1. INTRODUCTION:

The Siddha System of Medicine (SSM) encompasses essential principles, a profound understanding of anatomy, physiology, and pathology, and a comprehensive collection of medicinal substances. It encompasses a variety of internal treatments and external therapies as well, showcasing proficiency in iatrochemistry well before the emergence of modern science.

As recorded in literature, the use of Siddha medicine for healing and promoting human health dates back to a period before 4000 BCE¹. In accordance with the Panchabootha Pancheekaranam principle in SSM, all entities in the universe, including the human body, are comprised of five basic elements. This theory asserts that the human body, its sustenance, and medicinal substances closely mirror the universe due to this essential combination. As per SSM, the human body comprises 96 primary components encompassing physical, physiological, psychological, and intellectual aspects. Within this framework, the medical perspective assigns particular importance to three humors (Vātam, pittam,and kapam) and seven bodily tissues (Cāram, ceṉṉīr, ūṉ, koḻuppu, eṉpu, mūlai, and cukkilam/curōṇitam)². In the pharmacological dimension of SSM, formulations are derived from herbs, and minerals sources, encompassing 32 varieties of internal and 32 varieties of external remedies. These manifest in diverse forms like Cūraṇam, māttirai, ney, parpam, centūram and kaṭṭu³.

In the current global context, there is a growing demand for herbal medicines due to their established safety and efficacy. Folk medicine is currently undergoing rigorous scientific scrutiny these days, involving extensive research into various herbals and their medicinal properties. Chromatographic and spectral fingerprint analyses are pivotal techniques in the quality control of herbal drugs. Thin-layer chromatography (TLC) serves as the preliminary step in identifying phytochemical components within a sample. High-performance thin-layer chromatography (HPTLC) goes a step further by producing an electronic chromatographic fingerprint image and a densitogram to pinpoint marker compounds in plant samples. Both methods are known for their efficiency, speed, reliability, and reproducibility^4-6. HPTLC, which stands for High-performance thin-layer chromatography, is a valuable method employed in the analysis of herbal and medicinal plants. It plays a important role in standardizing herbal materials, essential for identifying and assessing the purity and quality of herbal drugs. HPTLC fingerprinting allows for the estimation of phytochemicals in these plants, providing a chemical fingerprint and biomarker insights. This analysis helps quantify pharmacologically active constituents, revealing the therapeutic potential of phytochemicals in herbal medicines. It also identifies the number of constituents present, authenticated by marker compounds, ensuring the efficacy of herbal plants in traditional Indian medicine. HPTLC's popularity is due to its simplicity and reliability, serving as a preferred method for qualitative analysis of herbal plant constituents⁷.

Antioxidants are act as a shield for the cells and tissues to prevent from oxidative harm triggered by range of oxidizing agents such as reactive oxygen species (ROS), reactive nitrogen species (RNS), and free radicals⁸. Antioxidants possess a range of biological functions including antimutagenic, anticarcinogenic, antiaging, anti-inflammatory, and etc⁹. Swasakasa Nei (SKN), a polyherbal formulation documented in the Siddha shastric text “Cikicca rattiṉa tīpam eṉṉum vaittiyanūl,” is recommended for addressing a wide range of health conditions, including allergen-triggered asthma and eosinophilia. The recommended dosage entails 1 to 3 ml, taken twice daily along with adjuvant Kaṟkaṇṭu cūraṇam. SKN comprises key ingredients such as Argemone mexicana Linn and Calotropis procera Linn¹⁰. It is widely acknowledged that medicinally significant species possess a diverse range of secondary metabolites, with certain compounds being responsible for their biological activity. To confirm this, current research focuses on identifying distinct metabolites within the traditional siddha herbal formulation SKN. It was accomplished through comprehensive analysis involving phytochemical assessment, biochemical examination, TLC and HPTLC analyses as well as in-vitro antioxidant activity.

2. MATERIAL AND METHODS:

2.1. Ingredients in the Swasakasa Nei (SKN)

Ingredients of the SKN were listed in Table No: 1.

2.2. Collection of the Ingredients:

The raw drug materials (items 1 and 2) were gathered from wastelands, cultivated fields, and roadside areas in Kallakurichi, Thirupathur, and Coimbatore Districts. Third one was procured from a reputable ghee supplier like Aavin store in Chennai.

