Analytical Standardization of Vasaguduchyadi Kvatha Churna Using Gallic Acid as a Phytomarker

Pushpendra Kumar¹, Umakant Sahu², Harkesh Dadsena³, Lokprabha Hirwani⁴, Chhavi Rahangdale⁵, Abhishek Nand⁶, Bhoomika Swarnkar⁷, Yashika Israni⁸, Narendra Kumar⁹, Vishal Jain^10, *

^1-10Department of Pharmacognosy, University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India -492010 

¹pushpendrakumar0311@gmail.com, ²uksahu28@gmail.com, ³harkeshdadsena2000@gmail.com, ⁴hirwanilokprabha2@gmail.com, ⁵chhavirahangdale1236@gmail.com, ⁶bhoomikaswarnkar67@gmail.com, ⁷abhisheknand008@gmail.com, ⁸yashikaisrani2003@gmail.com, ⁹narendrakumar.ch1789@gmail.com, ¹⁰vishaljain123@gmail.com

Graphical Abstract

Abstract:

According to Ayurveda, a harmonious relationship with nature is fundamental for maintaining optimal health. The increasing acceptance and widespread use of herbal remedies highlight the growing relevance of this ancient medical system in enhancing resistance against various diseases. In this context, the present study aims to scientifically validate a classical Ayurvedic formulation, Vasaguduchyadi Kvatha Churna (VGKC), along with its marketed preparation, with its major marker, phenol derivative gallic acid. The work focused on the comprehensive quality evaluation, and standardization was carried out through physicochemical assessments, including extractive values that are soluble in water and alcohol, total ash value, acid insoluble ash value, and loss after drying and preliminary phytochemical screening such as alkaloids, steroids, terpenoids, flavonoids, phenolic content, saponins, and glycosides. High-performance thin-layer chromatography (HPTLC) is an accurate and sensitive method for the identification and quantification of selected marker phenol content, gallic acid, in VGKC and their marketed formulation with a solvent system consisting of toluene: ethyl acetate: methanol: formic acid (4:4:2:0.3 v/v/v). Rf value 0.62 for gallic acid was noted, and it precisely matches the matching bands in Vasaguduchyadi Kvatha Churna. This standardization method has ensured the safety, quality, and consistency of the Vasaguduchyadi Kvatha Churna.

Keywords: Vasaguduchyadi Kvatha Churna, HPTLC, Standardization, Marketed formulation, Gallic acid.

Introduction:

Ayurveda is the oldest, most commonly recognized, practiced, and prosperous indigenous medical system in India, which has an extensive record of conventional medical treatments that uses six systems (Kizhakkeveettil et al., 2023). The Sanskrit term for the traditional Indian medical system is Ayurveda, which means "the knowledge (Skt. Veda) for longevity (Skt. Ayush)” (Verma et al., 2024). Ayurveda is a comprehensive medical philosophy and practice that includes both prescriptive and preventative elements. According to Ayurveda, "Chikitsa Chatushpada" is the key to any treatment's effectiveness. "Chatushpada" signifies the four pillars of treatment, and "Chikitsa" means treatment, which is the result of efforts. Chatushpada are the mutually dependent Dravya (medicine), Rogi (patient), Upastha (medical attendant), and Bhishag (physician) (Kumar, 2021). The public's interest in Ayurveda for ongoing and lifelong illnesses has naturally grown, as for the World Health Organization (WHO), herbal treatments are used by 80% of the population globally for their main healthcare needs. Nearly all of the statements in the classical Ayurvedic texts were derived via Ayurvedic examination techniques, such as direct observation (pratyaksh), inference (anuman), the ancient authoritative literature (aptopadesh), and reasoning (yukti) (Patel et al., 2021). The development of standardization parameters for the Ayurvedic formulation, such as organoleptic parameters, physicochemical parameters, preliminary phytochemical screening, and sophisticated methods like High Performance Thin Layer Chromatography (HPTLC) for the determination of proper consistency of the Ayurvedic formulation. Vasaguduchyadi Kvatha Churna (VGKC) is a polyherbal classical Ayurvedic formulation. It is mentioned in Astanghridya, Chikitsasthana, Adhyaya 16:13, in the management of Pandu (anemia), Raktapitta (bleeding), and Kamala (jaundice), when administered along with honey (Anonymous., 2003). In Pandu (anemia), Raktapitta (bleeding), and Kamala (jaundice) diseases, there is a predominant Pitta dosha. Acharya Charka mentioned that diet and drugs having Madhura, Tikta, Kashaya, Virya, Laghu & Mandura properties are useful in the management of Pandu Roga (Srikanth & Vedi, 2019). Herbs present in Vasaguduchyadi Kvatha Churna will detract Pitta dosha, thus resolving an obstruction and normalizing the Gati (movement) of Agni (digestive fire), as well as blood-purifying, anti-inflammatory, and hepatoprotective actions due to Rasayana-like (rejuvenative) properties. Much research has revealed that the components of Vasaguduchyadi Kvatha churna and its phytochemicals have hepatoprotective, antioxidant, detoxifying, pro-hematopoietic, antiviral, antimicrobial, and immunomodulatory properties (Nair Reshma.M. & Hameed Shahul, 2024). VGKC consists of eight herbal drugs: dried root of Adhatoda vasika (Vasa), stem of Tinospora cordifolia (Guduchi), fruit pulp of Terminalia chebula (Haritaki) and Terminalia berrilica (Bibhitaki), Embelica officinalae (Amla), rhizome of Picrorrhiza kurroa (Katuka), whole plant of Andrographis paniculata (Bhuinimba), and bark of Azadirachta indica (Neemba) (Anonymous., 2003).

