Validating the Consistency and Quality of “Vaankumari Legiyam” A Classical Siddha Polyherbal Formulation Through Advanced Analytical Methods

Validating the Consistency and Quality of “Vaankumari Legiyam” A Classical Siddha Polyherbal Formulation Through Advanced Analytical Methods | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Validating the Consistency and Quality of “Vaankumari Legiyam” A Classical Siddha Polyherbal Formulation Through Advanced Analytical Methods Sujit Dash This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4594047/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Among Siddha's internal medicine dosage forms, "Legiyam" stands out as a widely recognized medicinal confectionary. This preparation involves heating decoctions, juices, milk, and sugar to form a syrupy consistency. Drug powders and other ingredients are added, and the mixture is cooled before incorporating ghee and honey. "Vaankumaria legiyam," a classic Siddha polyherbal formulation in confectionary form, contains various herbal ingredients known for addressing numerous health concerns, including digestion, diabetes, and skin issues.Commercially available formulations often lack proper standardization, leading to safety and efficacy concerns. In this context, the present study aims to prepare an in-house Siddha formulation using standard raw materials outlined in the Siddha Pharmacopoeia of India. Comparative phytochemical studies was conducted using modern analytical techniques such as AAS, FTIR, HPTLC, and GCMS. The Vaankumari legiyam formulations were tested for heavy metals and trace elements, revealing concentrations below regulatory limits. High-Performance Thin-Layer Chromatography (HPTLC) and Fourier Transform Infrared Spectroscopy (FTIR) analyzed the phytochemical profile, confirming the product's authenticity. Gas Chromatography-Mass Spectrometry (GC-MS) identified and quantified volatile organic compounds, confirming the in-house preparation's reproducibility in capturing aromatic and volatile constituents. These tests confirmed the product's safety for human consumption.The current research has shed light on the potential phytoconstituents responsible for the formulations’ claimed therapeutic effects and facilitates quality assessment. The comprehensive analysis of both marketed and in-house formulations of Vaankumari legiyam through various techniques, including phytochemical, physicochemical, AAS, HPTLC, FTIR, and GCMS, has demonstrated consistent and equal results. Integrative & Complementary Medicine Vaankumari legiyam Siddha Polyherbal formulation AAS FTIR HPTLC and GCMS. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Vaankumari legiyam is a traditional Siddha polyherbal formulation used in various ailments, including sarva roganoigal, diabetes, high cholesterol, insect bites, and PCOS treatment [ 1 , 2 ]. However, traditional formulations often lack proper standardization, leading to safety and efficacy issues. The quality and composition of these ingredients can vary depending on the source, the growing conditions, and the processing methods used [ 3 ]. This can lead to variations in the quality, safety, and efficacy of Siddha formulations. Standardization can help to ensure that Siddha formulations are made to a consistent quality and that they meet the expected standards. Standardization can also help to facilitate research on Siddha formulations. By having standardized formulations, researchers can be sure that they are comparing the same product across different studies. To address this, an in-house formulation will be prepared using standard raw materials and modern analytical techniques like AAS, FTIR, HPTLC, and GCMS. This will help identify potential phytoconstituents responsible for treating various diseases and assess their quality. The aim is to ensure that all ingredients are present in the finished product and that consumers have access to information about the product's quality and safety. 2. Materials and Methods 2.1. Procurement of Samples and authentication The marketed Vaankumari legiyam (Confectionery) formulation was used in the present study having (Batch No. LEU 21019). The Vaankumari legiyam was procured from online e-commerce gateway. A systematic profiling of Siddha formulation Vaankumari legiyam which was carried out by using authenticated medicinal plant parts for the preparation of the in-house formulation. The plant materials/parts for the preparation of in-house Vaankumari legiyam were purchased from Rana Ayurveda, Banka Bazar, Cuttack-753002, Odisha, India and authenticated by Dr. Amar Kumar Rana (B.A.M.S), Cuttack,Odisha 2.2. Organoleptic evaluation of Vaankumari legiyam Organoleptic evaluation is crucial for assessing the quality and standardization of polyherbal formulations. It involves examining color, size, shape, and presence for extraneous material or foreign particles. This assessment helps determine sensory properties, authenticity, and consistency, ensuring the reliability and effectiveness of polyherbal formulations. Integrating organoleptic evaluation into quality control processes promotes their acceptance and integration into mainstream healthcare practices [ 4 ]. 2.3. Physicochemical evaluation of Vaankumari legiyam Physicochemical testing is crucial for quality control and standardization of polyherbal formulations, as it helps identify active ingredients, physical and chemical properties, and stability. It ensures the safety and efficacy of these formulations. To determine the quality of Vaankumari legiyam, tests were conducted, including loss on drying, total ash, acid insoluble ash, pH, reducing sugar, alcohol-soluble extractive, and water-soluble extractive. These tests help ensure the safety and efficacy of polyherbal formulations[ 5 , 6 , 7 ].. 2.4. Preliminary phytochemical evaluation of Vaankumari legiyam Therapeutic activity of vegetable drugs depends upon the type of constituents present in them. Plant material was screened for the presence of phytoconstituent(s) using different chemical tests. Powder drug and different extracts were screened for different phytoconstituents [ 8 , 9 ]. 2.5. Biochemical analysis of acid and basic radicals of Vaankumari legiyam Polyherbal formulations in traditional medicine require quality control and standardization to ensure safety and effectiveness. Inorganic salts contain acid radicals that donate protons, causing an excess of positively charged ions. Basic radicals, such as hydroxide, carbonate, and bicarbonate, modulate pH, solubility, and biological activity. Spectroscopic techniques like AAS and ICP-MS are used to isolate and identify specific components, while acid-base titration techniques determine the overall amount of acid or base in the formulation [ 10 , 11 , 12 ]. 