Introduction

Ayurvedic medicine, with its rich historical foundation and holistic principles, encompasses a wide range of polyherbal formulations that have demonstrated efficacy in managing diverse health conditions. These formulations, composed of multiple medicinal plants, are abundant in phytoconstituents and bioactive compounds that contribute to their therapeutic potential. Comprehensive characterization and quantification of these bioactive constituents are essential to substantiate their pharmacological effects and ensure the safety and efficacy of these traditional remedies. One of the major challenges in Ayurvedic pharmaceutics is the standardization of complex multi-component formulations. Unlike conventional pharmaceuticals that typically involve a single active ingredient, Ayurvedic preparations contain numerous synergistic compounds, making it difficult to isolate and quantify specific bioactive markers for quality control. This complexity poses significant hurdles in ensuring batch-to-batch consistency and therapeutic reliability.

Standardization plays a pivotal role in modernizing and validating Ayurvedic formulations. It enables the scientific evaluation of therapeutic claims, facilitates the monitoring of active constituents to ensure safety, and supports regulatory compliance for wider clinical acceptance and commercialization. Developing robust analytical methods to identify and quantify key marker compounds is thus indispensable for integrating traditional knowledge with contemporary quality assurance standards1.

Standardization is essential for minimizing variability in the composition of herbal formulations, thereby ensuring consistent therapeutic outcomes and reducing potential safety risks for consumers. Ayurvedic formulations typically incorporate one or more medicinal herbs, each containing distinct phytochemical markers that contribute to their therapeutic efficacy. The JKC formulation, for instance, comprises Andrographis paniculata, Cyperus rotundus, Piper longum, Piper nigrum, Zingiber officinale, Hedyotis corymbosa, and Picrorhiza kurroa. Key phytoconstituents such as andrographolide, piperine, picroside-I, picroside-II, and alpha-cyperone are present in these botanicals and can serve as reliable analytical markers for the standardization and quality control of the JKC formulation2,3,4.

High-Performance Thin-Layer Chromatography (HPTLC) has proven to be a robust and versatile analytical tool for the standardization of complex herbal formulations. Its ability to simultaneously separate, identify, and quantify multiple phytoconstituents makes it particularly advantageous for analyzing multi-component systems such as polyherbal preparations. HPTLC offers high resolution, enhanced sensitivity, and reproducibility while accommodating the inherent complexity and variability of herbal matrices5. These attributes make it especially suitable for ensuring the quality, consistency, and authenticity of traditional medicinal products6.

The medicinal plants incorporated in the Jwarahara Kwatha Choornam (JKC) formulation exhibit substantial therapeutic potential and have been extensively documented in classical Ayurvedic texts such as the Charaka Samhita7, and Bhava Prakash8. These botanicals have long been employed in traditional formulations for managing a wide spectrum of ailments, particularly those related to fever, liver function, and digestion.

Andrographis paniculata, a key component of JKC, is traditionally recognized for its antipyretic, hepatoprotective, and digestive properties. It features prominently in formulations such as Katuki Choorna, where it is used in combination with Picrorhiza kurroa to promote liver detoxification and digestive health. It is also a principal constituent in Amritarishta, a fermented formulation prescribed for chronic fevers, immune enhancement, and gastrointestinal support. Similarly, Mahasudarshana Choorna9 and Kalmegh Churna are classical preparations that utilize these herbs for managing febrile and hepatic disorders.

In addition, Piper longum (long pepper) and Piper nigrum (black pepper) are widely acknowledged in Ayurvedic pharmacopeia for their roles in enhancing digestion, metabolism, and respiratory function. These pungent spices are integral to formulations such as Trikatu10,, Pippalyadi Churna, Sitopaladi Churna11, and Pippali Rasayana, which are commonly indicated for respiratory ailments including colds and coughs. Piper nigrum is also included in Vyoshadi Vatakam12, noted for its efficacy in respiratory disorders, and in Dashamoola13, valued for its anti-inflammatory and analgesic actions.

