennai”. (Certified No. NISMB6652024, GUN/AUT/07/24).

The ingredients are depicted in Table 1.

Cinnabar (Lingam) powder and Plumbago zeylanica (Chitramoolam) root bark are powdered in the ratio 1:4. Then Burnt in the sublimation apparatus, resultant mercury residue (Linga Rasam) is collected from the upper portion.

Camphor was mixed with tender coconut water in a clay pot. mercuric chloride, it was tied in cloth and hung in the pot, ensuring it did not contact the surface of water. The pot was boiled for 30 min until one-third of the water evaporated.

Betel leaf and pepper are ground and mixed in 1.3 L water, calomel is immersed in water and heated till 3/4th volume of water remains. It is then washed and dried.

First, the Rasam is ground with the juice of betel leaves till they completely blend. After that, Veeram and Pooram are added to it. Then Clove (Kirambu) and Cardamom (Elam) were dry roasted and powdered. Add both powders and Varagarisi flour to the above mixture and grind them well. A small proportion of castor oil is added and triturated until it reaches the mezhugu (semisolid/wax) consistency. Then the medicine is stored in an airtight container.

Following AYUSH-PLIM guidelines, Vaalai Rasa Mezhugu (VRM) was evaluated for its physicochemical properties and active components using HPTLC (Indian Pharmacopeia, 2014; LOHARet al., 2008). Various tests including pH, loss on drying at 105°C, total ash, water-soluble ash, acid-soluble ash, acid-insoluble ash and both water and alcohol-soluble extracts were conducted at the “Siddha Central Research, Anna Govt. Hospital Campus, Arumbakkam, Institute in Chennai”. Results were noted in (Tables 3, 4).

Weigh 2 g of VRM in an evaporating dish, heat at 105ºC for 5 hr, then weigh again to determine moisture content.

Dissolve 5 g of VRM in 25 mL of distilled water, filter and measure pH after letting it sit for 30 min.

Incinerate 2 g of VRM in a silica dish at 400ºC until it turns white, indicating carbon’s absence. The % of total ash was then calculated concerning the weight of the air-dried drug.

The ash was boiled in 25 mL water for 5 min and then filtered to separate the insoluble residue. The residue was washed with hot water, incinerated at 450ºC for 15 min and weighed. The water-soluble ash percentage was calculated by subtracting the residue weight from the total ash weight and then expressed as a % of the air-dried sample.

The total ash sample was treated with 25 mL of hydrochloric acid and boiled for 6 min. The resulting mixture was filtered using ash-free filter paper. Residual insoluble material was rinsed with hot water and then incinerated in a muffle furnace until constant weight was achieved. The weight of the air-dried ash was subsequently used to determine the percentage of acid-insoluble ash content.

5 g of VRM were soaked in 100 mL chloroform-water (1:1) for 24 hr with intermittent shaking (6 hr) and settling (18 hr). The filtered solution (25 mL) was evaporated to dryness at 105ºC and weighed. The % of the water-soluble extract was calculated based on the initial air-dried VRM weight.

VRM was subjected to solvent extraction using 100 mL of alcohol. The sealed mixture underwent intermittent agitation for 6 hr, followed by 18 hr of stagnation. The filtered extract (25 mL) was evaporated and dried at 105ºC to constant weight. The % of alcohol-soluble extract was then calculated relative to the air-dried drug’s initial weight.

The Vaalai Rasa Mezhgu test drug underwent High-Performance Thin Layer Chromatography (HPTLC) techniques to produce its unique fingerprint.

The HPTLC method is a sophisticated and automated separation technique that has evolved from traditional TLC. Using autosamplers and specially coated HPTLC plates enables precise and sensitive separation of components on both qualitative and quantitative levels. High-Performance Thin-Layer Chromatography (HPTLC) is a valuable tool for cost-effective and efficient analysis of plant materials. It offers high selectivity, sensitivity, speed and a streamlined sample preparation process, making it ideal for routine quality control checks. This method generates a chromatographic fingerprint of phytochemicals, helping verify herbal products’ identity and purity.

Chromatogram development was conducted in CAMAG Twin Trough chambers. The elution method was based on how well the sample components adhere to the stationary phase. After the elution, the plates were taken out and dried. They were then scanned at 366 nm using UV light. CAMAG software was employed to analyze the scanned data, resulting in a chromatographic fingerprint for each sample, with the respective R_f values recorded (Hapuarachchiet al., 2020; Subramanian et al., 2023). The HPTLC of VRM is shown in Figure 1.