2.3. Preparation of the Swasakasa Nei:

Apertures, numbering four to five, were meticulously introduced at the lowermost section of the mud pot, subsequently accompanied by the attachment of iron wires, thereby facilitating the channelling of oil into a dedicated receptacle. Cow’s ghee was applied onto both sides of Veḷḷerukku leaves. The Piram'mataṇṭu root, quantified at 2¼ balam, underwent a meticulous division into diminutive fragments, which were subsequently enveloped in cow’s ghee. Within the aforementioned mud pot, stratification began with Veḷḷerukku leaves, followed by successive layers of the Piram'mataṇṭu root, a sequential repetition that persisted until the mud pot achieved saturation with these alternating layers of unprocessed substances. The pot’s orifice was methodically veiled through a combination of mud and cloth, with an underlying porcelain vessel strategically situated to capture the effluent oil. Superimposed atop this porcelain receptacle were cow dung cakes, subjected to a controlled process of incineration, ultimately resulting in the extraction of oil (referred to as Kuḻittayilam) due to the heat generated by the Puṭam¹⁰.

2.4. Preliminary Phytochemical Analysis:

Phytochemical Analysis were done by using standard methodologies for various phytochemicals mentioned below Alkaloids, Carbohydrates, Coumarins, Cyanins (Anthocyanins), Flavonoids, Glycosides, Phenols, Proteins, Saponins, Steroids, Tannins, Triterpenoids^11-13.

2.5. Biochemical analysis:

The qualitative biochemical chemical analysis of Swasakasa Nei was carried out at the Biochemical Laboratory of the National Institute of Siddha in Chennai using established methodologies. This comprehensive analysis encompassed a wide array of parameters. For acid radicals, tests were conducted for Chloride, Phosphate, Carbonate, Nitrate, Sulphide, Fluoride, Oxalate, Nitrite, and Borate. In the case of basic radicals, examinations covered Copper, Aluminium, Iron, Zinc, Calcium, Magnesium, Ammonium, Potassium, Sodium, Mercury, Arsenic, and Lead. Additionally, miscellaneous tests were performed, including assessments for Reducing sugar, alkaloids, Tannic acid, Unsaturated compounds, Amino acids etc.

2.6. Chromatographic identification & fingerprinting analysis by HPTLC:

2.6.1. Materials required:

i) Camag Linomat 5 sample applicator

ii) Syringe for application of sample on TLC plate Camag TLC Scanner 4

iii) HPTLC 60F 254 silica gel glass-backed layers (Merck)

iv) TLC developing chamber (Twint rough Chamber)

2.6.2. Method:

The HPTLC plates had been developed within a horizontal chamber, specifically the Camag 20 x 10 chamber, after being saturated with the same mobile phase. The chamber had been optimally saturated with the mobile phase for a duration of 20 minutes at room temperature. The chromatogram run had covered a length of 80mm. After development, the layers had been dried in an oven at temperatures ranging from 100 to 105°C for a period of 15 minutes before being subjected to detection. Initially, the separated components had been visually observed. Subsequently, the layers had been allowed to air-dry for 30 minutes before undergoing analysis using the appropriate detection method. For fingerprinting, a Camag TLC scanner 3, linked to win CATS software, had been configured to operate at 350nm, following an initial multi-wavelength scan ranging from 250 to 400nm in the absorption mode¹⁴.

2.7. Antioxidants Activity:

SKN was dissolved in an ethanol solution, and subsequent assays were conducted in a dose-dependent manner. The DPPH radical scavenging capacity of SKN was assessed following the method outlined by Suaib Luqman et al. with slight adjustments¹⁵. The Griess reaction method was employed to measure the nitric oxide radical scavenging activity of SKN¹⁶.

2.8. Statistical Analysis:

Statistical analysis, including the calculation of IC50 values and graph generation for all assays, was conducted using GraphPad Prism software.

3. RESULT:

3.1. Phytochemical Evaluation of Swasakasa Nei:

The preliminary phytochemicals test results reveal the presence of Alkaloids, Flavonoids Glycosides, Coumarin, and Phenols. The observed results were tabulated in Table no 2. These phytochemicals are responsible for potential therapeutic properties of SKN.

Table 2. Result of Phytochemical evaluation of Swasakasa Nei

S. No	Test	Observation
1.	Alkaloids	+Positive
2.	Anthocyanin	-Negative
3.	Betacyanin	-Negative
4.	Coumarin	+Positive
5.	Flavonoids	+Positive
6.	Glycosides	+Positive
7.	Phenols	+Positive
8.	Protein	-Negative
9.	Saponins	-Negative
10.	Steroids	-Negative
11.	Sugar	-Negative
12.	Tannins	-Negative
13.	Triterpenoids	-Negative

3.2. Biochemical Analysis of Swasakasa Nei:

Biochemical analysis of Acid radicals, Basic radicals and miscellaneous substances reveals the test drug SKN contains Carbonate, Aluminium, Iron and Alkaloid. The observed results were documented and presented in Tables 3, 4, and 5.

