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Open Access Full Text Article   Research Article

A Physicochemical and Chromatographic Fingerprint Study on Tilvak Ghrita: A Polyherbal Formulation

Dr. Rushikesh Shivtare ¹, Prof. (Dr.) Harish Kumar Singhal ²

¹ PG Scholar, P. G. Department of Kaumarbhritya, Postgraduate Institute of Ayurveda, Dr. S. R. Rajasthan Ayurved University, Jodhpur, Rajasthan

² Professor & H.O.D., P. G. Department of Kaumarbhritya, Postgraduate Institute of Ayurveda, Dr. S. R. Rajasthan Ayurved University, Jodhpur, Rajasthan

 

 

INTRODUCTION 

Ayurveda, India's traditional system of medicine, promotes the use of polyherbal formulations to improve therapeutic efficacy through reciprocal interactions between medicinal herbs. Classical Ayurvedic pharmaceutics (Bhaishajya Kalpana) discusses several dosage forms, with Ghrita Kalpana (medicated ghee preparations) being considered superior due to its stability, extended shelf life and ability to extract and administer both lipid- and water-soluble active ingredients. Ghrita is defined as Yogavahi, which means it enhances the pharmacological activity of medications processed with it and allows for deeper tissue penetration (Sukshma strotogami), particularly into the brain system ^{1 2} . 

 The formulation is explained in authoritative Ayurvedic books naming Ashtanga Hridaya, showing its clinical importance and long-term therapy³. From a modern pharmacological perspective, ghrita-based formulations are known for enhancing phyto-constituent bioavailability due to the lipid matrix, which helps intestine absorption and transport across biological 

membranes. Studies have shown that lipid-based herbal formulations can improve the distribution of active chemicals to target organs, including the brain, supporting the traditional use of ghrita in neurological and systemic illnesses⁴. Despite broad traditional use, rising global demand for Ayurvedic medicines has generated concerns about quality control, standardization, and batch-to-batch consistency of polyherbal formulations. Variations in raw ingredients, processing methods and storage conditions can have a considerable impact on the physicochemical and medicinal properties of such preparations. Thus, comprehensive analytical evaluation is required to assure identification, purity, safety and reproducibility in compliance with current pharmacopeial standards⁵. The current analytical study of Tilvak Ghrita was conducted to assess its physicochemical parameters and quality utilizing standard analytical techniques. Such research are critical for establishing reference standards, scientifically confirming classical formulations and promoting their wider adoption in evidence-based Ayurvedic practice and integrative healthcare systems.

Aims and Objectives

Aim

To establish the analytical quality parameters of Tilvak Ghrita, a classical Ayurvedic formulation, with special reference to its potential role in the management of Vata Vyadhi (Pakshaghat).

Objectives

• To identify and authenticate the raw drugs used in the preparation of Tilvak Ghrita in accordance with Ayurvedic Pharmacopoeia of India (API) standards.
• To prepare Tilvak Ghrita in a Good Manufacturing Practices (GMP) certified pharmacy following classical Ayurvedic   procedures.
• To evaluate the organoleptic characteristics of Tilvak Ghrita, including appearance, color, odor and taste.
• To assess the physicochemical parameters of Tilvak Ghrita as per API guidelines.
• To develop a thin-layer chromatographic (TLC) fingerprint of Tilvak Ghrita for quality control and standardization.

MATERIAL AND METHOD

Procurement of Raw Materials: - The raw drug was collected from the local market of Jaipur, Rajasthan and after being examined by the Department of Dravya-guna, after that Tilvak Ghrita was prepared under aseptic condition in the Nagarjuna pharmacy of PGIA, Dr. S. R. Rajasthan Ayurved University, Jodhpur under the supervision of competent authority.

Preparation of Tilvak Ghrita: -

 All procedures adhered strictly to Standard Operating Procedures (SOPs) to ensure reproducibility, quality and consistency. Each ingredient was measured according to the classical prescription, cleaned, dried, pulverized, and sieved to obtain a course powder, Murchhita Ghrita was heated in a large-mouthed container until fumes disappeared, after partial cooling, the specified Kwatha Dravya was added, Dadhi (curd) was added after half of Ghrita Paka and cooking continued, the process was repeated for a day until Ghrita reached Sidhhi Lakshana⁶. After the preparation of medicine drug was packaged in a sterile, airtight container labelled with the date of manufacture (14/07/2025) and batch number (102/2025). 

 

 

Table 1: Ingredients and Properties of Tilvak Ghrita

 

 

 

Parameters Studied in Tilvak ghrita

The analytical evaluation of Tilvak Ghrita was carried out following the guidelines outlined in the “Protocol for Testing of Ayurvedic, Siddha, and Unani Medicines”, published    by    the   National     Institute    of     Science 

 

Communication and Information Resources (NISCAIR), CSIR, and issued by the Department of Ayurveda, Yoga, Naturopathy, Unani, Siddha & Homeopathy (AYUSH), Government of India⁷.

