Phytochemical profiling of the Siddha Formulation Vasampathi Chooranam (VC): A Scientific Basis for Its Traditional Use in Treating Malignant Fungating Wounds

*Corresponding author: Roobhini Saravanan Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Phytochemical profiling of the Siddha Formulation Vasampathi Chooranam (VC): A Scientific Basis for Its Traditional Use in Treating Malignant Fungating Wounds Roobhini Saravanan * and Thomas Muthusamy Walter Department of Gunapadam marunthakaviyal, Government Siddha Medical College, Palayamkottai, Tirunelveli, Tamilnadu, India. World Journal of Advanced Research and Reviews, 2025, 27(03), 774–788 Publication history: Received on 02 August 2025; revised on 07 September 2025; accepted on 10 September 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.27.3.3144 Abstract Background and Aim: The Siddha system of medicine utilizes the polyherbal formulation, Vasampathi Chooranam (VC), to treat various ailments, including " vippuruthi," or Malignant fungating wounds. Despite its traditional use, a scientific validation of its active components and therapeutic mechanisms is lacking. This study aims to provide a comprehensive phytochemical profile of VC using modern analytical techniques to establish a scientific basis for its traditional application. Materials and Methods: The VC formulation was prepared according to the classical Siddha text “ Pararasasekara vaithiyam”. Its phytochemical composition was investigated using a three-stage approach: preliminary qualitative phytochemical screening identified major classes of compounds present in this formulation. High-Performance ThinLayer Chromatography (HPTLC) was employed to create a characteristic chromatographic fingerprint, identifying key compounds like gallic acid and quercetin. Finally, Gas Chromatography-Mass Spectrometry (GC-MS) was performed to identify specific volatile and semi-volatile compounds. Results and Discussion: The qualitative analysis confirmed the presence of tannins, phenols, terpenoids, and alkaloids, which are known for their antibacterial and anti-inflammatory properties. The HPTLC analysis further specified the presence of compounds such as ellagic acid, boswellic acid, and quercetin, linking the formulation's traditional use to their documented therapeutic effects. The GC-MS analysis identified specific antibacterial compounds, including 2- (Acetoxymethyl)-3-(methoxycarbonyl)biphenylene and Silicic acid, diethylbis(trimethylsilyl)ester, providing molecular-level evidence that supports the formulation's efficacy against infection-related pus. Conclusion: This study provides a robust scientific foundation for the traditional use of Vasampathi Chooranam. The synergistic presence of compounds with confirmed antibacterial, anti-inflammatory validates its application for Malignant fungating wounds forming cancers. The detailed phytochemical profile serves as a critical benchmark for quality control and paves the way for future pharmacological and clinical investigations into the formulation's therapeutic potential. Keywords: Siddha Medicine; Vasampathi Chooranam; Phytochemical Analysis; HPTLC; GC-MS; Malignant Fungating Wounds 1 Introduction Traditional systems of medicine, such as the Siddha system originating in ancient South India, possess a rich repository of knowledge regarding natural remedies. These systems utilize intricate formulations derived from herbs, minerals, World Journal of Advanced Research and Reviews, 2025, 27(03), 774–788 775 and animal products to address a vast spectrum of diseases. There is a need for scientific validation in integrating these traditional practices into modern healthcare. The Siddha formulation Vasampathi Chooranam (VC) is a Herbomineral formulation with a long history of use for various chronic wounds and cancers like “seezh vippuruthi ( Pus outbreak)”. It is a term that describes malignant fungating wounds, which occur when cancer cells spread to the skin, affect an estimated 5-14% of patients with advanced cancer. These incurable wounds typically appear in the final six months of a patient's life, often serving as