Cyperus rotundus is a pharmacologically versatile herb traditionally valued in Ayurveda for its cooling, carminative, and anti-inflammatory properties. It is employed in the management of digestive disturbances, menstrual irregularities, febrile conditions, and inflammatory disorders. Key formulations incorporating Cyperus rotundus include Mustarishta, used for digestive ailments and fever, and Dashamoola Kwatha, indicated for inflammatory and respiratory conditions14.

Picrorhiza kurroa (Katuki) is well-recognized for its hepatoprotective, digestive, and anti-inflammatory actions. It is a critical ingredient in several classical formulations such as Mahasudarshana Choorna, Arogyavardhini Vati, Jatyadi Ghrita, Tiktadya Ghrita, Punarnavasava, and Nimbadi Churna, all of which are traditionally prescribed for liver disorders, chronic inflammation, and gastrointestinal dysfunction15.

Zingiber officinale (Śuṇṭhī or dry ginger) holds a prominent place in Ayurvedic therapeutics due to its potent digestive stimulant, anti-inflammatory, and respiratory-supportive effects. It is a common constituent in numerous formulations targeting conditions ranging from dyspepsia to bronchitis16.

Hedyotis corymbosa (Parpaṭaka) is another botanically significant herb noted for its cooling, anti-inflammatory, and detoxifying properties17. It is frequently used in polyherbal preparations such as Arogyavardhini Vati, Parpatadi Kwatha, Chandanasava, and Parpataka Churna, where it aids in the treatment of fevers, gastrointestinal disturbances, inflammatory disorders, and skin diseases18,19.

Andrographolide (AG), the principal bioactive diterpenoid compound of Andrographis paniculata, is structurally characterized by a labdane-type diterpenoid ring system20. It has been extensively studied for its broad pharmacological profile, exhibiting potent antimicrobial, anti-inflammatory21, antioxidant, and hepatoprotective properties22, which contribute to its traditional use in treating febrile and hepatic disorders.

Piperine (PP), an alkaloid abundantly present in Piper nigrum and Piper longum, is recognized for its diverse pharmacodynamic effects23. It demonstrates significant analgesic, immunomodulatory24, anti-asthmatic, anti-inflammatory25, antioxidant26, and antimicrobial activities27. In addition, piperine enhances the bioavailability of various phytochemicals, making it a valuable synergist in polyherbal formulations.

α-Cyperone (AC), a sesquiterpene compound isolated from Cyperus rotundus, serves as a key phytochemical marker for this species28. AC has been reported to exert anti-inflammatory effects through mechanisms involving the destabilization of microtubule fibers in neural tissue, as demonstrated by Azimi et al.29. It also possesses antifungal and anti-capsular properties, underscoring its therapeutic potential in infectious and inflammatory conditions30.

Picroside-I (P-I) and Picroside-II (P-II) are key iridoid glycosides found in Picrorhiza kurroa (Katuki), widely recognized for their diverse pharmacological properties. These markers have been frequently employed for quality assessment and standardization of P. kurroa-based formulations. Singh et al. (2013) reported the simultaneous estimation of P-I and P-II using a mobile phase of chloroform: methanol (82:18 v/v), with retention times of 0.60 and 0.43, respectively, indicating their distinct chromatographic behavior31. Additionally, Tiwari et al. (2012) developed an HPTLC-densitometric method for the simultaneous quantification of these compounds, further supporting their use as reliable phytochemical markers32. More recently, Rizvi et al.. (2023) evaluated the antiviral potential of P. kurroa in a Syrian hamster model, demonstrating promising therapeutic activity against SARS-CoV-2 infection33.