Table 3. Result of Acid radicals evaluation

S. No.	Test	Observation
1.	Chloride	-Negative
2.	Phosphate	-Negative
3.	Carbonate	+Positive
4.	Nitrate	-Negative
5.	Sulphide	-Negative
6.	Fluoride and Oxalate	-Negative
7.	Nitrite	-Negative
8.	Borate	-Negative

Table 4. Result of Acid Basic evaluation

S. No	Experiment
1.	Aluminium	+Positive
2.	Ammonium	-Negative
3.	Arsenic	-Negative
4.	Calcium	-Negative
5.	Copper	+Positive
6.	Iron	+Positive
7.	Lead	-Negative
8.	Magnesium	-Negative
9.	Mercury	-Negative
10.	Potassium	-Negative
11.	Sodium	-Negative
12	Zinc	-Negative

Table 5. Result of Miscellaneous tests

S. No	Experiment
1.	Reducing sugar	-Negative
2.	Alkaloids	+Positive
3.	Tannic acid	-Negative
4.	Unsaturated compounds	-Negative
5.	Amino acids	-Negative

3.3. TLC and HPTLC finger print analysis of the Swasakasa Nei:

In this current study, we examined the patterns of phytochemical constituents by analyzing the color zones in the HPTLC chromatogram under different lighting conditions (UV short, UV long and white light) after derivatization. The HPTLC fingerprints we obtained can be used to accurately identify the ingredients and their phytochemical components.

HPTLC fingerprint analysis of SKN yielded noteworthy findings regarding its composition. The analysis unveiled distinct peaks [Figure 1], each of which carries significance in identifying the constituents of SKN. In the first track, observed at a wavelength of 245 nm, two prominent peaks were clearly visible. These peaks had Retention Factor (Rf) values that fell within the range of 0.597 to 0.962 [Figure 2]. These Rf values are crucial in chromatography as they help pinpoint the relative mobility of compounds within the chromatogram. In this case, these two peaks corresponded to the presence of two versatile phyto components within the SKN sample.

Moving to the second track at the same wavelength (245 nm), a more complex picture emerged. Here, three prominent peaks were detected, and their Rf values spanned from 0.593 to 0.940 [Figure 3]. This suggests the presence of three distinct phyto components within SKN. The use of multiple wavelengths in HPTLC analysis can provide deeper insights into the composition of a sample. Unfortunately, when the analysis was conducted in track 1 at 366 nm, no peaks were observed [Figure 4]. This may indicate that certain compounds or phyto components present in SKN are not as readily detectable at this wavelength. However, when shifting to track 2 at 366 nm, a different story unfolded. Here, four prominent peaks, as evidenced in figure 5, were clearly discernible. These peaks had Rf values ranging from 0.604 to 0.928, indicating the presence of four distinct versatile phytocomponents within SKN. Figure 6(a) depicts a TLC plate under illumination at a wavelength of 366 nm. Figure 6(b) illustrates the same TLC plate after undergoing derivatization.

These results indicate the presence of various phytochemicals in the formulation and its constituent ingredients, as evidenced by the multiple peaks with different Rf values. In summary, the HPTLC fingerprint analysis of SKN provides valuable insights into its composition. The varying Rf values and the presence or absence of peaks at different wavelengths indicate the diverse nature of phyto components within SKN, contributing to a more comprehensive understanding of its chemical makeup.

a b

Figure 6. (a) TLC plate at 366nm of wavelength, (b) TLC plate after derivatization

3.4. Antioxidants activity:

3.4.1. DPPH Radical Scavenging Assay:

The DPPH Radical Scavenging Assay was conducted on SKN and compared to standard Ascorbic acid across concentrations ranging from 0 to 100μg/ml. At the highest concentration (100 μg/ml), SKN exhibited an inhibition rate of 61.56%, whereas Ascorbic acid demonstrated an inhibition rate of 91.38% at the same concentration. The IC50 value for Ascorbic acid was determined to be 24.97, while for SKN, it was 75.34. These findings were graphically presented in Figure 7.