a grim reminder of their approaching end. Because of their severe nature, these wounds require palliative care to help manage symptoms and reduce suffering. These malignant fungating wounds are characterized by a range of distressing symptoms, including pus, a foul smell, pain, bleeding, and tissue death (necrosis). They also produce large amounts of fluid (exudate) and are prone to significant microbial growth. Studies show that a high concentration of bacteria (over 105/g) can intensify pain and exudate. The presence of anaerobic bacteria in particular is linked to severe odor and a larger amount of exudate. Furthermore, bacterial byproducts like DMTS and putrescine are known to contribute to the wound's smell and the deterioration of the surrounding skin. The conventional treatment includes incision and drainage (I&D) of any abscesses, wound debridement to remove dead tissue, antibiotics, and appropriate wound care and dressings. Current research on treating these wounds is limited, with no single best approach established. This research aims to bridge this gap by conducting a comprehensive phytochemical analysis of Vasampathi Chooranam. Our study will employ a multi-pronged analytical approach to thoroughly characterize the formulation's chemical composition. The initial step involves a qualitative phytochemical analysis, which will screen for the presence of major classes of bioactive compounds. This includes identifying key constituents such as alkaloids, known for their diverse pharmacological activities; flavonoids, which are potent antioxidants and anti-inflammatory agents; tannins, recognized for their astringent and antimicrobial properties; and saponins, which possess immunomodulatory and cytotoxic effects. This preliminary screening will provide a foundational understanding of the formulation's chemical richness. Following this, we will utilize High-Performance Thin-Layer Chromatography (HPTLC) to generate a unique and reproducible chromatographic fingerprint of Vasampathi Chooranam. This technique is invaluable for quality control, as it provides a visual profile of the compounds present. By establishing a characteristic HPTLC fingerprint, we can create a standardized benchmark for future batches of the formulation, ensuring consistency and authenticity. This is a critical step towards developing a reliable and reproducible herbal product, which is essential for its acceptance in a modern scientific context. Finally, we will perform Gas Chromatography-Mass Spectrometry (GC-MS) analysis to identify and characterize the volatile and semi-volatile compounds within Vasampathi Chooranam. Many of the biological activities of herbal medicines are attributed to these compounds, which often include essential oils, terpenes, and fatty acids. GC-MS will provide a detailed mass spectral library of these components, allowing for their precise identification that could be responsible for its purported anticancer and antimicrobial effects against Malignant fungating wounds. By integrating these robust analytical methods, this study seeks to provide a strong scientific foundation for the traditional use of Vasampathi Chooranam. The data obtained from the qualitative analysis, HPTLC fingerprinting, and GC-MS profiling will not only validate its phytochemical composition but also serve as a crucial first step toward isolating and investigating the therapeutic potential of its active compounds against cancer. The findings of this research will contribute to the scientific understanding of this Siddha formulation and pave the way for its future pharmacological and clinical evaluation. 