Analytical study of Tilvak Ghrita

Place of work 

Cultivator Phyto Lab Pvt. Ltd. Sonamukhi Nagar, Sangaria Fanta, Jodhpur. Sample registration no. CPL/O/25/12/02361. Sample sent to lab date and start of analysis 09/12/2025 and completed 15/12/25 in 7 days⁸. 

Analytical study was done under following headings-

1. Physio-chemical Parameters
2. Chromatographic fingerprint –TLC

Place of Work and Sample Details

The analyses were performed at Cultivator Phyto Lab Pvt. Ltd., Sonamukhi Nagar, Sangaria Fanta, Jodhpur. Sample Registration No. CPL/O/25/12/02361/2, bearing Sample Code CPL/O/25/12/02361, was sent to the laboratory on 09/12/2025. The analysis was initiated on 10/12/2025 and successfully completed on 15/12/2025. The total duration of the analysis was 7 days.

RESULT 

Physiochemical parameters 

Physicochemical parameters refer to the physical and chemical characteristics of Ayurvedic formulations. These parameters are crucial for assessing the quality, consistency, and stability of the formulations. Parameters include rancidity, saponification value, iodine value, refractive index, acid value, moisture etc.

 

 

 Table 4: Physicochemical Parameters of Tilvak Ghrita

 

 