2.6. Heavy metal analysis by AAS (Atomic absorption Spectroscopy) of Vaankumari legiyam The study used Atomic Absorption Spectrometry (AAS) to analyze heavy metals in the classical polyherbal formulation Vaankumari legiyam. The method involved a wet digestion method, where 2 g of sample was weighed, concentrated nitric acid added, and the solution was heated for 2 hours. Standard solutions were prepared, and the wavelength was fixed between 185–900 nm. Test samples were analyzed against the standard, and the results were plotted against the concentration of each standard solution. The study aimed to determine the concentration of heavy metals in the polyherbal formulation Vaankumari legiyam [ 13 , 14 ]. 2.7. High performance thin layer chromatography analysis of Vaankumari legiyam HPTLC is a valuable tool for evaluating polyherbal formulations, providing a chromatographic fingerprint of phytochemicals. Samples were prepared and filtered through a 0.45-micron syringe filter. HPTLC analysis was performed on Merck TLC plates Al silica gel 60 F 254. The extracts were spotted on a silica gel 60 F 254 plate, developed using methanol solvent, and visualized using Visualiser-1 A and CAMAG Scanner 4 B software [ 15 , 16 ]. 2.8. Fourier transform infra-red spectroscopy of Vaankumari legiyam FTIR is a crucial technique for identifying functional groups, determining material quality and consistency, and determining the number of compounds present in a sample. It uses infrared to absorb and transmit infrared, creating a molecular fingerprint of the sample. FTIR is fast, sensitivity, mechanically simple, and internally calibrated. The Central Instrumental Laboratory (CIL) at IPT Salipur conducted FTIR (ATR) analysis using a Bruker FT-IR Spectrometer with Attenuated Total Reflectance technology (ATR) [ 17 ]. 2.9. Gas chromatography- thin layer chromatography of Vaankumari legiyam GCMS is essential for analyzing plant-derived components, ionizing compounds and measuring mass numbers. The EI method, which produces ions, offers high stability and reproducibility. Methanol was extracted from Vaankumari legiyam, and GCMS analysis was performed using a Shimadzu GCMS-QP2020 and Quadrupole MS [ 18 ]. 3. Results and Discussion 3.1. Organoleptic evaluation of Vaankumari legiyam Organoleptic analysis is crucial for standardizing polyherbal formulations, as it helps determine the physical traits and quality of plant materials used in the formulation. The sensory qualities of plant extracts reveal information about their chemical makeup, such as taste, odour, and appearance. The organoleptic evaluation parameters are cited in Table 1. Organoleptic evaluationattributes can affect a product's acceptability, and organoleptic analysis is essential for determining consistency and homogeneity. The market formulation is standardized due to similarities in taste, color, odour, and appearance with the in-house formulation [19]. 3.2. Physicochemical evaluation of Vaankumari legiyam Physicochemical evaluation is crucial for standardizing Vaankumari legiyam formulations, as it helps identify and authenticate each ingredient. The selected and preferred formulation were subjected to various evaluations and analytical studies such as; Loss on drying, Total Ash, Acid insoluble ash, pH, Reducing Sugar, Total Sugar, Water soluble extractive, Alcohol soluble extractive. The physicochemical data obtained from the above studies are shown in Table 2. Physicochemical evaluation of Vaankumari legiyam helped establish quality standards for each ingredient, ensuring consistency and quality. The market formulation is standardized due to its comparable physicochemical parameters with the in-house formulation, ensuring safety, efficacy, and quality. The study demonstrates the importance of physicochemical evaluation in ensuring the safety, efficacy, and quality of Vaankumari legiyam formulations [20]. 3.3. Preliminary phytochemical evaluation of Vaankumari legiyam The Vaankumari legiyam formulation underwent standardization methods, which included preliminary phytochemical analysis on VLMF and VLIF to identify phytoconstituents. This analysis aimed to ensure consistency and quality in the active constituents, setting specifications for raw materials and finished products. The results of these analyses are presented in Table 3. Preliminary phytochemical evaluation of Vaankumari legiyam process confirmed the genuineness of the formulation by revealing the presence of comparable phytoconstituents in both marketed and in-house formulations, thus establishing its standardized status [21]. 3.4. Heavy metal analysis by AAS (Atomic absorption Spectroscopy) of Vaankumari legiyam AAS results have been displayed in Figure 1a,1b,1c,2a,2b,2c&Table 4which revealed the concentration level of arsenic and mercury was below detection limit (BDL) in both VLMF and VLIF. Results were compared with the allowable limit acceptability intake of the World Health Organization (WHO) and American Herbal Products Association (AHPA). Concentration of heavy metal (As and Hg) in ppb/mean medicines should not exceed 10 µg/g or ppm, and for mercury, the limit is 0.1 µg/g or 0.1 ppm. In the herbal sector, heavy metal contamination of plant extracts is a regular issue that might jeopardize the product's safety and effectiveness.According to WHO standards, acceptable levels of arsenic in herbal medicines should not exceed 10 µg/g or ppm, and for mercury, the limit is 0.1 µg/g or 0.1 ppm [22, 23]. A recent study discovered that VLMF and VLIF herbal formulations contained undetectable levels of arsenic and mercury. This absence of heavy metals indicates that these herbal products are safe for consumers, as excessive exposure to arsenic and mercury can lead to various health issues, including skin problems, neurological disorders, and more.The AAS protocol successfully assessed heavy metal and trace element concentrations in herbal products and phytopharmaceuticals. It utilized AAS spectra to analyze elemental composition, identifying contaminants. Results show that the marketed formulation meets standards and matches the in-house formulation. Arsenic and Mercury levels are below WHO limits, suggesting no heavy metal toxicity risk for users in healthcare applications. 