Numerous Ayurvedic research studies have described a variety of illnesses; however, the physicochemical and analytical properties of VGKC have not been standardized. In order to determine the standardized parameter for the VGKC in terms of its physicochemical and phytochemical characteristics as well as the qualitative and quantitative estimation of the marker compound using high-performance thin-layer chromatography, this study was carried out.

Materials and methods:

Chemicals and reagents:

The standard marker compound used is gallic acid (Batch no. MCR-18404), which is obtained from Molychem Pvt. Ltd., Mumbai. All the solvents, chemicals, and reagents were used, such as toluene, ethyl acetate, methanol, Dragendroffs reagent obtained from Molychem Pvt. Ltd., Mumbai; petroleum ether and ferric chloride from Fisher Scientific Pvt. Ltd., Mumbai; formic acid and lead acetate from Loba Chemie Pvt. Ltd., Mumbai; ethanol from Bengal Chemicals & Pharmaceuticals Ltd., chloroform purchased from Numex Chemical Products (India), benzene from Rankem, picric acid from Laboratory Rasayan, Vanillin sulphuric acid from Pallav Chemicals & Solvents Pvt. Ltd, hydrochloric acid from Emparta, sulphuric acid from Avantor Performance Materials India Ltd. Mumbai, ammonium hydroxide solution from Merok Life Science Pvt. Ltd. Mumbai, utilized in the estimation of were Analytical Grade. Pre-coated TLC Aluminum sheets (Batch no.-HX38268454) silica gel 60F254 (20 x10 cm, 0.2 mm thick) was obtained from Merck Ltd, Mumbai.

 Instruments:

A CAMAG HPTLC system comprising a CAMAG TLC SCANNER 3 and LINOMAT 5 automatic sample applicator fitted with a 100 μL syringe, TLC plates pre-coated with silica gel 60F254, 20×10 cm (Merck), visionCATS software, and TLC CAMAG Visualizer 2 were used.

 Collection of herbal raw material:

The dried root of Adhatoda vasika, stem of Tinospora cordifolia, fruit pulp of Terminalia chebula, Terminalia berrilica, Embelica officinalae, rhizome of Picrorrhiza kurroa, whole plant of Andrographis paniculate and bark of Azadirachta indica were procured from an established drug supplier from Raipur, Chhattisgarh, and authenticated by Dr. P.K. Joshi, Department of Dravyaguna, Government Ayurvedic College, Raipur, Chhattisgarh.