DISCUSSION

Organoleptic evaluation is a sensory assessment of a material based on its physical properties, such as appearance, texture, color, odor, and taste. The organoleptic examination serves different functions in the manufacture of Tilvak Ghrita. It is critical to ensure that the preparation has a consistent appearance, texture, and taste when evaluating quality. Variations in these factors could indicate batch-to-batch discrepancies or a departure from standard Ayurvedic practices¹⁰. Rancidity in it, which is predominantly induced by Ghrita oxidation and hydrolysis, has a substantial impact on its sensory properties and medicinal efficacy. Proper storage, packaging, and quality control techniques are critical for preventing rancidity and maintaining the formulation's potency and effectiveness. Regular organoleptic and chemical testing can assist identify early symptoms of spoiling and guarantee that Tilvak Ghrita is safe and good for consumption¹¹. The saponification value of long-chain fatty acids, which are found in fat, is low, whereas that of short-chain fatty acids (SCFAs) is high¹².  It has been demonstrated that short-chain fatty acids are a vital source of energy for colonocytes, especially those in the distal colon¹³. The histological, endoscopic, and metabolic similarities between diversion colitis and ulcerative colitis indicate that a dietary SCFA shortage may play a role in the etiology of both diseases. Short chain fatty acids are easily absorbed; there may be a protective advantage if SCFA production is raised and SCFAs, particularly butyrate, are delivered more effectively¹⁴ to the distal colon. It has a greater saponification value. Ghrita’s, which are esters, hydrolyze in the presence of an alkali (due to the alkaline nature of Kalka Dravya or the other Drava Dravya used in the Snehapaka procedure), resulting in the production of fatty acid (short chain). This shows that Tilvak Ghrita contains higher short-chain fatty acids. As a result, easily absorbed and digested, has a preventative effect, and improves intestine and overall health. The amount of unsaturated fatty substance in the Ghrita is determined by the iodine levels. The amount of unsaturated bonds in the fat increases with the iodine numbers. Unsaturated fat supplementation increases overall dietary energy intake to the necessary amounts without negatively affecting blood lipid levels. It also improves nutritional status and lowers systemic inflammation. Polyunsaturated fatty acids, which provide health benefits like controlling blood cholesterol levels, are abundant in lipids with high Iodine levels determine the quantity of unsaturated fat in Ghrita. The amount of unsaturated bonds in the fat increases as the iodine concentration rises. Unsaturated fat supplementation raises overall dietary energy intake to the required levels while having no deleterious effects on blood lipid levels. It also enhances nutritional status and reduces systemic inflammation. Polyunsaturated fatty acids, which give health benefits such as lowering blood cholesterol levels, are prevalent in lipids with a high iodine value¹⁵. The increased iodine value suggests that it contains more unsaturated fatty acids. This analytical value demonstrates that Tilvak Ghrita improves nutritional status and lowers systemic inflammation without having a negative effect on blood lipids despite the fatty acid. The increasing unsaturation of the Ghrita can be the result of the Snehapaka process. The refractive index is an important optical property that measures how light is distorted as it passes through a substance. The refractive index of Tilvak Ghrita can provide useful information about the formulation’s composition, purity and quality. This feature can be especially useful in determining the consistency of substances, such as Ghrita’s lipid matrix. The role of the refractive index in quality control is an important consideration. Studies have indicated that the refractive index of oils and fats, such as ghee, can be used as an indicator of purity and adulteration¹⁶.  Variations in the refractive index may indicate the presence of impurities like too much water, artificial additives or inferior raw ingredients. Therefore, maintaining the formulation's intended therapeutic qualities would depend in large part on the refractive index being consistent throughout batches. The acid value, which is related to the stability of the Ghrita, indicates the amount of free fatty acid (FFA) in the Ghrita. For the Ghrita, the production of free fatty acids may be a crucial indicator of rancidity. Triglycerides hydrolyse to produce FFA, which can be accelerated by the Ghrita’s contact with moisture¹⁷. The Ghrita’s stability, flavour, and shelf life are all impacted by its fatty acid composition. The presence of FFA in the Ghrita signifies its purity or individuality¹⁸. Tilvak Ghrita contains a higher acid value. This shows that Ghrita undergoes hydrolysis during the Snehapaka process, which may be facilitated by the reactivity of Ghrita’s triglycerides with the active components of Tilvak Ghrita, resulting in glyserol and free fatty acids. Excessive free fatty acid levels (Acid Value) promote a reduction in Ghrita quality. This shows that its nutritional value, stability and shelf life are inferior to those of Go Ghrita. Its moisture content influences its shelf life, stability, and medicinal efficacy. Tilvak Ghrita is commonly made with Ghrita, which has a low moisture content (less than 0.2%). The presence of moisture can promote the growth of microbiological organisms, lowering the product's shelf life. Research on other Ayurvedic formulations has established a link between moisture content and spoilage and microbial growth¹⁹. Excess moisture can alter the balance of ingredients and possibly affect the pharmacological properties of Tilvak Ghrita. Moisture could potentially compromise the bioavailability of these compounds. Specific gravity is defined as the density of one substance divided by the density of another, usually water. The specific gravity of liquids and semi-solid preparations indicates the formulation’s relative density and purity. Because Tilvak, Trifala and Guru pachamoola extracts play a role in the composition of Ghrita, changes in specific gravity could suggest alterations in their concentrations or the presence of impurities, which may interfere with therapeutic efficacy²⁰. TLC can be used to separate and identify these compounds using their Rf (retention factor) values, which are unique to each one. Ghosal et al. (2000) confirmed the presence of these chemicals in the herbal extract by comparing their Rf values to recognized standards. TLC can be used to evaluate batch-to-batch consistency and predict the existence of bioactive chemicals. According to a study by Kaur et al. (2018) demonstrated that TLC was an effective method for analysing Chyawanprash (another Ayurvedic formulation) for its active principles, ensuring its consistency. By applying similar methods to Tilvak Ghrita, TLC can help confirm that each batch meets the desired specification for active compounds, ensuring therapeutic efficacy²¹.  TLC can be used to detect unwanted substances such as microbial contamination, solvent residues, or adulterants that may affect the quality and safety. In contrast to the target compounds expected Rf values, impurities may show up on the TLC plate as extra spots. In a study by Gawande et al. (2017), TLC was employed to monitor the purity of Ayurvedic Ghrita based formulations, helping to identify potential contaminants and adulterants²². A similar approach can be applied to Tilvak Ghrita to ensure that it remains free of foreign substances that could compromise its safety or effectiveness. The ‘fingerprint’ refers to the distinctive pattern of spots produced by the compounds present in the sample when subjected to TLC. This fingerprint can be used to compare samples of Tilvak Ghrita from various batches or even from different manufacturers to ensure authenticity and consistency²³.

CONCLUSION

The present analytical study concludes that Tilvak Ghrita exhibits well-defined organoleptic characteristics along with a comprehensive range of physicochemical parameters, including rancidity, moisture content, iodine value, refractive index, saponification value, and specific gravity. These parameters provide essential evidence regarding the quality, safety and efficacy of the formulation. Furthermore, chromatographic fingerprinting through Thin Layer Chromatography (TLC) confirmed the presence of multiple phytoconstituents. The study also establishes specific analytical markers that can serve as reference standards for the identification, quality control, and consistent production of this formulation.

Acknowledgement: The author expresses sincere gratitude to the supervisor, Prof. (Dr.) Harish Kumar Singhal, Head of the Post graduate Department of Kaumarbhritya, Post Graduate Institute of Ayurveda, Dr. Sarvepalli Radhakrishnan Rajasthan Ayurved University, Jodhpur, India, for his invaluable guidance, academic support, and constant encouragement throughout the course of this work. His profound knowledge and dedication to the field of Ayurvedic pediatrics significantly contributed to shaping the direction and depth of this research.

Source of Funding: None.

Conflict of Interest: The author declares that there are no conflicts of interest related to this study.

Use of Artificial Intelligence (AI): The AI tools did not contribute to the study design, data collection, data analysis, interpretation of results, or the generation of scientific conclusions. The author takes full responsibility for the content of the manuscript.

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