3.5. High performance thin layer chromatography analysis of Vaankumari legiyam This study utilizes High Performance Thin Layer Chromatography (HPTLC) technique to verify the authenticity of Siddha polyherbal formulation and the final product. The study conducted a comparative fingerprint analysis on samples VLMF and VLIF using mobile Phase A and mobile Phase B. Results showed 2 bands at the same R f under 254nm, 4 bands under 366nm, and 5 bands under R white light after derivatization with Anisaldehyde sulphuric acid reagent. The results are shown in Figure 3a, 3b, 3c andTable 5. The HPTLC fingerprinting of VLMF showed 04 bands having maximum Rf value 0.43, 0.56, 0.68, 0.94 at 254nm and VLIF showed 02 bands having maximum Rf value 0.68, 0.94 at 254nm under Mobile phase A (Chloroform: Ethyl acetate: Formic acid 5:4:1 v/v/v). VLMF showed 05 bands having maximum Rf value 0.38, 0.49, 0.59, 0.67, 0.91 at R366 derivatized with ASR and VLIF showed also 05 bands having maximum Rf value 0.49, 0.56, 0.59, 0.67, 0.91 at R366 derivatized with ASR, VLMF showed 06 bands having maximum Rf value 0.90, 0.66, 0.59, 0.52, 0.49, 0.38 at Rwhitederivatized with ASR and VLIF showed also 06 bands having maximum Rf value 0.90, 0.66, 0.59, 0.57, 0.52, 0.49 at Rwhitederivatized with ASR under mobile phase B (Chloroform: Toluene: Ethanol 4:4:1 v/v/v). This HPTLC technique may be advantageous for both the identification and the quality aspect-evaluation of formulations containing different or diverse plants parts. The HPTLC study analyzed the similarities and differences between VLMF and VLIF herbal formulations. The marketed formulation showed common black color bands at the same Retention Factor (Rf) under 254nm with mobile phase A. Under 366nm, the plate developed with Mobile Phase B and derivatised with Anisaldehydesulphuric acid showed four pink-colored bands at the same Rf. Mobile Phase B showed five purple color bands at the same Rf. Two black color bands were found in VLMF under 254nm with mobile phase A, while two pink-colored bands were absent in VLIF. The Rf values provided information about the composition of the two formulations [24]. However, slight variations in Rf values can be expected due to batch-to-batch variations, raw material variations, or formulation process differences [25]. 3.6. Fourier transforms infra-red spectroscopy of Vaankumari legiyam In the present study, the content of Vaankumari legiyam marketed and in-houseformulationwere investigated using FTIR spectroscopy by monitoring different functional groups. Figure 4a & 4b shows the FTIR spectra obtained or achieved from the marketed and in-house Vaankumari legiyam in the 4000–500 cm -1 region. The VLMF and VLIF was identified to have important 12 and 10observed peaks respectively. There are the groups present in the polyherbal formulations of Vaankumari legiyam. The spectra were interpreted in context of comparative analysis of Vaankumari legiyam marketed and in-house Formulation. Both the samples were analyzed, evaluated for variation in the functional groups and bonding pattern or arrangement. The FTIR peaks of the spectra of the VLMF and VLIF were compared which are shown in Table 6 and Table 7. FTIR had been used to identify and measure the phytoconstituents present in the plant extracts used in the formulation, FTIR is a crucial tool for the standardization of polyherbal formulations. An infrared absorption spectrum specific to each phytoconstituent has used to identify and measure the component in a sample [26]. The purity and quality of the raw ingredients and the completed product has also been revealed by FTIR. The chemical make-up of the sample has been ascertained using FTIR spectra, as can any impurities or contaminants that may be present [27]. From the above results it is ascertained that the marketed formulation is a standardized one as because of there are no significance variations in important observed peak in cm -1 along with the functional group in VLMF and VLIF by use of advanced instruments. The functional groups identified from FTIR will give a better solution for future analysis and clinical trials. 3.7. Gas chromatography- thin layer chromatography of Vaankumari legiyam The GCMS chromatogram Figure 5a & 5b of methanolic extracts of Vaankumari legiyam marketed formulation revealed 76 and in-house formulation revealed 72 compounds respectively which belonged to various classes of secondary metabolites. The peak report of the chromatogram obtained with details of peak number, retention time, area percentage, name of the identified phytocomponent, its molecular formula and molecular weight, are presented in Table 8 & 9. The mass spectrum was obtained with mass/charge ratio on x-axis and relative intensity on y-axis. The comparative study of VLMF and VLIF formulations with respect to GCMS (Gas Chromatography-Mass Spectrometry) analysis focuses on the analysis and comparison of the chemical composition of the formulations. GCMS is a powerful technique that allows for the identification and quantification of a wide range of compounds present in complex mixtures [28]. In this study, both the marketed and in-house polyherbal formulations were subjected to GCMS analysis to determine their chemical profiles. The GCMS chromatograms obtained for each formulation were carefully examined and compared to identify any similarities or differences in the composition. The findings of the GCMS analysis revealed that both the marketed and in-house formulations contained a variety of bioactive compounds. The chromatograms exhibited RT, indicating the presence of similar compounds in both formulations. This suggests that the formulations may share common herbal ingredients. The appearance of peaks at different m/z ratio represents the smaller compounds, formed due to fragmentation of larger compounds. These spectra act as fingerprint of the compounds and are identified by matching with NIST data library [29, 30, 31]. 