In this context, the development of an HPTLC method for the standardization of Jwarahara Kwatha Choornam (JKC), a polyherbal Ayurvedic formulation, is both timely and essential. JKC is traditionally prescribed for managing chronic fever, cold, and malaria, as well as for enhancing digestion, appetite, blood detoxification, immune modulation, and protection against bacterial infections. Anecdotal and practitioner-based reports have also cited its supportive role during the COVID-19 pandemic.

JKC comprises seven medicinal plants—Cyperus rotundus (Musthakam), Piper longum (Pippali), Piper nigrum (Maricha), Zingiber officinale (Shunthi), Hedyotis corymbosa (Parpata), Andrographis paniculata (Kalmegh), and Picrorhiza kurroa (Katuki Rohini)—with the specific plant parts utilized outlined in Table 1. These botanicals contribute synergistically to the formulation’s therapeutic efficacy, aligning with Ayurvedic principles of holistic health management.

Table 1 List of plants and their parts used in JKC formulation.
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To ensure consistency, safety, and efficacy of JKC, the Central Council for Research in Ayurvedic Sciences (CCRAS), under the Ministry of AYUSH, Government of India, has undertaken initiatives to standardize this formulation34,35. The present study focuses on the development of a validated High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous estimation of five critical bioactive markers: Andrographolide (AG), Piperine (PP), Picroside-I (P-I), Picroside-II (P-II), and α-Cyperone (AC). The chemical structures of theses markers are represented in Fig. 1. This analytical advancement not only strengthens quality assurance for JKC but also establishes a methodological framework for standardizing other complex Ayurvedic polyherbal formulations.

Fig. 1
figure 1

Structures of the bioactive markers present in JKC formulation.

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Materials and methods

Plant materials and chemicals

All the standards Andrographolide (AG), Piperine (PP), Picroside-I (P-I), Picroside-II (P-II), and α-Cyperone (AC) were procured from a Sigma-Aldrich with documented purity. HPLC-grade Methanol, Ethyl acetate, Toluene, and Formic acid are procured from Merck and used to prepare the Mobile phase and sample solutions. Plant materials used in the preparation (Table 1) of JKC are collected from the Kerala region, India, and authenticated by Thulasi Radhakrishanan, Research Assistant (Botany) and voucher specimens are deposited in the herbarium for future reference with voucher specimen numbers from NARIP-IMR-JKC/001 to NARIP-IMR-JKC/007 (Table 1) at the National Ayurveda Research Institute for Panchakarma (NARIP), Cheruthuruthy. This study adhered to all relevant institutional, national, and international guidelines and legislation concerning the collection and study of wild plants. The research complies with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora (CITES) and relevant permission have obtained to collect the plant parts. The JKC formulation samples used in this study were procured from the NARIP Pharmacy, National Ayurveda Research Institute for Panchakarma (NARIP), CCRAS, Cheruthuruthy, Kerala. Three different batches were used for analysis: KL-JKC/2023-01, KL-JKC/2023-02, and KL-JKC/2023-03. These samples were stored under recommended conditions until further analysis.

Preparation of sample and standard solutions

Preparation of standard solutions

Stock solutions of Andrographolide (AG), Piperine (PP), Picroside-I (P-I), Picroside-II (P-II), and α-Cyperone (AC) were prepared by accurately weighing each standard and dissolving them in Methanol. The concentrations achieved were 2.5 mg/mL for AG and PP and 0.5 mg/mL for P-I, P-II, and AC. These stock solutions were then diluted to create a final marker mixture containing 0.5 mg/mL of AG and PP and 0.1 mg/mL of P-I, P-II, and AC.

Preparation of JKC sample solutions

Accurately weighed, 2 g of JKC powder was dissolved in 10 mL of Methanol and sonicated for 15 min at 40 °C. The resulting extract was then filtered through a 0.22 μm PTFE syringe filter, and the filtrate was used for HPTLC analysis.