2 Materials and methods 2.1 Procurement and Authentication of Drug The formulation of Vasampathi Chooranam was meticulously prepared following the standardized procedure detailed in the classical Siddha text, Pararasasekara Vaithiyam [1]. Prior to preparation, all raw ingredients underwent a purification process as described in the Siddha literature, Saraku Suthi Seimuraikal [2]. This entire process, from purification to final formulation, was authenticated by the Chief Consultant at the Walter Siddha Research Centre (https://walters.res.in/www.walters.res.in/index.html). The identity of the 18 raw drugs used in the formulation was confirmed, and their taxonomical classification is presented in Table 1. World Journal of Advanced Research and Reviews, 2025, 27(03), 774–788 776 Table 1 Taxonomical Classification of Raw Drugs [3] S.No INGREDIENTS (Tamil name / English name) BOTANICAL NAME PART USED QUANTITY 1 Vasmbu/ Sweet Flag Acorus calamus Rhizome 10 gm 2 Kadukkai/ Chebulic Myrobalan Terminalia chebula Fruit 10 gm 3 Thandrikai/ Belleric Myrobalan Terminalia Bellarica Fruit 10 gm 4 Nellikai/ Indian Gooseberry phyllanthus emblica Fruit 10 gm 5 Kundrikam/ Indian Frankincense Boswellia serrata Gum - resin 10 gm 6 Manjisti/ Indian Madder Rubia cordifolia Root 10 gm 7 Kirambu/Clove syzygium aromaticum Flower bud 10 gm 8 Kukkil/ Sal Tree shorea robusta Resin 10 gm 9 Vizhalarishi/ False Black Pepper Embelia ribes Fruit 10 gm 10 Induppu/ Rock Salt sodium chloride impura Salt 10 gm 11 Kothumalli/ Coriander coriandrum sativum Fruit 10 gm 12 Kostam/ Crepe Ginger costus speciosus Rhizome 10 gm 13 Seerakam/ Cumin cuminum cyminum Fruit 10 gm 14 Sukku/ Ginger zingiber officinale Rhizome 10 gm 15 Perunkurumpai/ Frangipani Vine chonemorpha fragnans Root 10 gm 16 Thakaram/ Sickle Senna cassia tora Seed 10 gm 17 Milagu/ Black Pepper Piper nigrum Fruit 10 gm 18 Thipilli/ Long Pepper Piper longum Fruit 10 gm Figure 1 Raw Drugs of the formulation VC World Journal of Advanced Research and Reviews, 2025, 27(03), 774–788 777 2.2 Preliminary Phytochemical Analysis: The powdered extract of Vasampathi Chooranam was subjected to a series of chemical tests to identify the major classes of phytochemicals.[4] These tests were performed as follows. ● Saponins: A small amount of the extract was mixed with distilled water and shaken vigorously. The formation of a persistent foam indicated the presence of saponins. ● Tannins and Phenols: The extract was dissolved in water, and a 5% alcoholic ferric chloride solution was added. A dark blue color confirmed the presence of tannins, while a dark blue or green color indicated the presence of phenols. ● Terpenoids: The extract was dissolved in chloroform, and concentrated sulfuric acid was carefully added. The formation of a dark brown precipitate suggested the presence of terpenoids. ● Steroids (Lieberman-Burchard Test): A small sample of the extract was combined with 2 ml of chloroform in a dry test tube. Acetic acid, acetic anhydride, and two drops of concentrated sulfuric acid were then added. A resulting green color confirmed the presence of steroids. ● Quinones: A few drops of concentrated sulfuric acid were added to the extract. The appearance of a red color indicated the presence of quinones. ● Glycosides: The extract was mixed with anthrone and concentrated sulfuric acid. The mixture was then heated in a water bath, and the appearance of a green color confirmed the presence of glycosides. ● Carbohydrates: The sample solution was treated with a few drops of α-naphthol followed by the careful addition of 2-3 ml of concentrated sulfuric acid. A reddish-violet or purple ring forming at the junction of the two liquids indicated the presence of carbohydrates.[5] ● Alkaloids (Dragendorff’s Test): The extract was warmed with 2% sulfuric acid for two minutes and filtered. A few drops of Dragendorff’s reagent were then added to the filtrate. The formation of an orange-red precipitate signified the presence of alkaloids.[6] ● Flavonoids: The extract was dissolved in alcohol, and a 10% sodium hydroxide or ammonia solution was added. A dark yellow color indicated the presence of flavonoids. ● Proteins (Biuret Test): The sample solution was treated with a sodium hydroxide solution, followed by a few drops of very dilute (1%) copper (II) sulfate solution. A purple color indicated the presence of proteins.