Preparation of VGKC:

There are 3 samples of in-house formulation prepared in the University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh. Vasaguduchyadi Kvatha Churna was prepared by equally weighing the parts of all eight above-mentioned plants. Then the plant parts were made into a coarse powder of 20 mesh sieves; they were mixed together and stored in an airtight container for further use, which are coded as per the preparation and location, VGKC-UIOP-I, VGKC-UIOP-II, and VGKC-UIOP-III and picked from the local market, which are coded VGKC-LM (Anonymous., 2003). A comparative study has been performed between marketed sample VGKC-LM and prepared samples VGKC-UIOP-I, VGKC-UIOP-II, and VGKC-UIOP-III in the University Institute of Pharmacy to evaluate each and every VGKC formulation's quality.

Figure 1. Preparation of VGKC (Vasaguduchyadi Kvath Churna)

Physicochemical parameters of VGKC and its raw materials:

The physicochemical analysis of Vasaguduchyadi Kvatha Churna and its raw materials was carried out in accordance with the general parameters of churna given in the Ayurvedic Pharmacopoeia of India (API, 2011), such as total ash (%), acid-insoluble ash (%), loss on drying (%), alcohol-soluble extractive (%) and water-soluble extractive (%) at the Department of Pharmacognosy Laboratory, University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh (Anonymous., 2001a, 2001b, 2001c, 2001d).

Preliminary phytochemical screening:

Preliminary phytochemical screening is a fundamental quality-control step for assessing the authenticity, consistency, and therapeutic potential of raw drugs and their formulations. In the present study, raw drugs of Vasaguduchyadi Kvatha Churna, such as the dried root of Vasaka (Adhatoda vasica), stem of Guduchi (Tinospora cordifolia), fruit pulp of Haritaki (Terminalia chebula), Bibhitaki (Terminalia berrilica), Amla (Embelica officinalae), rhizome of Katuka (Picrorrhiza kurroa), whole plant of Bhuinimba (Andrographis paniculate), and bark of Neemba (Azadirachta indica), and their combined formulation Vasaguduchyadi Kvatha Churna (VGKC-UIOP-I, VGKC-UIOP-II, VGKC-UIOP-III, and VGKC-LM) were evaluated for major phytoconstituents, including alkaloids, steroids and terpenoids, flavonoids, saponins, phenolic compounds and tannins, and glycosides. The tests were performed on petroleum ether, toluene, chloroform, ethyl acetate, methanol, ethanol, and water extract. For screening the phytochemicals, perform various types of tests, such as Dragendroffs test and Hager’s test for alkaloids; Salkowski test for steroids and terpenoids; ferric chloride and lead acetate test for flavonoids, phenolic compounds, and tannins; test for saponins; and Bontrager's test for glycosides (Maheshwaran et al., 2024).

Qualification of marker compound in VGKC by HPTLC analysis (Sahu et al., 2025; Sheikh et al., 2015)

Preparation of VGKC sample:

After being precisely weighed, one gram of powdered VGKC sample (VGKC-UIOP-I, VGKC-UIOP-II, VGKC-UIOP-III, and VGKC-LM) was transferred into four 10-milliliter volumetric flasks. Each volumetric flask received roughly 5 ml of methanol, which was sonicated afterwards for 10 minutes to get the volume up to the desired level. The solution's final concentration will be 100 mg/ml. After that, the solution is filtered through a 0.45 µm membrane filter paper to produce a clean solution, which is subsequently utilized for the HPTLC analysis.

 Preparation of standard solution of Gallic acid:

After precisely weighing 10 mg of the standard medication gallic acid and adding 50 ml of methanol to a 100 ml volumetric flask, the mixture was sonicated for 10 minutes and brought up to 100 ml with methanol. The resulting standard solution had a concentration of 100 µg/ml.

 Chromatographic condition:

The sample of methanolic extract of VGKC and standard gallic acid was spotted on pre-coated TLC aluminum sheets of silica gel 60F254 (20 x 10 cm, 0.2 mm thickness) as a 5 mm wide band by using the TLC applicator Linomat V, 10 mm from the bottom. The mobile phase is toluene: ethyl acetate: methanol: formic acid (4:4:2:0.3 v/v/v) was found to be the most fit for gallic acid. For twenty minutes, the plates were held in the twin trough chamber to reach saturation. Following development, the plates were allowed to air dry before being scanned with the CAMAG SCANNER 3 for gallic acid at 254 and 366 nm. Using the TLC CAMAG Visualizer 2, the plates were imaged at 254 and 366 nm.