4. Conclusion In conclusion, the comprehensive analysis of both marketed and in-house formulations of Vaankumari legiyam through various techniques, including phytochemical, physicochemical, AAS, HPTLC, FTIR, and GCMS, has demonstrated consistent and equal results. This rigorous standardization process reaffirms the authenticity and quality of the marketed product establishing its equivalence to the in-house formulation. Such meticulous evaluation ensures that consumers receive a reliable and effective herbal remedy while promoting trust in the herbal healthcare industry. While this study has laid a foundation for the standardization and quality assurance of Vaankumari Legiyam, further research in pharmacology could be undertaken to explore its therapeutic potential and mechanism of action more comprehensively. Declarations Acknowledgements The authors are grateful to the management members and Principal, Institute of Pharmacy & Technology, Salipur for providing all facilities and encouragement throughout the work. The authors would like to thanks the Central Analytical Facilities, Manipal University, Jaipur for carrying out the GC–MS, AAS analysis of Vaankumari Legiyam and Anchrom Enterprisesa (I) Pvt. Ltd. Mumbai for carrying out HPTLC analysis of Vaankumari Legiyam Ethical statement This article contain no studies with animals The authors declare that they have no conflicts of interest Author contributions Sujit Dash: Contributed to conceptualization, methodology, formal analysis, investigation, writing - original draft preparation, and supervision. Minaketan Sahoo: Contributed to methodology, investigation, and data curation. Amaresh Chandra Sahoo: Contributed to methodology, investigation, and data curation. Mrityunjay Baneerjee: Contributed to methodology, investigation, and data curation. Saroja Kumar Patro: Contributed to resources, writing - review and editing, and project administration. Prabhat Kumar Sahoo: Contributed to validation, writing - review and editing, and supervision. Priyanka Muduli: Contributed to investigation and data curation. References Anonymous. The Siddha Formulary of India part-1. 1st Edition. 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Vol. 9, No. 2, pp. 335-543, 2018. Kushwaha P, Yadav SS, Singh V, Dwivedi LK. GCMS analysis of bioactive compounds in methanoli extract of Ziziphus Mauritiana fruit. Int. J. Pharm. Sci. Res. Vol. 10, No. 6, pp. 2911-2916, 2019. Tables Tables 1 to 9 are available in the Supplementary Files section Additional Declarations The authors declare no competing interests. Supplementary Files Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4594047","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":315402074,"identity":"0fd1167b-190e-4294-af01-76d1ae69002a","order_by":0,"name":"Sujit 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12:09:41","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4594047/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4594047/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58654853,"identity":"27dbbe9b-ffc3-4137-ab25-b3f0fd6e6a87","added_by":"auto","created_at":"2024-06-19 11:09:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":214957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eStandard calibration curve for arsenic\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb \u003c/strong\u003eAbsorbance of VLMF for arsenic\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec \u003c/strong\u003eAbsorbance of VLIF for arsenic\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4594047/v1/5ee83260bd8df514326e5476.png"},{"id":58654852,"identity":"67fbb5be-0410-4756-ae23-edf1327753a7","added_by":"auto","created_at":"2024-06-19 11:09:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":282484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eStandard Calibration Curve for mercury\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb \u003c/strong\u003eAbsorbance of VLMF for mercury\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec \u003c/strong\u003eAbsorbance of VLIF for mercury\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4594047/v1/97c72ebb8436aabb640ad4a8.png"},{"id":58654851,"identity":"3430f20e-781f-4087-b91f-9f59e5cf0391","added_by":"auto","created_at":"2024-06-19 11:09:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":706789,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eHPTLC plate with R\u003csub\u003ef\u003c/sub\u003e tips:Development in Mobile Phase A, Image under R254, for Fingerprint profile (Track 1, 2, 3-VLMF, Track 4, 5, 6- VLIF)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e HPTLC plate with R\u003csub\u003ef\u003c/sub\u003e tips:Development in Mobile Phase B, derivatization with ASR: Image under R366, for Fingerprint profile(Track 1, 2, 3-VLMF, Track 4, 5, 6- VLIF)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec \u003c/strong\u003eHPTLC plate with R\u003csub\u003ef\u003c/sub\u003e tips:Development in Mobile Phase B, derivatization with ASR: Image under RWhite, for Fingerprint profile (Track 1, 2, 3-VLMF, Track 4, 5, 6- VLIF)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4594047/v1/e99b3aeb5c02fa580213a134.png"},{"id":58654855,"identity":"a3a0a783-8b8f-441b-9dd7-e0d0b91276a2","added_by":"auto","created_at":"2024-06-19 11:09:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":183992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eFTIR spectrum of VLMF\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb \u003c/strong\u003eFTIR spectrum of VLIF\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4594047/v1/e8ae1459cd427d954f18d7e5.png"},{"id":58654856,"identity":"4d6fc421-39b5-419a-84ea-93124afa1358","added_by":"auto","created_at":"2024-06-19 11:09:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":112410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eGC-MS Spectrum for VLMF\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb \u003c/strong\u003eGC-MS Spectrum for VLIF\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4594047/v1/b2fb787ac53d6a81dd85a9bf.png"},{"id":58655350,"identity":"1b04916c-d946-470e-9d27-b30016d894d5","added_by":"auto","created_at":"2024-06-19 11:17:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2514222,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4594047/v1/4c9c731b-00d0-433a-8876-8837e87f1de3.pdf"},{"id":58655348,"identity":"165d2752-bcc4-429f-9d75-52e6a4d57338","added_by":"auto","created_at":"2024-06-19 11:17:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":73922,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4594047/v1/0b092f5f6655d022d9655210.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eValidating the Consistency and Quality of “Vaankumari Legiyam” A Classical Siddha Polyherbal Formulation Through Advanced Analytical Methods\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eVaankumari legiyam is a traditional Siddha polyherbal formulation used in various ailments, including sarva roganoigal, diabetes, high cholesterol, insect bites, and PCOS treatment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, traditional formulations often lack proper standardization, leading to safety and efficacy issues. The quality and composition of these ingredients can vary depending on the source, the growing conditions, and the processing methods used [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This can lead to variations in the quality, safety, and efficacy of Siddha formulations. Standardization can help to ensure that Siddha formulations are made to a consistent quality and that they meet the expected standards. Standardization can also help to facilitate research on Siddha formulations. By having standardized formulations, researchers can be sure that they are comparing the same product across different studies. To address this, an in-house formulation will be prepared using standard raw materials and modern analytical techniques like AAS, FTIR, HPTLC, and GCMS. This will help identify potential phytoconstituents responsible for treating various diseases and assess their quality. The aim is to ensure that all ingredients are present in the finished product and that consumers have access to information about the product's quality and safety.