Chromatographic condition for HPTLC method

The experimental procedures were conducted using a CAMAG HPTLC system with a CAMAG Linomat-V applicator, TLC Scanner-IV, TLC Visualizer, and Vision CATS software. Precoated Silica gel 60 F254 HPTLC plates (20 cm x10 cm) have been used as the stationary phase, and Toluene: Ethyl acetate: Methanol: Formic Acid, (4:4:1:1 v/v/v/v) ratio has been used as the mobile phase. Standards and samples were applied with a 100 µL CAMAG Syringe on the TLC plate with the help of the Linomat-V applicator, and plates were developed in CAMAG Twin Trough Chamber (TTC), which is pre-saturated for 20 min with the optimized mobile phase at room temperature (25 ± 2 °C). Further, the plate was dried at room temperature for 5 min and then placed inside the CAMAG visualizer for detection. Densitometry scanning was performed at different wavelengths for different standards with TLC Scanner-IV, scanning at 229 nm for AG, 343 nm for PP, 279 nm for P-I, 264 nm for P-II, and 254 nm for AC. Further spectra of similar Rf values were documented by using the TLC scanner-IV. Further quantification is performed with the help of Vision Cats 3.1 Software.

Preparation of calibration curve for the markers

Calibration curves were constructed by plotting the peak areas of each standard against their corresponding concentrations. The concentration range for the calibration curve was selected based on preliminary experiments to ensure linearity. The linear regression equations and correlation coefficients (r²) were calculated to assess the linearity of the method.

Method validation

The developed HPTLC method was validated for the following parameters according to the International Conference on Harmonisation ICH Q2(R1) guidelines36 (ICH, 2005). The development of the HPTLC method includes specificity, linearity, range, accuracy, precision (repeatability and intermediate precision), detection limit (LOD), quantitation limit (LOQ), and robustness.

Linearity range of bioactive markers

A working standard marker mixture was prepared with 0.5 mg/mL concentrations for AG, PP and 0.1 mg/mL for P-I, P-II, and AC. The calibration range applied on the TLC plate included volumes of 1.0 µL, 2.0 µL, 4.0 µL, 6.0 µL, 8.0 µL, and 10.0 µL of the working standard marker mixture. These volumes correspond to a concentration range of 500 ng to 5000 ng for AG and PP; and 100 ng to 1000 ng for P-I, P-II, and AC standards. (See supplementary material)

Specificity

The specificity of the method was established by comparing the overlay spectra of the standards with those of the JKC samples having the same Retention factor (Rf) values. Additionally, peak purity was evaluated on the TLC plates (Fig. 2).

Fig. 2
figure 2

HPTLC plate @ 254 nm{ tracks: 1 to 6 are different dilutions of standard markers mixtures, Tracks: 7 to 9 are JKC B1 samples; Tracks: 10 to 12 JKC B2 samples; Tracks: 13 to 15 JKC B3 samples}.

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Precision

The precision of the method was evaluated in accordance with ICH Q2(R1) guidelines. Repeatability (intra-day precision) and intermediate precision (inter-day precision) were assessed by analyzing five replicates at three different concentration levels of the standard mixture. For inter-day studies, 4.0 µL, 6.0 µL, and 8.0 µL injection volumes were analyzed across three separate days to evaluate method reproducibility.

Accuracy

The accuracy of the method was evaluated in accordance with ICH Q2(R1) guidelines through recovery studies, by spiking known amounts of standards into the JKC sample matrix at three concentration levels (50%, 100%, and 150% of the target concentration). Each level was tested in triplicate, and the percentage recovery was calculated to assess the method’s accuracy and reliability.

Robustness

The Robustness of the method were validated by varying the saturation time of the mobile phase in the twin-trough chamber (TTC) prior to TLC plate development. One TLC plate was developed after a 20-minute saturation time, while another TLC plate was developed after a 30-minute saturation time.