Preparation of calibration curve:

The standard solution of gallic acid (1,2,3,4,5,6 μL/spot) was applied on the TLC plate, and further, it was developed and scanned as per the chromatographic condition mentioned above. The peak area was recorded, and a calibration curve of gallic acid was prepared by plotting peak area against concentration of gallic acid (Figures 4 and 5).

 Quantification of gallic acid:

The Gallic acid content in Vasaguduchyadi Kvatha Churna (VGKC-UIOP-I, VGKC-UIOP-II, VGKC-UIOP-III, VGKC-LM) was determined. The amounts of the methanolic extracts of Vasaguduchyadi Kvatha Churna (VGKC-UIOP-I, VGKC-UIOP-II, VGKC-UIOP-III, VGKC-LM) were applied as 1,1,1 μL/spot duplication. Gallic acid was spotted on TLC plates as 8 mm bands, with a minimum of 2 mm apart and 10 mm from the lower edge of the plates. A calibration graph was plotted to quantify the amount of the above-mentioned marker compound; Rf was observed, and a spectral match was also done to confirm the gallic acid in VGKC (Figure 6).

HPTLC Method validation (Pallavi & Jha, 2021):

 Linearity: The results are directly correlated with the sample quantity (within a certain range). It was determined by making a graph of the peaks vs. standardized quantity and examining the equation of the line and the correlation coefficient (R²).

Accuracy: The variance among the mean and the recognized real value, along with confidence intervals, or the proportion of recoveries from the assay of a known extra amount of analyte in the sample should be used to quantify accuracy. Before accuracy can be established, the specified range must be covered by a minimum of nine estimations covering at least three distinct concentration levels (e.g., three concentrations over three duplicates of the full analytical procedure). The percentage recovery was calculated by adding a predefined amount of standard to the sample and conducting recovery experiments in triplicate at three concentration levels: 80%, 100%, and 120%. After that, these samples were analyzed, and the results were compared to the predictions.

Precision: An analytical procedure's precision is a measure of its repeatability across time. The percentage relative standard deviation (%RSD) for a statistically significant number of samples is a common way to express it. Minimum, moderate, and extreme quality control are the various stages at which precision should be implemented, per the ICH guidelines. Accuracy under the same operating conditions over a short period of time is known as repeatability. Repeatability in an assay is also known as precision. The strategy was tested using at least nine occurrences that fell inside the specified range. Inter-assay precision was examined on three distinct days, whereas intra-day assay precision was tested on three occasions on similar days.

Specificity: Specificity is the ability to assess a substance despite the absence of elements that ought to be found in the tested matrix. The specificity of the process was ascertained by comparing the Rf value, and the peak purity of the process was ascertained by comparing the standard and sample spectra.

Limit of detection: Minimum amount of a substance being measured.

Limit of quantification: The minimum amount of a material that can be measured with reasonable precision and accuracy.

Robustness: The empirical technique's reliability, which gauges the degree to which it is by small but deliberate adjustments to technique variables, provides an indication of how dependable it will be under normal circumstances.

Results and discussion:

The percent loss after drying was determined for each batch of Vasaguduchyadi Kvatha Churna (VGKC-UIOP-I, VGKC-UIOP-II, and VGKC-UIOP-III), its marketed formulations (VGKC-UIOP-LM), and separately its raw materials Adhatoda vasika, Tinospora cordifolia, Terminalia chebula, Terminalia berrilica, Embelica officinalae, Picrorrhiza kurroa, Andrographis paniculate and Azadirachta indica as per the Ayurvedic Pharmacopeia of India. The results are recorded in Table 1.

Table 1. Physicochemical properties of VGKC and its raw drugs

To determine the presence of various phytoconstituents in formulations as well as in raw materials, a phytochemical investigation was performed. The tests were performed on petroleum ether, toluene, chloroform, ethyl acetate, methanol, ethanol, and water extract. The tests for the presence of phytoconstituents are recorded in Table 2.