Figure 7. DPPH Radical Scavenging Assay of Swasakasa Nei

3.4.2. Nitric oxide Scavenging Assay:

The Nitric oxide Scavenging Assay of SKN was evaluated and compared to standard Gallic acid across concentrations from 0 to 100 μg/ml. At the highest concentration (100 μg/ml), SKN showed an inhibition rate of 58.80%, while Gallic acid exhibited an inhibition rate of 86.60% at the same concentration. The IC50 value for Gallic acid was determined to be 34.65, while for SKN, it was 85.29. These findings were visually represented in Figure 8.

Figure 8. Nitric oxide Scavenging Assay of Swasakasa Nei

4. DISCUSSION:

The ingredients found in Swasakasa Nei consist of simple, effective, and readily available herbs, such as Brahmathandu ver (Argemone mexicana Linn) and Vellerukku ilai (Calotropis procera Linn). These ingredients possess various therapeutic properties, including Immunomodulatory, anti-inflammatory, anti-histaminic, anti-asthmatic, anti-spasmodic, anti-microbial, bronchodilator, expectorant, antioxidant, and anti-cancer activities. These effects are primarily attributed to the phytochemicals present in these ingredients. Various analytical studies reveals that the phytoconstituents of A. Mexicana include Cryptopine, Jatrorrhizine, Berberine, Argemexicaine A, Protopine, Chelerithrine, Columbamine, Sanguinarine, Arnottianamide, Argemexirine, Rutin, Coptisine, Oxyberberine, Dehydrocheilanthifoline, Nor-Sanguinarine, Angoline, (-)-Stylopine, Nor-Chelerythrine, Galanthamine, O-Methylzanthoxyline, Argemexicaine B, Thalifoline, Dihydrocoptisine, Dihydrosanguiranine, (+)-Cheilanthifoline, Magnoflorine, (-)-Tetrahydroberberine, Chelerythrine, Muramine, Palmatine, (±)-Tetrahydrocoptisine, Quercetin, Dehydrocorydalmine, Β-sitosterol, Sanguinarine, and Coptisine^17-19. C. Procera latex contains water, coagulate (0.8-2.5%), and compounds like calactin, calotropagenin, and calotoxin. Enzymes including Procerain and Procerain B exhibit clotting activity. The latex features amino acids, cardiac glycosides (e.g., calotropin), enzymes, and compounds like α-amyrin and β-sitosterol. Leaves contain cardenolides, tannins, glycosides, terpenoids, flavonoids, and alkaloids including β-amyrin and calotropin. Glycosides like calotoxin and calactin, along with the bitter compound Mudarine, are also present^20-30. In the present analysis, the results have demonstrated the abundant presence of constituents within the SKN preparation. Elevated levels of nitric oxide are integral to numerous inflammatory mechanisms. Excessive NO production can directly harm tissues and contribute to the vascular collapse observed in septic shock^{9, 31-40}. In this study, the ability of SKN to scavenge NO was investigated, and it was found that SKN exhibited notable scavenging activity against NO and also DPPH too.

5. CONCLUSION:

The study suggests that ensuring the quality assurance of the Siddha preparation, Swasakasa Nei (SKN), and its constituent elements can be achieved through a comprehensive evaluation of their phytochemical properties, biochemical characteristics, as well as TLC and HPTLC profiles. The existing HPTLC-fingerprinting profile, in conjunction with other scrutinized parameters, can be employed as a diagnostic tool to validate identity and gauge the quality and purity of the formulation in subsequent investigations. Moreover, the chromatographic fingerprint pattern will facilitate the identification of pharmacologically active and chemically distinctive constituents in both the formulation and the individual herbal components. The therapeutic potential of SKN could be attributed to its antioxidant properties. Moreover, in-depth examinations of specific biochemical or metabolic pathways could facilitate the identification of the cellular and molecular mechanisms of the SKN.

6. CONFLICT OF INTEREST:

The authors have stated that they do not have any conflicts of interest.

7. ACKNOWLEDGEMENTS:

The authors express their gratitude for the assistance and facilities extended by the National Institute of Siddha, located in Tambaram Sanatorium, Chennai, as well as ITC Labs (Interstellar Testing Centre Pvt. Ltd. Panchkula, Haryana).

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Received on 24.04.2024 Revised on 23.08.2024

Accepted on 02.11.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1765-1772.

DOI: 10.52711/0974-360X.2025.00253

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

S. No	Tamil Name	Scientific Name	Parts used	Quantity
1.	Piram'mataṇṭu	Argemone mexicana Linn	Root	2 ¼ palam (78.75 grams)
2.	Veḷḷerukku	Calotropis procera Linn	Riped leaf	60-80 Numbers
3.	Pacu ney	Cow’s ghee	Purified ghee	Required quantity