Limit of detection (LOD) and limit of quantification (LOQ)

The limit of detection (LOD) and limit of quantification (LOQ) for all standards were calculated based on the response’s standard deviation and the calibration curves’ slope. The LOD was defined as 3.3 times the ratio of the standard deviation (SD) of the y-intercept to the slope (S) of the calibration line, while the LOQ was defined as ten times the same ratio. LOD and LOQ of the standards were calculated by using the following formulae.

$$\:LOD=3.3\left(\frac{SD}{S}\right)$$
(1)
$$\:LOQ=10\left(\frac{SD}{S}\right)$$
(2)
$$\:LOD=Limit\:of\:Detection$$
$$\:LOQ=Limit\:of\:Quantification$$
$$\:SD=Standard\:deviation\:of\:Y-intercept$$
$$\:S=slope\:of\:the\:linear\:curve$$

Estimation of bioactive markers AG, PP, P-I, P-II, and AC in the JKC formulation

A carefully prepared sample solution with a concentration of 200 mg/mL from three different batches (B1, B2, and B3) of JKC was applied to a TLC plate alongside the different volumes of the standard marker mixture. The plate was developed using an optimized mobile phase of Toluene, Ethyl acetate, Methanol, and Formic acid in a (4:4:1:1 v/v/v/v) ratio (Fig. 2). After development, the TLC plate was scanned at different wavelengths specific to each marker: 229 nm for Andrographolide (AG), 343 nm for Piperine (PP), 279 nm for Picroside-I (P-I), 264 nm for Picroside-II (P-II), and 254 nm for α-Cyperone (AC). 2-D Denistograms and calibration curves of the markers at respective wavelengths are shown in supplementary data Annexure-I. Quantification was performed using Vision CATS software. All experiments were conducted in triplicate, and results were expressed as mean ± standard deviation (SD).

Results and observations

Method development

A robust and reproducible High-Performance Thin-Layer Chromatography (HPTLC) method was successfully developed and optimized for the simultaneous quantification of five key bioactive markers—Andrographolide, Piperine, Picroside-I, Picroside-II, and α-Cyperone—in the Ayurvedic polyherbal formulation Jwarahara Kwatha Choornam (JKC). Multiple mobile phase combinations were systematically evaluated to achieve optimal chromatographic performance. The mobile phase comprising Toluene: Ethyl Acetate: Methanol: Formic Acid in the ratio of 4:4:1:1 (v/v/v/v) was found to offer superior resolution, well-defined peak separation, and minimal baseline interference for all five analytes. The selection and precise ratio of solvents were critical to attaining distinct, non-overlapping Rf values, thereby ensuring accurate and simultaneous detection of the marker compounds within the complex polyherbal matrix.

Method validation

The calibration curves for all five bioactive standards—Andrographolide, Piperine, Picroside-I, Picroside-II, and α-Cyperone—demonstrated excellent linearity across the tested concentration ranges. The correlation coefficients (r²) for each marker compound ranged between 0.97 and 0.99, confirming a strong linear relationship between analyte concentration and corresponding peak area (Table 2). These results validate the method’s suitability for precise and quantitative analysis within the defined range, a critical requirement for the standardization of complex polyherbal formulations.

Table 2 Calibration parameters of 05 bioactive markers.
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Specificity, selectivity

The specificity of the developed HPTLC method was established by comparing the retention factor (Rf) values and peak purity profiles of the standard compounds with those observed in the Jwarahara Kwatha Choornam (JKC) formulation. The Rf values for Andrographolide (0.563 ± 0.005), Piperine (0.706 ± 0.015), Picroside-I (0.280 ± 0.0173), Picroside-II (0.180 ± 0.0115), and α-Cyperone (0.803 ± 0.005) were consistent across both standard and sample tracks, indicating the absence of co-eluting interfering substances and confirming the method’s high specificity. The spectral overlay and peak purity assessments further corroborated these findings. The corresponding 2D densitograms are presented in Figs. 3 and 4, while the chromatographic profile is illustrated in Fig. 2.