Figure 2. Physicochemical parameter of VGKC and its raw materials

Table 2. Preliminary Phytochemical screening of VGKC and its raw drugs

Figure 4. HPTLC fingerprint of VGKC and Gallic Acid

The HPTLC analysis of polyherbal formulation VGKC shows the relevant presence of various types of phytochemicals. The Rf value obtained for gallic acid is 0.62, respectively. The Rf values of the marker compounds with reference standards and polyherbal formulation were found comparable. The HPTLC analysis revealed that all the corresponding spectra of the test sample are overlapping with markers, confirming the presence. In the current investigation, we have examined the quantities of gallic acid quantified in VGKC-UIOP-I, VGKC-UIOP-II, VGKC-UIOP-III, and VGKC-LM. Estimation of the marker compound gallic acid using HPTLC ensures the quality of the product. The sample is extracted with a methanol and sonicated for 10 min and filtered with 0.45 μ membrane filter; the filtrate was used for the HPTLC study. The solvent system toluene: ethyl acetate: methanol: formic acid (4:4:2:0.3 v/v/v) showed better separation for gallic acid (Rf value 0.626 ± 0.005). Using visionCATS software, the plate was scanned at 254 and 366 nm for densitometric chromatographic analysis using a Camag Scanner IV. The existence and quantity of the marker in the sample are confirmed by the overlapping of all the relevant spectra. The linearity spectrum, precision, limitation of detecting (LOD), limitation of quantification (LOQ), and recovery of the method were all validated. With a correlation coefficient of 0.995, gallic acid showed a linearity range of 1 μg to 5 μg spot/1 (Figure 7). The linearity curves were depicted in these images, and all of the gallic acid peaks were visible upon derivatization. In terms of system precision, method precision, and middle precision for gallic acid, the techniques were determined to be accurate. The accuracy of the method was tested by undertaking recovery studies. Average recovery of Gallic acid was found to be 90.85%, 93.01%, 93.85%, respectively (Table 5.). Table 3 summarized the data related to method validation of HPTLC and various parameters of VGKC-UIOP-I, VGKC-UIOP-II, VGKC-UIOP-III, and VGKC-LM phytochemicals. The quantities of the marker compound gallic acid in the methanolic extract of VGKC-UIOP-I, VGKC-UIOP-II, VGKC-UIOP-III, and VGKC-LM were found to be 437 μg/ml, 413.3 μg/ml, 425.1 μg/ml, and 0 μg/ml, respectively.

Figure 5. 3D image of HPTLC fingerprint of VGKC and Gallic Acid

Figure 6. HPTLC Chromatogram of Methanolic Extract of VGKC-UIOP-I, VGKC-UIOP-II, VGKC-UIOP-III and VGKC-LM

Figure 7. HPTLC Chromatogram of standard marker compound Gallic Acid

Table 3. Method Validation Data of Standard

Table 4. Recovery Study of Standard

Table 5. Accuracy and Precision study

Conclusion:

Standardization parameters such as physicochemical parameters and preliminary phytochemical screening of a classical Ayurvedic formulation, VGKC, were found to be within the limits. An HPTLC method has been developed for the detection of gallic acid in VGKC, a polyherbal ayurvedic preparation. The suggested approach can be used for routine analysis, standardization, and quality control of ayurvedic formulation VGKC since it is straightforward, accurate, and reproducible. 

Abbreviation:

VGKC – Vasaguduchyadi Kvatha Churna

HPTLC – High Performance Thinn Layer Chromatography

VS – Vasa

GD – Guduchi

HR – Haritaki

BH – Bibhitaki

AM – Amla

KT – Katuka

BN – Bhuinimba

NE – Neemba

S.D. – Standard Deviation

 Conflict of interest:

The authors have no conflicts of interest regarding this investigation.

 Acknowledgement:

Authors are thankful to University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur (C.G.), for providing all resources for this work.

 References:

Anonymous. (2001a). The Ayurvedic Pharmacopeia of India, Part 1 (1st ed., Vol. 1, p. 143). Government of India, ministry of health and Family welfare, Department of Indian system of medicine & Homeopathy. Appendices 2.2.9. New Delhi: The controller of publications.

Anonymous. (2001b). The Ayurvedic Pharmacopeia of India, Part 1 (1st ed., Vol. 1, p. 143). Government of  India, ministry of health and Family welfare, Department of Indian system of  medicine & Homeopathy Appendices 2.2.3. New Delhi: The controller of  publications.