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Procurement of Samples and authentication\u003c/h2\u003e \u003cp\u003eThe marketed Vaankumari legiyam (Confectionery) formulation was used in the present study having (Batch No. LEU 21019). The Vaankumari legiyam was procured from online e-commerce gateway. A systematic profiling of Siddha formulation Vaankumari legiyam which was carried out by using authenticated medicinal plant parts for the preparation of the in-house formulation. The plant materials/parts for the preparation of in-house Vaankumari legiyam were purchased from Rana Ayurveda, Banka Bazar, Cuttack-753002, Odisha, India and authenticated by Dr. Amar Kumar Rana (B.A.M.S), Cuttack,Odisha\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Organoleptic evaluation of Vaankumari legiyam\u003c/h2\u003e \u003cp\u003eOrganoleptic evaluation is crucial for assessing the quality and standardization of polyherbal formulations. It involves examining color, size, shape, and presence for extraneous material or foreign particles. This assessment helps determine sensory properties, authenticity, and consistency, ensuring the reliability and effectiveness of polyherbal formulations. Integrating organoleptic evaluation into quality control processes promotes their acceptance and integration into mainstream healthcare practices [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Physicochemical evaluation of Vaankumari legiyam\u003c/h2\u003e \u003cp\u003ePhysicochemical testing is crucial for quality control and standardization of polyherbal formulations, as it helps identify active ingredients, physical and chemical properties, and stability. It ensures the safety and efficacy of these formulations. To determine the quality of Vaankumari legiyam, tests were conducted, including loss on drying, total ash, acid insoluble ash, pH, reducing sugar, alcohol-soluble extractive, and water-soluble extractive. These tests help ensure the safety and efficacy of polyherbal formulations[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]..\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Preliminary phytochemical evaluation of Vaankumari legiyam\u003c/h2\u003e \u003cp\u003eTherapeutic activity of vegetable drugs depends upon the type of constituents present in them. Plant material was screened for the presence of phytoconstituent(s) using different chemical tests. Powder drug and different extracts were screened for different phytoconstituents [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Biochemical analysis of acid and basic radicals of Vaankumari legiyam\u003c/h2\u003e \u003cp\u003ePolyherbal formulations in traditional medicine require quality control and standardization to ensure safety and effectiveness. Inorganic salts contain acid radicals that donate protons, causing an excess of positively charged ions. Basic radicals, such as hydroxide, carbonate, and bicarbonate, modulate pH, solubility, and biological activity. Spectroscopic techniques like AAS and ICP-MS are used to isolate and identify specific components, while acid-base titration techniques determine the overall amount of acid or base in the formulation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Heavy metal analysis by AAS (Atomic absorption Spectroscopy) of Vaankumari legiyam\u003c/h2\u003e \u003cp\u003eThe study used Atomic Absorption Spectrometry (AAS) to analyze heavy metals in the classical polyherbal formulation Vaankumari legiyam. The method involved a wet digestion method, where 2 g of sample was weighed, concentrated nitric acid added, and the solution was heated for 2 hours. Standard solutions were prepared, and the wavelength was fixed between 185\u0026ndash;900 nm. Test samples were analyzed against the standard, and the results were plotted against the concentration of each standard solution. The study aimed to determine the concentration of heavy metals in the polyherbal formulation Vaankumari legiyam [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. High performance thin layer chromatography analysis of Vaankumari legiyam\u003c/h2\u003e \u003cp\u003eHPTLC is a valuable tool for evaluating polyherbal formulations, providing a chromatographic fingerprint of phytochemicals. Samples were prepared and filtered through a 0.45-micron syringe filter. HPTLC analysis was performed on Merck TLC plates Al silica gel 60 F 254. The extracts were spotted on a silica gel 60 F 254 plate, developed using methanol solvent, and visualized using Visualiser-1 A and CAMAG Scanner 4 B software [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Fourier transform infra-red spectroscopy of Vaankumari legiyam\u003c/h2\u003e \u003cp\u003eFTIR is a crucial technique for identifying functional groups, determining material quality and consistency, and determining the number of compounds present in a sample. It uses infrared to absorb and transmit infrared, creating a molecular fingerprint of the sample. FTIR is fast, sensitivity, mechanically simple, and internally calibrated. The Central Instrumental Laboratory (CIL) at IPT Salipur conducted FTIR (ATR) analysis using a Bruker FT-IR Spectrometer with Attenuated Total Reflectance technology (ATR) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Gas chromatography- thin layer chromatography of Vaankumari legiyam\u003c/h2\u003e \u003cp\u003eGCMS is essential for analyzing plant-derived components, ionizing compounds and measuring mass numbers. The EI method, which produces ions, offers high stability and reproducibility. Methanol was extracted from Vaankumari legiyam, and GCMS analysis was performed using a Shimadzu GCMS-QP2020 and Quadrupole MS [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1. Organoleptic evaluation of Vaankumari legiyam\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOrganoleptic analysis is crucial for standardizing polyherbal formulations, as it helps determine the physical traits and quality of plant materials used in the formulation. The sensory qualities of plant extracts reveal information about their chemical makeup, such as taste, odour, and appearance.