Fig. 3
figure 3

HPTLC Chromatograms Marker mix (refernce) and JKC (sample) @ 254 nm.

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Fig. 4
figure 4

HPTLC Chromatogram comparison of JKC sample 3 batches with the five bioactive marker mixture @ 254 nm.

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Precision

The precision of the developed HPTLC method was assessed through intra-day and inter-day analyses by evaluating standard solutions at three different concentration levels within the established linear range. Intra-day precision was determined by repeated measurements on the same day, while inter-day precision involved analysis across three consecutive days. The method exhibited excellent repeatability and intermediate precision, with relative standard deviation (RSD) values consistently below 2% for all five bioactive markers, demonstrating the method’s robustness and reliability (Tables 3 and 4). Representative TLC plate images from the precision studies are provided in Annexure I (Supplementary Data).

Table 3 Inter-day precision studies of the bioactive markers.
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Table 4 Intra-day precision studies of the bioactive markers.
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Accuracy

The accuracy of the developed HPTLC method was validated through standard addition (recovery) studies. Known quantities of each reference standard were spiked into the Jwarahara Kwatha Choornam (JKC) matrix, and the resulting mixtures were analyzed under optimized chromatographic conditions. The percentage recoveries for all five bioactive markers ranged between 94% and 104%, reflecting the method’s high analytical accuracy and minimal matrix interference (Table 5). These results confirm the method’s suitability for reliable quantification in complex polyherbal formulations.

Table 5 Accuracy studies of the bioactive markers with the JKC formulation.
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Robustness

The robustness of the developed HPTLC method was evaluated by introducing deliberate minor variations in analytical conditions, specifically the saturation time of the mobile phase in the twin-trough chamber (TTC) prior to plate development. The method consistently yielded RSD values below 2%, indicating that such variations did not significantly affect the analytical performance, thereby confirming its robustness. Detailed results of the robustness evaluations are summarized in Table 6.

Table 6 Robustness studies of the bioactive markers.
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Limit of detection (LOD) and limit of quantification (LOQ)

The limits of detection (LOD) and limits of quantification (LOQ) for the five bioactive markers are detailed in Table 2, underscoring the high sensitivity of the developed HPTLC method. Specifically, the LODs and LOQs were determined as follows: Andrographolide (LOD: 0.7682 µg, LOQ: 2.3279 µg), Piperine (LOD: 0.8477 µg, LOQ: 2.5690 µg), Picroside-I (LOD: 0.1867 µg, LOQ: 0.5658 µg), Picroside-II (LOD: 0.0690 µg, LOQ: 0.2091 µg), and α-Cyperone (LOD: 0.1503 µg, LOQ: 0.4553 µg). These low detection and quantification thresholds reflect the method’s exceptional capability to accurately detect and measure trace levels of key phytoconstituents, establishing its robustness and reliability for stringent quality control and analytical evaluation of complex polyherbal formulations.

Essay and method application

The validated HPTLC method was successfully applied for the quantitative analysis of Jwarahara Kwatha Choornam (JKC) across three independent batches. The concentrations of the five targeted bioactive markers—Andrographolide (AG), Piperine (PP), Picroside-I (P-I), Picroside-II (P-II), and α-Cyperone (AC)—were found to be within the expected ranges, confirming the compositional consistency and quality of the formulation. In Batch-1, the concentrations (expressed as mean ± SD, n = 3) were determined to be: AG – 3.6384 ± 0.0234 mg/g, PP – 3.3604 ± 0.0792 mg/g, P-I – 0.1426 ± 0.0031 mg/g, P-II – 0.6025 ± 0.0198 mg/g, and AC – 0.2102 ± 0.0024 mg/g. Comparable results were obtained for Batch-2 and Batch-3, reinforcing the formulation’s batch-to-batch uniformity (Table 7). Furthermore, the 2D-densitograms of all three batches closely mirrored the reference marker mixture, supporting the analytical method’s specificity and the formulation’s consistency (Fig. 4). All analyses were performed in triplicate, and results are reported as mean ± standard deviation.