Figure 1:
HPTLC Results Demonstrating the Fingerprint of Vaalai Rasa Mezhugu (VRM).

Sigma standards for Arsenic (As), Lead (Pb), Cadmium (Cd) and Mercury (Hg) were utilized in this analysis. Atomic Absorption Spectrometry (AAS), specifically the AA 240 Series model, was selected for its reliability in detecting metals and metalloids in environmental samples. This method was applied to determine the overall concentrations of heavy metals-cadmium, lead, mercury and arsenic in the test sample. For sample preparation, digestion with 1 mol/L HCl was performed to measure mercury and arsenic levels, while 1 mol/L HNO₃ was used for lead and cadmium. Standard solutions of 100 ppm for As and Hg were prepared in 1 mol/L HCl, with Cd and Pb prepared in 1 mol/L HNO₃ (Mohammedet al., 2018).

The product’s sterility was assessed using the pour plate procedure. When a contaminated or non-sterile sample (formulation) comes into touch with a medium rich in nutrients, it encourages the growth of the organism. After the required amount of time, the organism’s growth is recognized by a distinctive pattern of colonies. The colonies are known as CFUs or colony-forming units. About 15 mL of melted agar was added to a sterile petri dish that had been inoculated with the test sample at 45ºC. The dish was tilted and swirled to mix the agar and sample properly. Till the agar completely gelled, it was kept undisturbed (about 10 min). After that, plates were turned over and incubated for 48 hr at 37°C before being left open for 72 hr to observe fungal development. The number of grown organism colonies was then determined for CFU (Liet al., 2003).

The test sample was inoculated into selective media “(Eosin Methylene Blue, Deoxycholate Citrate, Mannitol Salt and Cetrimide Agar)” using the pour plate technique. Plants exhibited visible growth after 24-72 hr of incubation at 37ºC. Distinct colony morphology and coloration patterns, specific to each medium, indicated the presence of targeted pathogens (Pour plate Method, 2022). Detailed methods of specific pathogen testing for VRM are summarized in Table 2.

Test samples were extracted using acetone and then mixed thoroughly for a short time. The liquid was allowed to filter before adding more acetone. The sample was heated in a rotary evaporator at a temperature not exceeding 40ºC until most of the solvent had evaporated. A few milliliters of toluene were then added to the remaining residue and it was heated again until all the acetone was eliminated. The toluene was used to dissolve the resulting residue and a membrane filter was employed to filter the solution (Kiehlbauchet al., 2000; WHO, 2007).

A standard solution of aflatoxins B1, G1, B2 and G2 was prepared at concentrations of 0.5 µg/mL for B1 and G1 and 0.1 µg/mL for B2 and G2 by dissolving the standards in a 9.8:0.2 chloroform-acetonitrile mixture. For the analysis, aflatoxin standards (2.5-10 μL) were applied to a pre-coated TLC plate. The test sample was then subjected to a solvent mixture of acetone, chloroform and isopropyl alcohol (85:10:5) in an unsaturated chamber until the solvent front reached 15 cm. After drying, the chromatogram was developed and examined under 365 nm UV light to visualize the separated spots (Luciana et al., 2001).

The organoleptic characters of VRM were done and noted in Table 3.

The physicochemical analysis of VRM was done and noted in Table 4.

HPTLC fingerprinting can be employed to create quality standards for polyherbal compositions. For the sample, the VRM fingerprint is mentioned in Figures 1–4.

HPTLC fingerprinting analysis of VRM included the generation of 3D chromatograms at wavelengths of 254, 366 and 520 nm. The HPTLC fingerprints were scanned at these wavelengths, with peaks at 254, 366 and 520 nm highlighted. Figures 2–4 display the respective chromatograms and noted peak details.

Figure 2:
HPTLC Finger Printing Analysis of VRM (a) 3D chromatogram of 254 nm, (b) HPTLC fingerprints scanning of 254 nm, (c) peak 254 nm.

Figure 3:
HPTLC Finger Printing Analysis of VRM 3D chromatogram of 366 nm, (b) HPTLC fingerprints scanning of 366 nm, (c) peak 366 nm.

Figure 4:
HPTLC Finger Printing Analysis of VRM 3D (post derivatized) chromatogram of 520 nm, (b) HPTLC fingerprints scanning of 520 nm, (c) peak 520 nm.