\u0026nbsp;The organoleptic evaluation parameters are cited in Table 1. Organoleptic evaluationattributes can affect a product\u0026apos;s acceptability, and organoleptic analysis is essential for determining consistency and homogeneity. The market formulation is standardized due to similarities in taste, color, odour, and appearance with the in-house formulation\u0026nbsp;[19].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Physicochemical evaluation of Vaankumari legiyam\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhysicochemical evaluation is crucial for standardizing Vaankumari legiyam formulations, as it helps identify and authenticate each ingredient. The selected and preferred formulation were subjected to various evaluations and analytical studies such as; Loss on drying, Total Ash, Acid insoluble ash, pH, Reducing Sugar, Total Sugar, Water soluble extractive, Alcohol soluble extractive. The physicochemical data obtained from the above studies are shown in Table 2. Physicochemical evaluation of Vaankumari legiyam\u0026nbsp;helped establish quality standards for each ingredient, ensuring consistency and quality. The market formulation is standardized due to its comparable physicochemical parameters with the in-house formulation, ensuring safety, efficacy, and quality. The study demonstrates the importance of physicochemical evaluation in ensuring the safety, efficacy, and quality of Vaankumari legiyam formulations [20].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Preliminary phytochemical evaluation of Vaankumari legiyam\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Vaankumari legiyam formulation underwent standardization methods, which included preliminary phytochemical analysis on VLMF and VLIF to identify phytoconstituents. This analysis aimed to ensure consistency and quality in the active constituents, setting specifications for raw materials and finished products. The results of these analyses are presented in Table 3.\u0026nbsp;Preliminary phytochemical evaluation of Vaankumari legiyam\u0026nbsp;process confirmed the genuineness of the formulation by revealing the presence of comparable phytoconstituents in both marketed and in-house formulations, thus establishing its standardized status [21].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Heavy metal analysis by AAS (Atomic absorption Spectroscopy) of Vaankumari legiyam\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAAS results have been displayed in Figure 1a,1b,1c,2a,2b,2c\u0026amp;Table 4which revealed the concentration level of arsenic and mercury was below detection limit (BDL) in both VLMF and VLIF. Results were compared with the allowable limit acceptability intake of the World Health Organization (WHO) and American Herbal Products Association (AHPA). Concentration of heavy metal (As and Hg) in ppb/mean\u0026nbsp;medicines should not exceed 10 \u0026micro;g/g or ppm, and for mercury, the limit is 0.1 \u0026micro;g/g or 0.1 ppm.\u0026nbsp;In the herbal sector, heavy metal contamination of plant extracts is a regular issue that might jeopardize the product\u0026apos;s safety and effectiveness.According to WHO standards, acceptable levels of arsenic in herbal medicines should not exceed 10 \u0026micro;g/g or ppm, and for mercury, the limit is 0.1 \u0026micro;g/g or 0.1 ppm [22, 23].\u0026nbsp;A recent study discovered that VLMF and VLIF herbal formulations contained undetectable levels of arsenic and mercury. This absence of heavy metals indicates that these herbal products are safe for consumers, as excessive exposure to arsenic and mercury can lead to various health issues, including skin problems, neurological disorders, and more.The AAS protocol successfully assessed heavy metal and trace element concentrations in herbal products and phytopharmaceuticals. It utilized AAS spectra to analyze elemental composition, identifying contaminants. Results show that the marketed formulation meets standards and matches the in-house formulation. Arsenic and Mercury levels are below WHO limits, suggesting no heavy metal toxicity risk for users in healthcare applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. High performance thin layer chromatography analysis of Vaankumari legiyam\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study utilizes High Performance Thin Layer Chromatography (HPTLC) technique to verify the authenticity of Siddha polyherbal formulation and the final product.\u003c/p\u003e\n\u003cp\u003eThe study conducted a comparative fingerprint analysis on samples VLMF and VLIF using mobile Phase A and mobile Phase B. Results showed 2 bands at the same R\u003csub\u003ef\u003c/sub\u003e under 254nm, 4 bands under 366nm, and 5 bands under R white light after derivatization with Anisaldehyde sulphuric acid reagent. The results are shown in Figure 3a, 3b, 3c andTable 5. The HPTLC fingerprinting of VLMF showed 04 bands having maximum Rf value 0.43, 0.56, 0.68, 0.94 at 254nm and VLIF showed 02 bands having maximum Rf value 0.68, 0.94 at 254nm under Mobile phase A (Chloroform: Ethyl acetate: Formic acid 5:4:1 v/v/v). VLMF showed 05 bands having maximum Rf value 0.38, 0.49, 0.59, 0.67, 0.91 at R366 derivatized with ASR and VLIF showed also 05 bands having maximum Rf value 0.49, 0.56, 0.59, 0.67, 0.91 at R366 derivatized with ASR, VLMF showed 06 bands having maximum Rf value 0.90, 0.66, 0.59, 0.52, 0.49, 0.38 at Rwhitederivatized with ASR and VLIF showed also 06 bands having maximum Rf value 0.90, 0.66, 0.59, 0.57, 0.52, 0.49 at Rwhitederivatized with ASR under mobile phase B (Chloroform: Toluene: Ethanol 4:4:1 v/v/v). This HPTLC technique may be advantageous for both the identification and the quality aspect-evaluation of formulations containing different or diverse plants parts. The HPTLC study analyzed the similarities and differences between VLMF and VLIF herbal formulations. The marketed formulation showed common black color bands at the same Retention Factor (Rf) under 254nm with mobile phase A. Under 366nm, the plate developed with Mobile Phase B and derivatised with Anisaldehydesulphuric acid showed four pink-colored bands at the same Rf. Mobile Phase B showed five purple color bands at the same Rf. Two black color bands were found in VLMF under 254nm with mobile phase A, while two pink-colored bands were absent in VLIF. The Rf values provided information about the composition of the two formulations [24]. \u0026nbsp;However, slight variations in Rf values can be expected due to batch-to-batch variations, raw material variations, or formulation process differences [25].