[7] 2.3 HPTLC Analysis High-Performance Thin-Layer Chromatography (HPTLC) was utilized to create a chromatographic fingerprint of the extract. ● Developing Solvent System: A range of solvent systems were tested to achieve optimal separation of the compounds. The system that provided the best resolution was selected for the analysis. ● Sample Application: The extracts were precisely applied as separate tracks onto pre-coated silica gel 60 F254 aluminum sheets. A CAMAG Automatic TLC Sampler 4 (ATS4) with a microliter syringe was used for this purpose, with each track having a width of 8 mm. ● Chromatogram Development: The plate with the applied samples was placed vertically in a pre-saturated CAMAG developing chamber. The mobile phase was allowed to ascend the plate by capillary action. ● Documentation and Densitometry: After the chromatogram was developed and air-dried, the plate was visualized and documented under ultraviolet (UV) light at 254 nm and 366 nm using a CAMAG Visualizer. The plate was then scanned at these same wavelengths using a TLC Scanner 4 to generate densitometric profiles. The WinCATs software associated with the scanner was used to record the Rf values and fingerprint data. ● Post-Chromatographic Derivatization: To visualize additional compounds, the plate was sprayed with a vanillin-sulfuric acid reagent. It was then heated to 105°C on a CAMAG TLC plate heater until colored bands appeared. The plate was then examined under white light, and the chromatograms were documented. The final scan was performed at 575 nm to capture the derivatized bands, and the corresponding Rf values and data were recorded.[8] 2.4 Gas Chromatography-Mass Spectrometry (GC-MS) analysis The volatile compounds present in the Vasampathi Chooranam sample were analyzed using a Gas ChromatographyMass Spectrometry (GC-MS) system. The setup included an Agilent 8890 GC-MS equipped with an AOC-20i auto-sampler and an Elite-5MS capillary column (30 × 0.25 μm ID × 0.25 μm df) [9]. World Journal of Advanced Research and Reviews, 2025, 27(03), 774–788 778 ● Operational Conditions: The instrument's electron ionization system was operated in electron impact mode at 70 eV. High-purity helium gas (99.99%) served as the carrier gas, flowing at a constant rate of 1.2 ml/min. A 1 μl sample was injected with a split ratio of 15:1 [10]. ● Temperature Profile: The injector temperature was maintained at 250°C, and the ion source was set to 230°C. The oven temperature began at 350°C, was ramped up at a rate of 5°C/min to 180°C (held for 3 min), and then increased again at 5°C/min to 300°C (held for 5 min) [11,12]. ● Mass Spectrometry Parameters: Mass spectra were recorded at 70 eV with a scan interval of 0.5 seconds across a mass range of 45 to 450 Da. The solvent delay was 3 minutes, and the total run time was 53.5 minutes [13]. ● Data Processing: A Turbo-Mass Gold-Perkin–Elmer mass detector was used to acquire the data. The relative abundance of each identified compound was determined by comparing its average peak area to the total peak area. The collected data was processed and analyzed using Turbo-Mass ver-5.2.28-29 software [14,15,16]. 3 Results 3.1 Phytochemical qualitative analysis The phytochemical qualitative analysis of the aqueous extract of Vasampathi Chooranam (VC) revealed a rich profile of secondary metabolites. Out of the eleven tests performed, seven yielded positive results, indicating a promising composition for the formulation. The findings, detailed in Table 2, suggest that VC is a complex mixture of bioactive compounds, which aligns with its traditional use and points to its potential therapeutic efficacy. Table 2 The results of phytochemical qualitative analysis of Vasampathi Chooranam Vasambathi Chooranam (VC) SI.No Tests Result 1 Saponins - 2 Tannins + 3 Phenols + 4 Terpenoids + 5 Alkaloids + 6 Flavanoids - 7 Steroids - 8 Glycosides + 9 Carbohydrates + 10 Quinones + 11 Proteins - 3.2 HPTLC Analysis The HPTLC analysis of the Vasampathi Chooranam (VC) extract was performed using a mobile phase consisting of Toluene: Ethyl acetate: Methanol (5:2.5:0.1), which provided good resolution. The chromatograms were visualized under UV light at 254 nm and 366 nm, and under visible light at 575 nm. The HPTLC analysis of the Vasampathi Chooranam (VC) extract was performed at two different concentrations (1–5 µl and 2–7 µl) to create a chromatographic