Table 7 Presence of bioactive markers in three batches of JKC formulation.
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Discussion

This study successfully developed and validated a novel High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous estimation of five bioactive markers—Andrographolide (AG), Piperine (PP), Picroside-I (P-I), Picroside-II (P-II), and α-Cyperone (AC)—in Jwarahara Kwatha Choornam (JKC), a classical Ayurvedic polyherbal formulation. The comprehensive validation, performed according to standard analytical guidelines, confirmed the method’s reliability, sensitivity, and precision for use in routine quality control.

All key validation parameters—including linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, robustness, and ruggedness—were systematically evaluated. The calibration plots for all five markers demonstrated excellent linearity (r² > 0.97), indicating a strong correlation between analyte concentration and peak area. The low LODs and LOQs further emphasized the method’s high sensitivity, enabling detection and quantification of marker compounds even at trace levels. This is particularly critical in the context of herbal formulations, where active constituents are often present in low concentrations yet play a vital therapeutic role.

The robustness of the method was affirmed by intentionally varying experimental parameters, such as the mobile phase saturation time, without observing significant deviations in results (RSD < 2%). These findings validate the method’s suitability for deployment in quality control laboratories where operational variability is a common challenge.

Application of the validated method to three independent batches of JKC revealed consistent concentrations of the bioactive markers, with values in Batch-1 (AG: 3.6384 ± 0.0234 mg/g, PP: 3.3604 ± 0.0792 mg/g, P-I: 0.1426 ± 0.0031 mg/g, P-II: 0.6025 ± 0.0198 mg/g, AC: 0.2102 ± 0.0024 mg/g) closely matching those observed in Batch-2 and Batch-3. This batch-to-batch uniformity underscores the reliability of the manufacturing process and supports the use of this method as a robust tool for ensuring formulation consistency.

Moreover, the method’s capability for simultaneous multi-marker analysis offers significant advantages in terms of efficiency and cost-effectiveness. Its rapid execution and minimal sample preparation requirements make it particularly attractive for large-scale routine analysis in the herbal pharmaceutical industry. The method holds potential for broader application in the standardization of other formulations containing similar phytochemical constituents, thereby contributing to enhanced quality control and regulatory compliance in traditional medicine systems.

A comparative analysis with previous literature further supports the novelty and utility of the developed method. For instance, Singh et al. (2013) reported Rf values of 0.60 and 0.43 for P-I and P-II, respectively, using a chloroform: methanol (82:18 v/v) mobile phase. In contrast, the present study optimized a different solvent system (Toluene: Ethyl Acetate: Methanol: Formic Acid, 4:4:1:1 v/v/v/v), yielding distinct and reproducible Rf values (0.28 for P-I and 0.18 for P-II). This highlights the critical role of mobile phase composition in achieving optimal resolution and reinforces the importance of tailoring chromatographic conditions to the complexity of polyherbal matrices.

Comparative analysis with previously published studies underscores the advancements achieved through the current HPTLC method developed for Jwarahara Kwatha Choornam (JKC). Hazra et al. (2019) reported the quantification of Piperine in various Ayurvedic churnas using a mobile phase composed of Toluene: Ethyl Acetate (7:3 v/v), with detection at 342 nm and an Rf value of 0.3937. In contrast, our optimized method identified Piperine at 343 nm with an Rf value of 0.706, demonstrating enhanced resolution and separation efficiency. Likewise, Rajopadhye et al. (2012) simultaneously estimated Piperine and Piperlongumine in Piper longum root extracts, reporting an Rf value of 0.51 for Piperine38. These discrepancies in Rf values highlight the influence of mobile phase composition, matrix complexity, and detection parameters on compound migration and detection, thus reinforcing the need for tailored method development for specific formulations.