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6. Fourier transforms infra-red spectroscopy of Vaankumari legiyam\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the present study, the content of\u0026nbsp;Vaankumari legiyam marketed and in-houseformulationwere investigated using FTIR spectroscopy by monitoring different functional groups. Figure 4a \u0026amp; 4b shows the FTIR spectra obtained or achieved from\u0026nbsp;the marketed and in-house Vaankumari legiyam\u0026nbsp;in the 4000\u0026ndash;500 cm\u003csup\u003e-1\u003c/sup\u003e region.\u0026nbsp;The VLMF and VLIF was identified to have important 12 and 10observed peaks respectively. There are the groups present in the polyherbal formulations of Vaankumari legiyam. The spectra were interpreted in context of comparative analysis of\u0026nbsp;Vaankumari legiyam marketed and in-house Formulation. Both the samples were analyzed, evaluated for variation in the functional groups and bonding pattern or arrangement. The FTIR peaks of the spectra of the VLMF and VLIF were compared which are shown in Table 6 and Table 7.\u0026nbsp;FTIR had been used to identify and measure the phytoconstituents present in the plant extracts used in the formulation, FTIR is a crucial tool for the standardization of polyherbal formulations. An infrared absorption spectrum specific to each phytoconstituent has used to identify and measure the component in a sample [26]. The purity and quality of the raw ingredients and the completed product has also been revealed by FTIR. The chemical make-up of the sample has been ascertained using FTIR spectra, as can any impurities or contaminants that may be present [27]. From the above results it is ascertained that the marketed formulation is a standardized one as because of\u0026nbsp;there are no significance variations in important observed peak in cm\u003csup\u003e-1\u003c/sup\u003e along with the functional group in VLMF and VLIF by use of advanced instruments.\u0026nbsp;The functional groups identified from FTIR will give a better solution for future analysis and clinical trials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7. Gas chromatography- thin layer chromatography of Vaankumari legiyam\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GCMS chromatogram Figure 5a \u0026amp; 5b of methanolic extracts of Vaankumari legiyam marketed formulation revealed 76 and in-house formulation revealed 72 compounds respectively which belonged to various classes of secondary metabolites. The peak report of the chromatogram obtained with details of peak number, retention time, area percentage, name of the identified phytocomponent, its molecular formula and molecular weight, are presented in Table 8 \u0026amp; 9. The mass spectrum was obtained with mass/charge ratio on x-axis and relative intensity on y-axis. The comparative study of VLMF and VLIF formulations with respect to GCMS (Gas Chromatography-Mass Spectrometry) analysis focuses on the analysis and comparison of the chemical composition of the formulations. GCMS is a powerful technique that allows for the identification and quantification of a wide range of compounds present in complex mixtures [28]. In this study, both the marketed and in-house polyherbal formulations were subjected to GCMS analysis to determine their chemical profiles. The GCMS chromatograms obtained for each formulation were carefully examined and compared to identify any similarities or differences in the composition. The findings of the GCMS analysis revealed that both the marketed and in-house formulations contained a variety of bioactive compounds. The chromatograms exhibited RT, indicating the presence of similar compounds in both formulations. This suggests that the formulations may share common herbal ingredients. The appearance of peaks at different m/z ratio represents the smaller compounds, formed due to fragmentation of larger compounds. These spectra act as fingerprint of the compounds and are identified by matching with NIST data library [29, 30, 31].\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn conclusion, the comprehensive analysis of both marketed and in-house formulations of Vaankumari legiyam through various techniques, including phytochemical, physicochemical, AAS, HPTLC, FTIR, and GCMS, has demonstrated consistent and equal results. This rigorous standardization process reaffirms the authenticity and quality of the marketed product establishing its equivalence to the in-house formulation. Such meticulous evaluation ensures that consumers receive a reliable and effective herbal remedy while promoting trust in the herbal healthcare industry. While this study has laid a foundation for the standardization and quality assurance of Vaankumari Legiyam, further research in pharmacology could be undertaken to explore its therapeutic potential and mechanism of action more comprehensively.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to the management members and Principal, Institute of Pharmacy \u0026amp; Technology, Salipur for providing all facilities and encouragement throughout the work. The authors would like to thanks the Central\u003cstrong\u003e\u0026nbsp;Analytical Facilities,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eManipal University, Jaipur for carrying out the GC\u0026ndash;MS, AAS analysis of Vaankumari Legiyam and Anchrom Enterprisesa (I) Pvt. Ltd. Mumbai for carrying out HPTLC analysis of Vaankumari Legiyam\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e This article contain no studies with animals\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSujit Dash: Contributed to conceptualization, methodology, formal analysis, investigation, writing - original draft preparation, and supervision. Minaketan Sahoo: Contributed to methodology, investigation, and data curation. Amaresh Chandra Sahoo: Contributed to methodology, investigation, and data curation. Mrityunjay Baneerjee: Contributed to methodology, investigation, and data curation. Saroja Kumar Patro: Contributed to resources, writing - review and editing, and project administration. Prabhat Kumar Sahoo: Contributed to validation, writing - review and editing, and supervision. Priyanka Muduli: Contributed to investigation and data curation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnonymous. The Siddha Formulary of India part-1. 1st Edition. Ministry of Health and Family Welfare, Department of Health, Government of India, Delhi, 1992, pp.117.