fingerprint. The mobile phase, a mixture of Toluene:Ethyl acetate:Formic acid (5:3:0.1), provided excellent separation and resolution of the compounds. The resulting chromatograms were documented under UV light at 254 nm and 366 nm, as well as under visible light at 575 nm, as shown in Figure 1. Under UV light at 254 nm, both concentrations revealed multiple distinct spots. The 1–5 µl concentration showed six spots with Rf values of 0.28, 0.34, 0.63, 0.74, 0.82, and 0.93. The 2–7 µl concentration also showed six spots, with Rf values of 0.25, 0.42, 0.63, 0.72, 0.79, and 0.90 (Figure 3). World Journal of Advanced Research and Reviews, 2025, 27(03), 774–788 779 When visualized under UV light at 366 nm, the chromatograms showed different profiles. The 1–5 µl concentration displayed seven spots with Rf values of 0.04, 0.11, 0.17, 0.31, 0.63, 0.78, and 0.89. The 2–7 µl concentration, on the other hand, showed six spots with Rf values of 0.09, 0.14, 0.28, 0.61, 0.76, and 0.87 (Figure 5). Finally, after post-chromatographic derivatization and visualization under visible light at 575 nm, the analysis revealed an even greater number of compounds. The 1–5 µl concentration produced twelve spots with a wide range of Rf values (0.01, 0.04, 0.07, 0.12, 0.16, 0.19, 0.28, 0.37, 0.55, 0.74, 0.84, and 0.94). The 2–7 µl concentration showed ten spots with Rf values of 0.02, 0.07, 0.25, 0.34, 0.45, 0.52, 0.62, 0.72, 0.82, and 0.92 (Figure 7). 3.3 Phytochemicals identified by HPTLC analysis HPTLC analysis identified several key compounds in the Vasampathi Chooranam extract. A spot with an Rf value of 0.28, observed in the 1–5 µl concentration under UV 254 nm, closely resembled gallic acid, a type of phenolic acid. Under UV 366 nm, the same concentration showed a spot with an Rf of 0.31, similar to the flavonoid rutin. At this same wavelength, spots with Rf values of 0.34 and 0.72 were observed, corresponding to the polyphenol ellagic acid and the phenolic compound boswellic acid, respectively. Furthermore, the 2–7 µl concentration, visualized under visible light at 575 nm using the same mobile phase (Toluene: Ethyl acetate: Formic acid at a ratio of 5:3:0.1), showed a spot with an Rf value of 0.76 that was very similar to the phenolic compound quercetin. Other identified compounds included ellagic acid (a tannin) at an Rf of 0.42, the alkaloid piperine at an Rf of 0.55, the steroid eugenol at an Rf of 0.79-0.80, and a triterpenoid at an Rf of 0.94. Figure 2 Compounds present in the HPTLC analysis in different wavelengths World Journal of Advanced Research and Reviews, 2025, 27(03), 774–788 780 Figure 3 HPTLC profile of Vasampathi Chooranam Figure 4 HPTLC finger print profiles and peak tables of VC at 254nm World Journal of Advanced Research and Reviews, 2025, 27(03), 774–788 781 Figure 5 HPTLC finger print profiles and peak tables of VC at 366nm Figure 6 HPTLC finger print profiles and peak tables of VC at 575nm 3.4 GC-MS Analysis for Identification of Bioactive Compounds To further identify the specific components of Vasampathi Chooranam (VC) and confirm the compound groups suggested by HPTLC, a Gas Chromatography-Mass Spectrometry (GC-MS) analysis was performed. This analysis was crucial for pinpointing individual compounds that may contribute to the formulation's traditional use against Malignant fungating wounds which often involves an antibacterial component. The GC-MS analysis revealed the presence of two specific compounds in the VC formulation. These compounds, along with their known medicinal properties, are as follows ● 2-(Acetoxymethyl)-3-(methoxycarbonyl)biphenylene ● Silicicacid, diethylbis(trimethylsilyl)ester World Journal of Advanced Research and Reviews, 2025, 27(03), 774–788 782 These identified compounds are known to possess significant biological activities. For instance, some of the detected compounds are reported to have antibacterial and antiviral effects. These include reported antibacterial and antiviral properties, which could be key to the formulation's efficacy against Malignant fungating wounds. Figure 7 Chromatogram of VCextract using Gas Chromatography-Mass Spectrometry