Anonymous. (2001c). The Ayurvedic Pharmacopeia of India, Part 1 (1st ed., Vol. 1, p. 143). Government of India, ministry of health and Family welfare, Department of Indian system of medicine & Homeopathy Appendices 2.2.4. New Delhi: The controller of publications.

Anonymous. (2001d). The Ayurvedic Pharmacopeia of India, Part 1 (1st ed., Vol. 1, p. 143). Government of India, ministry of health and Family welfare, Department of Indian system of medicine & Homeopathy Appendices 2.2.6 1st ed. New Delhi: The controller of publications.

Anonymous. (2003). The Ayurvedic Formulary of India. In The Ayurvedic Pharmacopeia of India, Part 1 (Second edition, p. 198). , Government of India, ministry of health and Family welfare, Department of Ayurveda, Controller of Publications Civil Lines, Delhi - 110054.

Kizhakkeveettil, A., Parla, J., Patwardhan, K., Sharma, A., & Sharma, S. (2023). History, Present and Prospect of Ayurveda. In History, Present and Prospect of World Traditional Medicine: In 2 Volumes (Vol. 2). https://doi.org/10.1142/9789811282171_0001

Kumar, J. (2021). An overview of Chikitsa Chatushpada. In Journal of Ayurveda and Integrated Medical Sciences (Vol. 6). www.jaims.in

Maheshwaran, L., Nadarajah, L., Senadeera, S. P. N. N., Ranaweera, C. B., Chandana, A. K., & Pathirana, R. N. (2024). Phytochemical Testing Methodologies and Principles for Preliminary Screening/ Qualitative Testing. Asian Plant Research Journal, 12(5). https://doi.org/10.9734/aprj/2024/v12i5267

Nair Reshma.M., & Hameed Shahul. (2024). A critical review on Vasaguduchyadi Kwatha. Kerala Journal of Ayurveda, 3(1). https://doi.org/10.55718/kja.219

Pallavi, R., & Jha, S. (2021). A validated quantification of gallic acid and ellagic acid in Triphala using a high-performance thin-layer chromatography method. Journal of Planar Chromatography - Modern TLC, 34(5), 447–453. https://doi.org/10.1007/s00764-021-00130-8

Patel, S., Baghel, G. S., Chauhan, N. S., Rathiya, S., Sahu, U., Parihar, A. S., Kumar, A., & Gupta, P. K. (2021). High-performance thin-layer chromatographic standardization and quantification of marker compounds in an Ayurvedic polyherbal formulation: Krishnadi Churna. Journal of Planar Chromatography - Modern TLC, 34(6), 493–502. https://doi.org/10.1007/s00764-021-00144-2

Sahu, U., Chauhan, N. S., Parihar, A. K. S., Karbhal, K. S., Inchulkar, S. R., Gupta, P. K., & Singh, R. K. (2025). Development of Simultaneous HPTLC Method and Validation for the Quality Assessment of Ayurvedic Formulation - Ayush Kvatha Churna by Using Marker Compound Rosmarinic Acid, Trans-Cinnamaldehyde and Piperine. Journal of Chromatographic Science, 63(3). https://doi.org/10.1093/chromsci/bmae019

Sheikh, Z. A., Shakeel, S., Gul, S., Zahoor, A., Khan, S. S., Zaidi, F. H., & Usmanghani, K. (2015). A novel HPTLC method for quantitative estimation of biomarkers in polyherbal formulation. Asian Pacific Journal of Tropical Biomedicine, 5(11). https://doi.org/10.1016/j.apjtb.2015.06.016

Srikanth, N., & Vedi, S. K. (2019). Management of Anaemia through Ayurveda Critical Appraisal of the role of Ayurveda Interventions in the management of Anaemia (Glimpses of National campaign on Anaemia control through Ayurveda) Technical Report. www.ccras.nic.in

Verma, S. K., Pandey, M., Sharma, A., & Singh, D. (2024). Exploring Ayurveda: principles and their application in modern medicine. Bulletin of the National Research Centre, 48(1). https://doi.org/10.1186/s42269-024-01231-0