\u003c/li\u003e\n\u003cli\u003eAbubakar MN, Majinda RRT. GC-MS analysis and preliminary antimicrobial activity of Albizia adianthifolia (Schumach) and Pterocarpus angolensis (DC). Medicines Vol. 3, No. 1, pp. 1-9, 2016.\u003c/li\u003e\n\u003cli\u003eAjmal SM, Rajeswari AR. Literature review on Siddha medicines available for the management of PCOS - A Review. International Journal of Current Research in Medical Sciences Vol. 3, No. 6, pp. 45-50, 2017.\u003c/li\u003e\n\u003cli\u003eBeg BM, Viquar U, Mohammad ARN, Habiba S, Munawwar HK. Standardization and HPTLC Fingerprinting of a Polyherbal Unani Formulation. 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Influence of inorganic salts on biomass production, biochemical composition and bioethanol production of Populus alba. iForest \u0026ndash; Biogeosciences and Forestry Vol. 13, No. 1, pp. 566-574, 2020.\u003c/li\u003e\n\u003cli\u003eQuds T, Ahmed M, Shakeel S, Jalbani N, Mazhar F, Azhar I. Determination of the heavy metal contents of frequently used herbal products in Pakistan. Tropical Journal of Pharmaceutical Research Vol. 20, No. 2, pp. 377-382, 2021.\u003c/li\u003e\n\u003cli\u003eKarayil S, Bhavani, Vivek. Heavy metal analysis from traditionally used herb Ceropegia juncea (Roxb.). IOSR Journal of Pharmacy Vol. 4, No. 12, pp. 7-11, 2014.\u003c/li\u003e\n\u003cli\u003eVajahath AA, Gobindaraj B, Saravanadevi MD, Velpandian V. Standardization of Siddha Poly-Herbal Formulation ThirinethiraChooranam by Modern Pharmaceutical Analytical Techniques. Int. J. Health Sci. Res. Vol. 12, No. 5, pp. 187-199, 2022.\u003c/li\u003e\n\u003cli\u003eSudha V, Niraimathi KL, Lavanya R, Raj David C, Brindha P. Chemical Standardization and in vitro Cytotoxic Studies on Nellikai Lehyam. Asian J. Chem. Vol. 26, No. 12, pp. 3679-3682, 2014.\u003c/li\u003e\n\u003cli\u003eVijaya RN, Abinaya R, Punithavalli V, Velpandian V. Standardization of Siddha polyherbal formulation \u0026ldquo;SiringipaerathiChooranam\u0026rdquo; through a scientific method \u0026ndash; FTIR (Fourier Transform Infrared Spectroscopy). Int. J. Curr. Res. Chem. Pharm. Sci. Vol. 6, No. 2, pp. 24-37, 2019.\u003c/li\u003e\n\u003cli\u003eRani SY, Mirunaleni P, Sathiyabama M, Suresh K, Meenakshi Sundaram M, Banumathi V. GC MS analysis of Thulasiennai \u0026ndash; A siddha polyherbal formulation. Int. J. Adv. Multidiscip. Res. Vol. 5, No. 7, pp. 65-69, 2018.\u003c/li\u003e\n\u003cli\u003eMunoz AM. Sensory evaluation in quality control: an overview, new developments and future opportunities. Food Qual. Prefer. 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J. Pharm. Sci. Res. Vol. 7, No. 2, pp. 97-106, 2016.\u003c/li\u003e\n\u003cli\u003ePattanayak P, Mohapatra P, Jena RK, Panda SK. Standardization of Sulaharan yoga: an ayurvedic tablet formulation. Indian J. Pharm. Sci. Vol. 73, No. 1, pp. 65-70, 2011.\u003c/li\u003e\n\u003cli\u003eKumar SS, Manoj P, Giridhar P. Fourier transform infrared spectroscopy (FTIR) analysis, chlorophyll content and antioxidant properties of native and defatted foliage of green leafy vegetables. J. Food Sci. Technol. Vol. 52, No. 12, pp. 8131-8139, 2015.\u003c/li\u003e\n\u003cli\u003eAlberto-Silva C, Malheiros FBM, Querobino SM. Fourier-transformed infrared spectroscopy, physicochemical and biochemical properties of chondroitin sulfate and glucosamine as supporting information on quality control of raw materials. Future J. Pharm. Sci. Vol. 6, No. 1, pp. 1-9, 2020.\u003c/li\u003e\n\u003cli\u003eKonappa N, Udayashankar AC, Krishnamurthy S, Pradeep CK, Chowdappa S, Jogaiah S. 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Vol. 10, No. 6, pp. 2911-2916, 2019.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 9 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Vaankumari legiyam, Siddha, Polyherbal formulation, AAS, FTIR, HPTLC, and GCMS.","lastPublishedDoi":"10.21203/rs.3.rs-4594047/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4594047/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmong Siddha's internal medicine dosage forms, \"Legiyam\" stands out as a widely recognized medicinal confectionary. This preparation involves heating decoctions, juices, milk, and sugar to form a syrupy consistency. Drug powders and other ingredients are added, and the mixture is cooled before incorporating ghee and honey. \"Vaankumaria legiyam,\" a classic Siddha polyherbal formulation in confectionary form, contains various herbal ingredients known for addressing numerous health concerns, including digestion, diabetes, and skin issues.Commercially available formulations often lack proper standardization, leading to safety and efficacy concerns. In this context, the present study aims to prepare an in-house Siddha formulation using standard raw materials outlined in the Siddha Pharmacopoeia of India. Comparative phytochemical studies was conducted using modern analytical techniques such as AAS, FTIR, HPTLC, and GCMS. The Vaankumari legiyam formulations were tested for heavy metals and trace elements, revealing concentrations below regulatory limits. High-Performance Thin-Layer Chromatography (HPTLC) and Fourier Transform Infrared Spectroscopy (FTIR) analyzed the phytochemical profile, confirming the product's authenticity. Gas Chromatography-Mass Spectrometry (GC-MS) identified and quantified volatile organic compounds, confirming the in-house preparation's reproducibility in capturing aromatic and volatile constituents. These tests confirmed the product's safety for human consumption.The current research has shed light on the potential phytoconstituents responsible for the formulations\u0026rsquo; claimed therapeutic effects and facilitates quality assessment. The comprehensive analysis of both marketed and in-house formulations of Vaankumari legiyam through various techniques, including phytochemical, physicochemical, AAS, HPTLC, FTIR, and GCMS, has demonstrated consistent and equal results.\u003c/p\u003e","manuscriptTitle":"Validating the Consistency and Quality of “Vaankumari Legiyam” A Classical Siddha Polyherbal Formulation Through Advanced Analytical Methods","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-19 11:09:07","doi":"10.21203/rs.3.rs-4594047/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7203ebc7-ecb5-47e3-a3b4-bd9a9869d985","owner":[],"postedDate":"June 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33351306,"name":"Integrative \u0026 Complementary Medicine"}],"tags":[],"updatedAt":"2024-06-19T11:09:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-19 11:09:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4594047","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4594047","identity":"rs-4594047","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}