The results of this investigation (Table 5) indicate that the sample contains no detectable traces of heavy metals, including mercury, arsenic, lead and cadmium.

Following the incubation period, no growth or colony formation was detected on any plates inoculated with the test sample. The results of the sterility testing for bacteria and fungi in VRM, as shown in Table 6 and Figure 5 revealed the following findings.

Figure 5:
Results of Sterility Testing for VRM.

In any of the plates the test material was put into, no growths or colonies were visible (Table 7 and Figure 6).

Figure 6:
Culture plate with specific pathogen medium (a) E. coli, (b) Salmonella, (c) Staphylococcus aureus, (d) Pseudomonas aeruginosa.

The results showed (Table 8) no traces of pesticide residues such as Organochlorine, Organophosphorus, Organo carbamates and pyrethroids in the sample provided for analysis.

The results indicated (Table 9) no spots on the TLC plates for the test sample when compared to the standards, confirming the absence of aflatoxin G1, G2, B1 and B2 in the sample.

In this study, the experimental drug Vaalai Rasa Mezhugu was prepared following established Siddha texts and included several purification processes as described in the literature. Physicochemical parameters were defined to evaluate the quality of the final product. The organoleptic characteristics of Vaalai Rasa Mezhugu verify the authenticity of both the raw materials and the final product, which is noted for its grey color, aromatic fragrance, sweet taste, semi-solid consistency, non-free-flowing nature and non-greasy texture. The moisture content of VRM is 6.8%, indicating its stability and shelf life. Additionally, the total ash value of VRM is 1.735%, representing the inorganic residue remaining after combustion, which is an important aspect of assessing the quality of crude drugs.

This analysis primarily ensures the quality of powdered medications. Assessing ash values helps confirm the absence of mineral contaminants such as earth, sand, or floor sweepings, as well as plant parts that should not be included, potential adulterants and degraded material (Shyamalaet al., 2021). The acid-insoluble ash value of VRM is 0.43%, while the water-soluble ash is 0.88%. A low acid-insoluble ash indicates minimal adulteration with siliceous substances (Shyamalaet al., 2021). The pH of the VRM is 6.25, reflecting its acidic nature. This acidity facilitates rapid absorption in the stomach when taken orally, making it suitable for oral administration. Solubility is essential for effective drug absorption in the gastrointestinal tract, as low oral bioavailability is often linked to poor solubility and permeability (Adithyaet al., 2024). The alcohol-soluble and water-soluble extractive values for the VRM formulation are 2.26% and 26.085%, respectively. The lower water-soluble extractive value suggests that water is less effective than ethanol in extracting active components of the drug. The HPTLC fingerprinting analysis of Vaalai Rasa Mezhugu (VRM) at 254 nm identifies eleven versatile phytocomponents, with R_f values spanning from 0.06 to 0.94. The peak with an R_f value of 0.58 shows the highest area percentage of 40.53%, indicating a high abundance of that compound. At 366 nm, there are 17 versatile phytocomponents present, with R_f values from 0.05 to 0.93 and the peak at 0.57 has the highest area percentage of 22.42%, reflecting the corresponding compound’s abundance. Finally, at 520 nm, twelve versatile phytocomponents are observed, with R_f values ranging from 0.03 to 0.89 and the peak with an R_f value of 0.33 has the highest area percentage of 42.47%, indicating a significant presence of that compound. The investigation results confirm that VRM is free from heavy metals such as arsenic and cadmium, ensuring the safety of the drug. Moreover, the levels of mercury and lead are below permissible limits, further ensuring safety. Microbiological analysis demonstrated no growth or colonies in plates inoculated with VRM, confirming the absence of viable microorganisms. The absence of pesticide residues such as organo-chlorine, organo-phosphorus and pyrethroids in VRM underscores its quality control.

In conclusion, the study on Vaalai Rasa Mezhugu (VRM) demonstrates the adherence to established Siddha practices and quality standards. These findings support the efficacy and safety of Vaalai Rasa Mezhugu, providing a solid foundation for its use in traditional medicine and encouraging further research into its pharmacological applications. Also this study will be serve as a reference in compiling the monograph of Vaalai Rasa Mezhgu in future.

The authors acknowledge the support and facilities provided by the National Institute of Siddha, Tambaram sanatorium and also, our Siddha Central Research Institute, Anna Govt. Hospital Campus, Arumbakkam, Chennai and Noble Research Solutions Chennai for their support in this study.