Similarly, Jain et al. (2010) reported Andrographolide (AG) detection in Andrographis paniculata at 229 nm with an Rf of 0.49 using a Chloroform: Toluene: Methanol (66:26:8 v/v) solvent system39. In contrast, our current method detected AG at an Rf of 0.563 under different chromatographic conditions, indicating that our optimized solvent system—Toluene: Ethyl Acetate: Methanol: Formic Acid (4:4:1:1 v/v/v/v)—offers improved separation and potentially greater analytical reproducibility.

Most prior studies have focused on the single or dual estimation of phytochemical markers, limiting their applicability for complex polyherbal formulations. The current study bridges this gap by enabling the simultaneous quantification of five pharmacologically significant bioactive markers—Picroside-I (P-I), Picroside-II (P-II), Piperine (PP), Andrographolide (AG), and α-Cyperone (AC)—within a single chromatographic run. The Rf values obtained for P-II, P-I, AG, PP, and AC were 0.18 ± 0.0115, 0.28 ± 0.0173, 0.563 ± 0.005, 0.706 ± 0.015, and 0.803 ± 0.005, respectively. These values were consistent across all three JKC batches, with spectral analysis confirming 100% concordance between the marker standards and corresponding bands in the formulation (Fig. 5a to e), thereby validating the specificity of the method.

Fig. 5
figure 5

a HPTLC spectral comparison with JKC formulation with similar Rf of AG (Andrographolide). b HPTLC spectral comparison with JKC formulation with similar Rf of PP (Piperine). c HPTLC spectral comparison with JKC formulation with similar Rf of P-I (Picroside-I). d HPTLC spectral comparison with JKC formulation with similar Rf of P-II (Picroside-II). e HPTLC spectral comparison with JKC formulation with similar Rf of AC (Alpha-Cyperone).

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The capability of this method to estimate multiple markers both simultaneously and individually offers a robust, high-throughput analytical strategy that enhances efficiency in routine quality assessment. It significantly reduces time and resource expenditure compared to conventional single-marker assays, without compromising accuracy or sensitivity. This is particularly beneficial for formulations like JKC, which incorporate multiple botanicals, each contributing specific pharmacological actions.

Moreover, the demonstrated robustness and ruggedness of the method—evidenced by low RSD values under varied conditions and across different analysts—support its broad applicability in diverse laboratory environments. These features enhance method transferability and facilitate its implementation in routine quality control settings.

Importantly, this work aligns with modern regulatory requirements for analytical method validation and quality assurance in herbal drug development. It provides a scientifically validated framework that not only ensures the standardization of JKC but also sets a precedent for the quality control of other complex Ayurvedic formulations. The approach is instrumental in safeguarding therapeutic efficacy, minimizing batch-to-batch variability, and mitigating adulteration risks—challenges that continue to affect the traditional medicine sector.

The current HPTLC method represents a significant advancement in phytopharmaceutical analysis, combining analytical rigor with practical utility, and holds considerable promise for widespread adoption in both research and industrial quality assurance workflows.

Conclusions

The development and validation of a HPTLC method for quantifying Andrographolide, Piperine, Picroside-I, Picroside-II, and α-Cyperone in Jwarahara Kwatha Choornam (JKC) represents a significant advancement in the standardization of Ayurvedic formulations. This method demonstrates high sensitivity, specificity, linearity, precision, and accuracy, making it robust for routine quality control. Ensuring consistent identification and quantification of key phytoconstituents, it enhances the quality, safety, and efficacy of JKC, supporting traditional claims and meeting regulatory standards for herbal medicines. Furthermore, this study showcases HPTLC as a cost-effective tool for standardizing other multi-herb formulations in Ayurveda, bridging traditional knowledge and modern pharmaceutical practices. Future work on stability studies and method transfer will further validate this approach, advancing the scientific integrity of Ayurvedic therapeutics.